G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
v-raf-1 murine leukemia viral oncogene homolog 1
G00000185 (Mus musculus)

Databases (7)

ENSG00000132155 (Ensembl human gene)
5894 (Entrez Gene)
48 (G2Cdb plasticity & disease)
RAF1 (GeneCards)
164760 (OMIM)
Marker Symbol
HGNC:9829 (HGNC)
Protein Sequence
P04049 (UniProt)

Synonyms (3)

  • CRAF
  • Raf-1
  • c-Raf

Diseases (4)

Disease Nervous effect Mutations Found Literature Mutations Type Genetic association?
D00000180: Gilles de la Tourette syndrome Y Y (8902885) Translocation (T) N
D00000028: Lung cancer N Y (7888083) No mutation found (N) N
D00000063: Urinary bladder cancer N Y (11389083) Deletion (D) Y
D00000177: Autism Y Y (16648338) Single nucleotide polymorphism (SNP) N
D00000177: Autism Y Y (16263864) Single nucleotide polymorphism (SNP) Y
D00000177: Autism Y Y (16205742) Single nucleotide polymorphism (SNP) N
D00000177: Autism Y Y (15056512) Single nucleotide polymorphism (SNP) Y


  • Lack of association between autism and SLC25A12.

    Rabionet R, McCauley JL, Jaworski JM, Ashley-Koch AE, Martin ER, Sutcliffe JS, Haines JL, DeLong GR, Abramson RK, Wright HH, Cuccaro ML, Gilbert JR and Pericak-Vance MA

    Center for Human Genetics, Department of Medicine, Duke University Medical Center, 595 LaSalle St., Durham, NC 27710, USA.

    Objective: Autism has a strong, complex genetic component, most likely involving several genes. Multiple genomic screens have shown evidence suggesting linkage to chromosome 2q31-q33, which includes the SLC25A12 gene. Recently, an association between autism risk and two single nucleotide polymorphisms (SNPs) in SLC25A12 was reported. This study aimed to test for association in SLC25A12 in an independent data set of 327 families with autistic offspring.

    Method: The authors analyzed two SNPs that were significant in the previous study group, as well as seven additional SNPs within the gene. Association analyses for individual SNPs as well as haplotypes were performed.

    Results: There was no evidence of an association between SLC25A12 and autism.

    Conclusions: These results suggest that SLC25A12 is not a major contributor to autism risk in these families.

    Funded by: NCRR NIH HHS: RR-00095; NINDS NIH HHS: NS-26630, NS-36768

    The American journal of psychiatry 2006;163;5;929-31

  • SLC25A12 and CMYA3 gene variants are not associated with autism in the IMGSAC multiplex family sample.

    Blasi F, Bacchelli E, Carone S, Toma C, Monaco AP, Bailey AJ, Maestrini E and International Molecular Genetic Study of Autism Consortium (IMGSAC)

    Department of Biology, University of Bologna, Bologna, Italy.

    Autism is a severe neurodevelopmental disorder with a complex genetic predisposition. Linkage findings from several genome scans suggest the presence of an autism susceptibility locus on chromosome 2q24-q33, making this region the focus of candidate gene and association studies. Recently, significant association with autism has been reported for single-nucleotide polymorphisms (SNPs) in the SLC25A12 and CMYA3 genes on chromosome 2q. We attempted to replicate these findings in the collection of families from the International Molecular Genetic Study of Autism Consortium (IMGSAC), using the transmission disequilibrium test and case-control comparison. Our study failed to reveal any significant association for the SNPs tested at either locus, suggesting that these variants are unlikely to play a major role in genetic susceptibility to autism in our sample.

    Funded by: Telethon: GGP030227; Wellcome Trust

    European journal of human genetics : EJHG 2006;14;1;123-6

  • Confirmation of association between autism and the mitochondrial aspartate/glutamate carrier SLC25A12 gene on chromosome 2q31.

    Segurado R, Conroy J, Meally E, Fitzgerald M, Gill M and Gallagher L

    Department of Genetics, Trinity College, Dublin 2, Ireland. rsegurdo@tcd.ie

    Objective: Autism is a neurodevelopmental disorder with childhood onset and a known major genetic component. A recent study identified a highly significant association between autism and a two-single-nucleotide-polymorphism haplotype in the SLC25A12 gene, with a homozygote genotype relative risk between 2.4 and 4.8. The authors' goal was to investigate this association with autism in Irish affected child-parent trios because replication in an independent sample is essential in the validation of such potentially important findings.

    Method: Markers rs2056202 and rs2292813 were genotyped in a total of 158 trios (442 individuals). The Transmission Disequilibrium Test was used to examine these markers for association with autism.

    Results: In agreement with the recent study, the authors found significant association between autism and the C alleles of both rs2056202 and rs2292813 as well as the two-marker haplotype.

    Conclusions: These findings provide replication of the association between autism and SLC25A12.

    The American journal of psychiatry 2005;162;11;2182-4

  • Linkage and association of the mitochondrial aspartate/glutamate carrier SLC25A12 gene with autism.

    Ramoz N, Reichert JG, Smith CJ, Silverman JM, Bespalova IN, Davis KL and Buxbaum JD

    Laboratory of Molecular Neuropsychiatry, Department of Psychiatry, Mount Sinai School of Medicine, New York, NY 10029, USA.

    Objective: Autism/autistic disorder (MIM number 209850) is a complex, largely genetic psychiatric disorder. The authors recently mapped a susceptibility locus for autism to chromosome region 2q24-q33 (MIM number 606053). In the present study, genes across the 2q24-q33 interval were analyzed to identify an autism susceptibility gene in this region.

    Method: Mutation screening of positional candidate genes was performed in two stages. The first stage involved identifying, in unrelated subjects showing linkage to 2q24-q33, genetic variants in exons and flanking sequence within candidate genes and comparing the frequency of the variants between autistic and unrelated nonautistic subjects. Two single nucleotide polymorphisms (SNPs) that showed evidence for divergent distribution between autistic and nonautistic subjects were identified, both within SLC25A12, a gene encoding the mitochondrial aspartate/glutamate carrier (AGC1). In the second stage, the two SNPs in SLC25A12 were further genotyped in 411 autistic families, and linkage and association tests were carried out in the 197 informative families.

    Results: Linkage and association were observed between autistic disorder and the two SNPs, rs2056202 and rs2292813, found in SLC25A12. Using either a single affected subject per family or all affected subjects, evidence for excess transmission was found by the Transmission Disequilibrium Test for rs2056202, rs2292813, and a two-locus G*G haplotype. Similar results were observed using TRANSMIT for the analyses. Evidence for linkage was supported by linkage analysis with the two SNPs, with a maximal multipoint nonparametric linkage score of 1.57 and a maximal multipoint heterogeneity lod score of 2.11. Genotype relative risk could be estimated to be between 2.4 and 4.8 for persons homozygous at these loci.

    Conclusions: A strong association of autism with SNPs within the SLC25A12 gene was demonstrated. Further studies are needed to confirm this association and to decipher any potential etiological role of AGC1 in autism.

    Funded by: NIMH NIH HHS: U54 MH066673; OMHHE CDC HHS: MN-066673

    The American journal of psychiatry 2004;161;4;662-9

  • High-throughput tissue microarray analysis of 3p25 (RAF1) and 8p12 (FGFR1) copy number alterations in urinary bladder cancer.

    Simon R, Richter J, Wagner U, Fijan A, Bruderer J, Schmid U, Ackermann D, Maurer R, Alund G, Knönagel H, Rist M, Wilber K, Anabitarte M, Hering F, Hardmeier T, Schönenberger A, Flury R, Jäger P, Fehr JL, Schraml P, Moch H, Mihatsch MJ, Gasser T and Sauter G

    Institute for Pathology and Urologic Clinics, University of Basel, Schoenbeinstrasse 40, CH-4031 Basel, Switzerland.

    Studies by comparative genomic hybridization revealed that the chromosomal regions 3p25 and 8p11-p12 are recurrently amplified in bladder cancer. To investigate the prevalence of DNA copy number alterations in these chromosomal regions and study their clinical significance, we used probes for the RAF1 (3p25) and FGFR1 (8p12) genes for fluorescence in situ hybridization. A tissue microarray containing 2317 tumors was analyzed. The analysis revealed RAF1 amplification in 4.0% and FGFR1 amplification in 3.4% of interpretable tumors. In addition, deletions were found at the 3p25 locus in 2.2% and at the 8p11-12 locus in 9.9% of interpretable tumors. Both amplifications and deletions of RAF1 and FGFR1 were significantly associated with high tumor grade (P < 0.0001), advanced stage (P < 0.0001), and poor survival (P < 0.05) if tumors of all of the stages where analyzed together. RAF1 amplifications were associated with subsequent tumor progression in pT1 carcinomas (P < 0.05). The marked differences in the frequency of all of the analyzed changes between pTa grade 1/grade 2 and pT1-4 carcinomas support the concept of these tumor groups representing different tumor entities.

    Cancer research 2001;61;11;4514-9

  • Linkage analysis and exclusion of regions of chromosomes 3 and 8 in Gilles de la Tourette syndrome following the identification of a balanced reciprocal translocation 46 XY, t(3:8)(p21.3 q24.1) in a case of Tourette syndrome.

    Brett PM, Curtis D, Robertson MM, Dahlitz M and Gurling HM

    Academic Department of Psychiatry, University College London Medical School, UK.

    Gilles de la Tourette syndrome (GTS) and related disorders such as chronic multiple tics and obsessive compulsive behaviour are likely to be genetically transmitted with a Mendelian autosomal dominant mode of transmission. Following our discovery of a patient with GTS who also carried a balanced translocation 46 XY, t(3:8) (p21.3 q24.1), a linkage study of several families was performed covering the areas on chromosomes 3 and 8 implicated by the cytogenetic abnormality in this unique GTS patient. A positive multipoint lod score of 2.9 was obtained on chromosome 3 with markers at the loci RAF1, THRB and D3S11. Subsequently, the genetic map of this region was improved and new polymorphic markers close to our original three markers were identified. With the new map the maximum two-point lod with any marker was reduced to 1.77 at RAF1, and the FASTMAP approximate multipoint lod excluded the likely region of the breakpoint. After constructing a somatic cell hybrid, the original three markers were mapped relative to the break point of the translocation and to other new markers. It was confirmed that the original markers were at least 20 cM away from the position of the break point. In addition, we traced further family members of our translocation GTS proband, and identified affected individuals who did not possess the translocation. We concluded that the translocation was not responsible for the GTS symptoms in our affected proband.

    Psychiatric genetics 1996;6;3;99-105

  • Absence of activating mutations of the RAF1 protooncogene in human lung cancer.

    Miwa W, Yasuda J, Yashima K, Makino R and Sekiya T

    Oncogene Division, National Cancer Center Research Institute, Tokyo, Japan.

    Recently, the RAF protein has been demonstrated to be a direct effector of RAS protein in a RAS-mediated signal transduction pathway. Activations of the RAF1 gene by small mutations, such as point mutations in the kinase domain and a tetrapeptide insertion into conserved region 2, have been suggested from analyses of chemically induced lung cancers in mice and by site-directed mutagenesis. We investigated the presence of small mutations of the RAF1 gene in human lung carcinomas, especially in those not carrying the mutated RAS gene, expecting that aberrations of the RAF1 gene might play a role complementary to RAS gene mutations in tumorigenesis. Single-strand conformation polymorphism (SSCP) analysis of polymerase chain reaction products of DNA samples from 140 patients revealed no tumor specific mutations of the RAF1 gene in any of these specimens. This result suggests that mutations of the RAF1 gene are not involved in tumorigenesis in human lung.

    Biological chemistry Hoppe-Seyler 1994;375;10;705-9

Literature (312)

Pubmed - human_disease

  • SLC25A12 and CMYA3 gene variants are not associated with autism in the IMGSAC multiplex family sample.

    Blasi F, Bacchelli E, Carone S, Toma C, Monaco AP, Bailey AJ, Maestrini E and International Molecular Genetic Study of Autism Consortium (IMGSAC)

    Department of Biology, University of Bologna, Bologna, Italy.

    Autism is a severe neurodevelopmental disorder with a complex genetic predisposition. Linkage findings from several genome scans suggest the presence of an autism susceptibility locus on chromosome 2q24-q33, making this region the focus of candidate gene and association studies. Recently, significant association with autism has been reported for single-nucleotide polymorphisms (SNPs) in the SLC25A12 and CMYA3 genes on chromosome 2q. We attempted to replicate these findings in the collection of families from the International Molecular Genetic Study of Autism Consortium (IMGSAC), using the transmission disequilibrium test and case-control comparison. Our study failed to reveal any significant association for the SNPs tested at either locus, suggesting that these variants are unlikely to play a major role in genetic susceptibility to autism in our sample.

    Funded by: Telethon: GGP030227; Wellcome Trust

    European journal of human genetics : EJHG 2006;14;1;123-6

  • Confirmation of association between autism and the mitochondrial aspartate/glutamate carrier SLC25A12 gene on chromosome 2q31.

    Segurado R, Conroy J, Meally E, Fitzgerald M, Gill M and Gallagher L

    Department of Genetics, Trinity College, Dublin 2, Ireland. rsegurdo@tcd.ie

    Objective: Autism is a neurodevelopmental disorder with childhood onset and a known major genetic component. A recent study identified a highly significant association between autism and a two-single-nucleotide-polymorphism haplotype in the SLC25A12 gene, with a homozygote genotype relative risk between 2.4 and 4.8. The authors' goal was to investigate this association with autism in Irish affected child-parent trios because replication in an independent sample is essential in the validation of such potentially important findings.

    Method: Markers rs2056202 and rs2292813 were genotyped in a total of 158 trios (442 individuals). The Transmission Disequilibrium Test was used to examine these markers for association with autism.

    Results: In agreement with the recent study, the authors found significant association between autism and the C alleles of both rs2056202 and rs2292813 as well as the two-marker haplotype.

    Conclusions: These findings provide replication of the association between autism and SLC25A12.

    The American journal of psychiatry 2005;162;11;2182-4

  • High-throughput tissue microarray analysis of 3p25 (RAF1) and 8p12 (FGFR1) copy number alterations in urinary bladder cancer.

    Simon R, Richter J, Wagner U, Fijan A, Bruderer J, Schmid U, Ackermann D, Maurer R, Alund G, Knönagel H, Rist M, Wilber K, Anabitarte M, Hering F, Hardmeier T, Schönenberger A, Flury R, Jäger P, Fehr JL, Schraml P, Moch H, Mihatsch MJ, Gasser T and Sauter G

    Institute for Pathology and Urologic Clinics, University of Basel, Schoenbeinstrasse 40, CH-4031 Basel, Switzerland.

    Studies by comparative genomic hybridization revealed that the chromosomal regions 3p25 and 8p11-p12 are recurrently amplified in bladder cancer. To investigate the prevalence of DNA copy number alterations in these chromosomal regions and study their clinical significance, we used probes for the RAF1 (3p25) and FGFR1 (8p12) genes for fluorescence in situ hybridization. A tissue microarray containing 2317 tumors was analyzed. The analysis revealed RAF1 amplification in 4.0% and FGFR1 amplification in 3.4% of interpretable tumors. In addition, deletions were found at the 3p25 locus in 2.2% and at the 8p11-12 locus in 9.9% of interpretable tumors. Both amplifications and deletions of RAF1 and FGFR1 were significantly associated with high tumor grade (P < 0.0001), advanced stage (P < 0.0001), and poor survival (P < 0.05) if tumors of all of the stages where analyzed together. RAF1 amplifications were associated with subsequent tumor progression in pT1 carcinomas (P < 0.05). The marked differences in the frequency of all of the analyzed changes between pTa grade 1/grade 2 and pT1-4 carcinomas support the concept of these tumor groups representing different tumor entities.

    Cancer research 2001;61;11;4514-9

  • Linkage analysis and exclusion of regions of chromosomes 3 and 8 in Gilles de la Tourette syndrome following the identification of a balanced reciprocal translocation 46 XY, t(3:8)(p21.3 q24.1) in a case of Tourette syndrome.

    Brett PM, Curtis D, Robertson MM, Dahlitz M and Gurling HM

    Academic Department of Psychiatry, University College London Medical School, UK.

    Gilles de la Tourette syndrome (GTS) and related disorders such as chronic multiple tics and obsessive compulsive behaviour are likely to be genetically transmitted with a Mendelian autosomal dominant mode of transmission. Following our discovery of a patient with GTS who also carried a balanced translocation 46 XY, t(3:8) (p21.3 q24.1), a linkage study of several families was performed covering the areas on chromosomes 3 and 8 implicated by the cytogenetic abnormality in this unique GTS patient. A positive multipoint lod score of 2.9 was obtained on chromosome 3 with markers at the loci RAF1, THRB and D3S11. Subsequently, the genetic map of this region was improved and new polymorphic markers close to our original three markers were identified. With the new map the maximum two-point lod with any marker was reduced to 1.77 at RAF1, and the FASTMAP approximate multipoint lod excluded the likely region of the breakpoint. After constructing a somatic cell hybrid, the original three markers were mapped relative to the break point of the translocation and to other new markers. It was confirmed that the original markers were at least 20 cM away from the position of the break point. In addition, we traced further family members of our translocation GTS proband, and identified affected individuals who did not possess the translocation. We concluded that the translocation was not responsible for the GTS symptoms in our affected proband.

    Psychiatric genetics 1996;6;3;99-105

  • Absence of activating mutations of the RAF1 protooncogene in human lung cancer.

    Miwa W, Yasuda J, Yashima K, Makino R and Sekiya T

    Oncogene Division, National Cancer Center Research Institute, Tokyo, Japan.

    Recently, the RAF protein has been demonstrated to be a direct effector of RAS protein in a RAS-mediated signal transduction pathway. Activations of the RAF1 gene by small mutations, such as point mutations in the kinase domain and a tetrapeptide insertion into conserved region 2, have been suggested from analyses of chemically induced lung cancers in mice and by site-directed mutagenesis. We investigated the presence of small mutations of the RAF1 gene in human lung carcinomas, especially in those not carrying the mutated RAS gene, expecting that aberrations of the RAF1 gene might play a role complementary to RAS gene mutations in tumorigenesis. Single-strand conformation polymorphism (SSCP) analysis of polymerase chain reaction products of DNA samples from 140 patients revealed no tumor specific mutations of the RAF1 gene in any of these specimens. This result suggests that mutations of the RAF1 gene are not involved in tumorigenesis in human lung.

    Biological chemistry Hoppe-Seyler 1994;375;10;705-9

Pubmed - other

  • Tumor spectrum in children with Noonan syndrome and SOS1 or RAF1 mutations.

    Denayer E, Devriendt K, de Ravel T, Van Buggenhout G, Smeets E, Francois I, Sznajer Y, Craen M, Leventopoulos G, Mutesa L, Vandecasseye W, Massa G, Kayserili H, Sciot R, Fryns JP and Legius E

    Department of Human Genetics, University of Leuven, Leuven, Belgium.

    Noonan syndrome (NS) is an autosomal dominant disorder caused by mutations in PTPN11, KRAS, SOS1, and RAF1. We performed SOS1, RAF1, BRAF, MEK1, and MEK2 mutation analysis in a cohort of 102 PTPN11- and KRAS-negative NS patients and found pathogenic SOS1 mutations in 10, RAF1 mutations in 4, and BRAF mutations in 2 patients. Three novel SOS1 mutations were found. One was classified as a rare benign variant and the other remains unclassified. We confirm a high prevalence of pulmonic stenosis and ectodermal abnormalities in SOS1-positive patients. Three patients with SOS1 mutations presented with tumors (embryonal rhabdomyosarcoma, Sertoli cell testis tumor, and granular cell tumors of the skin). One patient with a RAF1 mutation had a lesion suggestive for a giant cell tumor. This is the first report describing different tumor types in NS patients with germ line SOS1 mutations.

    Genes, chromosomes & cancer 2010;49;3;242-52

  • Proapoptotic kinase MST2 coordinates signaling crosstalk between RASSF1A, Raf-1, and Akt.

    Romano D, Matallanas D, Weitsman G, Preisinger C, Ng T and Kolch W

    Proteomics and Signalling Networks Group, The Beatson Institute for Cancer Research, London, United Kingdom.

    Mammalian MST kinases function in stress-induced apoptosis to limit tumor progression. However, there is limited understanding about MST2 control by key regulators of cell division and survival. Raf-1 binds and inhibits MST2 kinase, whereas dissociation from Raf-1 and binding to tumor suppressor protein RASSF1A activates MST2. Akt phosphorylates MST2 in response to mitogens, oncogenic Ras, or depletion of tumor suppressor phosphatase and tensin homologue deleted on chromosome 10. We identified T117 and T384 as Akt phosphorylation sites in MST2. Mutation of these sites inhibited MST2 binding to Raf-1 kinase but enhanced binding to tumor suppressor RASSF1A, accentuating downstream c-Jun NH(2)-terminal kinase and p38 mitogen-activated protein kinase signaling and promoting apoptosis. We determined that MST2 phosphorylation by Akt limits MST2 activity in two ways: first, by blocking its binding to RASSF1A and by promoting its association into the Raf-1 inhibitory complex, and second, by preventing homodimerization of MST2, which is needed for its activation. Dissociation of the Raf-1-MST2 complex promoted mitogenic signaling and coordinately licensed apoptotic risk. Using Ras effector domain mutants, we found that Akt is essential to prevent MST2 activation after mitogenic stimulation. Our findings elucidate how MST2 serves as a hub to integrate biological outputs of the Raf-1 and Akt pathways.

    Funded by: Cancer Research UK: A3677

    Cancer research 2010;70;3;1195-203

  • BAG-1 overexpression attenuates luminal apoptosis in MCF-10A mammary epithelial cells through enhanced RAF-1 activation.

    Anderson LR, Sutherland RL and Butt AJ

    Cancer Research Program, Garvan Institute of Medical Research, Darlinghurst, Sydney, New South Wales 2010, Australia.

    Although the multi-functional, prosurvival protein, Bcl-2-associated anthanogene 1 (BAG-1) is frequently overexpressed in breast cancers, its role in the development or maintenance of the malignant state remains unclear. Here, we have used the established MCF-10A 3-dimensional (3D) model of mammary morphogenesis as a biologically relevant system to determine how BAG-1 expression may influence the development of breast cancer. When cultured in 3D, MCF-10A cells undergo a highly regulated morphogenic program leading to the development of polarized acinar structures containing a central, hollow lumen formed, in part, through the induction of BIM-dependent apoptosis. BAG-1 overexpression resulted in an attenuation of this normal apoptotic program characterized by a significantly increased number of acini with filled lumens-a phenotype commonly observed in ductal carcinoma in situ. BAG-1's effects were associated with an activation of RAF-1-a known binding partner of BAG-1, enhanced signaling through the MAP kinase pathway and a decrease in BIM expression. Reversal of the BAG-1-associated survival phenotype by the mitogen-activated kinase/ERK kinase inhibitor, U0126, implicates the RAF-1-extracellular signal-regulated kinase signaling pathway as a major mediator of BAG-1's effects in this model. As BAG-1 expression is often elevated in preinvasive breast cancers, these findings support a possible role for BAG-1 as an early contributor to the malignant process in the breast.

    Oncogene 2010;29;4;527-38

  • c-Kit mutants require hypoxia-inducible factor 1alpha to transform melanocytes.

    Monsel G, Ortonne N, Bagot M, Bensussan A and Dumaz N

    INSERM U976, Hôpital Saint Louis, Paris, France.

    Many studies have highlighted the critical role of c-Kit in normal melanocyte development but its role in melanoma development remains unclear. Although c-Kit expression is often lost during melanoma progression, a subset of melanoma has been found to overexpress c-Kit and mutations activating c-Kit have recently been identified in some acral and mucosal melanoma. To address the role of these c-Kit mutants in the transformation of melanocytes, we characterized the physiological responses of melanocytes expressing the most frequent c-Kit mutants found in melanoma (K642E and L576P) and a novel mutant we identified in an acral melanoma. We analysed signaling pathways activated downstream of c-Kit and showed that all three mutants led to a strong activation of the phosphatidyl-inositol-3 kinase (PI3K) pathway but only weak activation of the Ras/Raf/Mek/Erk pathway, which was not sufficient to promote uncontrolled melanocyte proliferation and transformation. However, in hypoxic conditions or coexpressed with a constitutively active form of hypoxia-inducible factor 1alpha (HIF-1alpha), c-Kit mutants activate the Ras/Raf/Mek/Erk pathway, stimulate proliferation and transform melanocytes. Proliferation of melanocytes transformed by these mutants was specifically inhibited by imatinib. These results show for the first time that melanocytes require a specific epigenetic environment to be transformed by c-Kit mutants and highlight a distinct molecular mechanism of melanocyte transformation.

    Oncogene 2010;29;2;227-36

  • Semaphorin 4D signaling requires the recruitment of phospholipase C gamma into the plexin-B1 receptor complex.

    Swiercz JM, Worzfeld T and Offermanns S

    Institute of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, D-69120 Heidelberg, Germany. jakub.swiercz@mpi-bn.mpg.de

    The semaphorin 4D (Sema4D) receptor plexin-B1 constitutively interacts with particular Rho guanine nucleotide exchange factors (RhoGEFs) and thereby mediates Sema4D-induced RhoA activation, a process which involves the tyrosine phosphorylation of plexin-B1 by ErbB-2. It is, however, unknown how plexin-B1 phosphorylation regulates RhoGEF activity. We show here that activation of plexin-B1 by Sema4D and its subsequent tyrosine phosphorylation creates docking sites for the SH2 domains of phospholipase Cgamma (PLCgamma). PLCgamma is thereby recruited into the plexin-B1 receptor complex and via its SH3 domain activates the Rho guanine nucleotide exchange factor PDZ-RhoGEF. PLCgamma-dependent RhoGEF activation is independent of its lipase activity. The recruitment of PLCgamma has no effect on the R-Ras GTPase-activating protein activity of plexin-B1 but is required for Sema4D-induced axonal growth cone collapse as well as for the promigratory effects of Sema4D on cancer cells. These data demonstrate a novel nonenzymatic function of PLCgamma as an important mechanism of plexin-mediated signaling which links tyrosine phosphorylation of plexin-B1 to the regulation of a RhoGEF protein and downstream cellular processes.

    Molecular and cellular biology 2009;29;23;6321-34

  • C-Raf inhibits MAPK activation and transformation by B-Raf(V600E).

    Karreth FA, DeNicola GM, Winter SP and Tuveson DA

    Li Ka Shing Centre, Cambridge Research Institute, Cancer Research UK, Robinson Way, Cambridge CB2 0RE, UK.

    Activating B-Raf mutations that deregulate the MAPK pathway commonly occur in cancer. Whether additional proteins modulate the enzymatic activity of oncogenic B-Raf is unknown. Here we show that the proto-oncogene C-Raf paradoxically inhibits B-Raf(V600E) kinase activity through the formation of B-Raf(V600E)-C-Raf complexes. Although all Raf family members associate with oncogenic B-Raf, this inhibitory effect is specific to C-Raf. Indeed, a B-Raf(V600E) isoform with impaired ability to interact with C-Raf exhibits elevated oncogenic potential. Human melanoma cells expressing B-Raf(V600E) display a reduced C-Raf:B-Raf ratio, and further suppression of C-Raf increases MAPK activation and proliferation. Conversely, ectopic C-Raf expression lowers ERK phosphorylation and proliferation. Moreover, both oncogenic Ras and Sorafenib stabilize B-Raf(V600E)-C-Raf complexes, thereby impairing MAPK activation. This inhibitory function of C-Raf on B-Raf(V600E)-mediated MAPK activation may explain the lack of co-occurrence of B-Raf(V600E) and oncogenic Ras mutations, and influence the successful clinical development of small molecule inhibitors for B-Raf(V600E)-driven cancers.

    Funded by: Cancer Research UK

    Molecular cell 2009;36;3;477-86

  • The C-terminus of Raf-1 acts as a 14-3-3-dependent activation switch.

    Dhillon AS, Yip YY, Grindlay GJ, Pakay JL, Dangers M, Hillmann M, Clark W, Pitt A, Mischak H and Kolch W

    Department of Biochemistry and Molecular Biology, Bio21 Institute of Molecular Bioscience and Biotechnology, University of Melbourne, 30 Flemington Road, Parkville, VIC 3010, Australia. adhillon@unimelb.edu.au

    The Raf-1 protein kinase is a major activator of the ERK MAPK pathway, which links signaling by a variety of cell surface receptors to the regulation of cell proliferation, survival, differentiation and migration. Signaling by Raf-1 is regulated by a complex and poorly understood interplay between phosphorylation events and protein-protein interactions. One important mode of Raf-1 regulation involves the phosphorylation-dependent binding of 14-3-3 proteins. Here, we have examined the mechanism whereby the C-terminal 14-3-3 binding site of Raf-1, S621, controls the activation of MEK-ERK signaling. We show that phosphorylation of S621 turns over rapidly and is enriched in the activated pool of endogenous Raf-1. The phosphorylation on this site can be mediated by Raf-1 itself but also by other kinase(s). Mutations that prevent the binding of 14-3-3 proteins to S621 render Raf-1 inactive by specifically disrupting its capacity to bind to ATP, and not by gross conformational alteration as indicated by intact MEK binding. Phosphorylation of S621 correlates with the inhibition of Raf-1 catalytic activity in vitro, but 14-3-3 proteins can completely reverse this inhibition. Our findings suggest that 14-3-3 proteins function as critical cofactors in Raf-1 activation, which induce and maintain the protein in a state that is competent for both ATP binding and MEK phosphorylation.

    Funded by: Cancer Research UK

    Cellular signalling 2009;21;11;1645-51

  • Ras classical effectors: new tales from in silico complexes.

    Fuentes G and Valencia A

    Structural Computational Biology Group, Spanish National Cancer Research Center (CNIO), C/ Melchor Fernández Almagro, 3, 28029 Madrid, Spain. gfuentes@bii.a-star.edu.sg

    Components of signal transduction pathways have evolved as connected hubs, recognizing several binding partners with remarkable affinities and specificities. Ras is one of these hubs, sensitive to rapid and subtle changes, thus enabling the correct transfer of information. The dynamic nature of such systems makes their structural characterization challenging, despite the vast amount of experimental data available. These data, however, can be used as a restraint for generating comprehensive models of the association of Ras with its effectors. We believe that by following this type of approach, the derived 3D models can provide atomistic understanding of important biological issues, such as how Ras discriminates between the Ras binding domains of its various effectors. The modeled binding interfaces could be used as the starting points for selective modulations of interactions and pathways using small molecules, peptides or mutagenesis.

    Trends in biochemical sciences 2009;34;11;533-9

  • Diacylglycerol kinase eta augments C-Raf activity and B-Raf/C-Raf heterodimerization.

    Yasuda S, Kai M, Imai S, Takeishi K, Taketomi A, Toyota M, Kanoh H and Sakane F

    Department of Biochemistry, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo 060-8556.

    The Ras/B-Raf/C-Raf/MEK/ERK signaling cascade is critical for the control of many fundamental cellular processes, including proliferation, survival, and differentiation. This study demonstrated that small interfering RNA-dependent knockdown of diacylglycerol kinase eta (DGKeta) impaired the Ras/B-Raf/C-Raf/MEK/ERK pathway activated by epidermal growth factor (EGF) in HeLa cells. Conversely, the overexpression of DGKeta1 could activate the Ras/B-Raf/C-Raf/MEK/ERK pathway in a DGK activity-independent manner, suggesting that DGKeta serves as a scaffold/adaptor protein. By determining the activity of all the components of the pathway in DGKeta-silenced HeLa cells, this study revealed that DGKeta activated C-Raf but not B-Raf. Moreover, this study demonstrated that DGKeta enhanced EGF-induced heterodimerization of C-Raf with B-Raf, which transmits the signal to C-Raf. DGKeta physically interacted with B-Raf and C-Raf, regulating EGF-induced recruitment of B-Raf and C-Raf from the cytosol to membranes. The DGKeta-dependent activation of C-Raf occurred downstream or independently of the already known C-Raf modifications, such as dephosphorylation at Ser-259, phosphorylation at Ser-338, and interaction with 14-3-3 protein. Taken together, the results obtained strongly support that DGKeta acts as a novel critical regulatory component of the Ras/B-Raf/C-Raf/MEK/ERK signaling cascade via a previously unidentified mechanism.

    The Journal of biological chemistry 2009;284;43;29559-70

  • A short hairpin DNA analogous to miR-125b inhibits C-Raf expression, proliferation, and survival of breast cancer cells.

    Hofmann MH, Heinrich J, Radziwill G, Radziwil G and Moelling K

    University of Zurich, Zurich, Switzerland.

    The noncoding RNA miR-125b has been described to reduce ErbB2 protein expression as well as proliferation and migration of cancer cell lines. As additional target of miR-125b, we identified the c-raf-1 mRNA by sequence analysis. We designed a short hairpin-looped oligodeoxynucleotide (ODN) targeted to the same 3' untranslated region of c-raf-1 mRNA as miR-125b. The fully complementary ODN antisense strand is linked to a second strand constituting a partially double-stranded structure of the ODN. Transfection of the c-raf-1-specific ODN (ODN-Raf) in a breast cancer cell line reduced the protein levels of C-Raf, ErbB2, and their downstream effector cyclin D1 similar to miR-125b. MiR-125b as well as ODN-Raf showed no effect on the c-raf-1 mRNA level in contrast to small interfering RNA. Unlike miR-125b, ODN-Raf induced a cytopathic effect. This may be explained by the structural properties of ODN-Raf, which can form G-tetrads. Thus, the short hairpin-looped ODN-Raf, targeting the same region of c-raf-1 as miR-125b, is a multifunctional molecule reducing the expression of oncoproteins and stimulating cell death. Both features may be useful to interfere with tumor growth.

    Molecular cancer research : MCR 2009;7;10;1635-44

  • Carbohydrate-specific signaling through the DC-SIGN signalosome tailors immunity to Mycobacterium tuberculosis, HIV-1 and Helicobacter pylori.

    Gringhuis SI, den Dunnen J, Litjens M, van der Vlist M and Geijtenbeek TB

    Center of Infection and Immunity Amsterdam, University of Amsterdam, Amsterdam, The Netherlands. s.i.gringhuis@amc.uva.nl

    Cooperation between different innate signaling pathways induced by pattern-recognition receptors (PRRs) on dendritic cells (DCs) is crucial for tailoring adaptive immunity to pathogens. Here we show that carbohydrate-specific signaling through the C-type lectin DC-SIGN tailored cytokine production in response to distinct pathogens. DC-SIGN was constitutively associated with a signalosome complex consisting of the scaffold proteins LSP1, KSR1 and CNK and the kinase Raf-1. Mannose-expressing Mycobacterium tuberculosis and human immunodeficiency virus type 1 (HIV-1) induced the recruitment of effector proteins to the DC-SIGN signalosome to activate Raf-1, whereas fucose-expressing pathogens such as Helicobacter pylori actively dissociated the KSR1-CNK-Raf-1 complex from the DC-SIGN signalosome. This dynamic regulation of the signalosome by mannose- and fucose-expressing pathogens led to the enhancement or suppression of proinflammatory responses, respectively. Our study reveals another level of plasticity in tailoring adaptive immunity to pathogens.

    Funded by: PHS HHS: HHSN266200400091C

    Nature immunology 2009;10;10;1081-8

  • The variant rs1867277 in FOXE1 gene confers thyroid cancer susceptibility through the recruitment of USF1/USF2 transcription factors.

    Landa I, Ruiz-Llorente S, Montero-Conde C, Inglada-Pérez L, Schiavi F, Leskelä S, Pita G, Milne R, Maravall J, Ramos I, Andía V, Rodríguez-Poyo P, Jara-Albarrán A, Meoro A, del Peso C, Arribas L, Iglesias P, Caballero J, Serrano J, Picó A, Pomares F, Giménez G, López-Mondéjar P, Castello R, Merante-Boschin I, Pelizzo MR, Mauricio D, Opocher G, Rodríguez-Antona C, González-Neira A, Matías-Guiu X, Santisteban P and Robledo M

    Hereditary Endocrine Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain.

    In order to identify genetic factors related to thyroid cancer susceptibility, we adopted a candidate gene approach. We studied tag- and putative functional SNPs in genes involved in thyroid cell differentiation and proliferation, and in genes found to be differentially expressed in thyroid carcinoma. A total of 768 SNPs in 97 genes were genotyped in a Spanish series of 615 cases and 525 controls, the former comprising the largest collection of patients with this pathology from a single population studied to date. SNPs in an LD block spanning the entire FOXE1 gene showed the strongest evidence of association with papillary thyroid carcinoma susceptibility. This association was validated in a second stage of the study that included an independent Italian series of 482 patients and 532 controls. The strongest association results were observed for rs1867277 (OR[per-allele] = 1.49; 95%CI = 1.30-1.70; P = 5.9x10(-9)). Functional assays of rs1867277 (NM_004473.3:c.-283G>A) within the FOXE1 5' UTR suggested that this variant affects FOXE1 transcription. DNA-binding assays demonstrated that, exclusively, the sequence containing the A allele recruited the USF1/USF2 transcription factors, while both alleles formed a complex in which DREAM/CREB/alphaCREM participated. Transfection studies showed an allele-dependent transcriptional regulation of FOXE1. We propose a FOXE1 regulation model dependent on the rs1867277 genotype, indicating that this SNP is a causal variant in thyroid cancer susceptibility. Our results constitute the first functional explanation for an association identified by a GWAS and thereby elucidate a mechanism of thyroid cancer susceptibility. They also attest to the efficacy of candidate gene approaches in the GWAS era.

    PLoS genetics 2009;5;9;e1000637

  • Sorafenib induces growth inhibition and apoptosis in human synovial sarcoma cells via inhibiting the RAF/MEK/ERK signaling pathway.

    Peng CL, Guo W, Ji T, Ren T, Yang Y, Li DS, Qu HY, Li X, Tang S, Yan TQ and Tang XD

    Musculoskeletal Tumor Center, Peking University Peoples' Hospital, Beijing, China.

    Synovial sarcoma is a soft tissue sarcoma with poor prognosis and lack of response to conventional cytotoxic chemotherapy. The regulatory mechanisms for the rapid proliferation of synovial sarcoma cells and the particular aggressiveness of this sarcoma remain poorly understood. Mitogen-activated protein kinase (MAPK) cascades have been shown to play important roles in synovial sarcoma survival. Sorafenib (Nexavar, BAY 43-9006), a potent recombinant activated factor (RAF) inhibitor, inhibits the MAPK signaling pathway both in vitro and in vivo. In this study, we examined the inhibitory proliferation effects of sorafenib in synovial sarcoma growth and evaluated whether sorafenib modulates MAPK and tumor apoptosis cascades in human synovial sarcoma cell lines SW982 and HS-SY-II. Our results indicated that sorafenib effectively inhibited cellular proliferation and induces apoptosis of these two cells. Sorafenib inhibited the phosphorylation of MEK and ERK, downregulated cyclin D1 and Rb levels, caused G(1) arrest and S phase decrease, and induced apoptosis as confirmed by flow cytometry and the TUNEL assay. Furthermore, Bcl-xl and Mcl-1 levels significantly decreased, whereas expression levels of the proteins bcl-2 and bax were unchanged in response to sorafenib treatment in SW982 and HS-SY-II cells. In conclusion, our findings demonstrate that sorafenib is effective for growth inhibition of synovial sarcoma cell lines in vitro and suggest that sorafenib may be a new therapeutic option for patients with synovial sarcoma.

    Cancer biology & therapy 2009;8;18;1729-36

  • Sorafenib inhibits non-small cell lung cancer cell growth by targeting B-RAF in KRAS wild-type cells and C-RAF in KRAS mutant cells.

    Takezawa K, Okamoto I, Yonesaka K, Hatashita E, Yamada Y, Fukuoka M and Nakagawa K

    Department of Medical Oncology, Kinki University School of Medicine, Sakai Hospital, Osaka, Japan.

    Sorafenib is a multikinase inhibitor whose targets include B-RAF and C-RAF, both of which function in the extracellular signal-regulated kinase (ERK) signaling pathway but which also have distinct downstream targets. The relative effects of sorafenib on B-RAF and C-RAF signaling in tumor cells remain unclear, however. We have now examined the effects of sorafenib as well as of B-RAF or C-RAF depletion by RNA interference on cell growth and ERK signaling in non-small cell lung cancer (NSCLC) cell lines with or without KRAS mutations. Sorafenib inhibited ERK phosphorylation in cells with wild-type KRAS but not in those with mutant KRAS. Despite this difference, sorafenib inhibited cell growth and induced G(1) arrest in both cell types. Depletion of B-RAF, but not that of C-RAF, inhibited ERK phosphorylation as well as suppressed cell growth and induced G(1) arrest in cells with wild-type KRAS. In contrast, depletion of C-RAF inhibited cell growth and induced G(1) arrest, without affecting ERK phosphorylation, in cells with mutant KRAS; depletion of B-RAF did not induce G(1) arrest in these cells. These data suggest that B-RAF-ERK signaling and C-RAF signaling play the dominant roles in regulation of cell growth in NSCLC cells with wild-type or mutant KRAS, respectively. The G(1) arrest induced by either C-RAF depletion or sorafenib in cells with mutant KRAS was associated with down-regulation of cyclin E. Our results thus suggest that sorafenib inhibits NSCLC cell growth by targeting B-RAF in cells with wild-type KRAS and C-RAF in those with mutant KRAS.

    Cancer research 2009;69;16;6515-21

  • Retinoic acid induces nuclear accumulation of Raf1 during differentiation of HL-60 cells.

    Smith J, Bunaciu RP, Reiterer G, Coder D, George T, Asaly M and Yen A

    Department of Biomedical Sciences, T4-008 VRT, Cornell University, Ithaca, NY 14853, USA.

    All trans-retinoic acid (RA) is a standard therapeutic agent used in differentiation induction therapy treatment of acute promyelocytic leukemia (APL). RA and its metabolites use a diverse set of signal transduction pathways during the differentiation program. In addition to the direct transcriptional targets of the nuclear RAR and RXR receptors, signals derived from membrane receptors and the Raf-MEK-ERK pathway are required. Raf1 phosphorylation and the prolonged activation of Raf1 persisting during the entire differentiation process are required for RA-dependent differentiation of HL-60 cells. Here we identify a nuclear redistribution of Raf1 during the RA-induced differentiation of HL-60 cells. In addition, the nuclear accumulation of Raf1 correlates with an increase in Raf1 phosphorylated at serine 621. The serine 621 phosphorylated Raf1 is predominantly localized in the nucleus. The RA-dependent nuclear accumulation of Raf1 suggests a novel nuclear role for Raf1 during the differentiation process.

    Funded by: NCI NIH HHS: CA33505, R01 CA033505-25A2, R01 CA033505-27

    Experimental cell research 2009;315;13;2241-8

  • Loss of RAF kinase inhibitor protein is a somatic event in the pathogenesis of therapy-related acute myeloid leukemias with C-RAF germline mutations.

    Zebisch A, Haller M, Hiden K, Goebel T, Hoefler G, Troppmair J and Sill H

    Division of Hematology, Medical University of Graz, Graz, Austria.

    We recently described oncogenic and anti-apoptotic C-RAF germline mutations in patients with therapy-related acute myeloid leukemia (t-AML). Activation of the RAF effector ERK was restricted to transformed cells, suggesting the requirement for cooperating events in leukemogenesis. Western blot analysis of blast cells from patients with C-RAF germline mutations revealed loss of the tumor and metastasis suppressor RAF kinase inhibitor protein (RKIP). Immunohistochemistry of the patients' primary tumors revealed normal RKIP expression levels, indicating that the loss of RKIP is a somatic, t-AML-specific event. In focus formation assays, the oncogenic potential of human mutant C-RAF was strongly influenced by expression levels of RKIP. Although the number of colonies formed by C-RAF(S427G) was significantly increased by RKIP silencing, the opposite was observed after RKIP overexpression. These results show that the loss of RKIP is a functional somatic event in carriers of C-RAF germline mutations, which contributes to the development of t-AML.

    Funded by: Austrian Science Fund FWF: W 1101; Cancer Research UK

    Leukemia 2009;23;6;1049-53

  • Combined targeting of BRAF and CRAF or BRAF and PI3K effector pathways is required for efficacy in NRAS mutant tumors.

    Jaiswal BS, Janakiraman V, Kljavin NM, Eastham-Anderson J, Cupp JE, Liang Y, Davis DP, Hoeflich KP and Seshagiri S

    Department of Molecular Biology, Genentech Inc., South San Francisco, California, United States of America.

    Background: Oncogenic RAS is a highly validated cancer target. Attempts at targeting RAS directly have so far not succeeded in the clinic. Understanding downstream RAS-effectors that mediate oncogenesis in a RAS mutant setting will help tailor treatments that use RAS-effector inhibitors either alone or in combination to target RAS-driven tumors.

    In this study, we have investigated the sufficiency of targeting RAS-effectors, RAF, MEK and PI3-Kinase either alone or in combination in RAS mutant lines, using an inducible shRNA in vivo mouse model system. We find that in colon cancer cells harboring a KRAS(G13D) mutant allele, knocking down KRAS alone or the RAFs in combination or the RAF effectors, MEK1 and MEK2, together is effective in delaying tumor growth in vivo. In melanoma cells harboring an NRAS(Q61L) or NRAS(Q61K) mutant allele, we find that targeting NRAS alone or both BRAF and CRAF in combination or both BRAF and PIK3CA together showed efficacy.

    Our data indicates that targeting oncogenic NRAS-driven melanomas require decrease in both pERK and pAKT downstream of RAS-effectors for efficacy. This can be achieved by either targeting both BRAF and CRAF or BRAF and PIK3CA simultaneously in NRAS mutant tumor cells.

    PloS one 2009;4;5;e5717

  • Oncogenic RAF1 rearrangement and a novel BRAF mutation as alternatives to KIAA1549:BRAF fusion in activating the MAPK pathway in pilocytic astrocytoma.

    Jones DT, Kocialkowski S, Liu L, Pearson DM, Ichimura K and Collins VP

    Department of Pathology, Division of Molecular Histopathology, University of Cambridge, Cambridge, Cambridgeshire, UK. davidjones@cantab.net

    Pilocytic astrocytomas (PAs), WHO malignancy grade I, are the most frequently occurring central nervous system tumour in 5- to 19-year-olds. Recent reports have highlighted the importance of MAPK pathway activation in PAs, particularly through a tandem duplication leading to an oncogenic BRAF fusion gene. Here, we report two alternative mechanisms resulting in MAPK activation in PAs. Firstly, in striking similarity to the common BRAF fusion, tandem duplication at 3p25 was observed, which produces an in-frame oncogenic fusion between SRGAP3 and RAF1. This fusion includes the Raf1 kinase domain, and shows elevated kinase activity when compared with wild-type Raf1. Secondly, a novel 3 bp insertion at codon 598 in BRAF mimics the hotspot V600E mutation to produce a transforming, constitutively active BRaf kinase. Although these two alterations are not common, they bring the number of cases with an identified 'hit' on the Ras/Raf-signalling pathway to 36 from our series of 44 (82%), confirming its central importance to the development of pilocytic astrocytomas.

    Funded by: Cancer Research UK: A6618

    Oncogene 2009;28;20;2119-23

  • Epithelial sodium channel regulated by differential composition of a signaling complex.

    Soundararajan R, Melters D, Shih IC, Wang J and Pearce D

    Division of Nephrology, Department of Medicine, University of California, San Francisco, CA 94143, USA.

    Hormonal control of transepithelial sodium (Na(+)) transport utilizes phosphatidylinositide 3'-kinase (PI3K) and Raf-MAPK/ERK kinase (MEK)-ERK-dependent signaling pathways, which impact numerous cell functions. How signals transmitted by these pathways are sorted and appropriately transmitted to alter Na(+) transport without altering other physiologic processes is not well understood. Here, we report the identification of a signaling complex that selectively modulates the cell surface expression of the epithelial sodium channel (ENaC), an ion channel that is essential for fluid and electrolyte balance in mammals. Raf-1 and the ubiquitin ligase, Nedd4-2, are constitutively-expressed inhibitory components of this ENaC regulatory complex, which interact with, and decrease the expression of, cell surface ENaC. The activities of Nedd4-2 and Raf-1 are inhibited cooperatively by the PI3K-dependent kinase serum- and glucocorticoid-induced kinase 1 (SGK1), and the Raf-1-interacting protein glucocorticoid-induced leucine zipper (GILZ1), which are aldosterone-stimulated components of the complex. Together, SGK1 and GILZ1 synergistically stimulate ENaC cell surface expression. Interestingly, GILZ1 and SGK1 do not have synergistic, and in fact have opposite, effects on an unrelated activity, FKHRL1-driven gene transcription. Together, these data suggest that GILZ1 and SGK1 provide a physical and functional link between the PI3K- and Raf-1-dependent signaling modules and represent a unique mechanism for specifically controlling Na(+) transport without inappropriately activating other cell functions.

    Funded by: NIDDK NIH HHS: K01-DK078679, R01-DK56695

    Proceedings of the National Academy of Sciences of the United States of America 2009;106;19;7804-9

  • Germline BRAF mutations in Noonan, LEOPARD, and cardiofaciocutaneous syndromes: molecular diversity and associated phenotypic spectrum.

    Sarkozy A, Carta C, Moretti S, Zampino G, Digilio MC, Pantaleoni F, Scioletti AP, Esposito G, Cordeddu V, Lepri F, Petrangeli V, Dentici ML, Mancini GM, Selicorni A, Rossi C, Mazzanti L, Marino B, Ferrero GB, Silengo MC, Memo L, Stanzial F, Faravelli F, Stuppia L, Puxeddu E, Gelb BD, Dallapiccola B and Tartaglia M

    IRCCS, San Giovanni Rotondo, Dipartimento di Medicina Sperimentale e Patologia, Università La Sapienza, Rome, Italy.

    Noonan, LEOPARD, and cardiofaciocutaneous syndromes (NS, LS, and CFCS) are developmental disorders with overlapping features including distinctive facial dysmorphia, reduced growth, cardiac defects, skeletal and ectodermal anomalies, and variable cognitive deficits. Dysregulated RAS-mitogen-activated protein kinase (MAPK) signal traffic has been established to represent the molecular pathogenic cause underlying these conditions. To investigate the phenotypic spectrum and molecular diversity of germline mutations affecting BRAF, which encodes a serine/threonine kinase functioning as a RAS effector frequently mutated in CFCS, subjects with a diagnosis of NS (N=270), LS (N=6), and CFCS (N=33), and no mutation in PTPN11, SOS1, KRAS, RAF1, MEK1, or MEK2, were screened for the entire coding sequence of the gene. Besides the expected high prevalence of mutations observed among CFCS patients (52%), a de novo heterozygous missense change was identified in one subject with LS (17%) and five individuals with NS (1.9%). Mutations mapped to multiple protein domains and largely did not overlap with cancer-associated defects. NS-causing mutations had not been documented in CFCS, suggesting that the phenotypes arising from germline BRAF defects might be allele specific. Selected mutant BRAF proteins promoted variable gain of function of the kinase, but appeared less activating compared to the recurrent cancer-associated p.Val600Glu mutant. Our findings provide evidence for a wide phenotypic diversity associated with mutations affecting BRAF, and occurrence of a clinical continuum associated with these molecular lesions.

    Funded by: NHLBI NIH HHS: HL074728, HL71207, P50 HL074728, R01 HL071207, R01 HL071207-07; NICHD NIH HHS: HD01294, K24 HD001294; Telethon: GGP07115

    Human mutation 2009;30;4;695-702

  • Akt regulates vitamin D3-induced leukemia cell functional differentiation via Raf/MEK/ERK MAPK signaling.

    Wang J, Zhao Y, Kauss MA, Spindel S and Lian H

    Department of Molecular Biology and Genetics, Cornell University, New York 14853, USA. jw99@cornell.edu

    1,25-dihydroxyvitamin D3 (vitamin D3) induces differentiation of HL-60 human myeloid leukemia cells; however, the signaling mechanism governing these effects is not fully clear. Here, we show that vitamin D3 induced functional differentiation by Akt through Raf/MEK/ERK MAPK signaling. Vitamin D3 downregulated Akt, weakened Akt-Raf1 interaction, and subsequently activated the Raf/MEK/ERK MAPK pathway. Pharmacological inhibition of MEK/ERK crippled differentiation in response to vitamin D3. Ectopic overexpression of Akt inhibited MAPK signaling, downregulated cyclin-dependent kinase (CDK) inhibitors p21(Wip1/Cip1) and p27(Kip1) and blunted differentiation in response to vitamin D3 while knockdown of Akt by RNA interference gave reverse effects. Furthermore, knockdown of the CDK inhibitors by siRNA crippled the recruitment of retinoblastoma protein (Rb) from the Raf1-Rb complex and Rb hypophosphorylation, and abolished differentiation in response to vitamin D3. Vitamin D3-induced MAPK signaling mediated upregulation of the CDK inhibitors and Rb, disassociation of Raf1 and Rb, and dephosphorylation of Rb, resulting in Rb binding to transcription factor E2F1 and subsequent differentiation. Finally, knockdown of Rb by siRNA prevented vitamin D3-induced differentiation. Mutating Rb at Ser795 evokes its association with E2F1, indicating the critical role of Rb Ser795 in regulating cell differentiation. Taken together, our data suggest that vitamin D3-triggered differentiation of human myeloid leukemia cells depends on downregulation of Akt, which dissociates from Raf1 and activates MAPK signaling leading to CDK inhibitor upregulation, Raf1 disassociation from Rb, and Rb upregulation and hypophosphorylation coupled to E2F1 binding.

    European journal of cell biology 2009;88;2;103-15

  • Cognitive profile of disorders associated with dysregulation of the RAS/MAPK signaling cascade.

    Cesarini L, Alfieri P, Pantaleoni F, Vasta I, Cerutti M, Petrangeli V, Mariotti P, Leoni C, Ricci D, Vicari S, Selicorni A, Tartaglia M, Mercuri E and Zampino G

    Pediatric Neurology Unit, Catholic University, Rome, Italy.

    Mutations in genes coding for transducers participating in the RAS/MAPK pathway have been identified as the molecular cause underlying a group of clinically related developmental disorders with cognitive deficits of variable severity. To determine the spectrum of cognitive defects associated with dysregulation of this signal cascade, we studied the profile of cognitive abilities in patients with mutations affecting the PTPN11, SOS1, HRAS, KRAS, BRAF, RAF1, and MEK1 genes and phenotype-genotype correlations. Our findings support the observation that heterogeneity in cognitive abilities can be at least partially ascribed to the individual affected genes and type of mutation involved. While mutations affecting transducers upstream of RAS were less frequently associated with mental retardation, mutations in downstream components of the pathway were generally associated with a more severe cognitive impairment. Among patients with a heterozygous PTPN11 mutation, the T468M substitution was associated with a mean IQ significantly higher compared to that of individuals carrying the N308D change. Our study provides insights on the range of cognitive abilities in patients with gene mutations causing dysregulation of RAS signaling suggesting that the presence and severity of cognitive involvement can be predicted in part by the gene involved.

    Funded by: Telethon: GGP07115

    American journal of medical genetics. Part A 2009;149A;2;140-6

  • Dectin-1 directs T helper cell differentiation by controlling noncanonical NF-kappaB activation through Raf-1 and Syk.

    Gringhuis SI, den Dunnen J, Litjens M, van der Vlist M, Wevers B, Bruijns SC and Geijtenbeek TB

    Department of Molecular Cell Biology and Immunology, Vrije University Medical Center, 1007 MC Amsterdam, The Netherlands.

    The C-type lectin dectin-1 activates the transcription factor NF-kappaB through a Syk kinase-dependent signaling pathway to induce antifungal immunity. Here we show that dectin-1 expressed on human dendritic cells activates not only the Syk-dependent canonical NF-kappaB subunits p65 and c-Rel, but also the noncanonical NF-kappaB subunit RelB. Dectin-1, when stimulated by the beta-glucan curdlan or by Candida albicans, induced a second signaling pathway mediated by the serine-threonine kinase Raf-1, which integrated with the Syk pathway at the point of NF-kappaB activation. Raf-1 antagonized Syk-induced RelB activation by promoting sequestration of RelB into inactive p65-RelB dimers, thereby altering T helper cell differentiation. Thus, dectin-1 activates two independent signaling pathways, one through Syk and one through Raf-1, to induce immune responses.

    Nature immunology 2009;10;2;203-13

  • CRAF inhibition induces apoptosis in melanoma cells with non-V600E BRAF mutations.

    Smalley KS, Xiao M, Villanueva J, Nguyen TK, Flaherty KT, Letrero R, Van Belle P, Elder DE, Wang Y, Nathanson KL and Herlyn M

    The Wistar Institute, Philadelphia, PA, USA. k.smalley@mac.com

    Here, we identify a panel of melanoma lines with non-V600E mutations in BRAF. These G469E- and D594G-mutated melanomas were found to exhibit constitutive levels of phospho-extracellular signal-regulated kinase (pERK) and low levels of phospho-mitogen-activated protein kinase/ERK kinase (pMEK) and were resistant to MEK inhibition. Upon treatment with the CRAF inhibitor sorafenib, these lines underwent apoptosis and associated with mitochondrial depolarization and relocalization of apoptosis-inducing factor, whereas the BRAF-V600E-mutated melanomas did not. Studies have shown low-activity mutants of BRAF (G469E/D594G) instead signal through CRAF. Unlike BRAF, CRAF directly regulates apoptosis through mitochondrial localization where it binds to Bcl-2 and phosphorylates BAD. The CRAF inhibitor sorafenib was found to induce a time-dependent reduction in both BAD phosphorylation and Bcl-2 expression in the D594G/G469E lines only. Knockdown of CRAF using a lentiviral shRNA suppressed both Bcl-2 expression and induced apoptosis in the D594G melanoma line but not in a V600E-mutated line. Finally, we showed in a series of xenograft studies that sorafenib was more potent at reducing the growth of tumors with the D594G mutation than those with the V600E mutation. In summary, we have identified a group of melanomas with low-activity BRAF mutations that are reliant upon CRAF-mediated survival activity.

    Funded by: NCI NIH HHS: P01 CA025874, P01 CA025874-20A1, P01 CA114046, P01 CA114046-01A2, P50 CA093372, P50 CA093372-01, R01 CA047159, R01 CA047159-15, R01 CA076674, R01 CA076674-02, R01 CA080999, R01 CA080999-01

    Oncogene 2009;28;1;85-94

  • How does arrestin assemble MAPKs into a signaling complex?

    Song X, Coffa S, Fu H and Gurevich VV

    Department of Pharmacology, Vanderbilt University, Nashville, Tennessee 37232, USA.

    Arrestins bind active phosphorylated G protein-coupled receptors, precluding G protein activation and channeling signaling to alternative pathways. Arrestins also function as mitogen-activated protein kinase (MAPK) scaffolds, bringing together three components of MAPK signaling modules. Here we have demonstrated that all four vertebrate arrestins interact with JNK3, MKK4, and ASK1, but only arrestin3 facilitates JNK3 activation. Thus, the functional specificity of arrestins is not determined by differential binding of the kinases. Using receptor binding-impaired mutant, we have shown that free arrestin3 readily promotes JNK3 phosphorylation. We identified key arrestin-binding elements in JNK3 and ASK1 and investigated the molecular interactions of arrestin2 and arrestin3 and their individual domains with the components of the two MAPK cascades, ASK1-MKK4-JNK3 and c-Raf-1-MEK1-ERK2. We found that both arrestin domains interact with all six kinases. These findings shed new light on the mechanism of arrestin-mediated MAPK activation and the spatial arrangement of the three kinases on arrestin molecule.

    Funded by: NEI NIH HHS: EY011500; NIGMS NIH HHS: GM077561, GM081756

    The Journal of biological chemistry 2009;284;1;685-95

  • RAF may induce cell proliferation through hypermethylation of tumor suppressor gene promoter in gastric epithelial cells.

    Wang X, Sun DF, Lu R, Chen ZF, Chen YX and Fang JY

    Shanghai Jiao-Tong University School of Medicine Renji Hospital, Shanghai Institute of Digestive Disease, Shanghai, China.

    The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK-MAPK) is critical in human malignancies. It remained to be established whether DNA methyltransferases (Dnmt) and proliferating cell nuclear antigen (PCNA) involved in DNA methylation during RAF-transformed cell proliferation. The plasmid of constitutively active RAF was used to transfect gastric cell GES-1 and cancer cell AGS. RAF promoted cell proliferation, growth in soft agar and induced cell cycle progress faster than empty plasmid by accelerating G1/S transition in both cell lines, a massive induction of cyclin D1 and PCNA expression was observed, along with reduced expression of p16INK4A, p21WAF1 and p27KIP1. Methylation-specific polymerase chain reaction and bisulfite sequencing showed that the promoter of p16INK4A was methylated in RAF-transformed cells, treatment with 5-aza-dC or PD98059 restored the expression of p16INK4A, increased p21WAF1 and p27KIP1 partially, associated with upregulation of the activity of Dnmt in RAF-transformed cell GES-1, and also decreased the hypermethylation status of p16INK4A, but not all CpG islands of p21WAF1 and p27KIP1. These data suggest that RAF may induce cell proliferation through hypermethylation of tumor suppressor gene p16INK4A, while the epigenetic inactivation of p21WAF1 and p27KIP1 may be not a key factor in RAF-transformed cells.

    Cancer science 2009;100;1;117-25

  • Cancer genomics identifies regulatory gene networks associated with the transition from dysplasia to advanced lung adenocarcinomas induced by c-Raf-1.

    Rohrbeck A and Borlak J

    Department of Molecular Medicine, Fraunhofer Institute of Toxicology and Experimental Medicine, Hannover, Germany.

    Background: Lung cancer is a leading cause of cancer morbidity. To improve an understanding of molecular causes of disease a transgenic mouse model was investigated where targeted expression of the serine threonine kinase c-Raf to respiratory epithelium induced initially dysplasia and subsequently adenocarcinomas. This enables dissection of genetic events associated with precancerous and cancerous lesions.

    By laser microdissection cancer cell populations were harvested and subjected to whole genome expression analyses. Overall 473 and 541 genes were significantly regulated, when cancer versus transgenic and non-transgenic cells were compared, giving rise to three distinct and one common regulatory gene network. At advanced stages of tumor growth predominately repression of gene expression was observed, but genes previously shown to be up-regulated in dysplasia were also up-regulated in solid tumors. Regulation of developmental programs as well as epithelial mesenchymal and mesenchymal endothelial transition was a hall mark of adenocarcinomas. Additionally, genes coding for cell adhesion, i.e. the integrins and the tight and gap junction proteins were repressed, whereas ligands for receptor tyrosine kinase such as epi- and amphiregulin were up-regulated. Notably, Vegfr- 2 and its ligand Vegfd, as well as Notch and Wnt signalling cascades were regulated as were glycosylases that influence cellular recognition. Other regulated signalling molecules included guanine exchange factors that play a role in an activation of the MAP kinases while several tumor suppressors i.e. Mcc, Hey1, Fat3, Armcx1 and Reck were significantly repressed. Finally, probable molecular switches forcing dysplastic cells into malignantly transformed cells could be identified.

    This study provides insight into molecular pertubations allowing dysplasia to progress further to adenocarcinoma induced by exaggerted c-Raf kinase activity.

    PloS one 2009;4;10;e7315

  • Cell type-specific importance of ras-c-raf complex association rate constants for MAPK signaling.

    Kiel C and Serrano L

    European molecular biology laboratory-centre for genomic regulation (CRG), systems biology unit, university pompeu fabra (UPF), Universitat Pompeu Fabra, Dr. Aiguader 88, 08003 Barcelona, Spain. christina.kiel@crg.es

    We generated 17 c-Raf (RAF proto-oncogene serine-threonine protein kinase) mutants with altered Ras-Raf association and dissociation rates to investigate the role of electrostatically driven Ras-Raf association rates on epidermal growth factor (EGF)-activated mitogen-activated protein kinase (MAPK) signal transduction. Some of these mutants had compensating changes in association and dissociation rates, enabling the effects of changes in association rate to be distinguished from those of changes in affinity. In rabbit kidney (RK13) cells, these mutants affected downstream signaling, with changes in Ras-c-Raf association rates having a greater effect on MAPK signaling than did similar changes in dissociation rates. Mutants with compensating decreases in both association and dissociation rates stimulated less extracellular signal-regulated kinase (ERK)-dependent reporter activity than did wild-type c-Raf, whereas the converse was true for mutants with increased association and dissociation rates. In marked contrast, the mutants had little or no effect on signaling in human embryonic kidney (HEK) 293 cells. These two cell lines also showed distinct patterns of EGF-dependent ERK phosphorylation and signaling: ERK activation and signaling were transient in HEK293 cells and sustained in RK13 cells, with the difference resulting from the lack of negative feedback from ERK to Sos (Son of Sevenless) in the latter. Computer simulation revealed that, in the presence of negative feedback, changes in the rate of Ras-c-Raf binding have little effect on ERK activation. Thus, EGF-MAPK activation kinetics and feedback regulation is cell type specific and depends on the network topology.

    Science signaling 2009;2;81;ra38

  • Role of phosphatidic acid in the coupling of the ERK cascade.

    Kraft CA, Garrido JL, Fluharty E, Leiva-Vega L and Romero G

    Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.

    The production of phosphatidic acid plays a crucial role in the activation of the ERK cascade. This role was linked to the binding of phosphatidate to a specific polybasic site within the kinase domain of Raf-1. Here we show that phosphatidate promotes ERK phosphorylation in intact cells but does not activate Raf in vitro. The kinase suppressor of Ras (KSR) contains a sequence homologous to the phosphatidate binding site of Raf-1. Direct binding of phosphatidate to synthetic peptides derived from the sequences of the binding domains of Raf-1 and KSR was demonstrated by spectroscopic techniques. The specificity of these interactions was confirmed using synthetic lipids and mutated peptides in which the core of the phosphatidic acid binding domain was disrupted. Insulin and exogenous dioleoyl phosphatidate induced a rapid translocation of a mouse KSR1-EGFP construct to the plasma membrane of HIRcB cells. Mutation of two arginines located in the core of the putative phosphatidate binding site abolished dioleoyl phosphatidate- and insulin-induced translocation of KSR1. Overexpression of the mutant KSR1 in HIRcB cells inhibited insulin-dependent MEK and ERK phosphorylation. The addition of dioleoyl phosphatidate or insulin increased the co-localization of KSR1 and H-Ras and promoted the formation of plasma membrane patches enriched in both proteins and phosphatidic acid. These results, in conjunction with our previous work, suggest the formation of phosphatidate-enriched membrane microdomains that contain all components of the ERK cascade. We propose that these domains act as molecular scaffolds in the coupling of signaling events.

    Funded by: NIDDK NIH HHS: R01-DK-54782; NIGMS NIH HHS: T32-GM-54813

    The Journal of biological chemistry 2008;283;52;36636-45

  • Host alpha-adducin is redistributed and localized to the inclusion membrane in chlamydia- and chlamydophila-infected cells.

    Chu HG, Weeks SK, Gilligan DM and Rockey DD

    Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331-4804, USA.

    A large-scale analysis of proteins involved in host-cell signalling pathways was performed using chlamydia-infected murine cells in order to identify host proteins that are differentially activated or localized following infection. Two proteins whose distribution was altered in Chlamydia trachomatis-infected cells relative to mock-infected cells were the actin-binding protein adducin and the regulatory kinase Raf-1. Immunoblot analysis with antibodies to both phosphorylated and non-phosphorylated forms of these proteins demonstrated that the abundance of each protein was markedly reduced in the cytosolic fraction of C. trachomatis- and Chlamydophila caviae-infected cells, but the total cellular protein abundance remained unaffected by infection. Fluorescence microscopy of chlamydia-infected cells using anti-alpha-adducin antibodies demonstrated labelling at or near the chlamydial inclusion membrane. Treatment of infected cells with nocodazole or cytochalasin D did not affect alpha-adducin that was localized to the margins of the inclusion. The demonstration of alpha-adducin and Raf-1 redistribution within cells infected by different chlamydiae provides novel opportunities for analysis of host-pathogen interactions in this system.

    Funded by: NIAID NIH HHS: AI031448, AI48769

    Microbiology (Reading, England) 2008;154;Pt 12;3848-55

  • Multiple genetic variants along candidate pathways influence plasma high-density lipoprotein cholesterol concentrations.

    Lu Y, Dollé ME, Imholz S, van 't Slot R, Verschuren WM, Wijmenga C, Feskens EJ and Boer JM

    Division of Human Nutrition, Wageningen University, Wageningen, The Netherlands. kevin.lu@wur.nl

    The known genetic variants determining plasma HDL cholesterol (HDL-C) levels explain only part of its variation. Three hundred eighty-four single nucleotide polymorphisms (SNPs) across 251 genes based on pathways potentially relevant to HDL-C metabolism were selected and genotyped in 3,575 subjects from the Doetinchem cohort, which was examined thrice over 11 years. Three hundred fifty-three SNPs in 239 genes passed the quality-control criteria. Seven SNPs [rs1800777 and rs5882 in cholesteryl ester transfer protein (CETP); rs3208305, rs328, and rs268 in LPL; rs1800588 in LIPC; rs2229741 in NRIP1] were associated with plasma HDL-C levels with false discovery rate (FDR) adjusted q values (FDR_q) < 0.05. Five other SNPs (rs17585739 in SC4MOL, rs11066322 in PTPN11, rs4961 in ADD1, rs6060717 near SCAND1, and rs3213451 in MBTPS2 in women) were associated with plasma HDL-C levels with FDR_q between 0.05 and 0.2. Two less well replicated associations (rs3135506 in APOA5 and rs1800961 in HNF4A) known from the literature were also observed, but their significance disappeared after adjustment for multiple testing (P = 0.008, FDR_q = 0.221 for rs3135506; P = 0.018, FDR_q = 0.338 for rs1800961, respectively). In addition to replication of previous results for candidate genes (CETP, LPL, LIPC, HNF4A, and APOA5), we found interesting new candidate SNPs (rs2229741 in NRIP1, rs3213451 in MBTPS2, rs17585739 in SC4MOL, rs11066322 in PTPN11, rs4961 in ADD1, and rs6060717 near SCAND1) for plasma HDL-C levels that should be evaluated further.

    Journal of lipid research 2008;49;12;2582-9

  • X-linked and cellular IAPs modulate the stability of C-RAF kinase and cell motility.

    Dogan T, Harms GS, Hekman M, Karreman C, Oberoi TK, Alnemri ES, Rapp UR and Rajalingam K

    Emmy Noether Group of DFG, Institut für Biochemie II, Goethe University Medical School, Frankfurt, Germany.

    Inhibitor of apoptosis proteins (IAP) are evolutionarily conserved anti-apoptotic regulators. C-RAF protein kinase is a direct RAS effector protein, which initiates the classical mitogen-activated protein kinase (MAPK) cascade. This signalling cascade mediates diverse biological functions, such as cell growth, proliferation, migration, differentiation and survival. Here we demonstrate that XIAP and c-IAPs bind directly to C-RAF kinase and that siRNA-mediated silencing of XIAP and c-IAPs leads to stabilization of C-RAF in human cells. XIAP binds strongly to C-RAF and promotes the ubiquitylation of C-RAF in vivo through the Hsp90-mediated quality control system, independently of its E3 ligase activity. In addition, XIAP or c-IAP-1/2 knockdown cells showed enhanced cell migration in a C-RAF-dependent manner. XIAP promotes binding of CHIP (carboxy terminal Hsc70-interacting protein), a chaperone-associated ubiquitin ligase, to the C-RAF-Hsp90 complex in vivo. Interfering with CHIP expression resulted in stabilization of C-RAF and enhanced cell migration, as observed in XIAP knockdown cells. Our data show an unexpected role of XIAP and c-IAPs in the turnover of C-RAF protein, thereby modulating the MAPK signalling pathway and cell migration.

    Funded by: PHS HHS: GMO76167

    Nature cell biology 2008;10;12;1447-55

  • p21 activated kinase 5 activates Raf-1 and targets it to mitochondria.

    Wu X, Carr HS, Dan I, Ruvolo PP and Frost JA

    Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, 6431 Fannin St., Houston, Texas 77030, USA.

    Raf-1 is an important effector of Ras mediated signaling and is a critical regulator of the ERK/MAPK pathway. Raf-1 activation is controlled in part by phosphorylation on multiple residues, including an obligate phosphorylation site at serine 338. Previously PAK1 and casein kinase II have been implicated as serine 338 kinases. To identify novel kinases that phosphorylate this site, we tested the ability of group II PAKs (PAKs 4-6) to control serine 338 phosphorylation. We observed that all group II PAKs were efficient serine 338 kinases, although only PAK1 and PAK5 significantly stimulated Raf-1 kinase activity. We also showed that PAK5 forms a tight complex with Raf-1 in the cell, but not A-Raf or B-Raf. Importantly, we also demonstrated that the association of Raf-1 with PAK5 targets a subpopulation of Raf-1 to mitochondria. These data indicate that PAK5 is a potent regulator of Raf-1 activity and may control Raf-1 dependent signaling at mitochondria.

    Funded by: NCI NIH HHS: 5R01CA116356-02, R01 CA116356, R01 CA116356-02

    Journal of cellular biochemistry 2008;105;1;167-75

  • Cross-talk between aryl hydrocarbon receptor and mitogen-activated protein kinase signaling pathway in liver cancer through c-raf transcriptional regulation.

    Borlak J and Jenke HS

    Fraunhofer Institute of Toxicology and Experimental Medicine, Nikolai-Fuchs-Str.1, 30625 Hannover, Germany. borlak@item.fraunhofer.de

    c-raf is a serine-threonine kinase and a downstream effector of ras signaling. This kinase plays an essential role in cell proliferation, differentiation, and apoptosis. In the past, we reported induction of c-raf gene expression in rat liver cancer on treatment with a mixture of aryl hydrocarbon receptor (AhR) agonists. This prompted our interest in investigating the role of AhR in the transcriptional regulation of c-raf. Initially, we cloned the rat c-raf promoter and sequenced the genomic DNA and cDNA by Southern blotting and capillary electrophoresis. Then, a genetic algorithm was applied to search for putative AhR-binding sites. DNA-binding activity of AhR was confirmed by electromobility shift assay. We also studied c-raf gene expression in rat hepatoma cell lines with functional and/or devoid AhR and in primary human and rat hepatocyte cultures. Overall, we identified five and three AhR-binding sites in the human and rat c-raf gene, respectively. Treatment of hepatocyte cultures with the AhR antagonist resveratrol reduced DNA binding of AhR. Only rat hepatoma cells with functional AhR responded to 1 nmol/L 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment with >10-fold c-raf mRNA induction. Treatment of human and rat hepatocyte cultures with various AhR-activating chemicals resulted in induction of c-raf gene expression, albeit at different levels. Taken collectively, we show AhR to be a master regulator of c-raf and propose cross-talk between AhR and the mitogen-activated protein kinase signaling pathway in chemically induced hepatocarcinogenesis.

    Molecular cancer research : MCR 2008;6;8;1326-36

  • RAS signaling in colorectal carcinomas through alteration of RAS, RAF, NF1, and/or RASSF1A.

    Ahlquist T, Bottillo I, Danielsen SA, Meling GI, Rognum TO, Lind GE, Dallapiccola B and Lothe RA

    Department of Cancer Prevention, Institute for Cancer Research, Norwegian Radium Hospital, Rikshospitalet University Hospital, Oslo, Norway.

    More than half of all colorectal carcinomas are known to exhibit an activated mitogen-activated protein kinase pathway. The NF1 gene, a negative regulator of KRAS, has not previously been examined in a series of colorectal cancer. In the present study, primary colorectal carcinomas stratified according to microsatellite instability status were analyzed. The whole coding region of NF1 was analyzed for mutations using denaturing high-performance liquid chromatography and sequencing, and the copy number alterations of NF1 were examined using multiple ligation-dependent probe amplification and real-time polymerase chain reaction. The mutational hot spots in KRAS and BRAF were sequenced, and promoter hypermethylation status of RASSF1A was assessed with a methylation-specific polymerase chain reaction. One sample had two missense mutations in NF1, whereas nine additional tumors had intronic mutations likely to affect exon splicing. Interestingly, 8 of these 10 tumors were microsatellite-unstable. Four other tumors showed a duplication of NF1. Mutations in KRAS and BRAF were mutually exclusive and were present at 40% and 22%, respectively. RASSF1A was hypermethylated in 31% of the samples. We show that the RAS signaling network is extensively dysregulated in colorectal carcinomas, because more than 70% of the tumors had an alteration in one or more of the four examined components.

    Neoplasia (New York, N.Y.) 2008;10;7;680-6, 2 p following 686

  • GRP78 and Raf-1 cooperatively confer resistance to endoplasmic reticulum stress-induced apoptosis.

    Shu CW, Sun FC, Cho JH, Lin CC, Liu PF, Chen PY, Chang MD, Fu HW and Lai YK

    Institute of Biotechnology, Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.

    The chaperone glucose-regulated protein, 78/immunoglobulin binding protein (GRP78/Bip), protects cells from cytotoxicity induced by DNA damage or endoplasmic reticulum (ER) stress. In this study, we showed that GRP78 is a major inducible protein in human non-small cell lung cancer H460 cells treated with ER stress inducers, including A23187 and thapsigargin. AEBSF, an inhibitor of serine protease, diminished GRP78 induction, enhanced mitochondrial permeability, and augmented apoptosis in H460 cells during ER stress. Simultaneously, AEBSF promoted Raf-1 degradation and suppressed phosphorylation of Raf-1 at Ser338 and/or Tyr340 during ER stress. Coimmunoprecipitation assays and subcellular fractionations showed that GRP78 associated and colocalized with Raf-1 on the outer membrane of mitochondria, respectively. While treatment of cells with ER stress inducers inactivated BAD by phosphorylation at Ser75, a Raf-1 phosphorylation site; AEBSF attenuated phosphorylation of BAD, leading to cytochrome c release from mitochondria. Additionally, overexpression of GRP78 and/or Raf-1 protected cells from ER stress-induced apoptosis. Taken together, our results indicate that GRP78 may stabilize Raf-1 to maintain mitochondrial permeability and thus protect cells from ER stress-induced apoptosis.

    Journal of cellular physiology 2008;215;3;627-35

  • Isoform-specific interaction of C-RAF with mitochondria.

    Galmiche A, Fueller J, Santel A, Krohne G, Wittig I, Doye A, Rolando M, Flatau G, Lemichez E and Rapp UR

    Institut für Medizinische Strahlenkunde und Zellforschung, University of Würzburg, Würzburg, Germany. antoine.galmiche@mail.uni-wuerzburg.de

    The proteins of the RAF family (A-RAF, B-RAF, and C-RAF) are serine/threonine kinases that play important roles in development, mature cell regulation, and cancer. Although it is widely held that their localization on membranes is an important aspect of their function, there are few data that address this aspect of their mode of action. Here, we report that each member of the RAF family exhibits a specific distribution at the level of cellular membranes and that C-RAF is the only isoform that directly targets mitochondria. We found that the RAF kinases exhibit intrinsic differences in terms of mitochondrial affinity and that C-RAF is the only isoform that binds this organelle efficiently. This affinity is conferred by the C-RAF amino-terminal domain and does not depend on the presence of RAS GTPases on the surface of mitochondria. Finally, we analyzed the consequences of C-RAF activation on mitochondria and observed that this event dramatically changes their morphology and their subcellular distribution. Our observations indicate that: (i) RAF kinases exhibit different localizations at the level of cellular membranes; (ii) C-RAF is the only isoform that directly binds mitochondria; and (iii) through its functional coupling with MEK, C-RAF regulates the shape and the cellular distribution of mitochondria.

    The Journal of biological chemistry 2008;283;21;14857-66

  • A small molecule disruptor of Rb/Raf-1 interaction inhibits cell proliferation, angiogenesis, and growth of human tumor xenografts in nude mice.

    Kinkade R, Dasgupta P, Carie A, Pernazza D, Carless M, Pillai S, Lawrence N, Sebti SM and Chellappan S

    Drug Discovery Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA.

    Although it is well established that cyclin-dependent kinases phosphorylate and inactivate Rb, the Raf-1 kinase physically interacts with Rb and initiates the phosphorylation cascade early in the cell cycle. We have identified an orally active small molecule, Rb/Raf-1 disruptor 251 (RRD-251), that potently and selectively disrupts the Rb/Raf-1 but not Rb/E2F, Rb/prohibitin, Rb/cyclin E, and Rb/HDAC binding. The selective inhibition of Rb/Raf-1 binding suppressed the ability of Rb to recruit Raf-1 to proliferative promoters and inhibited E2F1-dependent transcriptional activity. RRD-251 inhibited anchorage-dependent and anchorage-independent growth of human cancer cells and knockdown of Rb with short hairpin RNA or forced expression of E2F1 rescued cells from RRD-251-mediated growth arrest. P.o. treatment of mice resulted in significant tumor growth suppression only in tumors with functional Rb, and this was accompanied by inhibition of angiogenesis, inhibition of proliferation, decreased phosphorylated Rb levels, and inhibition of Rb/Raf-1 but not Rb/E2F1 binding in vivo. Thus, selective targeting of Rb/Raf-1 interaction seems to be a promising approach for developing novel chemotherapeutic agents.

    Funded by: NCI NIH HHS: CA118210, CA63136, P01 CA118210, P01 CA118210-03, R01 CA063136

    Cancer research 2008;68;10;3810-8

  • Glutamate accelerates RPE cell proliferation through ERK1/2 activation via distinct receptor-specific mechanisms.

    García S, López E and López-Colomé AM

    Instituto de Fisiología Celular, Departamento de Neurociencias, Universidad Nacional Autónoma de México, Mexico, Mexico.

    The proliferation and migration of Retinal Pigment Epithelium cells resulting from an epithelial-mesenchymal transition plays a key role in proliferative vitreoretinopathy, which leads to retinal detachment and the loss of vision. In neurons, glutamate has been shown to activate the Ras/Raf/MEK/ERK cascade, which participates in the regulation of proliferation, differentiation, and survival processes. Although glutamate-stimulation and the activation of ERK1/2 by different stimuli have been shown to promote RPE cell proliferation, the signaling pathway(s) linking these effects has not been established. We analyzed the molecular mechanisms leading to glutamate-induced proliferation by determining ERK1/2 and CREB phoshporylation in chick RPE cells in primary culture and the human-derived RPE cell line ARPE-19. This study shows for the first time, that glutamate promotes RPE cell proliferation by activating two distinct signaling pathways linked to selective glutamate receptor subtypes. Results demonstrate that glutamate stimulates RPE cell proliferation as well as ERK and CREB phosphorylation. These effects were mimicked by the mGluR agonist ACPD and by NMDA, and were prevented by the respective receptor inhibitors MCPG and MK-801, indicating a cause-effect relationship between these processes. Whereas mGluR promoted proliferation by activating the MEK/ERK/CREB cascade, NMDA stimulated proliferation through the MEK-independent activation of Ca(2+)/calmodulin-dependent kinases. The blockage of both signaling pathways to proliferation by KN-62 suggests the involvement of CaMKs in the control of glutamate-induced proliferation at a common step, downstream of CREB, possibly the regulation of cell cycle progression. Based on these findings, the participation of glutamate in the development of PVR can be considered.

    Journal of cellular biochemistry 2008;104;2;377-90

  • The RKIP (Raf-1 Kinase Inhibitor Protein) conserved pocket binds to the phosphorylated N-region of Raf-1 and inhibits the Raf-1-mediated activated phosphorylation of MEK.

    Rath O, Park S, Tang HH, Banfield MJ, Brady RL, Lee YC, Dignam JD, Sedivy JM, Kolch W and Yeung KC

    The Beatson institute for Cancer Research, Cancer Research UK Garscube Estate, Bearsden, Glasgow G61 1BD, UK.

    The Raf-MEK-ERK pathway regulates many fundamental biological processes, and its activity is finely tuned at multiple levels. The Raf kinase inhibitory protein (RKIP) is a widely expressed negative modulator of the Raf-MEK-ERK signaling pathway. We have previously shown that RKIP inhibits the phosphorylation of MEK by Raf-1 through interfering with the formation of a kinase-substrate complex by direct binding to both Raf-1 and MEK. Here, we show that the evolutionarily conserved ligand-binding pocket of RKIP is required for its inhibitory activity towards the Raf-1 kinase mediated activation of MEK. Single amino acid substitutions of two of the conserved residues form the base and the wall of the pocket confers a loss-of-function phenotype on RKIP. Loss-of-function RKIP mutants still appear to bind to Raf-1. However the stability of the complexes formed between mutants and the N-region Raf-1 phosphopeptide were drastically reduced. Our results therefore suggest that the RKIP conserved pocket may constitute a novel phosphoamino-acid binding motif and is absolutely required for RKIP function.

    Funded by: Cancer Research UK; NIGMS NIH HHS: R01 GM064767-01, R01 GM064767-02, R01 GM064767-03, R01 GM064767-04, R01 GM064767-05, R01 GM064767-06, R01 GM64767

    Cellular signalling 2008;20;5;935-41

  • Constitutive activation of Raf-1 induces glioma formation in mice.

    Lyustikman Y, Momota H, Pao W and Holland EC

    Department of Cancer Biology and Genetics, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.

    In human glioblastoma multiforme (GBM), RAS activity is upregulated in the majority of the tumors. Furthermore, the levels of phospho-mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/ERK), a downstream effector of RAS, are also increased. In mice, activated KRas cooperates with the loss of INK4a-ARF locus or with activated Akt to induce gliomas, confirming an important role for this pathway in glioma biology. However, to correctly target therapies against the RAS signaling pathway, it is necessary to identify the effectors that contribute to RAS-mediated gliomagenesis. In this study, we investigated the contribution of RAF signaling in glioma oncogenesis. We find that the levels of RAF-1 and BRAF proteins and RAF kinase activity are increased in human GBM samples. We confirm the importance of this finding by demonstrating a causal role for a constitutively active Raf-1 mutant in glioma formation in mice. Specifically, we find that activated Raf-1 cooperates with Arf loss or Akt activation to generate gliomas similar to activated KRas under the same conditions. Our study suggests that the oncogenic effect of KRas in glioma formation may be transduced at least in part through Raf signaling and that therapeutic targeting of this pathway may be beneficial in glioma treatment.

    Funded by: NCI NIH HHS: R01 CA100688, R01-CA100688

    Neoplasia (New York, N.Y.) 2008;10;5;501-10

  • Genotype-phenotype analysis and natural history of left ventricular hypertrophy in LEOPARD syndrome.

    Limongelli G, Sarkozy A, Pacileo G, Calabrò P, Digilio MC, Maddaloni V, Gagliardi G, Di Salvo G, Iacomino M, Marino B, Dallapiccola B and Calabrò R

    Monaldi Hospital, Second University of Naples, Naples, Italy. limongelligiuseppe@libero.it

    Because it is unclear whether the genotype may influence the clinical course in patients with LEOPARD syndrome (LS), we analyzed clinical and molecular predictors of adverse cardiac events in patients with left ventricular hypertrophy (LVH). A comprehensive cardiovascular evaluation, including baseline electrocardiogram, echocardiography, exercise test and 24 hr Holter monitoring at the time of clinical diagnosis and during follow-up was conducted on 24 patients referred to our departments. Phenotypical examination and diagnosis were performed by expert clinical geneticists. The entire PTPN11 and RAF1 coding regions were screened for mutations by DHPLC analysis, followed by sequencing. Patients without PTPN11 mutations (34%) showed a higher frequency of family history of sudden death (P = 0.007), increased left atrial dimensions (P = 0.05), bradyarrhythmias (P = 0.04), episodes of supraventricular tachycardias (P = 0.06), atrial fibrillation (P = 0.009), and nonsustained ventricular tachycardias (P = 0.05) during Holter monitoring. Six patients (25%) had adverse cardiac events during follow-up (including sudden deaths, resuscitated cardiac arrest, septal myectomy, and heart failure). LVH, New York Heart Association Class, left ventricular outflow tract obstruction, and nonsustained ventricular tachycardias were associated to adverse cardiac events. Of note, three patients with mutations in exon 13 showed a severe obstructive cardiomyopathy, with serious cardiac complications during follow-up (heart failure, septal myectomy, and sudden death). In conclusion, patients with LVH associated with LS seem to carry a relatively high risk of adverse (arrhythmic and nonarrhythmic) events. Further genotype-phenotype studies are warranted to fully elucidate the impact of the genotype on the natural history of patients with LS and LVH.

    American journal of medical genetics. Part A 2008;146A;5;620-8

  • Inhibition of Raf-1 alters multiple downstream pathways to induce pancreatic beta-cell apoptosis.

    Alejandro EU and Johnson JD

    Laboratory of Molecular Signaling in Diabetes, Diabetes Research Group, Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.

    The serine threonine kinase Raf-1 plays a protective role in many cell types, but its function in pancreatic beta-cells has not been elucidated. In the present study, we examined whether primary beta-cells possess Raf-1 and tested the hypothesis that Raf-1 is critical for beta-cell survival. Using reverse transcriptase-PCR, Western blot, and immunofluorescence, we identified Raf-1 in human islets, mouse islets, and in the MIN6 beta-cell line. Blocking Raf-1 activity using a specific Raf-1 inhibitor or dominant-negative Raf-1 mutants led to a time- and dose-dependent increase in cell death, assessed by real-time imaging of propidium iodide incorporation, TUNEL, PCR-enhanced DNA laddering, and Caspase-3 cleavage. Although the rapid increase in apoptotic cell death was associated with decreased Erk phosphorylation, studies with two Mek inhibitors suggested that the classical Erk-dependent pathway could explain only part of the cell death observed after inhibition of Raf-1. An alternative Erk-independent pathway downstream of Raf-1 kinase involving the pro-apoptotic protein Bad has recently been characterized in other tissues. Inhibiting Raf-1 in beta-cells led to a striking loss of Bad phosphorylation at serine 112 and an increase in the protein levels of both Bad and Bax. Together, our data strongly suggest that Raf-1 signaling plays an important role regulating beta-cell survival, via both Erk-dependent and Bad-dependent mechanisms. Conversely, acutely inhibiting phosphatidylinositol 3-kinase Akt had more modest effects on beta-cell death. These studies identify Raf-1 as a critical anti-apoptotic kinase in pancreatic beta-cells and contribute to our understanding of survival signaling in this cell type.

    Funded by: NIDDK NIH HHS: F31 DK079346, F31 DK079346-01, F31DK079346

    The Journal of biological chemistry 2008;283;4;2407-17

  • The SH3 domain of Lck modulates T-cell receptor-dependent activation of extracellular signal-regulated kinase through activation of Raf-1.

    Li M, Ong SS, Rajwa B, Thieu VT, Geahlen RL and Harrison ML

    Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, Hansen Life Sciences Building, 201 South University Street, West Lafayette, IN 47907, USA.

    Engagement of the T-cell antigen receptor (TCR) results in the proximal activation of the Src family tyrosine kinase Lck. The activation of Lck leads to the downstream activation of the Ras/Raf/MEK/ERK signaling pathway (where ERK is extracellular signal-related kinase). Under conditions of weak, but not strong, stimulation through the TCR, a version of Lck that contains a single point mutation in the SH3 (Src homology 3) domain (W97ALck) fails to support the activation of ERK, despite initiating signaling through the TCR, as demonstrated by the robust activation of ZAP-70, PLC-gamma, and Ras. We determined that the signaling lesion in W97ALck-expressing cells lies at the level of Raf-1 activation and is dependent on the presence of tyrosines 340/341 in the Raf-1 sequence. These data demonstrate a second function for Lck in TCR-mediated signaling to ERK. Additionally, we found that a significant fraction of Lck is localized to the Golgi apparatus and that, compared with wild-type Lck, W97ALck displays aberrant Golgi membrane localization. Our results support a model where under conditions of weak stimulation through the TCR, in addition to activated Ras, Golgi apparatus-localized Lck is needed for the full activation of Raf-1.

    Funded by: NCI NIH HHS: P30 CA023168; NIGMS NIH HHS: R01 GM048099, R01 GM48099

    Molecular and cellular biology 2008;28;2;630-41

  • Gastrin transactivates the chromogranin A gene through MEK-1/ERK- and PKC-dependent phosphorylation of Sp1 and CREB.

    Cramer T, Jüttner S, Plath T, Mergler S, Seufferlein T, Wang TC, Merchant J and Höcker M

    Laboratory for Angiogenesis and Tumor Metastasis, Charité - Universitätsmedizin Berlin, Campus Virchow-Klinikum, Germany.

    Our previous work revealed that gastrin regulates chromogranin A (CgA) transcription through enhanced binding of Sp1, CREB and Egr-1 to a proximal gastrin-responsive promoter element (Gas-RE). Here, we provide a detailed characterization of the signalling pathways transmitting the effect of gastrin on the CgA promoter. Gastrin treatment of gastric AGS-B cells potently stimulated MEK-1 as well as MAP kinases ERK-1/-2, JNK and p38 in a time-dependent manner. Interruption of ERK-1/-2/MEK-1 pathways abolished the transactivating effect of gastrin, whereas blockade of JNK or p38 activity was without effect. Functional promoter analysis revealed that the minimal element CgA-85/-64 was sufficient and necessary to confer MEK-1/ERK responsiveness. Analysis of proximal signalling pathways showed that activation of the MEK-1/ERK-1/2 module by gastrin does not require Ras, PI3-kinase or intracellular calcium signals, but depends on activation of kinases of the PKC family. This report demonstrates that a pathway comprising PKCs>Raf-1>MEK-1>ERK-1/-2 mediates the effect of gastrin on the CgA promoter, and strongly suggests that enhanced phosphorylation of Sp1 and CREB is crucial for CgA transactivation through the G protein-coupled CCK-B/gastrin receptor.

    Funded by: NIDDK NIH HHS: R0 1 DK 48077, R01 DK045729, R37 DK045729

    Cellular signalling 2008;20;1;60-72

  • Overexpression of human Raf-1 enhances radiosensitivity in fission yeast, Schizosaccharomyces pombe.

    Lee M

    Department of Biology, College of Natural Sciences, University of Incheon, Incheon, Korea. mikelee@incheon.ac.kr

    Recently we isolated Rad24, a 14-3-3 homologue, which is essential for DNA damage checkpoint, as a Raf-1 interacting protein by screening a Schizosaccharomyces pombe (S. pombe) cDNA library. Raf-1 was also found to recognize Cdc25 that is sequestered and inactivated by Rad24. In the present study, experiments were performed to determine the effect of overexpression of Raf-1 proteins on asynchronously growing S. pombe cells. The overexpression of Rad24 induced elongated cell morphology and reduction in growth rate, resulting in cell cycle arrest while the overexpression of catalytically active Raf-1 led to a decrease in cell size at division in S. pombe. However, the active Raf-1 failed to rescue the growth arrest induced by Rad24 overexpression. In addition, the cells carrying catalytically active Raf-1 were significantly more radiosensitive than those from a normal control as assessed by ultraviolet sensitivity assay, suggesting that constitutive overproduction of Raf-1 kinase can revert DNA replication checkpoint arrest caused by UV irradiation. Taken together, these data suggest that Raf-1 may interfere with the role of Rad24 by competing with Rad24 for binding to Cdc25 in DNA repair, bypassing the checkpoint pathway through Cdc25 activation.

    Cell biochemistry and function 2008;26;1;125-31

  • Morphoproteomic profile of mTOR, Ras/Raf kinase/ERK, and NF-kappaB pathways in human gastric adenocarcinoma.

    Feng W, Brown RE, Trung CD, Li W, Wang L, Khoury T, Alrawi S, Yao J, Xia K and Tan D

    Department of Pathology, MD Anderson Cancer Center, Houston, TX 77030, USA.

    Preclinical studies using human gastric adenocarcinoma (GAC) cell lines have shown that the mammalian target of rapamycin (mTOR) inhibitor, rapamycin, can inhibit tumor growth and that the extracellular signal-regulated kinase (ERK) of the Ras/Raf kinase/ERK pathway is related to chemoresistance and apoptosis. We examined the state of activation of components of mTOR, Ras/Raf kinase/ERK, and nuclear factor (NF)-kappaB signal transduction pathways, as well as cell cycle protein analyte correlates in GAC cases. Formalin-fixed paraffin-embedded tissue microarray blocks containing samples from 210 cases of GAC were examined. Immunohistochemistry was utilized to detect the following antigens: S100P, upstream stimulator of ERK, and NF-kappaB pathways; phosphorylated (p)-mTOR (Ser 2448), p-ERK-1/2 (Thr 202/Tyr 204), and one of their common down-stream effectors, p-p70S6K(Thr 389); p-NF-kappaBp65(Ser 536); and cell cycle associated proteins, Ki-67, and S phase kinase-associated protein (Skp)2. Immunoreactivity (0 to 4+) of protein expression and compartmentalization were assessed by bright-field microscopy. The majority of cases showed positive (1+ to 4+) cytoplasmic/plasmalemmal p-mTOR (88%), and moderate-strong (2+ to 4+) nuclear p-p70S6K (93%) and nuclear S100P (81%) expression. A subset of cases exhibited moderate-strong nuclear p-ERK-1/2 (15%) and p-NF-kappaBp65 (36%) expression. The majority of cases showed concomitant moderate-strong (2+ to 4+) nuclear Ki-67 (71%) and Skp2 (68%). Nuclear expression levels of p-ERK-1/2 and p-NF-kappaBp65, of p-p70S6K and p-NF-kappaB, and of Ki-67 and Skp2, respectively, showed significant linear correlations in GAC (p <0.001). Additionally, there were statistically significant differences in the mean expression levels of p-ERK-1/2 and p-NF-kappaBp65 in diffuse vs intestinal types of GAC, with higher levels of both in the diffuse type ( p = 0.001 and p <0.0001, respectively). In summary, morphoproteomic analysis reveals constitutive activation of mTOR and to some extent, Ras/Raf kinase and NF-kappaB pathways in GAC, as evidenced by increased cytoplasmic p-mTOR, nuclear translocation of p-p70S6K and p-ERK-1/2 phosphorylated at putative sites of activation (Ser 2448, Thr 389, and Thr 202/Tyr 204, respectively), as well as correlative expression of cell cycle analytes, Ki-67, and Skp2. These results suggest that a prospective study is warranted to evaluate the use of morphoproteomic profiling of individual patients with GAC in order to design combinatorial treatment strategies that target the mTOR, Ras/Raf kinase/ERK, and/or NF-kappaB pathways.

    Annals of clinical and laboratory science 2008;38;3;195-209

  • PTPN11, SOS1, KRAS, and RAF1 gene analysis, and genotype-phenotype correlation in Korean patients with Noonan syndrome.

    Ko JM, Kim JM, Kim GH and Yoo HW

    Department of Pediatrics, Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea.

    After 2006, germline mutations in the KRAS, SOS1, and RAF1 genes were reported to cause Noonan syndrome (NS), in addition to the PTPN11 gene, and now we can find the etiology of disease in approximately 60-70% of NS cases. The aim of this study was to assess the correlation between phenotype and genotype by molecular analysis of the PTPN11, SOS1, KRAS, and RAF1 genes in 59 Korean patients with NS. We found disease-causing mutations in 30 (50.8%) patients, which were located in the PTPN11 (27.1%), SOS1 (16.9%), KRAS (1.7%), and RAF1 (5.1%) genes. Three novel mutations (T59A in PTPN11, K170E in SOS1, S259T in RAF1) were identified. The patients with PTPN11 mutations showed higher prevalences of patent ductus arteriosus and thrombocytopenia. The patients with SOS1 mutations had a lower prevalence of delayed psychomotor development. All patients with RAF1 mutations had hypertrophic cardiomyopathy. Typical facial features and auxological parameters were, on statistical analysis, not significantly different between the groups. The molecular defects of NS are genetically heterogeneous and involve several genes other than PTPN11 related to the RAS-MAPK pathway.

    Journal of human genetics 2008;53;11-12;999-1006

  • Site-specific phosphorylation of raf in cells containing oncogenic ras-p21 is likely mediated by jun-N-terminal kinase.

    Adler V, Bowne W, Michl J, Sookraj KA, Ikram K, Pestka S, Izotova L, Zenilman M, Friedman FK, Qu Y and Pincus MR

    Department of Pathology, SUNY Downstate Medical Center, Brooklyn, New York, USA.

    In a study of interactions between the raf-MEK-MAPK (ERK) and JNK-jun pathways, we found previously that JNK can induce phosphorylation of raf but not vice versa. In this study, we investigate the nature of the JNK-induced phosphorylation of raf. In in vitro experiments in which immunobead-bound raf is phosphorylated by activated JNK, we find strong phosphorylation signals at raf-Ser259 and Ser338. The Ser259 phosphorylation is surprising since it is associated with inhibition of migration of raf to the cell membrane where it can interact with ras-p21. We also find that in oocytes induced to mature with oncogenic ras-p21, which induces high levels of phosphorylated JNK and MAPK, the same pattern of phosphorylation of raf occurs. In contrast, in oocytes induced to mature with insulin, which requires activation of wild-type ras-p21, phosphorylation of raf-Ser338 but not raf-Ser259 occurs. In oncogenic ras-transformed human pancreatic cancer MIA-PaCa-2 cells, phosphorylation of both raf serines occurs. Treatment of these cells with the ras peptide, PNC-2 attached to a penetrating sequence that blocks JNK and MAPK phosphorylation and induces tumor cell necrosis, results in a marked decrease in phosphorylation of raf-Ser259, but not that of raf-Ser338. These results suggest that oncogenic ras-p21 induces phosphorylation of both raf-Ser259 and Ser338 and that raf-Ser 259 phosphorylation may be effected by activated JNK.

    Annals of clinical and laboratory science 2008;38;1;47-56

  • Low-dose radiation stimulates the proliferation of normal human lung fibroblasts via a transient activation of Raf and Akt.

    Kim CS, Kim JK, Nam SY, Yang KH, Jeong M, Kim HS, Kim CS, Jin YW and Kim J

    Radiation Health Research Institute, Korea Hydro and Nuclear Power Co., Ltd., Seoul 132-703, Korea.

    The biological effects of low-dose radiation have been investigated and debated for more than a century, but its cellular effects and regulatory mechanisms remain poorly understood. This study shows the human cellular responses to low-dose radiation in CCD-18 Lu cells, which are derived from normal human lung fibroblasts. We examined a colony-forming assay for cell survival by ionizing radiation. Live cell counting and cell cycle analysis were measured for cell proliferation and cell cycle progression following low-dose irradiation. We examined Raf and Akt phosphorylation to determine the proliferation mechanism resulting from low-dose radiation. We also observed that p53 and p21 were related to cell cycle response. We found that 0.05 Gy of ionizing radiation enhanced cell proliferation and did not change the progression of the cell cycle. In addition, 0.05 Gy of ionizing radiation transiently activated Raf and Akt, but did not change phospho-p53, p53 and p21 in CCD-18 Lu cells. However, 2 Gy of ionizing radiation induced cell cycle arrest, phosphorylation of p53, and expression of p53 and p21. The phosphorylation of Raf and Akt proteins induced by 0.05 Gy of ionizing radiation was abolished by pre-treatment with an EGFR inhibitor, AG1478, or a PI3k inhibitor, LY294002. Cell proliferation stimulated by 0.05 Gy of ionizing radiation was blocked by the suppression of Raf and Akt phosphorylation with these inhibitors. These results suggest that 0.05 Gy of ionizing radiation stimulates cell proliferation through the transient activation of Raf and Akt in CCD-18 Lu cells.

    Molecules and cells 2007;24;3;424-30

  • A novel tandem affinity purification strategy for the efficient isolation and characterisation of native protein complexes.

    Gloeckner CJ, Boldt K, Schumacher A, Roepman R and Ueffing M

    Institute of Human Genetics, GSF-National Research Center for Environment and Health, Neuherberg, Germany.

    Isolation and dissection of native multiprotein complexes is a central theme in functional genomics. The development of the tandem affinity purification (TAP) tag has enabled an efficient and large-scale purification of native protein complexes. However, the TAP tag features a size of 21 kDa and requires time consuming cleavage. By combining a tandem Strep-tag II with a FLAG-tag we were able to reduce the size of the TAP (SF-TAP) tag to 4.6 kDa. Both moieties have a medium affinity and avidity to their immobilised binding partners. This allows the elution of SF-tagged proteins under native conditions using desthiobiotin in the first step and the FLAG octapeptide in the second step. The SF-TAP protocol represents an efficient, fast and straightforward purification of protein complexes from mammalian cells within 2.5 h. The power of this novel method is demonstrated by the purification of Raf associated protein complexes from HEK293 cells and subsequent analysis of their protein interaction network by dissection of interaction patterns from the Raf binding partners MEK1 and 14-3-3.

    Proteomics 2007;7;23;4228-34

  • RAF-1 over-expression does condition survival of patients affected by aggressive mantle cell lymphoma.

    Nagy B, Galimberti S, Benedetti E, Caracciolo F, Pacini S, Vilpo J, Ferrer A, Elonen E, Franssila K, Knuutila S and Petrini M

    Leukemia research 2007;31;11;1595-7

  • Ubiquitination of beta-arrestin links seven-transmembrane receptor endocytosis and ERK activation.

    Shenoy SK, Barak LS, Xiao K, Ahn S, Berthouze M, Shukla AK, Luttrell LM and Lefkowitz RJ

    Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA. sudha@receptor-biol.duke.edu

    Beta-arrestin2 and its ubiquitination play crucial roles in both internalization and signaling of seven-transmembrane receptors (7TMRs). To understand the connection between ubiquitination and the endocytic and signaling functions of beta-arrestin, we generated a beta-arrestin2 mutant that is defective in ubiquitination (beta-arrestin2(0K)), by mutating all of the ubiquitin acceptor lysines to arginines and compared its properties with the wild type and a stably ubiquitinated beta-arrestin2-ubiquitin (Ub) chimera. In vitro translated beta-arrestin2 and beta-arrestin2(0K) displayed equivalent binding to recombinant beta(2)-adrenergic receptor (beta(2)AR) reconstituted in vesicles, whereas beta-arrestin2-Ub bound approximately 4-fold more. In cellular coimmunoprecipitation assays, beta-arrestin2(0K) bound nonreceptor partners, such as AP-2 and c-Raf and scaffolded phosphorylated ERK robustly but displayed weak binding to clathrin. Moreover, beta-arrestin2(0K) was recruited only transiently to activated receptors at the membrane, did not enhance receptor internalization, and decreased the amount of phosphorylated ERK assimilated into isolated beta(2)AR complexes. Although the wild type beta-arrestin2 formed ERK signaling complexes with the beta(2)AR at the membrane, a stably ubiquitinated beta-arrestin2-Ub chimera not only stabilized the ERK signalosomes but also led to their endosomal targeting. Interestingly, in cellular fractionation assays, the ubiquitination state of beta-arrestin2 favors its distribution in membrane fractions, suggesting that ubiquitination increases the propensity of beta-arrestin for membrane association. Our findings suggest that although beta-arrestin ubiquitination is dispensable for beta-arrestin cytosol to membrane translocation and its "constitutive" interactions with some cytosolic proteins, it nevertheless is a prerequisite both for the formation of tight complexes with 7TMRs in vivo and for membrane compartment interactions that are crucial for downstream endocytic and signaling processes.

    Funded by: NHLBI NIH HHS: HL080525, HL16037, R01 HL016037, R01 HL080525, R01 HL080525-02; NIDDK NIH HHS: DK55524, R01 DK055524, R56 DK055524

    The Journal of biological chemistry 2007;282;40;29549-62

  • B- and C-RAF display essential differences in their binding to Ras: the isotype-specific N terminus of B-RAF facilitates Ras binding.

    Fischer A, Hekman M, Kuhlmann J, Rubio I, Wiese S and Rapp UR

    Institut für Medizinische Strahlenkunde und Zellforschung, University of Wuerzburg, 97078 Wuerzburg, Germany.

    Recruitment of RAF kinases to the plasma membrane was initially proposed to be mediated by Ras proteins via interaction with the RAF Ras binding domain (RBD). Data reporting that RAF kinases possess high affinities for particular membrane lipids support a new model in which Ras-RAF interactions may be spatially restricted to the plane of the membrane. Although the coupling features of Ras binding to the isolated RAF RBD were investigated in great detail, little is known about the interactions of the processed Ras with the functional and full-length RAF kinases. Here we present a quantitative analysis of the binding properties of farnesylated and nonfarnesylated H-Ras to both full-length B- and C-RAF in the presence and absence of lipid environment. Although isolated RBD fragments associate with high affinity to both farnesylated and nonfarnesylated H-Ras, the full-length RAF kinases revealed fundamental differences with respect to Ras binding. In contrast to C-RAF that requires farnesylated H-Ras, cytosolic B-RAF associates effectively and with significantly higher affinity with both farnesylated and nonfarnesylated H-Ras. To investigate the potential farnesyl binding site(s) we prepared several N-terminal fragments of C-RAF and found that in the presence of cysteine-rich domain only the farnesylated form of H-Ras binds with high association rates. The extreme N terminus of B-RAF turned out to be responsible for the facilitation of lipid independent Ras binding to B-RAF, since truncation of this region resulted in a protein that changed its kinase properties and resembles C-RAF. In vivo studies using PC12 and COS7 cells support in vitro results. Co-localization measurements using labeled Ras and RAF documented essential differences between B- and C-RAF with respect to association with Ras. Taken together, these data suggest that the activation of B-RAF, in contrast to C-RAF, may take place both at the plasma membrane and in the cytosolic environment.

    The Journal of biological chemistry 2007;282;36;26503-16

  • Spatial regulation of Raf kinase signaling by RKTG.

    Feng L, Xie X, Ding Q, Luo X, He J, Fan F, Liu W, Wang Z and Chen Y

    Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Shanghai 200031, China.

    Subcellular compartmentalization has become an important theme in cell signaling such as spatial regulation of Ras by RasGRP1 and MEK/ERK by Sef. Here, we report spatial regulation of Raf kinase by RKTG (Raf kinase trapping to Golgi). RKTG is a seven-transmembrane protein localized at the Golgi apparatus. RKTG expression inhibits EGF-stimulated ERK and RSK phosphorylation, blocks NGF-mediated PC12 cell differentiation, and antagonizes Ras- and Raf-1-stimulated Elk-1 transactivation. Through interaction with Raf-1, RKTG changes the localization of Raf-1 from cytoplasm to the Golgi apparatus, blocks EGF-stimulated Raf-1 membrane translocation, and reduces the interaction of Raf-1 with Ras and MEK1. In RKTG-null mice, the basal ERK phosphorylation level is increased in the brain and liver. In RKTG-deleted mouse embryonic fibroblasts, EGF-induced ERK phosphorylation is enhanced. Collectively, our results reveal a paradigm of spatial regulation of Raf kinase by RKTG via sequestrating Raf-1 to the Golgi apparatus and thereby inhibiting the ERK signaling pathway.

    Proceedings of the National Academy of Sciences of the United States of America 2007;104;36;14348-53

  • Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy.

    Pandit B, Sarkozy A, Pennacchio LA, Carta C, Oishi K, Martinelli S, Pogna EA, Schackwitz W, Ustaszewska A, Landstrom A, Bos JM, Ommen SR, Esposito G, Lepri F, Faul C, Mundel P, López Siguero JP, Tenconi R, Selicorni A, Rossi C, Mazzanti L, Torrente I, Marino B, Digilio MC, Zampino G, Ackerman MJ, Dallapiccola B, Tartaglia M and Gelb BD

    Center for Molecular Cardiology, Department of Pediatrics and Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, New York 10029, USA.

    Noonan and LEOPARD syndromes are developmental disorders with overlapping features, including cardiac abnormalities, short stature and facial dysmorphia. Increased RAS signaling owing to PTPN11, SOS1 and KRAS mutations causes approximately 60% of Noonan syndrome cases, and PTPN11 mutations cause 90% of LEOPARD syndrome cases. Here, we report that 18 of 231 individuals with Noonan syndrome without known mutations (corresponding to 3% of all affected individuals) and two of six individuals with LEOPARD syndrome without PTPN11 mutations have missense mutations in RAF1, which encodes a serine-threonine kinase that activates MEK1 and MEK2. Most mutations altered a motif flanking Ser259, a residue critical for autoinhibition of RAF1 through 14-3-3 binding. Of 19 subjects with a RAF1 mutation in two hotspots, 18 (or 95%) showed hypertrophic cardiomyopathy (HCM), compared with the 18% prevalence of HCM among individuals with Noonan syndrome in general. Ectopically expressed RAF1 mutants from the two HCM hotspots had increased kinase activity and enhanced ERK activation, whereas non-HCM-associated mutants were kinase impaired. Our findings further implicate increased RAS signaling in pathological cardiomyocyte hypertrophy.

    Funded by: NHLBI NIH HHS: HL066681, HL074728, HL71207; NICHD NIH HHS: HD01294, HD042569; NIDDK NIH HHS: DK57683; NIGMS NIH HHS: T32 GM072474; Telethon: GGP04172, GGP07115

    Nature genetics 2007;39;8;1007-12

  • Germline gain-of-function mutations in RAF1 cause Noonan syndrome.

    Razzaque MA, Nishizawa T, Komoike Y, Yagi H, Furutani M, Amo R, Kamisago M, Momma K, Katayama H, Nakagawa M, Fujiwara Y, Matsushima M, Mizuno K, Tokuyama M, Hirota H, Muneuchi J, Higashinakagawa T and Matsuoka R

    International Research and Educational Institute for Integrated Medical Sciences (IREIIMS), Tokyo Women's Medical University, Tokyo 162-8666, Japan.

    Noonan syndrome is characterized by short stature, facial dysmorphia and a wide spectrum of congenital heart defects. Mutations of PTPN11, KRAS and SOS1 in the RAS-MAPK pathway cause approximately 60% of cases of Noonan syndrome. However, the gene(s) responsible for the remainder are unknown. We have identified five different mutations in RAF1 in ten individuals with Noonan syndrome; those with any of four mutations causing changes in the CR2 domain of RAF1 had hypertrophic cardiomyopathy (HCM), whereas affected individuals with mutations leading to changes in the CR3 domain did not. Cells transfected with constructs containing Noonan syndrome-associated RAF1 mutations showed increased in vitro kinase and ERK activation, and zebrafish embryos with morpholino knockdown of raf1 demonstrated the need for raf1 for the development of normal myocardial structure and function. Thus, our findings implicate RAF1 gain-of-function mutations as a causative agent of a human developmental disorder, representing a new genetic mechanism for the activation of the MAPK pathway.

    Nature genetics 2007;39;8;1013-7

  • Hepatitis B virus X protein stimulates the mitochondrial translocation of Raf-1 via oxidative stress.

    Chen J and Siddiqui A

    Department of Medicine, SCRB 409, University of California-San Diego, 9500 Gilman Drive, MC0711, La Jolla, CA 92093, USA, and Liver Disease Research Center, Second Xiangya Hospital, Changsha, China.

    The human hepatitis B virus (HBV) X protein (HBx) plays a crucial role(s) in the viral life cycle and contributes to the onset of hepatocellular carcinoma (HCC). HBx caused the mitochondrial translocation of Raf-1 kinase either alone or in the context of whole-viral-genome transfections. Mitochondrial translocation of Raf-1 is mediated by HBx-induced oxidative stress and was dependent upon the phosphorylation of Raf-1 at the serine338/339 and Y340/341 residues by p21-activated protein kinase 1 and Src kinase, respectively. These studies provide an insight into the mechanisms by which HBV induces intracellular events relevant to liver disease pathogenesis, including HCC.

    Funded by: NCI NIH HHS: CA64415

    Journal of virology 2007;81;12;6757-60

  • Raf-1 kinase mediates adenylyl cyclase sensitization by TNF-alpha in human airway smooth muscle cells.

    Osawa Y, Yim PD, Xu D, Panettieri RA and Emala CW

    Department of Anesthesiology, College of Physicians and Surgeons of Columbia University, New York, New York 10032, USA.

    Tumor necrosis factor (TNF)-alpha is a potent inflammatory cytokine implicated in the exacerbation of asthma. Chronic exposure to TNF-alpha has been reported to induce G protein-coupled receptor desensitization, but adenylyl cyclase sensitization, in airway smooth muscle cells by an unknown mechanism. Cyclic AMP, which is synthesized by adenylyl cyclases in response to G protein-coupled receptor signals, is an important second messenger involved in the regulation of the airway muscle proliferation, migration, and tone. In other cell types, TNF-alpha receptors transactivate the EGF receptor, which activates raf-1 kinase. Further studies in transfected cells show that raf-1 kinase can phosphorylate and activate some isoforms of adenylyl cyclase. Cultured human airway smooth muscle cells were treated with TNF-alpha in the presence or absence of inhibitors of prostaglandin signaling, protein kinases, or G(i) proteins. TNF-alpha caused a significant dose- (1-10 ng/ml) and time-dependent (24 and 48 h) increase in forskolin-stimulated adenylyl cyclase activity, which was abrogated by pretreatment with GW5074 (a raf-1 kinase inhibitor), was partially inhibited by an EGF receptor inhibitor, but was unaffected by pertussis toxin. TNF-alpha also increased phosphorylation of Ser(338) on raf-1 kinase, indicative of activation. IL-1beta and EGF sensitization of adenylyl cyclase activity was also sensitive to raf-1 kinase inhibition by GW5074. Taken together, these studies link two signaling pathways not previously characterized in human airway smooth muscle cells: TNF-alpha transactivation of the EGF receptor, with subsequent raf-1 kinase-mediated activation of adenylyl cyclase.

    Funded by: NHLBI NIH HHS: HL-58519

    American journal of physiology. Lung cellular and molecular physiology 2007;292;6;L1414-21

  • Human Raf-1 proteins associate with Rad24 and Cdc25 in cell-cycle checkpoint pathway of fission yeast, Schizosaccharomyces pombe.

    Lee M and Yoo HS

    Department of Biology, College of Natural Sciences, University of Incheon, Incheon, Korea. mikelee@incheon.ac.kr

    Raf-1 is a serine/threonine protein kinase that connects cell surface receptor signals to nuclear transcription factors. By screening Schizosaccharomyces pombe (S. pombe) cDNA library, we isolated Rad24, which is a 14-3-3 homolog that is important in the DNA damage checkpoint in S. pombe, as a Raf-1 interacting protein. The interaction found in yeast was confirmed by co-immunoprecipitation. Furthermore, Cdc25, which has been known to bind to Rad24, also associated with Raf-1 and was phosphorylated in vitro by catalytically active Raf-1. However, in the presence of Raf-1, an interaction between Rad24 and Cdc25 was inhibited in triple hybrid assay, indicating that Raf-1 inhibits the interaction between Rad24 and Cdc25. An in vitro competition assay showed that the binding of Cdc25 and of Rad24 to Raf-1 is mutually exclusive. Western blots of whole cell lysates probed with polyclonal antibodies specific for tyrosine-15-phosphorylated Cdc2 showed that overproduction of Rad24 led to the dephosphorylation of tyrosine residue on Cdc2, which is known to be activated through dephosphorylation by Cdc25 phosphatase. Unexpectedly, overexpression of catalytically inactive mutant protein of Raf-1, S624A, also caused tyrosine dephosphorylation of Cdc2. Thus, these data suggest that Raf-1 may interfere with the role of Rad24 by competing with Rad24 for binding to Cdc25 or a direct phosphorylation of Cdc25, bypassing the checkpoint pathway in DNA repair through Cdc25 activation.

    Journal of cellular biochemistry 2007;101;2;488-97

  • C-Raf antagonizes apoptosis induced by IFN-alpha in human lung cancer cells by phosphorylation and increase of the intracellular content of elongation factor 1A.

    Lamberti A, Longo O, Marra M, Tagliaferri P, Bismuto E, Fiengo A, Viscomi C, Budillon A, Rapp UR, Wang E, Venuta S, Abbruzzese A, Arcari P and Caraglia M

    Department of Biochemistry and Medical Biotechnology, Federico II University of Naples, Naples, Italy.

    Interferon alpha (IFNalpha) induces both apoptosis and a counteracting epidermal growth factor Erk-dependent survival response in cancer cells. In this report, IFNalpha increased eukaryotic elongation factor 1A (eEF-1A) protein expression by inhibition of eEF-1A degradation via a proteasome-dependent pathway. The reduction of the expression level of eEF-1A by RNA interference enhanced the apoptosis induced by IFNalpha on the same cells. Moreover, IFNalpha induced the phosphorylation of both serine and threonine in eEF-1A. These effects were paralleled by an increased co-immunoprecipitation and colocalization of eEF-1A with C-Raf. The suppression of C-Raf kinase activity with the inhibitor BAY 43-9006 completely antagonized the increase of both eEF-1A phosphorylation and expression and of C-Raf/eEF-1A colocalization induced by IFNalpha and enhanced apoptosis and eEF-1A ubiquitination. Cell transfection with the mutated K48R ubiquitin increased EF-1A expression and desensitized tumor cells to the modulating effects of IFNalpha. The dynamic simulation of 3Dstructure of eEF-1A identified putative serine and threonine phosphorylation sites. In conclusion, the interaction between eEF-1A and C-Raf increases eEF-1A stability and induces a survival activity.

    Cell death and differentiation 2007;14;5;952-62

  • C-type lectin DC-SIGN modulates Toll-like receptor signaling via Raf-1 kinase-dependent acetylation of transcription factor NF-kappaB.

    Gringhuis SI, den Dunnen J, Litjens M, van Het Hof B, van Kooyk Y and Geijtenbeek TB

    Department of Molecular Cell Biology and Immunology, VU University Medical Center, 1007 MB Amsterdam, The Netherlands.

    Adaptive immune responses by dendritic cells (DCs) are critically controlled by Toll-like receptor (TLR) function. Little is known about modulation of TLR-specific signaling by other pathogen receptors. Here, we have identified a molecular signaling pathway induced by the C-type lectin DC-SIGN that modulates TLR signaling at the level of the transcription factor NF-kappaB. We demonstrated that pathogens trigger DC-SIGN on human DCs to activate the serine and threonine kinase Raf-1, which subsequently leads to acetylation of the NF-kappaB subunit p65, but only after TLR-induced activation of NF-kappaB. Acetylation of p65 both prolonged and increased IL10 transcription to enhance anti-inflammatory cytokine responses. We demonstrated that different pathogens such as Mycobacterium tuberculosis, M. leprae, Candida albicans, measles virus, and human immunodeficiency virus-1 interacted with DC-SIGN to activate the Raf-1-acetylation-dependent signaling pathway to modulate signaling by different TLRs. Thus, this pathway is involved in regulation of adaptive immunity by DCs to bacterial, fungal, and viral pathogens.

    Funded by: NIAID NIH HHS: N01 AI-25469; PHS HHS: HHSN266200400091C

    Immunity 2007;26;5;605-16

  • Systematic identification of cellular signals reactivating Kaposi sarcoma-associated herpesvirus.

    Yu F, Harada JN, Brown HJ, Deng H, Song MJ, Wu TT, Kato-Stankiewicz J, Nelson CG, Vieira J, Tamanoi F, Chanda SK and Sun R

    Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California, United States of America.

    The herpesvirus life cycle has two distinct phases: latency and lytic replication. The balance between these two phases is critical for viral pathogenesis. It is believed that cellular signals regulate the switch from latency to lytic replication. To systematically evaluate the cellular signals regulating this reactivation process in Kaposi sarcoma-associated herpesvirus, the effects of 26,000 full-length cDNA expression constructs on viral reactivation were individually assessed in primary effusion lymphoma-derived cells that harbor the latent virus. A group of diverse cellular signaling proteins were identified and validated in their effect of inducing viral lytic gene expression from the latent viral genome. The results suggest that multiple cellular signaling pathways can reactivate the virus in a genetically homogeneous cell population. Further analysis revealed that the Raf/MEK/ERK/Ets-1 pathway mediates Ras-induced reactivation. The same pathway also mediates spontaneous reactivation, which sets the first example to our knowledge of a specific cellular pathway being studied in the spontaneous reactivation process. Our study provides a functional genomic approach to systematically identify the cellular signals regulating the herpesvirus life cycle, thus facilitating better understanding of a fundamental issue in virology and identifying novel therapeutic targets.

    Funded by: NCI NIH HHS: CA 32737, CA83525, CA91791, P01 CA032737, R01 CA091791; NIDCR NIH HHS: DE14153, R01 DE014153

    PLoS pathogens 2007;3;3;e44

  • Raf 1 represses expression of the tight junction protein occludin via activation of the zinc-finger transcription factor slug.

    Wang Z, Wade P, Mandell KJ, Akyildiz A, Parkos CA, Mrsny RJ and Nusrat A

    Epithelial Pathobiology Research Unit, Department of Pathology, Emory University, Atlanta, GA 30322, USA.

    Although dysregulation of tight junction (TJ) proteins is observed in epithelial malignancy, their participation in epithelial transformation is poorly understood. Recently we demonstrated that expression of oncogenic Raf 1 in Pa4 epithelial cells disrupts TJs and induces an oncogenic phenotype by downregulating expression of the TJ protein, occludin. Here we report the mechanism by which Raf 1 regulates occludin expression. Raf 1 inhibited occludin transcription by repressing a minimal segment of the occludin promoter in concert with upregulation of the transcriptional repressor, Slug without influencing the well-documented transcriptional repressor, Snail. Overexpression of Slug in Pa4 cells recapitulated the effect of Raf 1 on occludin expression, and depletion of Slug by small interfering RNA abrogated the effect of Raf 1 on occludin. Finally, chromatin immunoprecipitation assays and site-directed mutagenesis demonstrated a direct interaction between Slug and an E-box within the minimal Raf 1-responsive segment of the occludin promoter. These findings support a role of Slug in mediating Raf 1-induced transcriptional repression of occludin and subsequent epithelial to mesenchymal transition.

    Funded by: NIDDK NIH HHS: DK 59888, DK 61379

    Oncogene 2007;26;8;1222-30

  • Oncogenic tyrosine kinase NPM/ALK induces activation of the MEK/ERK signaling pathway independently of c-Raf.

    Marzec M, Kasprzycka M, Liu X, Raghunath PN, Wlodarski P and Wasik MA

    Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

    The mechanisms of cell transformation mediated by the highly oncogenic, chimeric NPM/ALK tyrosine kinase remain only partially understood. Here we report that cell lines and native tissues derived from the NPM/ALK-expressing T-cell lymphoma (ALK+ TCL) display phosphorylation of the extracellular signal-regulated protein kinase (ERK) 1/2 complex. Transfection of BaF3 cells with NPM/ALK induces phosphorylation of EKR1/2 and of its direct activator mitogen-induced extracellular kinase (MEK) 1/2. Depletion of NPM/ALK by small interfering RNA (siRNA) or its inhibition by WHI-154 abrogates the MEK1/2 and ERK1/2 phosphorylation. The NPM/ALK-induced MEK/ERK activation is independent of c-Raf as evidenced by the lack of MEK1/2 and ERK1/2 phosphorylation upon c-Raf inactivation by two different inhibitors, RI and ZM336372, and by its siRNA-mediated depletion. In contrast, ERK1/2 activation is strictly MEK1/2 dependent as shown by suppression of the ERK1/2 phosphorylation by the MEK1/2 inhibitor U0126. The U0126-mediated inhibition of ERK1/2 activation impaired proliferation and viability of the ALK+ TCL cells and expression of antiapoptotic factor Bcl-xL and cell cycle-promoting CDK4 and phospho-RB. Finally, siRNA-mediated depletion of both ERK1 and ERK2 inhibited cell proliferation, whereas depletion of ERK 1 (but not ERK2) markedly increased cell apoptosis. These findings identify MEK/ERK as a new signaling pathway activated by NPM/ALK and indicate that the pathway represents a novel therapeutic target in the ALK-induced malignancies.

    Funded by: NCI NIH HHS: R01-CA89194, R01-CA96856

    Oncogene 2007;26;6;813-21

  • Large-scale mapping of human protein-protein interactions by mass spectrometry.

    Ewing RM, Chu P, Elisma F, Li H, Taylor P, Climie S, McBroom-Cerajewski L, Robinson MD, O'Connor L, Li M, Taylor R, Dharsee M, Ho Y, Heilbut A, Moore L, Zhang S, Ornatsky O, Bukhman YV, Ethier M, Sheng Y, Vasilescu J, Abu-Farha M, Lambert JP, Duewel HS, Stewart II, Kuehl B, Hogue K, Colwill K, Gladwish K, Muskat B, Kinach R, Adams SL, Moran MF, Morin GB, Topaloglou T and Figeys D

    Protana, Toronto, Ontario, Canada.

    Mapping protein-protein interactions is an invaluable tool for understanding protein function. Here, we report the first large-scale study of protein-protein interactions in human cells using a mass spectrometry-based approach. The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. Large-scale immunoprecipitation of Flag-tagged versions of these proteins followed by LC-ESI-MS/MS analysis resulted in the identification of 24,540 potential protein interactions. False positives and redundant hits were filtered out using empirical criteria and a calculated interaction confidence score, producing a data set of 6463 interactions between 2235 distinct proteins. This data set was further cross-validated using previously published and predicted human protein interactions. In-depth mining of the data set shows that it represents a valuable source of novel protein-protein interactions with relevance to human diseases. In addition, via our preliminary analysis, we report many novel protein interactions and pathway associations.

    Molecular systems biology 2007;3;89

  • Distribution, levels and phosphorylation of Raf-1 in Alzheimer's disease.

    Mei M, Su B, Harrison K, Chao M, Siedlak SL, Previll LA, Jackson L, Cai DX and Zhu X

    Department of Pathology, Case Western Reserve University, and MetroHealth Medical Center, Cleveland, Ohio 44106, USA.

    Extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein kinase pathway, has been increasingly implicated in the pathogenesis of Alzheimer's disease due to its critical role in brain function. While we previously demonstrated that ERK is activated in Alzheimer's disease, the upstream cascade leading to its activation had not been fully examined. In this study, we focused on Raf-1, one of the physiological activators of the ERK pathway. Raf-1 is activated by phosphorylation at Ser338 and Tyr340/341 and inhibited by phosphorylation at Ser259. Interestingly, phosphorylation at all three sites on Raf-1 was increased as evidenced by both immunocytochemistry and immunoblot analysis in Alzheimer's disease brains compared to age-matched controls. Both phospho-Raf-1 (Ser259) and phospho-Raf-1 (Ser338) were localized to intracytoplasmic granular structures, whereas phospho-Raf-1 (Tyr340/341) was localized to neurofibrillary tangles and granules in pyramidal neurons in Alzheimer's disease hippocampus. There is extensive overlap between phospho-Raf-1 (Ser338) and phospho-Mek1/2, the downstream effector of Raf-1, suggestive of a mechanistic link. Additionally, increased levels of Raf-1 are associated with Ras and MEK1 in Alzheimer's disease as evidenced by its coimmunoprecipitation with Ras and Mek1, respectively. Based on these findings, we speculate that Raf-1 is activated to effectively mediate Ras-dependent signals in Alzheimer's disease.

    Journal of neurochemistry 2006;99;5;1377-88

  • Regulation of RKIP binding to the N-region of the Raf-1 kinase.

    Park S, Rath O, Beach S, Xiang X, Kelly SM, Luo Z, Kolch W and Yeung KC

    Medical University of Ohio, Department of Biochemistry and Cancer Biology, 3035 Arlington Avenue, Toledo, OH 43614-5804, USA.

    The Raf kinase inhibitory protein (RKIP) binds to Raf-1 interfering with binding of the MEK substrate and potentially also Raf-1 activation. In response to mitogen stimulation RKIP dissociates from Raf-1 and later re-associates. Here, using a combination of mutational approaches, biochemical studies, peptide arrays and plasmon surface resonance (BIAcore), we fine map and characterize a minimal 24 amino acid long RKIP binding domain in the Raf-1 N-region, which consists of constitutive elements at both flanks and a center element that is regulated by phosphorylation and enhances the re-binding of RKIP to Raf-1 in the later phase of mitogen stimulation.

    Funded by: NIGMS NIH HHS: R01 GM057959, R01 GM064767, R01 GM64767

    FEBS letters 2006;580;27;6405-12

  • Global, in vivo, and site-specific phosphorylation dynamics in signaling networks.

    Olsen JV, Blagoev B, Gnad F, Macek B, Kumar C, Mortensen P and Mann M

    Center for Experimental BioInformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, DK-5230 Odense, Denmark.

    Cell signaling mechanisms often transmit information via posttranslational protein modifications, most importantly reversible protein phosphorylation. Here we develop and apply a general mass spectrometric technology for identification and quantitation of phosphorylation sites as a function of stimulus, time, and subcellular location. We have detected 6,600 phosphorylation sites on 2,244 proteins and have determined their temporal dynamics after stimulating HeLa cells with epidermal growth factor (EGF) and recorded them in the Phosida database. Fourteen percent of phosphorylation sites are modulated at least 2-fold by EGF, and these were classified by their temporal profiles. Surprisingly, a majority of proteins contain multiple phosphorylation sites showing different kinetics, suggesting that they serve as platforms for integrating signals. In addition to protein kinase cascades, the targets of reversible phosphorylation include ubiquitin ligases, guanine nucleotide exchange factors, and at least 46 different transcriptional regulators. The dynamic phosphoproteome provides a missing link in a global, integrative view of cellular regulation.

    Cell 2006;127;3;635-48

  • Activation of Ras/Raf protects cells from melanoma differentiation-associated gene-5-induced apoptosis.

    Lin L, Su Z, Lebedeva IV, Gupta P, Boukerche H, Rai T, Barber GN, Dent P, Sarkar D and Fisher PB

    Department of Pathology, Herbert Irving Comprehensive Cancer Center, Columbia University Medical Center, College of Physicians and Surgeons, New York, NY 10032, USA.

    Melanoma differentiation-associated gene-5 (mda-5) was the first molecule identified in nature whose encoded protein embodied the unique structural combination of an N-terminal caspase recruitment domain and a C-terminal DExD/H RNA helicase domain. As suggested by its structure, cumulative evidences documented that ectopic expression of mda-5 leads to growth inhibition and/or apoptosis in various cell lines. However, the signaling pathways involved in mda-5-mediated killing have not been elucidated. In this study, we utilized either genetically modified cloned rat embryo fibroblast cells overexpressing different functionally and structurally distinct oncogenes or human pancreatic and colorectal carcinoma cells containing mutant active ras to resolve the role of the Ras/Raf signaling pathway in mda-5-mediated growth inhibition/apoptosis induction. Rodent and human tumor cells containing constitutively activated Raf/Raf/MEK/ERK pathways were resistant to mda-5-induced killing and this protection was antagonized by intervening in this signal transduction cascade either by directly inhibiting ras activity using an antisense strategy or by targeting ras-downstream factors, such as MEK1/2, with the pharmacological inhibitor PD98059. The present findings provide a further example of potential cross-talk between growth-inhibitory and growth-promoting pathways in which the ultimate balance of these factors defines cellular homeostasis, leading to survival or induction of programmed cell death.

    Funded by: NIGMS NIH HHS: GM068848

    Cell death and differentiation 2006;13;11;1982-93

  • Raf-1 signaling is required for the later stages of 1,25-dihydroxyvitamin D3-induced differentiation of HL60 cells but is not mediated by the MEK/ERK module.

    Wang X and Studzinski GP

    Department of Pathology and Laboratory Medicine, UMDNJ-New Jersey Medical School, Newark, New Jersey.

    We are interested in determining the signaling pathways for 1,25-dihydroxyvitamin D3 (1,25D)-induced differentiation of HL60 leukemic cells. One possible candidate is Raf-1, which is known to signal cell proliferation and neoplastic transformation through MEK, ERK, and downstream targets. It can also participate in the regulation of cell survival and various forms of cell differentiation, though the precise pathways are less well delineated. Here we report that Raf-1 has a role in monocytic differentiation of human myeloid leukemia HL60, which is not mediated by MEK and ERK, but likely by direct interaction with p90RSK. Specifically, we show that Raf-1 and p90RSK are increasingly activated in the later stages of differentiation of HL60 cells, at the same time as activation of MEK and ERK is decreasing. Transfection of a wild-type Raf-1 construct enhances 1,25D-induced differentiation, while antisense Raf-1 or short interfering (si) Raf-1 reduces 1,25D-induced differentiation. In contrast, antisense oligodeoxynucleotides (ODN) and siRNAs to MEK or ERK have no detectable effect on differentiation. In late stage differentiating cells Raf-1 and p90RSK are found as a complex, and inhibition of Raf-1, but not MEK or ERK expression reduces the levels of phosphorylated p90 RSK. These findings support the thesis that Raf-1 signals cell proliferation and cell differentiation through different intermediary proteins.

    Funded by: NCI NIH HHS: R01 CA044722, R01 CA044722-16, R01-CA44722-16

    Journal of cellular physiology 2006;209;2;253-60

  • In melanoma, RAS mutations are accompanied by switching signaling from BRAF to CRAF and disrupted cyclic AMP signaling.

    Dumaz N, Hayward R, Martin J, Ogilvie L, Hedley D, Curtin JA, Bastian BC, Springer C and Marais R

    Signal Transduction Team, The Institute for Cancer Research, Cancer Research UK Centre of Cell and Molecular Biology, London, United Kingdom.

    Melanocytes require the RAS/RAF/MEK/ERK and the cyclic AMP (cAMP) signaling pathways to maintain the fine balance between proliferation and differentiation. We have investigated how cross-talk between these pathways affects melanoma progression. We show that cAMP suppresses CRAF activity in melanocytes and that this is essential to suppress the oncogenic potential of CRAF in these cells. As a consequence, BRAF alone is responsible for signaling to MEK. However, when RAS is mutated in melanoma, the cells switch their signaling from BRAF to CRAF. This switch is accompanied by dysregulated cAMP signaling, a step that is necessary to allow CRAF to signal to MEK. Thus, a fundamental switch in RAF isoform usage occurs when RAS is mutated in melanoma, and this occurs in the context of disrupted cAMP signaling. These data have important implications for the development of therapeutic strategies to treat this life-threatening disease.

    Cancer research 2006;66;19;9483-91

  • Interaction and stabilization of X-linked inhibitor of apoptosis by Raf-1 protein kinase.

    Tian S, Mewani RR, Kumar D, Li B, Danner MT, Ahmad I, Rahman A, Notario V, Dritschilo A, Kasid UN and Gokhale PC

    Department of Radiation Medicine, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington DC 20057, USA.

    Raf-1 serine/threonine protein kinase plays an important role in cell growth, differentiation and cell survival. Recent reports using c-raf-1 gene-knockouts have observed MEK/ERK independent functions of Raf-1 in cell survival and protection from apoptosis. Raf-1 has also been shown to be involved in counteracting specific apoptotic pathways by restraining caspase activation, although the precise mechanism is unknown. XIAP is a potent inhibitor of apoptosis that blocks both the mitochondria and death receptor mediated pathways of apoptosis by directly binding to and inhibiting the initiator and effector caspases. In our efforts to understand the mechanism by which Raf-1 inhibits caspase activation, we discovered a novel interaction between Raf-1 and XIAP. In this study, we describe the physical interaction between Raf-1 and XIAP in vitro and in vivo in mammalian cells. We also demonstrate that Raf-1 phosphorylates XIAP in vitro and in vivo. Additionally, Raf-1 prevents XIAP degradation in response to different apoptotic triggers. Our studies identify XIAP as a new substrate of Raf-1 and provide potentially important insight into mechanisms underlying Raf-1 effects on cell survival.

    Funded by: NCI NIH HHS: 5 P01 CA074175

    International journal of oncology 2006;29;4;861-7

  • Rheb inhibits C-raf activity and B-raf/C-raf heterodimerization.

    Karbowniczek M, Robertson GP and Henske EP

    Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.

    The Ras-Raf-MEK signaling cascade is critical for normal development and is activated in many forms of cancer. We have recently shown that B-Raf kinase interacts with and is inhibited by Rheb, the target of the GTPase-activating domain of the tuberous sclerosis complex 2 gene product tuberin. Here, we demonstrate for the first time that activation of Rheb is associated with decreased B-Raf and C-Raf phosphorylation at residues Ser-446 and Ser-338, respectively, concomitant with a decrease in the activities of both kinases and decreased heterodimerization of B-Raf and C-Raf. Importantly, the impact of Rheb on B-Raf/C-Raf heterodimerization and kinase activity are rapamycin-insensitive, indicating that they are independent of Rheb activation of the mammalian target of rapamycin-Raptor complex. In addition, we found that Rheb inhibits the association of B-Raf with H-Ras. Taken together, these results support a central role of Rheb in the regulation of the Ras/B-Raf/C-Raf/MEK signaling network.

    Funded by: NIDDK NIH HHS: DK 51052

    The Journal of biological chemistry 2006;281;35;25447-56

  • A dimeric kinase assembly underlying autophosphorylation in the p21 activated kinases.

    Pirruccello M, Sondermann H, Pelton JG, Pellicena P, Hoelz A, Chernoff J, Wemmer DE and Kuriyan J

    Howard Hughes Medical Institute, Department of Molecular and Cell Biology, Department of Chemistry, University of California, Berkeley, CA 94720, USA.

    The p21-activated kinases (PAKs) are serine/threonine kinases that are involved in a wide variety of cellular functions including cytoskeletal motility, apoptosis, and cell cycle regulation. PAKs are inactivated by blockage of the active site of the kinase domain by an N-terminal regulatory domain. GTP-bound forms of Cdc42 and Rac bind to the regulatory domain and displace it, thereby allowing phosphorylation of the kinase domain and maximal activation. A key step in the activation process is the phosphorylation of the activation loop of one PAK kinase domain by another, but little is known about the underlying recognition events that make this phosphorylation specific. We show that the phosphorylated kinase domain of PAK2 dimerizes in solution and that this association is prevented by addition of a substrate peptide. We have identified a crystallographic dimer in a previously determined crystal structure of activated PAK1 in which two kinase domains are arranged face to face and interact through a surface on the large lobe of the kinase domain that is exposed upon release of the auto-inhibitory domain. The crystallographic dimer is suggestive of an engagement that mediates trans-autophosphorylation. Mutations at the predicted dimerization interface block dimerization and reduce the rate of autophosphorylation, supporting the role of this interface in PAK activation.

    Funded by: NCRR NIH HHS: RR15756; NIGMS NIH HHS: GM-08295

    Journal of molecular biology 2006;361;2;312-26

  • Nicotine induces cell proliferation by beta-arrestin-mediated activation of Src and Rb-Raf-1 pathways.

    Dasgupta P, Rastogi S, Pillai S, Ordonez-Ercan D, Morris M, Haura E and Chellappan S

    Department of Interdisciplinary Oncology, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa 33612, USA.

    Recent studies have shown that nicotine, a component of cigarette smoke, can stimulate the proliferation of non-neuronal cells. While nicotine is not carcinogenic by itself, it has been shown to induce cell proliferation and angiogenesis. Here we find that mitogenic effects of nicotine in non-small cell lung cancers (NSCLCs) are analogous to those of growth factors and involve activation of Src, induction of Rb-Raf-1 interaction, and phosphorylation of Rb. Analysis of human NSCLC tumors show enhanced levels of Rb-Raf-1 complexes compared with adjacent normal tissue. The mitogenic effects of nicotine were mediated via the alpha7-nAChR subunit and resulted in enhanced recruitment of E2F1 and Raf-1 on proliferative promoters in NSCLC cell lines and human lung tumors. Nicotine stimulation of NSCLC cells caused dissociation of Rb from these promoters. Proliferative signaling via nicotinic acetylcholine receptors (nAChRs) required the scaffolding protein beta-arrestin; ablation of beta-arrestin or disruption of the Rb-Raf-1 interaction blocked nicotine-induced proliferation of NSCLCs. Additionally, suppression of beta-arrestin also blocked activation of Src, suppressed levels of phosphorylated ERK, and abrogated Rb-Raf-1 binding in response to nicotine. It appears that nicotine induces cell proliferation by beta-arrestin-mediated activation of the Src and Rb-Raf-1 pathways.

    Funded by: NCI NIH HHS: CA63136, R01 CA063136

    The Journal of clinical investigation 2006;116;8;2208-2217

  • Three-dimensional culture regulates Raf-1 expression to modulate fibronectin matrix assembly.

    Winters BS, Raj BK, Robinson EE, Foty RA and Corbett SA

    Department of Surgery, Robert Wood Johnson Medical School, New Brunswick, NJ 08903, USA.

    Oncogenic transformation has been associated with decreased fibronectin (FN) matrix assembly. For example, both the HT-1080 fibrosarcoma and MAT-LyLu cell lines fail to assemble a FN matrix when grown in monolayer culture (2-dimensional [2D] system). In this study, we show that these cells regain the ability to assemble a FN matrix when they are grown as aggregates (3-dimensional [3D] system). FN matrix assembly in 3D correlates with decreased Raf-1 protein expression compared with cells grown in monolayer culture. This effect is associated with reduced Raf-1 mRNA levels as determined by quantitative RT-PCR and not proteasome-mediated degradation of endogenous Raf-1. Interestingly, transient expression of a Raf-1 promoter-reporter construct demonstrates increased Raf-1 promoter activity in 3D, suggesting that the transition to 3D culture may modulate Raf-1 mRNA stability. Finally, to confirm that decreased Raf-1 expression results in increased FN matrix assembly, we used both pharmacological and small interfering RNA knockdown of Raf-1. This restored the ability of cells in 2D culture to assemble a FN matrix. Moreover, overexpression of Raf-1 prevented FN matrix assembly by cells cultured in 3D, resulting in decreased aggregate compaction. This work provides new insight into how the cell microenvironment may influence Raf-1 expression to modulate cell-FN interactions in 3D.

    Funded by: NIGMS NIH HHS: GM-061487

    Molecular biology of the cell 2006;17;8;3386-96

  • Neuronal nitric oxide synthase-induced S-nitrosylation of H-Ras inhibits calcium ionophore-mediated extracellular-signal-regulated kinase activity.

    Raines KW, Cao GL, Lee EK, Rosen GM and Shapiro P

    Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201, USA.

    nNOS (neuronal nitric oxide synthase) is a constitutively expressed enzyme responsible for the production of NO* from L-arginine and O2. NO* acts as both an intra- and an inter-cellular messenger that mediates a variety of signalling pathways. Previous studies from our laboratory have demonstrated that nNOS production of NO* blocks Ca2+-ionophore-induced activation of ERK1/2 (extracellular-signal-regulated kinase 1/2) of the mitogen-activated protein kinases through a mechanism involving Ras G-proteins and Raf-1 kinase. Herein we describe a mechanism by which NO* blocks Ca2+-mediated ERK1/2 activity through direct modification of H-Ras. Ca2+-mediated ERK1/2 activation in NO*-producing cells could be restored by exogenous expression of constitutively active mitogen-activated protein kinase kinase 1. In contrast, exogenous expression of constitutively active mutants of Raf-1 and H-Ras only partially restored ERK1/2 activity, by 50% and 10% respectively. On the basis of these findings, we focused on NO*-mediated mechanisms of H-Ras inhibition. Assays for GTP loading and H-Ras interactions with the Ras-binding domain on Raf-1 demonstrated a decrease in H-Ras activity in the presence of NO*. We demonstrate that S-nitrosylation of H-Ras occurs in nNOS-expressing cells activated with Ca2+ ionophore. Mutation of a putative nitrosylation site at Cys118 inhibited S-nitrosylation and restored ERK1/2 activity by constitutively active H-Ras even in the presence of NO*. These findings indicate that intracellular generation of NO* by nNOS leads to S-nitrosylation of H-Ras, which interferes with Raf-1 activation and propagation of signalling through ERK1/2.

    Funded by: NCI NIH HHS: CA-10529; NIBIB NIH HHS: EB-2034, P41 EB002034; NIGMS NIH HHS: R25 GM055036, R25-GM55036

    The Biochemical journal 2006;397;2;329-36

  • Hyaluronan-CD44 interaction with leukemia-associated RhoGEF and epidermal growth factor receptor promotes Rho/Ras co-activation, phospholipase C epsilon-Ca2+ signaling, and cytoskeleton modification in head and neck squamous cell carcinoma cells.

    Bourguignon LY, Gilad E, Brightman A, Diedrich F and Singleton P

    Department of Medicine, University of California at San Francisco and Endocrine Unit (111N), Veterans Affairs Medical Center, San Francisco, California 94121, USA. lillyb@itsa.ucsf.edu

    In this study we have examined the interaction of CD44 (a major hyaluronan (HA) receptor) with a RhoA-specific guanine nucleotide exchange factor (leukemia-associated RhoGEF (LARG)) in human head and neck squamous carcinoma cells (HNSCC-HSC-3 cell line). Immunoprecipitation and immunoblot analyses indicate that CD44 and the LARG protein are expressed in HSC-3 cells and that these two proteins are physically associated as a complex. HA-CD44 binding induces LARG-specific RhoA signaling and phospholipase C epsilon (PLC epsilon) activity. In particular, the activation of RhoA-PLC epsilon by HA stimulates inositol 1,4,5-triphosphate production, intracellular Ca2+ mobilization, and the up-regulation of Ca2+/calmodulin-dependent kinase II (CaMKII), leading to phosphorylation of the cytoskeletal protein, filamin. The phosphorylation of filamin reduces its interaction with filamentous actin, promoting tumor cell migration. The CD44-LARG complex also interacts with the EGF receptor (EGFR). Most importantly, the binding of HA to the CD44-LARG-EGFR complex activates the EGFR receptor kinase, which in turn promotes Ras-mediated stimulation of a downstream kinase cascade including the Raf-1 and ERK pathways leading to HNSCC cell growth. Using a recombinant fragment of LARG (the LARG-PDZ domain) and a binding assay, we have determined that the LARG-PDZ domain serves as a direct linker between CD44 and EGFR. Transfection of the HSC-3 cells with LARG-PDZcDNA significantly reduces LARG association with CD44 and EGFR. Overexpression of the LARG-PDZ domain also functions as a dominant-negative mutant (similar to the PLC/Ca2+-calmodulin-dependent kinase II (CaMKII) and EGFR/MAPK inhibitor effects) to block HA/CD44-mediated signaling events (e.g. EGFR kinase activation, Ras/RhoA co-activation, Raf-ERK signaling, PLC epsilon-mediated inositol 1,4,5-triphosphate production, intracellular Ca2+ mobilization, CaMKII activity, filamin phosphorylation, and filamin-actin binding) and to abrogate tumor cell growth/migration. Taken together, our findings suggest that CD44 interaction with LARG and EGFR plays a pivotal role in Rho/Ras co-activation, PLC epsilon-Ca2+ signaling, and Raf/ERK up-regulation required for CaMKII-mediated cytoskeleton function and in head and neck squamous cell carcinoma progression.

    Funded by: NCI NIH HHS: R01 CA66163, R01 CA78633; NIAMS NIH HHS: P01 AR39448

    The Journal of biological chemistry 2006;281;20;14026-40

  • Lack of association between autism and SLC25A12.

    Rabionet R, McCauley JL, Jaworski JM, Ashley-Koch AE, Martin ER, Sutcliffe JS, Haines JL, DeLong GR, Abramson RK, Wright HH, Cuccaro ML, Gilbert JR and Pericak-Vance MA

    Center for Human Genetics, Department of Medicine, Duke University Medical Center, 595 LaSalle St., Durham, NC 27710, USA.

    Objective: Autism has a strong, complex genetic component, most likely involving several genes. Multiple genomic screens have shown evidence suggesting linkage to chromosome 2q31-q33, which includes the SLC25A12 gene. Recently, an association between autism risk and two single nucleotide polymorphisms (SNPs) in SLC25A12 was reported. This study aimed to test for association in SLC25A12 in an independent data set of 327 families with autistic offspring.

    Method: The authors analyzed two SNPs that were significant in the previous study group, as well as seven additional SNPs within the gene. Association analyses for individual SNPs as well as haplotypes were performed.

    Results: There was no evidence of an association between SLC25A12 and autism.

    Conclusions: These results suggest that SLC25A12 is not a major contributor to autism risk in these families.

    Funded by: NCRR NIH HHS: RR-00095; NINDS NIH HHS: NS-26630, NS-36768

    The American journal of psychiatry 2006;163;5;929-31

  • Collagen type I-mediated activation of ERK/MAP Kinase is dependent on Ras, Raf-1 and protein phosphatase 2A in Jurkat T cells.

    Chetoui N, Gendron S, Chamoux E and Aoudjit F

    Centre de Recherche en Rhumatologie et Immunologie, Centre Hospitalier Universitaire de Québec, Pavillon CHUL, and Faculté de Médecine, Université Laval, 2705, Blvd. Laurier, Local T1-49, Que., Canada G1V 4G2.

    Growing evidence indicates that interactions of T cells with extracellular matrix through beta1 integrins are important for the regulation of T cell-mediated immune responses and diseases. In this regard, we have recently demonstrated that collagen I (Coll I) through alpha2beta1 integrin inhibited Fas-induced apoptosis of T cells by activating a protein phosphatase 2A (PP2A)-dependent ERK/MAP Kinase pathway. As survival of T cells is critical for their functions, we further investigated the mechanisms underlying the activation of this pathway. Inhibition studies demonstrated that Coll I activates the ERK/MAP Kinase pathway in Jurkat T cells through the activation of Ras and Raf-1. Activation of PP2A was not necessary for the binding of Coll I to Jurkat T cells, but is required for the activation of Raf-1. In accordance, activation of Ras, Raf-1 and PP2A were also required for the ability of Coll I to protect Jurkat T cells from Fas-induced apoptosis. In contrast and despite its capacity to activate Ras, fibronectin (Fbn) failed to activate PP2A and Raf-1. These results might explain, at least in part, the weak ability of Fbn to activate ERK in T cells, supporting thus the differential signaling of beta1 integrin members in these cells. This study provides novel insights into the mechanisms by which beta1 integrins activate the ERK/MAP Kinase pathway in T cells, and is the first report to provide a role for PP2A in integrin-mediated ERK/MAP Kinase activation.

    Molecular immunology 2006;43;10;1687-93

  • Raf-1 is the predominant Raf isoform that mediates growth factor-stimulated growth in ovarian cancer cells.

    McPhillips F, Mullen P, MacLeod KG, Sewell JM, Monia BP, Cameron DA, Smyth JF and Langdon SP

    Cancer Research UK Centre, University of Edinburgh, Edinburgh EH4 2XR, UK.

    There is currently much interest in the role of the Raf family in cancer, particularly since mutated B-Raf has been shown to be oncogenic in certain disease types. In this study we have explored the expression, signaling and function of the three known Raf isoforms (Raf-1, A-Raf and B-Raf) in patients with ovarian cancer. While increased expression of Raf-1 was associated with poor survival, increased expression of B-Raf was associated with improved survival. Using a panel of ovarian cancer cell lines, all three isoforms were shown to be involved in growth factor initiated signaling. Antisense inhibition of function in ovarian cancer cell lines indicated that both Raf-1 and A-Raf, but not B-Raf, were linked to cell proliferation. Raf-1 (but not A-Raf or B-Raf) was also associated with reduced apoptosis. While individual Raf reduction by isoform-targeted antisense oligonucleotides (ODNs) produced growth inhibition in some cell lines, similar use of the MEK inhibitor UO126 produced growth inhibition in all cell lines tested. These data suggest that Raf-1 is the predominant Raf isoform responsible for regulating cellular growth in ovarian cancer cells and may be particularly important in high grade serous ovarian cancers.

    Carcinogenesis 2006;27;4;729-39

  • Mixed-lineage kinase 3 regulates B-Raf through maintenance of the B-Raf/Raf-1 complex and inhibition by the NF2 tumor suppressor protein.

    Chadee DN, Xu D, Hung G, Andalibi A, Lim DJ, Luo Z, Gutmann DH and Kyriakis JM

    Molecular Cardiology Research Institute, Tufts-New England Medical Center, Tufts University School of Medicine, 750 Washington Street, Boston, MA 02111, USA.

    The Ras --> Raf --> MEK1/2 --> extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway couples mitogenic signals to cell proliferation. B-Raf and Raf-1 function within an oligomer wherein they are regulated in part by mutual transactivation. The MAPK kinase kinase (MAP3K) mixed-lineage kinase 3 (MLK3) is required for mitogen activation of B-Raf and cell proliferation. Here we show that the kinase activity of MLK3 is not required for support of B-Raf activation. Instead, MLK3 is a component of the B-Raf/Raf-1 complex and is required for maintenance of the integrity of this complex. We show that the activation of ERK and the proliferation of human schwannoma cells bearing a loss-of-function mutation in the neurofibromatosis 2 (NF2) gene require MLK3. We find that merlin, the product of NF2, blunts the activation of both ERK and c-Jun N-terminal kinase (JNK). Finally, we demonstrate that merlin and MLK3 can interact in situ and that merlin can disrupt the interactions between B-Raf and Raf-1 or those between MLK3 and either B-Raf or Raf-1. Thus, MLK3 is part of a multiprotein complex and is required for ERK activation. The levels of this complex may be negatively regulated by merlin.

    Funded by: NCI NIH HHS: CA 112399; NIGMS NIH HHS: GM 46577

    Proceedings of the National Academy of Sciences of the United States of America 2006;103;12;4463-8

  • Gene expression profile by inhibiting Raf-1 protein kinase in breast cancer cells.

    Mewani RR, Tian S, Li B, Danner MT, Carr TD, Lee S, Rahman A, Kasid UN, Jung M, Dritschilo A and Gokhale PC

    Department of Radiation Medicine, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC 20057, USA.

    Raf-1 protein serine-threonine kinase plays an important role in cell growth, proliferation, and cell survival. Previously, we and others have demonstrated that antisense raf oligonucleotide-mediated inhibition of Raf-1 expression leads to tumor growth arrest, radiosensitization and chemosensitization in vivo. Raf-1 inhibition is also associated with apoptotic cell death. In this study, we inhibited Raf-1 using an antisense raf oligonucleotide (AS-raf-ODN) to identify downstream targets of Raf-1 using microarray gene expression analysis. Treatment of MDA-MB-231 breast cancer cells with 250 nM AS-raf-ODN led to significant inhibition of Raf-1 protein (75.2 +/- 9.6%) and c-raf-1 mRNA levels (86.2 +/- 3.3%) as compared to untreated control cells. The lipofectin control or mismatch oligonucleotide had no effect on Raf-1 expression. To determine the changes in gene expression profiles that were due to inhibition of Raf-1, we simultaneously compared the gene expression patterns in AS-raf-ODN treated cells with untreated control cells and cells treated with lipofectin alone or MM-ODN. A total of 17 genes (4 upregulated and 13 down-regulated) including c-raf-1 were identified that were altered after AS-raf-ODN treatment. Functional clustering analysis revealed genes involved in apoptosis (Bcl-XL), cell adhesion (paxillin, plectin, Rho GDIalpha, CCL5), metabolism (GM2A, SLC16A3, PYGB), signal transduction (protein kinase C nu), and transcriptional regulation (HMGA1), and membrane-associated genes (GNAS, SLC16A3). Real-time PCR, Northern analysis and Western analysis confirmed the microarray findings. Our study provides insight into Raf-1 related signaling pathways and a model system to identify potential target genes.

    Funded by: NCI NIH HHS: 5 P01 CA074175

    International journal of molecular medicine 2006;17;3;457-63

  • 14-3-3zeta interacts with human thromboxane receptors and is involved in the agonist-induced activation of the extracellular-signal-regulated kinase.

    Yan W, Ding Y and Tai HH

    Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0082, USA.

    Thromboxane receptor (TP) signaling results in a broad range of cellular responses including kinase activation and subsequent nuclear signaling events involved in cell transformation, proliferation, and cell survival. Proteins that may participate in the early signaling following receptor activation remain to be identified. We found that 14-3-3zeta is a novel protein interacting with TP intracellular loop 3 (i3) by yeast two-hybrid system. This interaction was further confirmed by GST pull-down and co-immunoprecipitation methods. Site-directed mutagenesis studies indicated that Pro-236 of the TP-i3 was involved in the binding to the 14-3-3zeta. Co-immunoprecipitation studies in the same cell lysate by TP antibody showed that TP binds not only with the 14-3-3zeta but also with the Raf-1. Our data also demonstrated that TP receptor activation induced by agonist rapidly recruited 14-3-3zeta and Raf-1 to form a complex with the TP on the plasma membrane. The significance of assembling this protein complex was examined by TP agonist-induced extracellular-signal-regulated kinase (ERK) phosphorylation in intact cells. TP agonist, I-BOP, induced ERK phosphorylation in HEK 293 cells expressing wild type TPalpha but significantly lower in those expressing TPalpha-P236V mutant. Attenuation of the expression of 14-3-3zeta by 14-3-3zeta siRNA decreased I-BOP-induced ERK phosphorylation indicating the involvement of the 14-3-3zeta in the signal transduction process. These results suggest that 14-3-3zeta may serve as a scaffold protein to form a protein complex consisting of TP, 14-3-3zeta, and Raf-1, and that this protein complex may be involved in the activation of ERK pathway following TP receptor activation.

    Funded by: NHLBI NIH HHS: HL 46296

    Biochemical pharmacology 2006;71;5;624-33

  • Chaperoning checkpoint kinase 1 (Chk1), an Hsp90 client, with purified chaperones.

    Arlander SJ, Felts SJ, Wagner JM, Stensgard B, Toft DO and Karnitz LM

    Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Graduate School.

    Checkpoint kinase 1 (Chk1), a serine/threonine kinase that regulates DNA damage checkpoints, is destabilized when heat shock protein 90 (Hsp90) is inhibited, suggesting that Chk1 is an Hsp90 client. In the present work we examined the interplay between Chk1 and Hsp90 in intact cells, identified a source of unchaperoned Chk1, and report the in vitro chaperoning of Chk1 in reticulocyte lysates and with purified chaperones and co-chaperones. We find that bacterially expressed Chk1 is post-translationally chaperoned to an active kinase. This reaction minimally requires Hsp90, Hsp70, Hsp40, Cdc37, and the protein kinase CK2. The co-chaperone Hop, although not essential for the activation of Chk1 in vitro, enhanced the chaperoning process, whereas the co-chaperone p23 did not stimulate the chaperoning reaction. Additionally, we found that the C-terminal regulatory domain of Chk1 affects the association of Chk1 with Hsp90. Collectively these results provide new insights into Hsp90-dependent chaperoning of a client kinase and identify a novel, biochemically tractable model system that will be useful to further dissect the Hsp90-dependent chaperoning of this important and ubiquitous class of Hsp90 clients.

    Funded by: NCI NIH HHS: CA104378; NIDDK NIH HHS: DK46249

    The Journal of biological chemistry 2006;281;5;2989-98

  • Erbin inhibits RAF activation by disrupting the sur-8-Ras-Raf complex.

    Dai P, Xiong WC and Mei L

    Program of Developmental Neurobiology, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, 30912, USA.

    Erbin is a member of the LAP (leucine-rich repeat (LRR) and PDZ domain) family. It inhibits Ras-mediated activation of ERK in response to growth factors. In this study, we investigated the mechanisms by which Erbin regulates the Ras-Raf-MEK pathway. The N-terminal LRR domain was necessary and sufficient to inhibit neuregulin-activated expression of epsilon416-Luc, a reporter of ERK activation. On the other hand, Erbin had no effect on Ras activation, but it attenuated neuregulin-induced Raf activation, suggesting that Erbin may regulate Raf activation by Ras. Via the LRR domain, Erbin interacts with Sur-8, a scaffold protein necessary for the Ras-Raf complex. Expression of Erbin attenuated the interaction of Sur-8 with active Ras and Raf. Moreover, Erbin-shRNA, which suppressed Erbin expression at mRNA and protein levels, increased the interaction of Sur-8 with Ras and Raf, ERK activation, and neuregulin-induced expression of endogenous acetylcholine receptor epsilon-subunit mRNA. These results demonstrate a regulatory role of Erbin in the Ras-Raf-MEK pathway, suggesting that Erbin may inhibit ERK activation by disrupting the Sur-8-Ras/Raf interaction.

    The Journal of biological chemistry 2006;281;2;927-33

  • ERK MAP kinase signaling in post-mortem brain of suicide subjects: differential regulation of upstream Raf kinases Raf-1 and B-Raf.

    Dwivedi Y, Rizavi HS, Conley RR and Pandey GN

    Department of Psychiatry, University of Illinois at Chicago, 1601 West Taylor Street, Chicago, IL 60612, USA. ydwivedi@psych.uic.edu

    The Raf kinases Raf-1 and B-Raf are upstream activators of the extracellular signal-regulated kinase (ERK)-signaling pathway and therefore participates in many physiological functions in brain, including neuronal survival and synaptic plasticity. Previously, we observed that activation of ERK-1/2, the downstream component of ERK signaling, is significantly reduced in post-mortem brain of suicide victims. The present study was undertaken to further examine whether suicide brain is also associated with abnormalities in upstream molecules in ERK signaling. The study was performed in prefrontal cortex (PFC) and hippocampus obtained from 28 suicide victims and 21 normal controls. mRNA levels of Raf-1, B-Raf, and cyclophilin were measured by quantitative RT-PCR. Protein levels of Raf-1 and B-Raf were determined by Western blot, whereas their catalytic activities were determined by immunoprecipitation and enzymatic assays. It was observed that the catalytic activity of B-Raf was significantly reduced in PFC and hippocampus of suicide subjects. This decrease was associated with a decrease in its protein, but not mRNA, level. On the other hand, catalytic activity, and mRNA and protein levels, of Raf-1 were not altered in post-mortem brain of suicide subjects. The observed changes were not related to confounding variables; however, Raf-1 showed a negative correlation with age. Also, the changes in B-Raf were present in all suicide subjects, irrespective of psychiatric diagnosis. Our results of selective reduction in catalytic activity and expression of B-Raf but not Raf-1 suggest that B-Raf may be playing an important role in altered ERK signaling in brain of suicide subjects, and thus in the pathophysiology of suicide.

    Funded by: NIMH NIH HHS: K01 MH 01836; PHS HHS: R01 68777

    Molecular psychiatry 2006;11;1;86-98

  • Positive regulation of Raf1-MEK1/2-ERK1/2 signaling by protein serine/threonine phosphatase 2A holoenzymes.

    Adams DG, Coffee RL, Zhang H, Pelech S, Strack S and Wadzinski BE

    Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.

    Protein serine/threonine phosphatase 2A (PP2A) regulates a wide variety of cellular signal transduction pathways. The predominant form of PP2A in cells is a heterotrimeric holoenzyme consisting of a scaffolding (A) subunit, a regulatory (B) subunit, and a catalytic (C) subunit. Although PP2A is known to regulate Raf1-MEK1/2-ERK1/2 signaling at multiple steps in this pathway, the specific PP2A holoenzymes involved remain unclear. To address this question, we established tetracycline-inducible human embryonic kidney 293 cell lines for overexpression of FLAG-tagged Balpha/delta regulatory subunits by approximately 3-fold or knock-down of Balpha by greater than 70% compared with endogenous levels. The expression of functional epitope-tagged B subunits was confirmed by the detection of A and C subunits as well as phosphatase activity in FLAG immune complexes from extracts of cells overexpressing the FLAG-Balpha/delta subunit. Western analysis of the cell extracts using phosphospecific antibodies for MEK1/2 and ERK1/2 demonstrated that activation of these kinases in response to epidermal growth factor was markedly diminished in Balpha knock-down cells but elevated in Balpha- and Bdelta-overexpressing cells as compared with control cells. In parallel with the activation of MEK1/2 and ERK1/2, the inhibitory phosphorylation site of Raf1 (Ser-259) was dephosphorylated in cells overexpressing Balpha or Bdelta. Pharmacological inhibitor studies as well as reporter assays for ERK-dependent activation of the transcription factor Elk1 revealed that the PP2A holoenzymes ABalphaC and ABdeltaC act downstream of Ras and upstream of MEK1 to promote activation of this MAPK signaling cascade. Furthermore both PP2A holoenzymes were found to associate with Raf1 and catalyze dephosphorylation of inhibitory phospho-Ser-259. Together these findings indicate that PP2A ABalphaC and ABdeltaC holoenzymes function as positive regulators of Raf1-MEK1/2-ERK1/2 signaling by targeting Raf1.

    Funded by: NCI NIH HHS: CA68485; NIDDK NIH HHS: 5T32DK07563, DK20593; NIGMS NIH HHS: GM51366, GM62265; NIMH NIH HHS: MH19732

    The Journal of biological chemistry 2005;280;52;42644-54

  • Wild-type and mutant B-RAF activate C-RAF through distinct mechanisms involving heterodimerization.

    Garnett MJ, Rana S, Paterson H, Barford D and Marais R

    Signal Transduction Team, The Institute of Cancer Research, Cancer Research UK Centre of Cell and Molecular Biology, London.

    The protein kinase B-RAF is mutated in approximately 7% of human cancers. Most mutations are activating, but, surprisingly, a small number have reduced kinase activity. However, the latter can still stimulate cellular signaling through the MEK-ERK pathway because they activate the related family member C-RAF. We examine the mechanism underlying C-RAF activation by B-RAF. We show that C-RAF is activated in the cytosol in a RAS-independent manner that requires activation segment phosphorylation and binding of 14-3-3 to C-RAF. We show that wild-type B-RAF forms a complex with C-RAF in a RAS-dependent manner, whereas the mutants bind independently of RAS. Importantly, we show that wild-type B-RAF can also activate C-RAF. Our data suggest that B-RAF activates C-RAF through a mechanism involving 14-3-3 mediated heterooligomerization and C-RAF transphosphorylation. Thus, we have identified a B-RAF-C-RAF-MEK-ERK cascade that signals not only in cancer but also in normal cells.

    Molecular cell 2005;20;6;963-9

  • Towards a proteome-scale map of the human protein-protein interaction network.

    Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, Li N, Berriz GF, Gibbons FD, Dreze M, Ayivi-Guedehoussou N, Klitgord N, Simon C, Boxem M, Milstein S, Rosenberg J, Goldberg DS, Zhang LV, Wong SL, Franklin G, Li S, Albala JS, Lim J, Fraughton C, Llamosas E, Cevik S, Bex C, Lamesch P, Sikorski RS, Vandenhaute J, Zoghbi HY, Smolyar A, Bosak S, Sequerra R, Doucette-Stamm L, Cusick ME, Hill DE, Roth FP and Vidal M

    Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115, USA.

    Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.

    Funded by: NCI NIH HHS: R33 CA132073; NHGRI NIH HHS: P50 HG004233, R01 HG001715, RC4 HG006066, U01 HG001715; NHLBI NIH HHS: U01 HL098166

    Nature 2005;437;7062;1173-8

  • Identification of Raf-1 S471 as a novel phosphorylation site critical for Raf-1 and B-Raf kinase activities and for MEK binding.

    Zhu J, Balan V, Bronisz A, Balan K, Sun H, Leicht DT, Luo Z, Qin J, Avruch J and Tzivion G

    Cardiovascular Research Institute, The Texas A&M University Health Science Center, Temple, TX 76504, USA.

    The Ras-Raf-MAPK cascade is a key growth-signaling pathway and its uncontrolled activation results in cell transformation. Although the general features of the signal transmission along the cascade are reasonably defined, the mechanisms underlying Raf activation remain incompletely understood. Here, we show that Raf-1 dephosphorylation, primarily at epidermal growth factor (EGF)-induced sites, abolishes Raf-1 kinase activity. Using mass spectrometry, we identified five novel in vivo Raf-1 phosphorylation sites, one of which, S471, is located in subdomain VIB of Raf-1 kinase domain. Mutational analyses demonstrated that Raf-1 S471 is critical for Raf-1 kinase activity and for its interaction with mitogen-activated protein kinase kinase (MEK). Similarly, mutation of the corresponding B-Raf site, S578, resulted in an inactive kinase, suggesting that the same Raf-1 and B-Raf phosphorylation is needed for Raf kinase activation. Importantly, the naturally occurring, cancer-associated B-Raf activating mutation V599E suppressed the S578A mutation, suggesting that introducing a charged residue at this region eliminates the need for an activating phosphorylation. Our results demonstrate an essential role of specific EGF-induced Raf-1 phosphorylation sites in Raf-1 activation, identify Raf-1 S471 as a novel phosphorylation site critical for Raf-1 and B-Raf kinase activities, and point to the possibility that the V599E mutation activates B-Raf by mimicking a phosphorylation at the S578 site.

    Funded by: NIGMS NIH HHS: R01 GM-067134, R01 GM067134

    Molecular biology of the cell 2005;16;10;4733-44

  • Functional interactions of Raf and MEK with Jun-N-terminal kinase (JNK) result in a positive feedback loop on the oncogenic Ras signaling pathway.

    Adler V, Qu Y, Smith SJ, Izotova L, Pestka S, Kung HF, Lin M, Friedman FK, Chie L, Chung D, Boutjdir M and Pincus MR

    QRNA Corporation, West 167th Street, New York, New York 10032, USA.

    In previous studies we have found that oncogenic (Val 12)-ras-p21 induces Xenopus laevis oocyte maturation that is selectively blocked by two ras-p21 peptides, 35-47, also called PNC-7, that blocks its interaction with raf, and 96-110, also called PNC-2, that blocks its interaction with jun-N-terminal kinase (JNK). Each peptide blocks activation of both JNK and MAP kinase (MAPK or ERK) suggesting interaction between the raf-MEK-ERK and JNK-jun pathways. We further found that dominant negative raf blocks JNK induction of oocyte maturation, again suggesting cross-talk between pathways. In this study, we have undertaken to determine where these points of cross-talk occur. First, we have immunoprecipitated injected Val 12-Ha-ras-p21 from oocytes and found that a complex forms between ras-p21 raf, MEK, MAPK, and JNK. Co-injection of either peptide, but not a control peptide, causes diminished binding of ras-p21, raf, and JNK. Thus, one site of interaction is cooperative binding of Val 12-ras-p21 to raf and JNK. Second, we have injected JNK, c-raf, and MEK into oocytes alone and in the presence of raf and MEK inhibitors and found that JNK activation is independent of the raf-MEK-MAPK pathway but that activated JNK activates raf, allowing for activation of ERK. Furthermore, we have found that constitutively activated MEK activates JNK. We have corroborated these findings in studies with isolated protein components from a human astrocyte (U-251) cell line; that is, JNK phosphorylates raf but not the reverse; MEK phosphorylates JNK but not the reverse. We further have found that JNK does not phosphorylate MAPK and that MAPK does not phosphorylate JNK. The stress-inducing agent, anisomycin, causes activation of JNK, raf, MEK, and ERK in this cell line; activation of JNK is not inhibitable by the MEK inhibitor, U0126, while activation of raf, MEK, and ERK are blocked by this agent. These results suggest that activated JNK can, in turn, activate not only jun but also raf that, in turn, activates MEK that can then cross-activate JNK in a positive feedback loop.

    Funded by: NCI NIH HHS: R01-CA46465, R01CA42500; NIAID NIH HHS: R01-AI36450, R01-AI43369

    Biochemistry 2005;44;32;10784-95

  • Prohibitin is required for Ras-induced Raf-MEK-ERK activation and epithelial cell migration.

    Rajalingam K, Wunder C, Brinkmann V, Churin Y, Hekman M, Sievers C, Rapp UR and Rudel T

    Department of Molecular Biology, Max Planck Institute for Infection Biology, Schumannstrasse 21/22, D-10117 Berlin, Germany.

    Ras proteins control the signalling pathways that are responsible for normal growth and malignant transformation. Raf protein kinases are direct Ras effector proteins that initiate the mitogen-activated protein kinase (MAPK) cascade, which mediates diverse biological functions such as cell growth, survival and differentiation. Here we show that prohibitin, a ubiquitously expressed and evolutionarily conserved protein is indispensable for the activation of the Raf-MEK-ERK pathway by Ras. The membrane targeting and activation of C-Raf by Ras needs prohibitin in vivo. In addition, direct interaction with prohibitin is required for C-Raf activation. C-Raf kinase fails to interact with the active Ras induced by epidermal growth factor in the absence of prohibitin. Moreover, in prohibitin-deficient cells the adhesion complex proteins cadherin and beta-catenin relocalize to the plasma membrane and thereby stabilize adherens junctions. Our data show an unexpected role of prohibitin in the activation of the Ras-Raf signalling pathway and in modulating epithelial cell adhesion and migration.

    Nature cell biology 2005;7;8;837-43

  • Apoptosis-linked gene-2 connects the Raf-1 and ASK1 signalings.

    Chen C and Sytkowski AJ

    Laboratory for Cell and Molecular Biology, Division of Hematology and Oncology, Beth Israel Deaconess Medical Center, Department of Medicine, Harvard Medical School, USA. cchen3@bidmc.harvard.edu

    Raf-1 plays important roles in cell proliferation, differentiation, and survival. However, the unique and essential function of Raf-1 is anti-apoptotic. The molecules that mediate Raf-1's anti-apoptotic function are not known. In the course of identifying new substrates of Raf-1, we found that the Raf-1 kinase domain interacted with apoptosis-linked gene-2 (ALG-2) in yeast two-hybrid system. Our further studies showed that Raf-1 phosphorylated ALG-2 in an in vitro kinase assay. We also found that apoptosis signal-regulating kinase 1 (ASK1) strongly phosphorylated ALG-2. Importantly, Raf-1 blocks the ASK1-dependent ALG-2 phosphorylation. Since ALG-2 associates with ASK1, and both ASK1 and ALG-2 are involved in apoptosis, our observations indicate that Raf-1 may mediate its anti-apoptotic function by interrupting ASK1-dependent phosphorylation of ALG-2.

    Funded by: NCI NIH HHS: R01 CA89204; NIDDK NIH HHS: K01 DK62113

    Biochemical and biophysical research communications 2005;333;1;51-7

  • p21-activated Kinase 1 (Pak1)-dependent phosphorylation of Raf-1 regulates its mitochondrial localization, phosphorylation of BAD, and Bcl-2 association.

    Jin S, Zhuo Y, Guo W and Field J

    Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

    Raf-1 protects cells from apoptosis, independently of its signals to MEK and ERK, by translocating to the mitochondria where it binds Bcl-2 and displaces BAD. However, the answer to the question of how Raf-1 is normally lured to the mitochondria and becomes activated remains elusive. p21-activated protein kinases (Paks) are serine/threonine protein kinases that phosphorylate Raf-1 at Ser-338 and Ser-339. Here we elucidate the molecular mechanism through which Pak1 signals to BAD through a Raf-1-activated pathway. Upon phosphorylation by Pak1, Raf-1 translocates to mitochondria and phosphorylates BAD at Ser-112. Moreover, the mitochondrial translocation of Raf-1 and the interaction between Raf-1 and Bcl-2 are regulated by Raf-1 phosphorylation at Ser-338/Ser-339. Notably, we show that formation of a Raf-1-Bcl-2 complex coincides with loss of an interaction between Bcl-2 and BAD. These signals are specific for Pak1, because Src-activated Raf-1 only stimulates the MAP kinase cascade. Thus, our data identify the molecular connections of a Pak1-Raf-1-BAD pathway that is involved in cell survival signaling.

    Funded by: NIGMS NIH HHS: GM48241, R01 GM048241

    The Journal of biological chemistry 2005;280;26;24698-705

  • Raf-1 is a binding partner of DSCR1.

    Cho YJ, Abe M, Kim SY and Sato Y

    Department of Vascular Biology, Institute of Development, Aging and Cancer, Tohoku University, Sendai 980-8575, Japan.

    Down syndrome critical region 1 (DSCR1) is recognized as an endogenous calcineurin inhibitor. DSCR1 is induced in endothelial cells and may play an important role in inflammation and angiogenesis. To address a novel function of DSCR1, we searched interacting partners of DSCR1. We performed pull-down analysis using DSCR1 as a bait and identified Raf-1 as a binding partner. The association of Raf-1 was confirmed by co-immunoprecipitation in GM7373 cells expressing green fluorescence protein tagged DSCR1. We determined two Raf-1 binding regions in DSCR1; one in the N-terminus and the other in the C-terminus regions. We further demonstrated that calpain cleaved DSCR1 and generated fragments with different binding affinity to Raf-1 or calcineurin. These results constitute the first demonstration of Raf-1 as a binding partner of DSCR1, and suggest a novel role of DSCR1.

    Archives of biochemistry and biophysics 2005;439;1;121-8

  • CNK1 is a scaffold protein that regulates Src-mediated Raf-1 activation.

    Ziogas A, Moelling K and Radziwill G

    Institute of Medical Virology, University of Zurich, Gloriastrasse 30, CH-8006 Zurich, Switzerland.

    Raf-1 is a regulator of cellular proliferation, differentiation, and apoptosis. Activation of the Raf-1 kinase activity is tightly regulated and involves targeting to the membrane by Ras and phosphorylation by various kinases, including the tyrosine kinase Src. Here we demonstrate that the connector enhancer of Ksr1, CNK1, mediates Src-dependent tyrosine phosphorylation and activation of Raf-1. CNK1 binds preactivated Raf-1 and activated Src and forms a trimeric complex. CNK1 regulates the activation of Raf-1 by Src in a concentration-dependent manner typical for a scaffold protein. Down-regulation of endogenously expressed CNK1 by small inhibitory RNA interferes with Src-dependent activation of ERK. Thus, CNK1 allows cross-talk between Src and Raf-1 and is essential for the full activation of Raf-1.

    The Journal of biological chemistry 2005;280;25;24205-11

  • Raf-1 expression may influence progression to androgen insensitive prostate cancer.

    Mukherjee R, Bartlett JM, Krishna NS, Underwood MA and Edwards J

    Endocrine Cancer Group, Division of Cancer Studies and Molecular Pathology, University Department of Surgery, Glasgow Royal Infirmary, Glasgow, United Kingdom.

    Background: Recent evidence has implicated the MAP kinase pathway with the development of androgen insensitive prostate cancer (AIPC). We have previously reported gene amplification of critical members of this pathway with the development of androgen insensitive disease.

    Methods: Protein expression of Raf-1 was analyzed using immunohistochemistry (IHC) in a database of 65 paired tumor specimens obtained before and after the development of AIPC and correlated with other members of the pathway.

    Results: Patients whose Raf-1 expression rose with development of AIPC had a significantly shorter median time to biochemical relapse compared to those whose expression fell or remained unchanged (1.16 vs. 2.62 years, P = 0.0005). In AIPC tumors, expression of Raf-1 correlated significantly with expression of HER2 and with expression of c-fos.

    Conclusions: We conclude that the HER2/Raf-1/AP-1 axis may promote the development of AIPC, leading to early relapse. Members of the pathway may act as novel therapeutic targets for patients.

    The Prostate 2005;64;1;101-7

  • Medullary thyroid cancer: the functions of raf-1 and human achaete-scute homologue-1.

    Chen H, Kunnimalaiyaan M and Van Gompel JJ

    Department of Surgery, University of Wisconsin Medical School, the University of Wisconsin Comprehensive Cancer Center, Madison, Wisconsin, USA. chen@surgery.wisc.edu

    Medullary thyroid cancer (MTC) is a prototypic neuroendocrine tumor of the thyroid C cells. Other than surgery, there are no curative therapies for MTC. In this review, we detail recent studies that suggest that targeting specific signaling pathways may be a viable strategy to control MTC tumor progression. Specifically, we discuss the role of the raf-1 and achaete-scute homologue-1 pathways in the MTC tumor growth and differentiation.

    Thyroid : official journal of the American Thyroid Association 2005;15;6;511-21

  • Raf-1 serine 338 phosphorylation plays a key role in adhesion-dependent activation of extracellular signal-regulated kinase by epidermal growth factor.

    Edin ML and Juliano RL

    Department of Pharmacology, University of North Carolina, Chapel Hill, 1017 Mary Ellen Jones Building, CB 7365, Chapel Hill, NC 27599-7365, USA.

    Activation of the extracellular signal-regulated kinase (ERK) 1/2 cascade by polypeptide growth factors is tightly coupled to adhesion to extracellular matrix in nontransformed cells. Raf-1, the initial kinase in this cascade, is intricately regulated by phosphorylation, localization, and molecular interactions. We investigated the complex interactions between Raf-1, protein kinase A (PKA), and p21-activated kinase (PAK) to determine their roles in the adhesion dependence of signaling from epidermal growth factor (EGF) to ERK. We conclude that Raf-1 phosphorylation on serine 338 (S338) is a critical step that is inhibited in suspended cells. Restoration of phosphorylation at S338, either by expression of highly active PAK or by expression of an S338 phospho-mimetic Raf-1 mutation, led to a partial rescue of ERK activation in suspended cells. Raf-1 inhibition in suspension was not due to excessive negative regulation on inhibitory sites S43 and S259, as these serines were largely dephosphorylated in suspended cells. Finally, strong phosphorylation of Raf-1 S338 provided resistance to PKA-mediated inhibition of ERK activation. Phosphorylation at Raf-1 S43 and S259 by PKA only weakly inhibited EGF activation of Raf-1 and ERK when cells maintained high Raf-1 S338 phosphorylation.

    Funded by: NHLBI NIH HHS: HL4500; NIGMS NIH HHS: GM26165

    Molecular and cellular biology 2005;25;11;4466-75

  • Targeted proteomic analysis of 14-3-3 sigma, a p53 effector commonly silenced in cancer.

    Benzinger A, Muster N, Koch HB, Yates JR and Hermeking H

    Molecular Oncology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried/Munich, Germany.

    To comprehensively identify proteins interacting with 14-3-3 sigma in vivo, tandem affinity purification and the multidimensional protein identification technology were combined to characterize 117 proteins associated with 14-3-3 sigma in human cells. The majority of identified proteins contained one or several phosphorylatable 14-3-3-binding sites indicating a potential direct interaction with 14-3-3 sigma. 25 proteins were not previously assigned to any function and were named SIP2-26 (for 14-3-3 sigma-interacting protein). Among the 92 interactors with known function were a number of proteins previously implicated in oncogenic signaling (APC, A-RAF, B-RAF, and c-RAF) and cell cycle regulation (AJUBA, c-TAK, PTOV-1, and WEE1). The largest functional classes comprised proteins involved in the regulation of cytoskeletal dynamics, polarity, adhesion, mitogenic signaling, and motility. Accordingly ectopic 14-3-3 sigma expression prevented cellular migration in a wounding assay and enhanced mitogen-activated protein kinase signaling. The functional diversity of the identified proteins indicates that induction of 14-3-3 sigma could allow p53 to affect numerous processes in addition to the previously characterized inhibitory effect on G2/M progression. The data suggest that the cancer-specific loss of 14-3-3 sigma expression by epigenetic silencing or p53 mutations contributes to cancer formation by multiple routes.

    Funded by: NCRR NIH HHS: RR11823-08

    Molecular & cellular proteomics : MCP 2005;4;6;785-95

  • Conserved docking site is essential for activation of mammalian MAP kinase kinases by specific MAP kinase kinase kinases.

    Takekawa M, Tatebayashi K and Saito H

    Division of Molecular Cell Signaling, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

    Mammalian mitogen-activated protein kinase (MAPK) cascades control various cellular events, ranging from cell growth to apoptosis, in response to external stimuli. A conserved docking site, termed DVD, is found in the mammalian MAP kinase kinases (MAPKKs) belonging to the three major subfamilies, namely MEK1, MKK4/7, and MKK3/6. The DVD sites bind to their specific upstream MAP kinase kinase kinases (MAPKKKs), including MTK1 (MEKK4), ASK1, TAK1, TAO2, MEKK1, and Raf-1. DVD site is a stretch of about 20 amino acids immediately on the C-terminal side of the MAPKK catalytic domain. Mutations in the DVD site strongly inhibited MAPKKs from binding to, and being activated by, their specific MAPKKKs, both in vitro and in vivo. DVD site mutants could not be activated by various external stimuli in vivo. Synthetic DVD oligopeptides inhibited specific MAPKK activation, both in vitro and in vivo, demonstrating the critical importance of the DVD docking in MAPK signaling.

    Molecular cell 2005;18;3;295-306

  • Rheb binds and regulates the mTOR kinase.

    Long X, Lin Y, Ortiz-Vega S, Yonezawa K and Avruch J

    Diabetes Unit and Medical Services and Department of Molecular Biology, Massachusetts General Hospital, Boston, 02114, USA.

    Background: The target of rapamycin (TOR), in complex with the proteins raptor and LST8 (TOR complex 1), phosphorylates the p70S6K and 4E-BP1 to promote mRNA translation. Genetic evidence establishes that TOR complex activity in vivo requires the small GTPase Rheb, and overexpression of Rheb can rescue TOR from inactivation in vivo by amino-acid withdrawal. The Tuberous Sclerosis heterodimer (TSC1/TSC2) functions as a Rheb GTPase activator and inhibits TOR signaling in vivo.

    Results: Here, we show that Rheb binds to the TOR complex specifically, independently of its ability to bind TSC2, through separate interactions with the mTOR catalytic domain and with LST8. Rheb binding to the TOR complex in vivo and in vitro does not require Rheb guanyl nucleotide charging but is modulated by GTP and impaired by certain mutations (Ile39Lys) in the switch 1 loop. Nucleotide-deficient Rheb mutants, although capable of binding mTOR in vivo and in vitro, are inhibitory in vivo, and the mTOR polypeptides that associate with nucleotide-deficient Rheb in vivo lack kinase activity in vitro. Reciprocally, mTOR polypeptides bound to Rheb(Gln64Leu), a mutant that is nearly 90% GTP charged, exhibit substantially higher protein kinase specific activity than mTOR bound to wild-type Rheb.

    Conclusions: The TOR complex 1 is a direct target of Rheb-GTP, whose binding enables activation of the TOR kinase.

    Funded by: NCI NIH HHS: CA-73818; NIDDK NIH HHS: DK-17776, DK007028

    Current biology : CB 2005;15;8;702-13

  • B-Raf and Raf-1 are regulated by distinct autoregulatory mechanisms.

    Tran NH, Wu X and Frost JA

    Department of Integrative Biology and Pharmacology, University of Texas Health Science Center, Houston, Texas 77030, USA.

    B-Raf is a key regulator of the ERK pathway and is mutationally activated in two-thirds of human melanomas. In this work, we have investigated the activation mechanism of B-Raf and characterized the roles of Ras and of B-Raf phosphorylation in this regulation. Raf-1 is regulated by an N-terminal autoinhibitory domain whose actions are blocked by interaction with Ras and subsequent phosphorylation of Ser(338). We observed that B-Raf also contains an N-terminal autoinhibitory domain and that the interaction of this domain with the catalytic domain was inhibited by binding to active H-Ras. However, unlike Raf-1, the phosphorylation of B-Raf at Ser(445) was constitutive and was only moderately increased by expression of constitutively active H-Ras or constitutively active PAK1. Ser(445) phosphorylation is important to the B-Raf activation mechanism, however, because mutation of this site to alanine increased the affinity of the regulatory domain for the catalytic domain and increased autoinhibition. Similarly, expression of constitutively active PAK1 also decreased auto-inhibition. B-Raf autoinhibition was negatively regulated by acidic substitutions at phosphorylation sites within the activation loop of B-Raf and by the oncogenic substitution V599E. However, these substitutions did not affect the ability of the regulatory domain to co-immunoprecipitate with the catalytic domain. These data demonstrate that B-Raf activity is autoregulated, that constitutive phosphorylation of Ser(445) primes B-Raf for activation, and that a key feature of phosphorylation within the activation loop or of oncogenic mutations within this region is to block autoinhibition.

    The Journal of biological chemistry 2005;280;16;16244-53

  • PARG1, a protein-tyrosine phosphatase-associated RhoGAP, as a putative Rap2 effector.

    Myagmar BE, Umikawa M, Asato T, Taira K, Oshiro M, Hino A, Takei K, Uezato H and Kariya K

    Division of Cell Biology, Graduate School of Medicine, University of the Ryukyus, 207 Uehara, Nishihara-cho, Okinawa 903-0215, Japan.

    Rap2 belongs to the Ras family of small GTP-binding proteins, but its specific signaling role is unclear. By yeast two-hybrid screening, we have found that the Caenorhabditis elegans ortholog of Rap2 interacts with a protein containing a Rho-GTPase-activating protein (RhoGAP) domain, ZK669.1a, whose human ortholog PARG1 exhibits RhoGAP activity in vitro. ZK669.1a and PARG1 share a homology region with previously unknown function, designated the ZK669.1a and PARG1 homology (ZPH) region. Here we show that the ZPH region of PARG1 mediates interaction with Rap2. PARG1 interacted with Rap2 in a GTP-dependent manner but not with Ras or Rap1. We also show that PARG1 and its mutant lacking the ZPH region induce typical cytoskeletal changes for Rho inactivation in fibroblasts. Rap2 suppressed this in vivo action of PARG1 but not that of the mutant PARG1. These results suggest that PARG1 is a putative specific effector of Rap2 to regulate Rho.

    Biochemical and biophysical research communications 2005;329;3;1046-52

  • Elastin peptides activate extracellular signal-regulated kinase 1/2 via a Ras-independent mechanism requiring both p110gamma/Raf-1 and protein kinase A/B-Raf signaling in human skin fibroblasts.

    Duca L, Lambert E, Debret R, Rothhut B, Blanchevoye C, Delacoux F, Hornebeck W, Martiny L and Debelle L

    Laboratoire de Biochimie, UMR CNRS 6198, IFR53 Biomolécules, Faculty of Sciences, Université de Reims Champagne Ardenne, Moulin de la Housse, BP 1039, 51687 Reims Cedex 2, France.

    Elastin peptides (EPs) produced during cancer progression bind to the elastin binding protein (EBP) found at the surface of dermal fibroblasts, leading to the expression of collagenase-1 gene. The production of this enzyme involved in stromal reaction is caused by the sustained activation of the extracellular signal-regulated kinases 1/2 (ERK1/2) pathway via cAMP/protein kinase A (PKA) and phosphatidylinositol 3-kinase (PI3K). However, the mechanism of these signaling events remains unknown. We show that kappa-elastin (kappaE), a commonly used EP, induces maximum phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK)1/2 and ERK1/2 after 30 min. The simultaneous inhibition of PKA and PI3K, by N-(2-(p-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide (H89) and 2-(4-morpholynil)-8-phenyl-4H-1-bemzopyran-4-one (LY294002), respectively, blocked MEK1/2 and ERK1/2 phosphorylation, as did lactose, an EBP antagonist. kappaE induced Raf-1 phosphorylation and activation in a PI3K-dependent manner. In our system, the PI3K p110gamma is expressed and activated by betagamma-derived subunits from a pertussis toxin-sensitive G protein after fibroblast stimulation. Pertussis toxin also blocks the Raf-1/MEK1/2/ERK1/2 phosphorylation cascade. In addition, we found that B-Raf is expressed in dermal fibroblasts and activated in a PKA-dependent manner after kappaE treatment, thereby integrating PKA signals to MEK1/2. It is noteworthy that Ras involvement was excluded because ERK1/2 activation by kappaE was not blocked in RasN17-transfected fibroblasts. Together, our results identify a novel Ras-independent ERK1/2 activation system in which p110gamma/Raf-1/MEK1/2 and PKA/B-Raf/MEK1/2 cooperate to activate ERK1/2. Thus, p110gamma and B-Raf seem to be important modulators of dermal fibroblasts physiology and should now qualify as therapeutic targets in strategies aiming at limiting elastin degradation contribution to cancer progression.

    Molecular pharmacology 2005;67;4;1315-24

  • Cooperation and selectivity of the two Grb2 binding sites of p52Shc in T-cell antigen receptor signaling to Ras family GTPases and Myc-dependent survival.

    Patrussi L, Savino MT, Pellegrini M, Paccani SR, Migliaccio E, Plyte S, Lanfrancone L, Pelicci PG and Baldari CT

    Department of Evolutionary Biology, University of Siena, Via Aldo Moro 2, Siena 53100, Italy.

    Shc proteins participate in a variety of processes regulating cell proliferation, survival and apoptosis. The two ubiquitously expressed isoforms, p52Shc/p46Shc, couple tyrosine kinase receptors to Ras by recruiting Grb2/Sos complexes to a membrane-proximal localization. Tyrosine residues 239/240 and 317 become phosphorylated following receptor engagement and, as such, form two Grb2 binding sites, which have been proposed to be differentially coupled to Myc-dependent survival and to fos-dependent proliferation, respectively. Here, we have addressed the individual function of YY239/240 and Y317 in T-cell antigen receptor (TCR) signaling. We show that p52Shc is phosphorylated on both YY239/240 and Y317 following TCR engagement. Mutation of either YY239/240 or Y317 results in impaired interaction with Grb2 and inhibition of Ras/MAP kinase activation and CD69 induction, supporting a role for both Grb2 binding sites in this function. Substitution of either YY239/240 or Y317 also results in a defective activation of Rac and the coupled stress kinases JNK and p38. Furthermore, mutation of Y317 or, to a larger extent, of YY239/240, results in increased activation-induced cell death, which in cells expressing the FF239/240 mutant is accompanied by impaired TCR-dependent c-myc transcription. The data underline a pleiotropic and nonredundant role of Shc, mediated by both YY239/240 and Y317, in T-cell activation and survival.

    Oncogene 2005;24;13;2218-28

  • An acylation cycle regulates localization and activity of palmitoylated Ras isoforms.

    Rocks O, Peyker A, Kahms M, Verveer PJ, Koerner C, Lumbierres M, Kuhlmann J, Waldmann H, Wittinghofer A and Bastiaens PI

    Department of Structural Biology, Max Planck Institute for Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.

    We show that the specific subcellular distribution of H- and Nras guanosine triphosphate-binding proteins is generated by a constitutive de/reacylation cycle that operates on palmitoylated proteins, driving their rapid exchange between the plasma membrane (PM) and the Golgi apparatus. Depalmitoylation redistributes farnesylated Ras in all membranes, followed by repalmitoylation and trapping of Ras at the Golgi, from where it is redirected to the PM via the secretory pathway. This continuous cycle prevents Ras from nonspecific residence on endomembranes, thereby maintaining the specific intracellular compartmentalization. The de/reacylation cycle also initiates Ras activation at the Golgi by transport of PM-localized Ras guanosine triphosphate. Different de/repalmitoylation kinetics account for isoform-specific activation responses to growth factors.

    Science (New York, N.Y.) 2005;307;5716;1746-52

  • Ras binding opens c-Raf to expose the docking site for mitogen-activated protein kinase kinase.

    Terai K and Matsuda M

    Department of Tumor Virology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.

    A key signalling molecule, c-Raf, is situated downstream from Ras and upstream from the mitogen-activated protein kinase kinase (MEK). We studied the mechanism underlying the signal transduction from Ras to MEK by using probes based on the principle of fluorescence resonance energy transfer. In agreement with previous models, it was found that c-Raf adopted two conformations: open active and closed inactive. Ras binding induced the c-Raf transition from closed to open conformation, which enabled c-Raf to bind to MEK. In the presence of a cytosolic Ras mutant, c-Raf bound to, but failed to phosphorylate, MEK in the cytoplasm. In contrast, the cytosolic Ras mutant significantly enhanced MEK phosphorylation by a membrane-targeted c-Raf. These results demonstrated the essential role of Ras-induced conformational change in MEK activation by c-Raf.

    EMBO reports 2005;6;3;251-5

  • Location and functional significance of retinol-binding sites on the serine/threonine kinase, c-Raf.

    Hoyos B, Jiang S and Hammerling U

    Immunology Program, Memorial Sloan Kettering Cancer, New York, New York, 10021, USA. b-hoyos@ski.mskcc.org

    Redox activations of serine/threonine kinases represent alternate pathways in which vitamin A plays a crucial co-factor role. Vitamin A binds the zinc finger domain of c-Raf with nanomolar affinity. The retinoid-binding site has been mapped within this structure by scanning mutagenesis. The deduced contact sites were found anchored on Phe-8, counting from the 1st conserved histidine of the zinc finger. These sites agreed with contact amino acids identified by computational docking. The boundaries of a related binding pocket were identified by mutagenesis and partially confirmed by docking trials in the protein kinase C-alpha C1A zinc finger. They comprised Phe-7, Phe-8, and Trp-22. This trio was absent from the alphaC1B domain, explaining why the latter did not bind retinol. Reconfiguring at a minimum the two corresponding amino acids of alphaC1B, Thr-7 and Tyr-22, to conform to alphaC1A converted this domain to a binder. Deletion of the predicted retinoid-binding site in the full-length molecule created a mutant c-Raf that was deficient in retinol-dependent redox activation but fully responsive to epidermal growth factor. Our findings indicate that ligation of retinol to a specific site embedded in the regulatory domain is an important feature of c-Raf regulation in the redox pathway.

    Funded by: NCI NIH HHS: CA 08748, CA49933, CA89362

    The Journal of biological chemistry 2005;280;8;6872-8

  • Apoptosis of hematopoietic cells induced by growth factor withdrawal is associated with caspase-9 mediated cleavage of Raf-1.

    Cornelis S, Bruynooghe Y, Van Loo G, Saelens X, Vandenabeele P and Beyaert R

    Unit of Molecular Signal Transduction in Inflammation, Department for Molecular Biomedical Research, VIB-Ghent University, Technologiepark 927, B-9052 Ghent-Zwijnaarde, Belgium.

    The Raf-1 serine/threonine kinase is a key protein that is implicated in the transmission of many growth and cell survival signals. In the present study we demonstrate that apoptosis of hematopoietic cells induced by IL-3-deprivation is associated with the cleavage of Raf-1, resulting in the separation of the N-terminal regulatory domain and the C-terminal kinase domain. Raf-1 cleavage specifically occurs upon triggering of the mitochondrial death pathway, and coincides with the activation of specific caspases. Moreover, Bcl-2 overexpression or treatment with the caspase inhibitor z-VAD.fmk completely prevented Raf-1 cleavage, whereas caspase inhibition by treatment of cells with Ac-DEVD.fmk or z-IETD.fmk, or CrmA overexpression had no effect. Furthermore, in vitro cleavage studies indicate that caspase-9, which is the apical protease in the mitochondrial death pathway, is able to cleave Raf-1 at position D279. Cell fractionation studies showed that the Raf-1 C-terminal fragment that is generated upon IL-3 withdrawal is localized predominantly to the mitochondria. In addition, constitutive expression of this C-terminal Raf-1 fragment fused to a mitochondrial targeting sequence in Ba/F3 pre-B cells significantly delays apoptosis induced by IL-3 withdrawal. These results suggest an important role for caspase-9 mediated cleavage of Raf-1 in the negative feedback regulation of hematopoietic cell apoptosis induced by growth factor withdrawal.

    Oncogene 2005;24;9;1552-62

  • Regulation of Raf-1 by direct feedback phosphorylation.

    Dougherty MK, Müller J, Ritt DA, Zhou M, Zhou XZ, Copeland TD, Conrads TP, Veenstra TD, Lu KP and Morrison DK

    Laboratory of Protein Dynamics and Signaling, National Cancer Institute-Frederick, Frederick, MD 21702, USA.

    The Raf-1 kinase is an important signaling molecule, functioning in the Ras pathway to transmit mitogenic, differentiative, and oncogenic signals to the downstream kinases MEK and ERK. Because of its integral role in cell signaling, Raf-1 activity must be precisely controlled. Previous studies have shown that phosphorylation is required for Raf-1 activation, and here, we identify six phosphorylation sites that contribute to the downregulation of Raf-1 after mitogen stimulation. Five of the identified sites are proline-directed targets of activated ERK, and phosphorylation of all six sites requires MEK signaling, indicating a negative feedback mechanism. Hyperphosphorylation of these six sites inhibits the Ras/Raf-1 interaction and desensitizes Raf-1 to additional stimuli. The hyperphosphorylated/desensitized Raf-1 is subsequently dephosphorylated and returned to a signaling-competent state through interactions with the protein phosphatase PP2A and the prolyl isomerase Pin1. These findings elucidate a critical Raf-1 regulatory mechanism that contributes to the sensitive, temporal modulation of Ras signaling.

    Funded by: NIGMS NIH HHS: R01GM58556

    Molecular cell 2005;17;2;215-24

  • Activation of Raf1 and the ERK pathway in response to l-ascorbic acid in acute myeloid leukemia cells.

    Park S, Park CH, Hahm ER, Kim K, Kimler BF, Lee SJ, Park HK, Lee SH, Kim WS, Jung CW, Park K, Riordan HD and Lee JH

    Samsung Medical Center and Sungkyunkwan University School of Medicine, Seoul 135-710, Korea. sypark21@yumc.yonsei.ac.kr

    L-ascorbic acid (LAA) shows cytotoxicity and induces apoptosis of malignant cells in vitro, but the mechanisms by which such effects occur have not been elucidated. In the present study, we provide evidence that the ERK MAP kinase pathway is activated in response to LAA (< 1 mM) in acute myeloid leukemia cell lines. LAA treatment of cells induces a dose-dependent phosphorylation of extracellular signal-regulated kinases (ERK) and results in activation of its catalytic domain. Our data also demonstrate that the small G protein Raf1 and MAPK-activated protein kinase 2 are activated by LAA as an upstream and a downstream regulator of ERK, respectively. Although the ERK pathway has been known to activate cell proliferation, pharmacologic inhibition of ERK reduces LAA-dependent apoptosis and growth inhibitory response of acute myeloid leukemia cell lines, suggesting that this signaling cascade positively regulates induction of apoptotic response by LAA.

    Cellular signalling 2005;17;1;111-9

  • Inhibition of gastric cancer angiogenesis by vector-based RNA interference for Raf-1.

    Meng F, Ding J, Liu N, Zhang J, Shao X, Shen H, Xue Y, Xie H and Fan D

    Institude of Digestive Disease, Xijing Hospital, The Fourth Military Medical University, Xi'an, Shaanxi Province, PR, China.

    Tumor angiogenesis plays an important role in the malignancy of solid tumors. A number of recent studies including our own have suggested that Raf-1 is involved in this process, and may be critical in regulating gene activation of several angiogenesis factors. To further define Raf-1 function in angiogenesis and to explore novel approaches to modulate angiogenesis, we employed the small interference RNA technique to knock-down gene expression of Raf-1 in gastric cancer cell line SGC7901 that expresses a high level of Raf-1. The protein level of Raf-1 in the SGC7901cells was decreased dramatically after transfection with a Raf-1 specific siRNA vector. Further study proved that, VEGF and HIF-1alpha, two angiogenesis promoting factors, were found to be downregulated. And we also find that Vector-based RNA interference for Raf-1 increases transfected gastric cell apoptosis and inhibits cellular proliferation. Our results suggest that Raf-1 may be involved in angiogenesis by controlling the expression of angiogenesis-related factors and provide a possible strategy for the treatment of tumor angiogenesis by targeting Raf-1.

    Cancer biology & therapy 2005;4;1;113-7

  • Protein kinase C and epidermal growth factor stimulation of Raf1 potentiates adenylyl cyclase type 6 activation in intact cells.

    Beazely MA, Alan JK and Watts VJ

    Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907-2091, USA.

    Adenylyl cyclase type 6 (AC6) activity is inhibited by protein kinase C (PKC) in vitro; however, in intact cells, PKC activation does not inhibit the activity of transiently expressed AC6. To investigate the effects of PKC activation on AC6 activity in intact cells, we constructed human embryonic kidney (HEK) 293 cells that stably express wild-type AC6 (AC6-WT) or an AC6 mutant lacking a PKC and cyclic AMP-dependent protein kinase (PKA) phosphorylation site, Ser674 (AC6-S674A). In contrast to in vitro observations, we observed a PKC-mediated enhancement of forskolin- and isoproterenol-stimulated cyclic AMP accumulation in HEK-AC6 cells. Phorbol 12-myristate 13-acetate also potentiated cyclic AMP accumulation in cells expressing endogenous AC6, including Chinese hamster ovary cells and differentiated Cath.a differentiated cells. In HEK-AC6-S674A cells, the potentiation of AC6 stimulation was significantly greater than in cells expressing AC6-WT. The positive effect of PKC activation on AC6 activity seemed to involve Raf1 kinase because the Raf1 inhibitor 3-(3,5-dibromo-4-hydroxybenzylidene-5-iodo-1,3-dihydro-indol-2-one (GW5074) inhibited the PKC potentiation of AC6 activity. Furthermore, the forskolin-stimulated activity of a recombinant AC6 in which the putative Raf1 regulatory sites have been eliminated was not potentiated by activation of PKC. The ability of Raf1 to regulate AC6 may involve a direct interaction because AC6 and a constitutively active Raf1 construct were coimmunoprecipitated. In addition, we report that epidermal growth factor receptor activation also enhances AC6 signaling in a Raf1-dependent manner. These data suggest that Raf1 potentiates drug-stimulated cyclic AMP accumulation in cells expressing AC6 after activation of multiple signaling pathways.

    Funded by: NIMH NIH HHS: MH 60397

    Molecular pharmacology 2005;67;1;250-9

  • Role of the kinase MST2 in suppression of apoptosis by the proto-oncogene product Raf-1.

    O'Neill E, Rushworth L, Baccarini M and Kolch W

    The Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Glasgow G61 1BD, UK.

    The ablation of the protein kinase Raf-1 renders cells hypersensitive to apoptosis despite normal regulation of extracellular signal-regulated kinases, which suggests that apoptosis protection is mediated by a distinct pathway. We used proteomic analysis of Raf-1 signaling complexes to show that Raf-1 counteracts apoptosis by suppressing the activation of mammalian sterile 20-like kinase (MST2). Raf-1 prevents dimerization and phosphorylation of the activation loop of MST2 independently of its protein kinase activity. Depletion of MST2 from Raf-1-/- mouse or human cells abrogated sensitivity to apoptosis, whereas overexpression of MST2 induced apoptosis. Conversely, depletion of Raf-1 from Raf-1+/+ mouse or human cells led to MST2 activation and apoptosis. The concomitant depletion of both Raf-1 and MST2 prevented apoptosis.

    Science (New York, N.Y.) 2004;306;5705;2267-70

  • Inhibition of gap junction activity through the release of the C1B domain of protein kinase Cgamma (PKCgamma) from 14-3-3: identification of PKCgamma-binding sites.

    Nguyen TA, Takemoto LJ and Takemoto DJ

    Department of Biochemistry and Division of Biology, Kansas State University, Manhattan, Kansas 66506, USA.

    We have shown previously that insulin-like growth factor-I or lens epithelium-derived growth factor increases the translocation of protein kinase Cgamma (PKCgamma)to the membrane and the phosphorylation of Cx43 by PKCgamma and causes a subsequent decrease of gap junction activity (Nguyen, T. A., Boyle, D. L., Wagner, L. M., Shinohara, T., and Takemoto, D. J. (2003) Exp. Eye Res. 76, 565-572; Lin, D., Boyle, D. L., and Takemoto, D. J. (2003) Investig. Ophthalmol. Vis. Sci. 44, 1160-1168). Gap junction activity in lens epithelial cells is regulated by PKCgamma-mediated phosphorylation of Cx43. PKCgamma activity is stimulated by growth factor-regulated increases in the synthesis of diacylglycerol but is inhibited by cytosolic docking proteins such as 14-3-3. Here we have identified two sites on the PKCgamma-C1B domain that are responsible for its interaction with 14-3-3epsilon. Two sites, C1B1 (residues 101-112) and C1B5 (residues 141-151), are located within the C1 domain of PKCgamma. C1B1 and/or C1B5 synthetic peptides can directly compete for the binding of 14-3-3epsilon, resulting in the release of endogenous cellular PKCgamma from 14-3-3epsilon, in vivo or in vitro, in activation of PKCgamma enzyme activity, phosphorylation of PKCgamma, in the subsequent translocation of PKCgamma to the membrane, and in inhibition of gap junction activity. Gap junction activity was decreased by at least 5-fold in cells treated with C1B1 or C1B5 peptides when compared with a control. 100 microM of C1B1 or C1B5 peptides also caused a 10- or 4-fold decrease of Cx43 plaque formation compared with control cells. The uptake of these synthetic peptides into cells was verified by using high pressure liquid chromatography and matrix-assisted laser desorption ionization time-of-flight-mass spectrometry. We have demonstrated that the activity and localization of PKCgamma are regulated by its binding to 14-3-3epsilon at the C1B domain of PKCgamma. Synthetic peptides corresponding to these regions of PKCgamma successfully competed for the binding of 14-3-3epsilon to endogenous PKCgamma, resulting in inhibition of gap junction activity. This demonstrates that synthetic peptides can be used to exogenously regulate gap junctions.

    Funded by: NEI NIH HHS: EY02932, EY13421

    The Journal of biological chemistry 2004;279;50;52714-25

  • Insulin-like growth factor 1 signaling in human gastrointestinal carcinoid tumor cells.

    Van Gompel JJ and Chen H

    Department of Surgery, University of Wisconsin Comprehensive Cancer Center, 600 Highland Avenue, Madison, WI 53792, USA.

    Background Insulin-like growth factor 1 (IGF-1) is an autocrine regulator of carcinoid tumors. Blockade of IGF-1 signaling has been proposed as a therapeutic target in the treatment of patients with carcinoid syndrome. We hypothesized that the induction of parallel raf-1/MEK1 pathways will block IGF-1-mediated chromogranin A (CgA) maintenance. Methods Human gastrointestinal carcinoid tumor cells (BON) were treated with IGF-1 (0-500 ng/mL). Raf-1/MEK1 activation was achieved with an estrogen-inducible raf-1 vector that was transduced into BON cells. Activation of IGF-1/raf-1 pathways was determined by phosphorylation of downstream targets p70s6 and ERK1/2. The secreted and intercellular levels of CgA were measured in conditioned media and whole cell extracts by Western and enzyme-linked immunosorbent assay analysis. Results IGF-1 and raf-1 pathways were activated successfully in BON cells, as shown by high levels of phosphorylated p70s6 and phosphorylated ERK1/2, respectively. Treatment of BON cells with IGF-1 stimulated the release of CgA, while high intracellular CgA levels were maintained. The activation of raf-1/MEK1 reversed the effect of IGF-1 treatment by the depletion of intracellular CgA. Conclusions The induction of the raf-1/MEK1 pathway blocks IGF-1-mediated intracellular neuroendocrine hormone regulation. Therefore, raf-1/MEK1 activation may be a viable method to block IGF-1-mediated cellular effects and serve as a therapeutic target in gastrointestinal carcinoid tumors.

    Funded by: NIDDK NIH HHS: R21 DK 063015-01, R21 DK 064735-01

    Surgery 2004;136;6;1297-302

  • Disruption of the Rb--Raf-1 interaction inhibits tumor growth and angiogenesis.

    Dasgupta P, Sun J, Wang S, Fusaro G, Betts V, Padmanabhan J, Sebti SM and Chellappan SP

    Department of Interdisciplinary Oncology, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, 12902 Magnolia Dr., Tampa, FL 33612, USA.

    The retinoblastoma tumor suppressor protein (Rb) plays a vital role in regulating mammalian cell cycle progression and inactivation of Rb is necessary for entry into S phase. Rb is inactivated by phosphorylation upon growth factor stimulation of quiescent cells, facilitating the transition from G(1) phase to S phase. Although the signaling events after growth factor stimulation have been well characterized, it is not yet clear how these signals contact the cell cycle machinery. We had found previously that growth factor stimulation of quiescent cells lead to the direct binding of Raf-1 kinase to Rb, leading to its inactivation. Here we show that the Rb-Raf-1 interaction occurs prior to the activation of cyclin and/or cyclin-dependent kinases and facilitates normal cell cycle progression. Raf-1-mediated inactivation of Rb is independent of the mitogen-activated protein kinase cascade, as well as cyclin-dependent kinases. Binding of Raf-1 seemed to correlate with the dissociation of the chromatin remodeling protein Brg1 from Rb. Disruption of the Rb-Raf-1 interaction by a nine-amino-acid peptide inhibits Rb phosphorylation, cell proliferation, and vascular endothelial growth factor-mediated capillary tubule formation. Delivery of this peptide by a carrier molecule led to a 79% reduction in tumor volume and a 57% reduction in microvessel formation in nude mice. It appears that Raf-1 links mitogenic signaling to Rb and that disruption of this interaction could aid in controlling proliferative disorders.

    Funded by: NCI NIH HHS: CA63136, R01 CA063136

    Molecular and cellular biology 2004;24;21;9527-41

  • Kinase suppressor of Ras-1 protects intestinal epithelium from cytokine-mediated apoptosis during inflammation.

    Yan F, John SK, Wilson G, Jones DS, Washington MK and Polk DB

    Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2576, USA.

    TNF plays a pathogenic role in inflammatory bowel diseases (IBDs), which are characterized by altered cytokine production and increased intestinal epithelial cell apoptosis. In vitro studies suggest that kinase suppressor of Ras-1 (KSR1) is an essential regulatory kinase for TNF-stimulated survival pathways in intestinal epithelial cell lines. Here we use a KSR1-deficient mouse model to study the role of KSR1 in regulating intestinal cell fate during cytokine-mediated inflammation. We show that KSR1 and its target signaling pathways are activated in inflamed colon mucosa. Loss of KSR1 increases susceptibility to chronic colitis and TNF-induced apoptosis in the intestinal epithelial cell. Furthermore, disruption of KSR1 expression enhances TNF-induced apoptosis in mouse colon epithelial cells and is associated with a failure to activate antiapoptotic signals including Raf-1/MEK/ERK, NF-kappaB, and Akt/protein kinase B. These effects are reversed by WT, but not kinase-inactive, KSR1. We conclude that KSR1 has an essential protective role in the intestinal epithelial cell during inflammation through activation of cell survival pathways.

    Funded by: NCI NIH HHS: CA-68485, P30 CA068485; NIDDK NIH HHS: DK-56008, DK-58404, P30 DK058404, R01 DK056008, Z01 DK056008

    The Journal of clinical investigation 2004;114;9;1272-80

  • A novel human phosphatidylethanolamine-binding protein resists tumor necrosis factor alpha-induced apoptosis by inhibiting mitogen-activated protein kinase pathway activation and phosphatidylethanolamine externalization.

    Wang X, Li N, Liu B, Sun H, Chen T, Li H, Qiu J, Zhang L, Wan T and Cao X

    Institute of Immunology, Zhejiang University, Hangzhou 310031, People's Republic of China.

    The phosphatidylethanolamine (PE)-binding proteins (PEBPs) are an evolutionarily conserved family of proteins with pivotal biological functions. Here we describe the cloning and functional characterization of a novel family member, human phosphatidylethanolamine-binding protein 4 (hPEBP4). hPEBP4 is expressed in most human tissues and highly expressed in tumor cells. Its expression in tumor cells is further enhanced upon tumor necrosis factor (TNF) alpha treatment, whereas hPEBP4 normally co-localizes with lysosomes, TNFalpha stimulation triggers its transfer to the cell membrane, where it binds to Raf-1 and MEK1. L929 cells overexpressing hPEBP4 are resistant to both TNFalpha-induced ERK1/2, MEK1, and JNK activation and TNFalpha-mediated apoptosis. Co-precipitation and in vitro protein binding assay demonstrated that hPEBP4 interacts with Raf-1 and MEK1. A truncated form of hPEBP4, lacking the PE-binding domain, maintains lysosomal co-localization but has no effect on cellular responses to TNFalpha. Given that MCF-7 breast cancer cells expressed hPEBP4 at a high level, small interfering RNA was used to silence the expression of hPEBP4. We demonstrated that down-regulation of hPEBP4 expression sensitizes MCF-7 breast cancer cells to TNFalpha-induced apoptosis. hPEBP4 appears to promote cellular resistance to TNF-induced apoptosis by inhibiting activation of the Raf-1/MEK/ERK pathway, JNK, and PE externalization, and the conserved region of PE-binding domain appears to play a vital role in this biological activity of hPEBP4.

    The Journal of biological chemistry 2004;279;44;45855-64

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • Raf-1 activation disrupts its binding to keratins during cell stress.

    Ku NO, Fu H and Omary MB

    Department of Medicine, VA Palo Alto Medical Center, 3801 Miranda Ave., 154J, Palo Alto, CA 94304, USA.

    Keratins 8 and 18 (K8/18) heteropolymers may regulate cell signaling via the known K18 association with 14-3-3 proteins and 14-3-3 association with Raf-1 kinase. We characterized Raf-keratin-14-3-3 associations and show that Raf associates directly with K8, independent of Raf kinase activity or Ras-Raf interaction, and that K18 is a Raf physiologic substrate. Raf activation during oxidative and toxin exposure in cultured cells and animals disrupt keratin-Raf association in a phosphorylation-dependent manner. Mutational analysis showed that 14-3-3 residues that are essential for Raf binding also regulate 14-3-3-keratin association. Similarly, Raf phosphorylation sites that are important for binding to 14-3-3 are also essential for Raf binding to K8/18. Therefore, keratins may modulate some aspects of Raf signaling under basal conditions via sequestration by K8, akin to Raf-14-3-3 binding. Keratin-bound Raf kinase is released upon Raf hyperphosphorylation and activation during oxidative and other stresses.

    Funded by: NIDDK NIH HHS: DK52951, DK56339; NIGMS NIH HHS: GM53165

    The Journal of cell biology 2004;166;4;479-85

  • Change in protein flexibility upon complex formation: analysis of Ras-Raf using molecular dynamics and a molecular framework approach.

    Gohlke H, Kuhn LA and Case DA

    Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

    Changes in flexibility upon protein-protein complex formation of H-Ras and the Ras-binding domain of C-Raf1 have been investigated using the molecular framework approach FIRST (Floppy Inclusion and Rigid Substructure Topology) and molecular dynamics simulations (MD) of in total approximately 35 ns length. In a computational time of about one second, FIRST identifies flexible and rigid regions in a single, static three-dimensional molecular framework, whose vertices represent protein atoms and whose edges represent covalent and non-covalent (hydrogen bond and hydrophobic) constraints and fixed bond angles within the protein. The two methods show a very good agreement with respect to the identification of changes in flexibility in both binding partners on a local scale. This implies that flexibility can be successfully predicted by identifying which bonds limit motion within a molecule and how they are coupled. In particular, as identified by MD, the beta-sheet in Raf shows considerably more pronounced orientational correlations in the bound state compared to the unbound state. Similarly, FIRST assigns the beta-sheet to the largest rigid cluster of the complex. Interestingly, FIRST allows us to identify that interactions across the interface (but not conformational changes upon complex formation) result in the observed rigidification. Since regions of the beta-sheet of Raf that do not interact directly with Ras become rigidified, this also demonstrates the long-range aspect to rigidity percolation. Possible implications of the change of flexibility of the Ras-binding domain of Raf on the activation of Raf upon complex formation are discussed. Finally, the sensitivity of FIRST results with respect to the representation of non-covalent interactions used as constraints is probed.

    Funded by: NCRR NIH HHS: RR12255; NIGMS NIH HHS: GM67249

    Proteins 2004;56;2;322-37

  • Regulation of B-Raf kinase activity by tuberin and Rheb is mammalian target of rapamycin (mTOR)-independent.

    Karbowniczek M, Cash T, Cheung M, Robertson GP, Astrinidis A and Henske EP

    Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.

    Tuberous sclerosis complex (TSC) is a tumor suppressor gene syndrome with manifestations that can include seizures, mental retardation, autism, and tumors in the brain, retina, kidney, heart, and skin. The products of the TSC1 and TSC2 genes, hamartin and tuberin, respectively, heterodimerize and inhibit the mammalian target of rapamycin (mTOR). We found that tuberin expression increases p42/44 MAPK phosphorylation and B-Raf kinase activity. Short interfering RNA down-regulation of tuberin decreased the p42/44 MAPK phosphorylation and B-Raf activity. Expression of Rheb, the target of the GTPase-activating domain of tuberin, inhibited wild-type B-Raf kinase but not activated forms of B-Raf. The interaction of endogenous Rheb with B-Raf was enhanced by serum and by Ras overexpression. A farnesylation-defective mutant of Rheb co-immunoprecipitated with and inhibited B-Raf but did not activate ribosomal protein S6 kinase, indicating that farnesylation is not required for B-Raf inhibition by Rheb and that B-Raf inhibition and S6 kinase activation are separable activities of Rheb. Consistent with this, inhibition of B-Raf and p42/44 MAPK by Rheb was resistant to rapamycin in contrast to Rheb activation of S6 kinase, which is rapamycin-sensitive. Taken together these data demonstrate that inhibition of B-Raf kinase via Rheb is an mTOR-independent function of tuberin.

    Funded by: NHLBI NIH HHS: HL 60746; NIDDK NIH HHS: DK 51052

    The Journal of biological chemistry 2004;279;29;29930-7

  • The scaffold protein CNK1 interacts with the tumor suppressor RASSF1A and augments RASSF1A-induced cell death.

    Rabizadeh S, Xavier RJ, Ishiguro K, Bernabeortiz J, Lopez-Ilasaca M, Khokhlatchev A, Mollahan P, Pfeifer GP, Avruch J and Seed B

    Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.

    The connector enhancer of KSR (CNK) is a multidomain scaffold protein discovered in Drosophila, where it is necessary for Ras activation of the Raf kinase. Recent studies have shown that CNK1 also interacts with RalA and Rho and participates in some aspects of signaling by these GTPases. Herein we demonstrate a novel aspect of CNK1 function, i.e. reexpression of CNK1 suppresses tumor cell growth and promotes apoptosis. As shown previously for apoptosis induced by Ki-Ras(G12V), CNK1-induced apoptosis is suppressed by a dominant inhibitor of the mammalian sterile 20 kinases 1 and (MST1/MST2). Immunoprecipitates of MST1 endogenous to LoVo colon cancer cells contain endogenous CNK1; however, no association of these two polypeptides can be detected in a yeast two-hybrid assay. CNK1 does, however, bind directly to the RASSF1A and RASSF1C polypeptides, constitutive binding partners of the MST1/2 kinases. Deletion of the MST1 carboxyl-terminal segment that mediates its binding to RASSF1A/C eliminates the association of MST1 with CNK1. Coexpression of CNK1 with the tumor suppressive isoform, RASSF1A, greatly augments CNK1-induced apoptosis, whereas the nonsuppressive RASSF1C isoform is without effect on CNK1-induced apoptosis. Overexpression of CNK1-(1-282), a fragment that binds RASSF1A but is not proapoptotic, blocks the apoptosis induced by CNK1 and by Ki-Ras(G12V). Thus, in addition to its positive role in the proliferative outputs of active Ras, the CNK1 scaffold protein, through its binding of a RASSF1A.MST complex, also participates in the proapoptotic signaling initiated by active Ras.

    The Journal of biological chemistry 2004;279;28;29247-54

  • Raf promotes human herpesvirus-8 (HHV-8/KSHV) infection.

    Akula SM, Ford PW, Whitman AG, Hamden KE, Shelton JG and McCubrey JA

    Department of Microbiology & Immunology, Brody School of Medicine, East Carolina University, Greenville, NC 27858, USA. akulas@mail.ecu.edu

    Human herpesvirus-8 (HHV-8/KSHV) is etiologically associated with Kaposi's sarcoma (KS) and other tumors. Constitutive activation of the mitogen-activated protein kinase (MAPK) signaling pathway has been associated with a variety of tumors, including AIDS-related KS. The oncoprotein Raf is situated at a pivotal position in regulating the MAPK pathway. Hence, we analysed the effect of oncoprotein Raf on HHV-8 infectious entry into target cells. Here we report Raf expression to significantly enhance HHV-8 infection of target cells. These findings implicate a role for Raf not only in the infectious entry of HHV-8 but also in modulating KS pathogenesis.

    Funded by: NCI NIH HHS: R01CA098195

    Oncogene 2004;23;30;5227-41

  • Identification and characterization of rain, a novel Ras-interacting protein with a unique subcellular localization.

    Mitin NY, Ramocki MB, Zullo AJ, Der CJ, Konieczny SF and Taparowsky EJ

    Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907-2054, USA.

    The Ras small GTPase functions as a signaling node and is activated by extracellular stimuli. Upon activation, Ras interacts with a spectrum of functionally diverse downstream effectors and stimulates multiple cytoplasmic signaling cascades that regulate cellular proliferation, differentiation, and apoptosis. In addition to the association of Ras with the plasma membrane, recent studies have established an association of Ras with Golgi membranes. Whereas the effectors of signal transduction by activated, plasma membrane-localized Ras are well characterized, very little is known about the effectors used by Golgi-localized Ras. In this study, we report the identification of a novel Ras-interacting protein, Rain, that may serve as an effector for endomembrane-associated Ras. Rain does not share significant sequence similarity with any known mammalian proteins, but contains a Ras-associating domain that is found in RalGDS, AF-6, and other characterized Ras effectors. Rain interacts with Ras in a GTP-dependent manner in vitro and in vivo, requires an intact Ras core effector-binding domain for this interaction, and thus fits the definition of a Ras effector. Unlike other Ras effectors, however, Rain is localized to perinuclear, juxta-Golgi vesicles in intact cells and is recruited to the Golgi by activated Ras. Finally, we found that Rain cooperates with activated Raf and causes synergistic transformation of NIH3T3 cells. Taken together, these observations support a role for Rain as a novel protein that can serve as an effector of endomembrane-localized Ras.

    Funded by: NCI NIH HHS: CA09634, CA42978; NIAMS NIH HHS: AR41115; NIGMS NIH HHS: GM08737

    The Journal of biological chemistry 2004;279;21;22353-61

  • STAT5 activation underlies IL7 receptor-dependent B cell development.

    Goetz CA, Harmon IR, O'Neil JJ, Burchill MA and Farrar MA

    Department of Laboratory Medicine and Pathology, Center for Immunology, Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA.

    Signals initiated by the IL7R are required for B cell development. However, the roles that distinct IL7R-induced signaling pathways play in this process remains unclear. To identify the function of the Raf and STAT5 pathways in IL7R-dependent B cell development, we used transgenic mice that express constitutively active forms of Raf (Raf-CAAX) or STAT5 (STAT5b-CA) throughout lymphocyte development. Both Raf-CAAX and STAT5b-CA mice exhibit large increases in pro-B cells. However, crossing the Raf-CAAX transgene onto the IL7R(-/-) background fails to rescue B cell development. In contrast, STAT5 activation selectively restores B cell expansion in IL7R(-/-) mice. Notably, the expansion of pro-B cells in STAT5b-CA mice correlated with an increase in cyclin D2, pim-1, and bcl-x(L) expression, suggesting that STAT5 directly affects pro-B cell proliferation and survival. In addition, STAT5 activation also restored B cell differentiation in IL7R(-/-) mice as determined by 1) the restoration of V(H) Ig gene rearrangement and 2) the appearance of immature and mature B cell subsets. These findings establish STAT5 as the key player entraining B cell development downstream of the IL7R.

    Funded by: NIAID NIH HHS: 2T32-AI07313, AI05737

    Journal of immunology (Baltimore, Md. : 1950) 2004;172;8;4770-8

  • Linkage and association of the mitochondrial aspartate/glutamate carrier SLC25A12 gene with autism.

    Ramoz N, Reichert JG, Smith CJ, Silverman JM, Bespalova IN, Davis KL and Buxbaum JD

    Laboratory of Molecular Neuropsychiatry, Department of Psychiatry, Mount Sinai School of Medicine, New York, NY 10029, USA.

    Objective: Autism/autistic disorder (MIM number 209850) is a complex, largely genetic psychiatric disorder. The authors recently mapped a susceptibility locus for autism to chromosome region 2q24-q33 (MIM number 606053). In the present study, genes across the 2q24-q33 interval were analyzed to identify an autism susceptibility gene in this region.

    Method: Mutation screening of positional candidate genes was performed in two stages. The first stage involved identifying, in unrelated subjects showing linkage to 2q24-q33, genetic variants in exons and flanking sequence within candidate genes and comparing the frequency of the variants between autistic and unrelated nonautistic subjects. Two single nucleotide polymorphisms (SNPs) that showed evidence for divergent distribution between autistic and nonautistic subjects were identified, both within SLC25A12, a gene encoding the mitochondrial aspartate/glutamate carrier (AGC1). In the second stage, the two SNPs in SLC25A12 were further genotyped in 411 autistic families, and linkage and association tests were carried out in the 197 informative families.

    Results: Linkage and association were observed between autistic disorder and the two SNPs, rs2056202 and rs2292813, found in SLC25A12. Using either a single affected subject per family or all affected subjects, evidence for excess transmission was found by the Transmission Disequilibrium Test for rs2056202, rs2292813, and a two-locus G*G haplotype. Similar results were observed using TRANSMIT for the analyses. Evidence for linkage was supported by linkage analysis with the two SNPs, with a maximal multipoint nonparametric linkage score of 1.57 and a maximal multipoint heterogeneity lod score of 2.11. Genotype relative risk could be estimated to be between 2.4 and 4.8 for persons homozygous at these loci.

    Conclusions: A strong association of autism with SNPs within the SLC25A12 gene was demonstrated. Further studies are needed to confirm this association and to decipher any potential etiological role of AGC1 in autism.

    Funded by: NIMH NIH HHS: U54 MH066673; OMHHE CDC HHS: MN-066673

    The American journal of psychiatry 2004;161;4;662-9

  • Mutation analysis of the BRAF, ARAF and RAF-1 genes in human colorectal adenocarcinomas.

    Fransén K, Klintenäs M, Osterström A, Dimberg J, Monstein HJ and Söderkvist P

    Division of Cell Biology, Floor 9, Department of Biomedicine and Surgery, Faculty of Health Sciences, Linköping University, SE-581 85 Linköping, Sweden. karf@liu.se

    Colorectal cancer is a multi-step process characterized by a sequence of genetic alterations in cell growth regulatory genes, such as the adenomatous polyposis coli, KRAS, p53 and DCC genes. In the present study mutation analysis was performed with SSCA/direct sequencing of the hot-spot regions in exons 11 and 15 for the BRAF gene and exons 1-2 for the KRAS gene in 130 primary colorectal cancer tumors and correlated with clinico-pathological and mutational data. We also performed mutation analysis of the corresponding conserved regions in the ARAF and RAF-1 genes. Mutations in the BRAF and KRAS genes were found in 11.5 and 40% of the tumors, respectively. One germline exonic and nine germline intronic genetic variants were found in the ARAF and RAF-1 genes. All of the BRAF mutations were located in the kinase domain of the conserved region 3 in exon 15 of the BRAF gene. One novel somatic mutation was also identified in the BRAF gene. The majority of the BRAF mutations were found in colon compared with rectal tumors (P = 0.014). In agreement with others, a statistically significant correlation between BRAF mutations and microsatellite instability could be found. A negative correlation was also evident between mutations in the BRAF and KRAS genes, which supports earlier studies where somatic mutations in these genes are mutually exclusive. Collectively, our results provide support for the idea that activation of the MAP kinase pathway, especially via BRAF and KRAS mutations, is of critical importance for the development of colorectal cancer.

    Carcinogenesis 2004;25;4;527-33

  • Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF.

    Wan PT, Garnett MJ, Roe SM, Lee S, Niculescu-Duvaz D, Good VM, Jones CM, Marshall CJ, Springer CJ, Barford D, Marais R and Cancer Genome Project

    Section of Structural Biology, The Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, UK.

    Over 30 mutations of the B-RAF gene associated with human cancers have been identified, the majority of which are located within the kinase domain. Here we show that of 22 B-RAF mutants analyzed, 18 have elevated kinase activity and signal to ERK in vivo. Surprisingly, three mutants have reduced kinase activity towards MEK in vitro but, by activating C-RAF in vivo, signal to ERK in cells. The structures of wild type and oncogenic V599EB-RAF kinase domains in complex with the RAF inhibitor BAY43-9006 show that the activation segment is held in an inactive conformation by association with the P loop. The clustering of most mutations to these two regions suggests that disruption of this interaction converts B-RAF into its active conformation. The high activity mutants signal to ERK by directly phosphorylating MEK, whereas the impaired activity mutants stimulate MEK by activating endogenous C-RAF, possibly via an allosteric or transphosphorylation mechanism.

    Cell 2004;116;6;855-67

  • Antisense oligonucleotide targeting of Raf-1: importance of raf-1 mRNA expression levels and raf-1-dependent signaling in determining growth response in ovarian cancer.

    Mullen P, McPhillips F, MacLeod K, Monia B, Smyth JF and Langdon SP

    Cancer Research UK, Edinburgh Oncology Unit, Western General Hospital, Edinburgh, United Kingdom.

    Purpose: We sought to identify determinants of growth response to the Raf-1-targeted antisense oligonucleotide (ASO; ISIS 5132) using a large panel of ovarian cancer cell lines.

    First-(ISIS 5132) and second-generation (ISIS 13650) anti-Raf 1 ASOs were compared with control oligonucleotides. Growth was assessed by cell counts; apoptosis was assessed by poly(ADP-ribose) polymerase cleavage; and cell cycle analysis was assessed by flow cytometry. Protein expression was detected by Western blot analysis, and mRNA expression was detected by quantitative reverse transcription-PCR. Raf-1 kinase activity was detected by anti-Raf-1 immunoprecipitation, followed by myelin basic protein phosphorylation.

    Results: A panel of 15 ovarian cancer cell lines was used to model a range of growth responses to ASOs targeting Raf-1 mRNA. Growth inhibition varied from 10% to >90% inhibition. Growth inhibition was associated with increased apoptosis and accumulation of cells in the G(2)-M and S phases of the cell cycle. Growth response was not related to level of Raf-1 protein expression, Raf-1 kinase activity, intracellular ASO uptake, or degree of Raf-1 protein inhibition. However, ASO growth response was associated with a high proportion of Raf-1 mRNA [relative to total (i.e., Raf-1 + A-Raf + B-Raf) Raf mRNA] and significantly higher Raf-1 kinase activity induction following growth factor (transforming growth factor alpha) stimulation in the cell lines consistent with dependency of these cell lines on Raf-1.

    Conclusions: These data indicate that ovarian cancers demonstrate differential sensitivity to ASOs targeted against Raf-1, and target expression levels and degree of utilization of Raf-1 signaling are implicated. Clinically sensitive tumors could feasibly be identified.

    Clinical cancer research : an official journal of the American Association for Cancer Research 2004;10;6;2100-8

  • The small GTP-binding protein, Rhes, regulates signal transduction from G protein-coupled receptors.

    Vargiu P, De Abajo R, Garcia-Ranea JA, Valencia A, Santisteban P, Crespo P and Bernal J

    Instituto de Investigaciones Biomédicas Alberto Sols. Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, 28029 Madrid, Spain.

    The Ras homolog enriched in striatum, Rhes, is the product of a thyroid hormone-regulated gene during brain development. Rhes and the dexamethasone-induced Dexras1 define a novel distinct subfamily of proteins within the Ras family, characterized by an extended variable domain in the carboxyl terminal region. We have carried this study because there is a complete lack of knowledge on Rhes signaling. We show that in PC12 cells, Rhes is targeted to the plasma membrane by farnesylation. We demonstrate that about 30% of the native Rhes protein is bound to GTP and this proportion is unaltered by typical Ras family nucleotide exchange factors. However, Rhes is not transforming in murine fibroblasts. We have also examined the role of Rhes in cell signaling. Rhes does not stimulate the ERK pathway. By contrast, it binds to and activates PI3K. On the other hand, we demonstrate that Rhes impairs the activation of the cAMP/PKA pathway by thyroid-stimulating hormone, and by an activated beta2 adrenergic receptor by a mechanism that suggests uncoupling of the receptor to its cognate heterotrimeric complex. Overall, our results provide the initial insights into the role in signal transduction of this novel Ras family member.

    Oncogene 2004;23;2;559-68

  • Ras regulates assembly of mitogenic signalling complexes through the effector protein IMP.

    Matheny SA, Chen C, Kortum RL, Razidlo GL, Lewis RE and White MA

    Department of Cell Biology, UT Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9039, USA.

    The signal transduction cascade comprising Raf, mitogen-activated protein (MAP) kinase kinase (MEK) and MAP kinase is a Ras effector pathway that mediates diverse cellular responses to environmental cues and contributes to Ras-dependent oncogenic transformation. Here we report that the Ras effector protein Impedes Mitogenic signal Propagation (IMP) modulates sensitivity of the MAP kinase cascade to stimulus-dependent activation by limiting functional assembly of the core enzymatic components through the inactivation of KSR, a scaffold/adaptor protein that couples activated Raf to its substrate MEK. IMP is a Ras-responsive E3 ubiquitin ligase that, on activation of Ras, is modified by auto-polyubiquitination, which releases the inhibition of Raf-MEK complex formation. Thus, Ras activates the MAP kinase cascade through simultaneous dual effector interactions: induction of Raf kinase activity and derepression of Raf-MEK complex formation. IMP depletion results in increased stimulus-dependent MEK activation without alterations in the timing or duration of the response. These observations suggest that IMP functions as a threshold modulator, controlling sensitivity of the cascade to stimulus and providing a mechanism to allow adaptive behaviour of the cascade in chronic or complex signalling environments.

    Nature 2004;427;6971;256-60

  • Protein kinase C switches the Raf kinase inhibitor from Raf-1 to GRK-2.

    Lorenz K, Lohse MJ and Quitterer U

    Institut für Pharmakologie und Toxikologie, Versbacher Strasse 9, D-97078 Würzburg, Germany.

    Feedback inhibition is a fundamental principle in signal transduction allowing rapid adaptation to different stimuli. In mammalian cells, the major feedback inhibitor for G-protein-coupled receptors (GPCR) is G-protein-coupled receptor kinase 2 (GRK-2), which phosphorylates activated receptors, uncouples them from G proteins and initiates their internalization. The functions of GRK-2 are indispensable and need to be tightly controlled. Dysregulation promotes disorders such as hypertension or heart failure. In our search for a control mechanism for this vital kinase, here we show that the Raf kinase inhibitor protein (RKIP) is a physiological inhibitor of GRK-2. After stimulation of GPCR, RKIP dissociates from its known target, Raf-1 (refs 6-8), to associate with GRK-2 and block its activity. This switch is triggered by protein kinase C (PKC)-dependent phosphorylation of the RKIP on serine 153. The data delineate a new principle in signal transduction: by activating PKC, the incoming receptor signal is enhanced both by removing an inhibitor from Raf-1 and by blocking receptor internalization. A physiological role for this mechanism is shown in cardiomyocytes in which the downregulation of RKIP restrains beta-adrenergic signalling and contractile activity.

    Nature 2003;426;6966;574-9

  • SIAH-1 interacts with CtIP and promotes its degradation by the proteasome pathway.

    Germani A, Prabel A, Mourah S, Podgorniak MP, Di Carlo A, Ehrlich R, Gisselbrecht S, Varin-Blank N, Calvo F and Bruzzoni-Giovanelli H

    Laboratory of Vascular Biology and Gene Therapy, Centro Cardiologico Fondazione-IRCCS, Via Parea 4, 20138 Milano, Italy.

    SIAH-1 and SIAH-2 are the human members of an evolutionary highly conserved E3 ligase family. SIAH-1 is a p53 and p21(Waf-1/Cip-1) induced gene during apoptosis and tumor suppression. In stable-transfected clones of MCF-7 cells, SIAH-1 overexpression was associated with apoptosis, mitotic alterations and p21(Waf-1/Cip-1) induction of expression. Using a two-hybrid screening, we identified here the transcriptional corepressor CtBP-interacting protein (CtIP) as a SIAH-1-interacting protein. CtIP has been proposed as a regulator of p21(Waf-1/Cip-1) gene transcription through a protein complex involving BRCA1. We demonstrate that SIAH-1 associates with CtIP both in vitro and in vivo. This interaction led to CtIP degradation by the ubiquitin-proteasome pathway. As expected, SIAH-1 induced p21(Waf-1/Cip-1) transcription in Jurkat-T cell. Surprisingly, a SIAH protein deleted of its RING finger, SIAH-1DeltaN, which is able to interact with CtIP but does not promote its degradation, also induced transcription from the p21(Waf-1) promoter in a similar extent as did SIAH-1. Our results suggest that p21(Waf-1/Cip-1) induction by SIAH-1 could not be mediated by CtIP degradation.

    Oncogene 2003;22;55;8845-51

  • The synergistic activation of Raf-1 kinase by phorbol myristate acetate and hydrogen peroxide in NIH3T3 cells.

    Lee M, Petrovics G and Anderson WB

    Korea Institute of Toxicology, Korea Research Institute of Chemical Technology, P.O. Box 123, Yusong, 305-600, Daejeon, Republic of Korea. mikelee@kitox.re.kr

    We have previously demonstrated that a 33kDa C-terminal fragment of c-Raf-1 underwent a mobility shift in response to hydrogen peroxide (H(2)O(2)) and phorbol myristate acetate (PMA), respectively. In this study, we have demonstrated that H(2)O(2) induced the activation of N-terminal deletion mutant as well as full length Raf-1 kinase. The pharmacological PKC activator PMA also induced a weak increase in Raf-1 kinase activity through PKC-epsilon activation as determined by the transient expression of dominant negative mutants of PKC-epsilon-K436R. Interestingly, H(2)O(2) produced synergistic increase of PMA-stimulated Raf-1 kinase activation after simultaneous treatment of PMA and H(2)O(2). This synergistic activation of Raf-1 kinase was further enhanced by cypermethrin (an inhibitor of protein phosphatase 2B) and dephostatin (tyrosine kinase inhibitor) implying an inhibitory role for these phosphatases in the Raf-1 signaling pathway. Taken together, our data suggest that the synergistic activation of Raf-1 kinase in response to PMA and H(2)O(2) occurs via mechanisms that involve an interaction of Raf-1 kinase and PKC-epsilon, along with a transient phosphorylation of both Raf-1 kinase and PKC.

    Biochemical and biophysical research communications 2003;311;4;1026-33

  • Identification of a novel human kinase supporter of Ras (hKSR-2) that functions as a negative regulator of Cot (Tpl2) signaling.

    Channavajhala PL, Wu L, Cuozzo JW, Hall JP, Liu W, Lin LL and Zhang Y

    Department of Inflammation, Wyeth Research, 200 Cambridge Park Drive, Cambridge, MA 02140, USA. pchannavajhala@wyeth.com

    Kinase suppressor of Ras (KSR) is an integral and conserved component of the Ras signaling pathway. Although KSR is a positive regulator of the Ras/mitogen-activated protein (MAP) kinase pathway, the role of KSR in Cot-mediated MAPK activation has not been identified. The serine/threonine kinase Cot (also known as Tpl2) is a member of the MAP kinase kinase kinase (MAP3K) family that is known to regulate oncogenic and inflammatory pathways; however, the mechanism(s) of its regulation are not precisely known. In this report, we identify an 830-amino acid novel human KSR, designated hKSR-2, using predictions from genomic data base mining based on the structural profile of the KSR kinase domain. We show that, similar to the known human KSR, hKSR-2 co-immunoprecipitates with many signaling components of the Ras/MAPK pathway, including Ras, Raf, MEK-1, and ERK-1/2. In addition, we demonstrate that hKSR-2 co-immunoprecipitates with Cot and that co-expression of hKSR-2 with Cot significantly reduces Cot-mediated MAPK and NF-kappaB activation. This inhibition is specific to Cot, because Ras-induced ERK and IkappaB kinase-induced NF-kappaB activation are not significantly affected by hKSR-2 co-expression. Moreover, Cot-induced interleukin-8 production in HeLa cells is almost completely inhibited by the concurrent expression of hKSR-2, whereas transforming growth factor beta-activated kinase 1 (TAK1)/TAK1-binding protein 1 (TAB1)-induced interleukin-8 production is not affected by hKSR-2 co-expression. Taken together, these results indicate that hKSR-2, a new member of the KSR family, negatively regulates Cot-mediated MAP kinase and NF-kappaB pathway signaling.

    The Journal of biological chemistry 2003;278;47;47089-97

  • Calcium/calmodulin-dependent protein kinase II binds to Raf-1 and modulates integrin-stimulated ERK activation.

    Illario M, Cavallo AL, Bayer KU, Di Matola T, Fenzi G, Rossi G and Vitale M

    Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università Federico II, Napoli, 80131 Italy.

    Integrin activation generates different signalings in a cell type-dependent manner and stimulates cell proliferation through the Ras/Raf-1/Mek/Erk pathway. In this study, we demonstrate that integrin stimulation by fibronectin (FN), besides activating the Ras/Erk pathway, generates an auxiliary calcium signal that activates calmodulin and the Ca2+/calmodulin-dependent protein kinase II (CaMKII). This signal regulates Raf-1 activation by Ras and modulates the FN-stimulated extracellular signal-regulated kinase (Erk-1/2). The binding of soluble FN to integrins induced increase of intracellular calcium concentration associated with phosphorylation and activation of CaMKII. In two different cell lines, inhibition of CaMKII activity by specific inhibitors inhibited Erk-1/2 phosphorylation. Whereas CaMK inhibition affected neither integrin-stimulated Akt phosphorylation nor p21Ras or Mek-1 activity, it was necessary for Raf-1 activity. FN-induced Raf-1 activity was abrogated by the CaMKII specific inhibitory peptide ant-CaNtide. Integrin activation by FN induced the formation of a Raf-1/CaMKII complex, abrogated by inhibition of CaMKII. Active CaMKII phosphorylated Raf-1 in vitro. This is the first demonstration that CaMKII interplays with Raf-1 and regulates Erk activation induced by Ras-stimulated Raf-1. These findings also provide evidence supporting the possible existence of cross-talk between other intracellular pathways involving CaMKII and Raf-1.

    The Journal of biological chemistry 2003;278;46;45101-8

  • Functional analysis of a phosphatidic acid binding domain in human Raf-1 kinase: mutations in the phosphatidate binding domain lead to tail and trunk abnormalities in developing zebrafish embryos.

    Ghosh S, Moore S, Bell RM and Dush M

    GlaxoSmithKline, Genetics Research, Research Triangle Park, North Carolina 27709, USA. sg45653@gsk.com

    Previously, we and others identified a 35-amino acid segment within human Raf-1 kinase that preferentially binds phosphatidic acid. The presence of phosphatidic acid was found to be necessary for the translocation of Raf-1 to the plasma membrane. We have now employed a combination of alanine-scanning and deletion mutagenesis to identify the critical amino acid residues in Raf-1 necessary for interaction with phosphatidic acid. Progressive mutations within a tetrapeptide motif (residues 398-401 of human Raf-1) reduced and finally eliminated binding of Raf-1 to phosphatidic acid. We then injected zebrafish embryos with RNA encoding wild-type Raf-1 kinase or a mutant version with triple alanine mutations in the tetrapeptide motif and followed the morphological fate of embryonic development. Embryos with mutant but not wild-type Raf-1 exhibited defects in posterior axis formation exemplified by bent trunk and tail structures. Molecular evidence for lack of signaling through mutated Raf-1 was obtained by aberrant in situ hybridization of the ntl (no tail) gene, which functions downstream of Raf-1. Our results demonstrate that a functional phosphatidate binding site is necessary for Raf-1 function in embryonic development.

    The Journal of biological chemistry 2003;278;46;45690-6

  • Human homologue of Drosophila CNK interacts with Ras effector proteins Raf and Rlf.

    Lanigan TM, Liu A, Huang YZ, Mei L, Margolis B and Guan KL

    Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606, USA.

    Connector enhancer of KSR (CNK) is a multidomain protein that participates in Ras signaling in Drosophila eye development. In this report we identify the human homologue of CNK, termed CNK2A, and a truncated alternatively spliced variant, CNK2B. We characterize CNK2 phosphorylation, membrane localization, and interaction with Ras effector molecules. Our results show that MAPK signaling appears to play a role in the phosphorylation of CNK2 in vivo. CNK2 is found in both membrane and cytoplasmic fractions of the cell. In MDCK cells, full-length CNK2 is localized to the lateral plasma membrane. Consistent with previous reports, we show CNK2 interacts with Raf. CNK2 interaction was mapped to the regulatory and kinase domains of Raf, as well as to the carboxyl-terminal half of CNK2. CNK2 also interacts with the Ral signaling components, Ral GTPase, and the RalGDS family member Rlf. CNK2 interaction was mapped to the GEF domain of Rlf. The ability of CNK2 to interact with both Ras effector proteins Raf and Rlf suggests that CNK2 may integrate signals between MAPK and Ral pathways through a complex interplay of components.

    Funded by: NIA NIH HHS: 5T32AG00114-17

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2003;17;14;2048-60

  • Regulation of Raf through phosphorylation and N terminus-C terminus interaction.

    Chong H and Guan KL

    Department of Biological Chemistry and the Institute of Gerontology, University of Michigan, Ann Arbor, Michigan 48109, USA.

    Raf kinase is a key component in regulating the MAPK pathway. B-Raf has been reported as an oncogene and is mutated in 60% of human melanomas. The main focus of Raf regulation studies has been on phosphorylation, dephosphorylation, and scaffolding proteins; however, Raf also has its own auto-regulatory domain. Removal of the N-terminal regulatory domain, initially discovered in the viral Raf oncogene (v-Raf), results in a kinase domain with high basal activity independent of Ras activation. In this report, we show that activating phosphorylations are still required for activity of the truncated C-terminal kinase domain (called 22W). The interaction between the N-terminal regulatory domain and the C-terminal kinase domain is disrupted by activated Ras. Mutations in the Ras binding domain, cysteine-rich domain, or S259A do not affect the inhibition of 22W by the N-terminal domain. When phosphomimetic residues are substituted at the activating sites (DDED) in 22W, this results in a higher basal activity that is no longer inhibited by expression of the N-terminal domain, although binding to the N-terminal domain still occurs. Although the interaction between 22W/DDED and the N-terminal domain may be in a different conformation, the interaction is still disrupted by activated Ras. These data demonstrate that N-terminal domain binding to the kinase domain inhibits the activity of the kinase domain. However, this inhibition is relieved when the C-terminal kinase domain is activated by phosphorylation.

    Funded by: NIA NIH HHS: T32-AG00114-18; NIGMS NIH HHS: 5-T32-GM07544

    The Journal of biological chemistry 2003;278;38;36269-76

  • Raf-1 activation suppresses neuroendocrine marker and hormone levels in human gastrointestinal carcinoid cells.

    Sippel RS, Carpenter JE, Kunnimalaiyaan M, Lagerholm S and Chen H

    Dept. of Surgery, Univ. of Wisconsin Medical School, University of Wisconsin Comprehensive Cancer Center, Madison 53792, USA.

    Gastrointestinal carcinoid cells secrete multiple neuroendocrine markers and hormones including 5-HT and chromogranin A. The intracellular signaling pathways that regulate production of bioactive molecules are not completely understood. Our aim was to determine whether activation of the raf-1/MEK/MAPK signal transduction pathway in carcinoid cells could modulate production of neuroendocrine markers and hormones. Human pancreatic carcinoid cells (BON) were stably transduced with an estrogen-inducible raf-1 construct creating BON-raf cells. Activation of raf-1 in BON-raf cells led to a marked induction of phosphorylated MEK and ERK1/2 within 48 h. Importantly, raf-1 activation resulted in morphological changes accompanied by a marked decrease in neuroendocrine secretory granules by electronmicroscopy. Moreover, induction of raf-1 in BON-raf cells led to significant reductions in 5-HT, chromogranin A, and synaptophysin levels. Furthermore, treatment of BON-raf cells with MEK inhibitors PD-98059 and U-0126 blocked raf-1-mediated morphological changes and hormone suppression but not ERK1/2 phosphorylation. These results show that raf-1 induction suppresses neuroendocrine marker and hormone production in human gastrointestinal carcinoid cells via a pathway dependent on MEK activation.

    Funded by: NCI NIH HHS: T32-CA-90217

    American journal of physiology. Gastrointestinal and liver physiology 2003;285;2;G245-54

  • Role of Raf in vascular protection from distinct apoptotic stimuli.

    Alavi A, Hood JD, Frausto R, Stupack DG and Cheresh DA

    Department of Immunology, Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.

    Raf kinases have been linked to endothelial cell survival. Here, we show that basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) differentially activate Raf, resulting in protection from distinct pathways of apoptosis in human endothelial cells and chick embryo vasculature. bFGF activated Raf-1 via p21-activated protein kinase-1 (PAK-1) phosphorylation of serines 338 and 339, resulting in Raf-1 mitochondrial translocation and endothelial cell protection from the intrinsic pathway of apoptosis, independent of the mitogen-activated protein kinase kinase-1 (MEK1). In contrast, VEGF activated Raf-1 via Src kinase, leading to phosphorylation of tyrosines 340 and 341 and MEK1-dependent protection from extrinsic-mediated apoptosis. These findings implicate Raf-1 as a pivotal regulator of endothelial cell survival during angiogenesis.

    Funded by: NCI NIH HHS: CA45726, CA50286, CA75924, P01 CA78045

    Science (New York, N.Y.) 2003;301;5629;94-6

  • Fibroblast quiescence in floating collagen matrices: decrease in serum activation of MEK and Raf but not Ras.

    Fringer J and Grinnell F

    Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9039, USA.

    Fibroblasts synthesize, organize, and maintain connective tissues during development and in response to injury and fibrotic disease. Studies on cells in three-dimensional collagen matrices have shown that fibroblasts switch between proliferative and quiescence phenotypes, depending upon whether matrices are attached or floating during matrix remodeling. Previous work showed that cell signaling through the ERK pathway was decreased in fibroblasts in floating matrices. In the current research, we extend the previous findings to show that serum stimulation of fibroblasts in floating matrices does not result in ERK translocation to the nucleus. In addition, there was decreased serum activation of upstream members of the ERK signaling pathway, MEK and Raf, even though Ras became GTP loaded. The findings suggest that quiescence of fibroblasts in floating collagen matrices may result from a defect in Ras coupling to its downstream effectors.

    Funded by: NIGMS NIH HHS: GM31321

    The Journal of biological chemistry 2003;278;23;20612-7

  • 1,25-dihydroxyvitamin D(3) increases human cystatin A expression by inhibiting the Raf-1/MEK1/ERK signaling pathway of keratinocytes.

    Takahashi H, Ibe M, Honma M, Ishida-Yamamoto A, Hashimoto Y and Iizuka H

    Department of Dermatology, Asahikawa Medical College, 2-1-1-1 Midorigaoka higashi, 078-8510, Asahikawa, Hokkaido, Japan. ht@asahikawa-med.ac.jp

    The active form of vitamin D(3), 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D3), regulates proliferation and differentiation of keratinocytes. Cystatin A, a cysteine proteinase inhibitor, is a cornified cell envelope constituent and a differentiation marker of keratinocytes. In the present study, we examined the effect of 1,25(OH)(2)D3 on the expression of cystatin A of cultured normal human keratinocytes (NHK). 1,25(OH)(2)D3 suppressed NHK proliferation in a dose-dependent manner with the maximal effect at 1x10(-7) M. It also stimulated cystatin A promoter activity and its expression with similar dose effects. The increased cystatin A was detected by 24 h and the effect was accompanied by the suppression of ERK activity. Cystatin A promoter activity was not affected by cotransfection of vitamin D(3) receptor or retinoid X receptor. Further analyses disclosed that the 12- o-tetradecanoylphorbol-13-acetate (TPA)-responsive element (TRE), T2 (-272 to -278), in cystatin A promoter is critical for the regulation by 1,25(OH)(2)D3. Transfection of the dominant-negative form of ERK adenovirus (Ad-dnERK) increased cystatin A promoter activity and its expression, which was markedly augmented by 1,25(OH)(2)D3 treatment. Transfection of the dominant-active form of Raf-1 (Ad-daRaf-1) or MEK1 (Ad-daMEK1) inhibited 1,25(OH)(2)D3-dependent cystatin A promoter activity and its expression. Consistent with these results, the MEK1 inhibitor, PD98059, further augmented 1,25(OH)(2)D3-induced cystatin A promoter activity and its expression. The present study demonstrated that the 1,25(OH)(2)D3-responsive element in the cystatin A gene is identical to the TRE, T2 (-272 to -278), and that the suppression of Raf-1/MEK1/ERK1,2 signaling pathway increases cystatin A expression of NHK.

    Archives of dermatological research 2003;295;2;80-7

  • Role of ERas in promoting tumour-like properties in mouse embryonic stem cells.

    Takahashi K, Mitsui K and Yamanaka S

    Laboratory of Animal Molecular Technology, Research and Education Center for Genetic Information, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan.

    Embryonic stem (ES) cells are pluripotent cells derived from early mammalian embryos. Their immortality and rapid growth make them attractive sources for stem cell therapies; however, they produce tumours (teratomas) when transplanted, which could preclude their therapeutic usage. Why ES cells, which lack chromosomal abnormalities, possess tumour-like properties is largely unknown. Here we show that mouse ES cells specifically express a Ras-like gene, which we have named ERas. We show that human HRasp, which is a recognized pseudogene, does not contain reported base substitutions and instead encodes the human orthologue of ERas. This protein contains amino-acid residues identical to those present in active mutants of Ras and causes oncogenic transformation in NIH 3T3 cells. ERas interacts with phosphatidylinositol-3-OH kinase but not with Raf. ERas-null ES cells maintain pluripotency but show significantly reduced growth and tumorigenicity, which are rescued by expression of ERas complementary DNA or by activated phosphatidylinositol-3-OH kinase. We conclude that the transforming oncogene ERas is important in the tumour-like growth properties of ES cells.

    Nature 2003;423;6939;541-5

  • Serine 338 phosphorylation is dispensable for activation of c-Raf1.

    Oehrl W, Rubio I and Wetzker R

    Institute for Molecular Cell Biology, University of Jena, 07747 Jena, Germany.

    Numerous extracellular agonists induce consecutive stimulation of Ras guanine nucleotide exchange factors, Ras and c-Raf1, as the starting point of the intracellular mitogen-activated protein kinase cascade. Recent data point to a more complex reaction pattern of this simple sequence. This study was aimed at elucidating the activation process of endogenous c-Raf1 in U937 cells. Treatment of permeabilized U937 cells with the nonhydrolyzable nucleotide guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) induced prolonged stimulation of Ras and c-Raf1 activity. Intriguingly, both signaling proteins expressed differential responses toward specific inhibitors of phosphoinositide 3-kinases and tyrosine kinases, which indicates diverse signaling reactions feeding into Ras and cRaf-1. Phosphorylation of c-Raf1 serine 338 by p21-activated kinase has been recently reported to contribute to phosphoinositide 3-kinase-dependent activation of c-Raf1. However, in U937 cells stimulation of c-Raf1 activity by GTPgammaS did not correlate with p21-activated kinase activity and Ser-338 phosphorylation. Thus Ser-338 phosphorylation appears dispensable for c-Raf1 activation under the conditions used. Together these data deny an essential role for serine 338 phosphorylation in c-Raf1 activation and disclose divergent signaling connections of Ras and c-Raf1 in U937 cells.

    The Journal of biological chemistry 2003;278;20;17819-26

  • Mammalian Sprouty4 suppresses Ras-independent ERK activation by binding to Raf1.

    Sasaki A, Taketomi T, Kato R, Saeki K, Nonami A, Sasaki M, Kuriyama M, Saito N, Shibuya M and Yoshimura A

    Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.

    The signalling cascade including Raf, mitogen-activated protein kinase (MAPK) kinase and extracellular-signal-regulated kinase (ERK) is important in many facets of cellular regulation. Raf is activated through both Ras-dependent and Ras-independent mechanisms, but the regulatory mechanisms of Raf activation remain unclear. Two families of membrane-bound molecules, Sprouty and Sprouty-related EVH1-domain-containing protein (Spred) have been identified and characterized as negative regulators of growth-factor-induced ERK activation. But the molecular functions of mammalian Sproutys have not been clarified. Here we show that mammalian Sprouty4 suppresses vascular epithelial growth factor (VEGF)-induced, Ras-independent activation of Raf1 but does not affect epidermal growth factor (EGF)-induced, Ras-dependent activation of Raf1. Sprouty4 binds to Raf1 through its carboxy-terminal cysteine-rich domain, and this binding is necessary for the inhibitory activity of Sprouty4. In addition, Sprouty4 mutants of the amino-terminal region containing the conserved tyrosine residue, which is necessary for suppressing fibroblast growth factor signalling, still inhibit the VEGF-induced ERK pathway. Our results show that receptor tyrosine kinases use distinct pathways for Raf and ERK activation and that Sprouty4 differentially regulates these pathways.

    Nature cell biology 2003;5;5;427-32

  • Down-regulation of Raf-1 kinase is associated with paclitaxel resistance in human breast cancer MCF-7/Adr cells.

    Lee M, Koh WS and Han SS

    Korea Institute of Toxicology, Korea Research Institute of Chemical Technology, P.O. Box 107,Yusong, Daejeon 305-600, South Korea. mikelee@kitox.re.kr

    Experiments were carried out to determine the role of Raf-1 kinase in the development of drug resistance and apoptosis induced by paclitaxel. In the present study, paclitaxel sensitivity, Raf-1 activity and mitogen-activated protein kinases activation were compared in two cell lines: parental human breast cancer cells and its drug resistant variant (MCF-7/Adr) cells. Paclitaxel treatment of parental MCF-7 cells caused a marked inhibition of Raf-1 kinase activity, concomitant with its mobility shift after 18 h exposure. In addition, paclitaxel greatly increased c-Jun N-terminal protein kinase (JNK) activity whereas showing a small enhancing effect on extracellular-regulated kinases (ERK) activity. Interestingly, MCF-7/Adr cells have lower basal Raf-1 activity, yet have much higher basal ERK activity than parental cells. However, it appeared that PD 98059, which turns off ERK through mitogen-activated protein kinase kinase (MEK) inhibition, enhanced basal Raf-1 kinase activity in MCF-7/Adr cells. Thus, the findings suggest that paclitaxel-induced apoptosis is mediated by JNK and occurs in parallel with suppression of the Raf-1 kinase activity in parental MCF-7 cells. In addition, down-regulation of Raf-1 kinase, which can be induced through the sustained ERK activation, may contribute to the development of acquired resistance in MCF-7/Adr cells.

    Cancer letters 2003;193;1;57-64

  • HIV-1 Nef control of cell signalling molecules: multiple strategies to promote virus replication.

    Greenway AL, Holloway G, McPhee DA, Ellis P, Cornall A and Lidman M

    Macfarlane Burnet Institute for Medical Research and Public Health, Cnr Commercial and Punt Roads, Melbourne, Victoria 3004, Australia. greenway@burnet.edu.au

    HIV-1 has at its disposal numerous proteins encoded by its genome which provide the required arsenal to establish and maintain infection in its host for a considerable number of years. One of the most important and enigmatic of these proteins is Nef. The Nef protein of HIV-1 plays a fundamental role in the virus life cycle. This small protein of approximately 27 kDa is required for maximal virus replication and disease progression. The mechanisms by which it is able to act as a positive factor during virus replication is an area of intense research and although some controversy surrounds Nef much has been gauged as to how it functions. Its ability to modulate the expression of key cellular receptors important for cell activation and control signal transduction elements and events by interacting with numerous cellular kinases and signalling molecules, including members of the Src family kinases, leading to an effect on host cell function is likely to explain at least in part its role during infection and represents a finely tuned mechanism where this protein assists HIV-1 to control its host.

    Journal of biosciences 2003;28;3;323-35

  • Phosphorylation of Raf-1 by p21-activated kinase 1 and Src regulates Raf-1 autoinhibition.

    Tran NH and Frost JA

    Department of Integrative Biology and Pharmacology, University of Texas Houston Health Science Center, Houston, Texas 77030, USA.

    Exposure of cells to mitogens or growth factors stimulates Raf-1 activity through a complex mechanism that involves binding to active Ras, phosphorylation on multiple residues, and protein-protein interactions. Recently it was shown that the amino terminus of Raf-1 contains an autoregulatory domain that can inhibit its activity in Xenopus oocytes. In the present work we show that expression of the Raf-1 autoinhibitory domain blocks extracellular signal-regulated kinase 2 activation by the Raf-1 catalytic domain in mammalian cells. We also show that phosphorylation of Raf-1 on serine 338 by PAK1 and tyrosines 340 and 341 by Src relieves autoinhibition and that this occurs through a specific decrease in the binding of the Raf-1 regulatory domain to its catalytic domain. In addition, we demonstrate that phosphorylation of threonine 491 and serine 494, two phosphorylation sites in the catalytic domain that are required for Raf-1 activation, is unlikely to regulate autoinhibition. These results demonstrate that the autoinhibitory domain of Raf-1 is functional in mammalian cells and that its interaction with the Raf-1 catalytic domain is regulated by phosphorylation of serine 338 and tyrosines 340 and 341.

    The Journal of biological chemistry 2003;278;13;11221-6

  • Novel raf kinase protein-protein interactions found by an exhaustive yeast two-hybrid analysis.

    Yuryev A and Wennogle LP

    Novartis Institute for Biomedical Research, Summit, NJ 07901, USA.

    We have performed an exhaustive unbiased yeast two-hybrid analysis to identify interaction partners of two human Raf kinase isoforms, A-Raf and C-Raf, using their N-terminal regulatory domain as "bait." A total of 20 different human proteins were found to interact with Raf isoforms. Several of these interactions were novel and an extensive bioinformatics evaluation was performed for each. The novel putative interactions include a signalosome component, TOPK/PBK kinase, and two new putative protein phosphatases. The cysteine-rich zinc-binding domain (CRD) of Raf was found to interact with all 20 proteins and to achieve isoform-specific interactions. Since similar putative CRDs are present in a variety of protein serine-threonine kinases, the data suggest that the CRD may function as a major protein-protein interaction domain of these kinases. We propose possible functional consequences of these novel Raf interactions.

    Genomics 2003;81;2;112-25

  • Erbin suppresses the MAP kinase pathway.

    Huang YZ, Zang M, Xiong WC, Luo Z and Mei L

    Department of Neurobiology, Civitan International Research Center, University of Alabama at Birmingham, 35294-0021, USA.

    We present evidence here that Erbin is a negative regulator of the Ras-Raf-Erk signaling pathway. Expression of Erbin decreases transcription of the AChR epsilon-subunit gene, an event that is mediated by Erk activation. Although it interacts with the ErbB2 C terminus through the PDZ domain, Erbin has no effect on ErbB2 tyrosine phosphorylation or binding to the adaptor proteins Shc and Grb2. In contrast, expression of Erbin greatly impairs activation of Erk, but not Akt, by ligands that activate receptor tyrosine kinases. Moreover, Erbin inhibits the Erk activation by active Ras, while it fails to do so in the presence of active Raf-1. Erbin associates with active Ras, but not inactive Ras nor Raf. Consistently, Erbin interferes with the interaction between Ras and Raf both in vivo and in vitro. Finally, overexpression of Erbin leads to inhibition of NGF-induced neuronal differentiation of PC12 cells, whereas down-regulation of endogenous Erbin by specific siRNA exhibits an opposite effect. Collectively, our study has identified Erbin as a novel suppressor of the Ras signaling by disrupting the Ras-Raf interaction.

    Funded by: NINDS NIH HHS: NS40480

    The Journal of biological chemistry 2003;278;2;1108-14

  • Usage of tautomycetin, a novel inhibitor of protein phosphatase 1 (PP1), reveals that PP1 is a positive regulator of Raf-1 in vivo.

    Mitsuhashi S, Shima H, Tanuma N, Matsuura N, Takekawa M, Urano T, Kataoka T, Ubukata M and Kikuchi K

    Division of Biochemical Oncology and Immunology, Institute for Genetic Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo 060-0815, Japan.

    Protein phosphatase type 1 (PP1), together with protein phosphatase 2A (PP2A), is a major eukaryotic serine/threonine protein phosphatase involved in regulation of numerous cell functions. Although the roles of PP2A have been studied extensively using okadaic acid, a well known inhibitor of PP2A, biological analysis of PP1 has remained restricted because of lack of a specific inhibitor. Recently we reported that tautomycetin (TC) is a highly specific inhibitor of PP1. To elucidate the biological effects of TC, we demonstrated in preliminary experiments that treatment of COS-7 cells with 5 microm TC for 5 h inhibits endogenous PP1 by more than 90% without affecting PP2A activity. Therefore, using TC as a specific PP1 inhibitor, the biological effect of PP1 on MAPK signaling was examined. First, we found that inhibition of PP1 in COS-7 cells by TC specifically suppresses activation of ERK, among three MAPK kinases (ERK, JNK, and p38). TC-mediated inhibition of PP1 also suppressed activation of Raf-1, resulting in the inactivation of the MEK-ERK pathway. To examine the role of PP1 in regulation of Raf-1, we overexpressed the PP1 catalytic subunit (PP1C) in COS-7 cells and found that PP1C enhanced activation of Raf-1 activity, whereas phosphatase-dead PP1C blocked Raf-1 activation. Furthermore, a physical interaction between PP1C and Raf-1 was also observed. These data strongly suggest that PP1 positively regulates Raf-1 in vivo.

    The Journal of biological chemistry 2003;278;1;82-8

  • Activation of the ras/raf-1 signal transduction pathway in carcinoid tumor cells results in morphologic transdifferentiation.

    Sippel RS and Chen H

    Department of Surgery, The University of Wisconsin Medical School, H4/750 Clinical Science Center, 600 Highland Avenue, Madison, WI 53792, USA.

    Background: Recent studies of neuroendocrine tumor cell lines suggest that ras/raf-1 activation could be detrimental to tumorigenesis. The mechanism by which it alters neuroendocrine tumor cells is unclear. We hypothesize that activation of the ras/raf signal transduction pathway may alter gastrointestinal carcinoid cells by inducing morphologic transdifferentiation.

    Methods: Pancreatic carcinoid (BON) cells were transduced in a stable manner with an estrogen inducible raf-1 fusion protein (creating "BON-raf cells"). BON and BON-raf cells were then treated with either control or 1 micromol/L estradiol (E2). Western blots were used to confirm the phosphorylation of extracellular signal-regulated kinase 1/2. Morphologic changes were evaluated using light and electron microscopy.

    Results: Western blots using antibodies against phosphorylated and unphosphorylated extracellular signal-regulated kinase 1/2. confirmed that phosphorylation was only present in the BON-raf E2 cells. BON cells treated with control and E2 and BON-raf cells treated with control all looked identical in culture. After treatment with E2 to induce raf-1, the BON-raf cells underwent dramatic morphologic changes. Under light and electron microscopy the cells became flatter and developed much sharper cellular borders mimicking cellular differentiation.

    Conclusions: Activation of the ras/raf-1 signal transduction pathway leads to prominent phenotypic changes that resemble differentiation of gastrointestinal carcinoid cells in vitro.

    Funded by: NCI NIH HHS: T32 CA 90217

    Surgery 2002;132;6;1035-9; discussion 1039

  • Phosphorylation of 338SSYY341 regulates specific interaction between Raf-1 and MEK1.

    Xiang X, Zang M, Waelde CA, Wen R and Luo Z

    Diabetes and Metabolism Research Unit, Endocrinology Section, Evans Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118, USA.

    The present study characterizes the interaction between the Raf-1 kinase domain and MEK1 and examines whether the magnitude of their interaction correlates to the ability of Raf to phosphorylate MEK1. Here we show that the minimal domain required for the Raf kinase activity starts from tryptophan 342. Maximal binding of the Raf kinase domain to MEK1 and its kinase activity are achieved upon phosphorylation of the region (338)SSYY(341) in response to 4beta-12-O-tetradecanoylphorbol-13-acetate (TPA), or mutation of Y340Y341 to aspartic acids. Conversely, the TPA-stimulated MEK binding and kinase activity are diminished when this region is deleted or Ser(338) and Ser(339) are mutated to alanines. We also show that the integrity of the Raf ATP-binding site is necessary for the interaction between Raf-1 and MEK1. Furthermore, two MEK-binding sites are identified; the first is localized between amino acids 325 and 349, and the second is within the region between amino acids 350 and 648. Separately, the binding of each site to MEK1 is weak, but in a cis context, they give rise to a much stronger association, which can be further stimulated by TPA. Finally, we find that tryptophan 342, which is conserved among the Raf family and other protein kinases, is essential for the Ser(338) phosphorylation of the full-length Raf and its binding to MEK1. Taken together, our results indicate that the phosphorylation of Ser(338) and Tyr(341) on Raf exerts an important effect on reconfiguring the two MEK-binding sites. As a result, these two sites coordinate to form a high affinity MEK-binding epitope, leading to a marked increase in Raf kinase activity.

    Funded by: NIGMS NIH HHS: GM 57959

    The Journal of biological chemistry 2002;277;47;44996-5003

  • Role of the 14-3-3 C-terminal loop in ligand interaction.

    Truong AB, Masters SC, Yang H and Fu H

    Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

    14-3-3 proteins are a family of conserved dimeric molecules that interact with a broad range of target proteins, most of which contain phosphoserine/threonine. The amphipathic groove of 14-3-3 is the main structural feature involved in mediating its associations. We have studied another domain of 14-3-3, the C-terminal loop, to determine what role it plays in ligand interaction. A truncated form of 14-3-3zeta lacking this C-terminal loop was generated and found to bind with higher affinity than the wild-type 14-3-3zeta protein to the ligands Raf-1 and Bad. Interestingly, the truncated 14-3-3zeta also showed increased association with the 14-3-3 binding-deficient Bad/S136A mutant. Taken together, these data support a role for the C-terminal loop as a general inhibitor of 14-3-3/ligand interactions. This may provide a mechanism by which inappropriate associations with 14-3-3 are prevented.

    Funded by: NIGMS NIH HHS: GM53165, GM60033

    Proteins 2002;49;3;321-5

  • Glucocorticoid-induced leucine zipper inhibits the Raf-extracellular signal-regulated kinase pathway by binding to Raf-1.

    Ayroldi E, Zollo O, Macchiarulo A, Di Marco B, Marchetti C and Riccardi C

    Department of Clinical and Experimental Medicine, Section of Pharmacology. Department of Drug Chemistry and Technology, University of Perugia, 06100 Perugia, Italy.

    Glucocorticoid-induced leucine zipper (GILZ) is a leucine zipper protein, whose expression is augmented by dexamethasone (DEX) treatment and downregulated by T-cell receptor (TCR) triggering. Stable expression of GILZ in T cells mimics some of the effects of glucocorticoid hormones (GCH) in GCH-mediated immunosuppressive and anti-inflammatory activity. In fact, GILZ overexpression inhibits TCR-activated NF-kappaB nuclear translocation, interleukin-2 production, FasL upregulation, and the consequent activation-induced apoptosis. We have investigated the molecular mechanism underlying GILZ-mediated regulation of T-cell activation by analyzing the effects of GILZ on the activity of mitogen-activated protein kinase (MAPK) family members, including Raf, MAPK/extracellular signal-regulated kinase (ERK) 1/2 (MEK-1/2), ERK-1/2, and c-Jun NH(2)-terminal protein kinase (JNK). Our results indicate that GILZ inhibited Raf-1 phosphorylation, which resulted in the suppression of both MEK/ERK-1/2 phosphorylation and AP-1-dependent transcription. We demonstrate that GILZ interacts in vitro and in vivo with endogenous Raf-1 and that Raf-1 coimmunoprecipitated with GILZ in murine thymocytes treated with DEX. Mapping of the binding domains and experiments with GILZ mutants showed that GILZ binds the region of Raf interacting with Ras through the NH(2)-terminal region. These data suggest that GILZ contributes, through protein-to-protein interaction with Raf-1 and the consequent inhibition of Raf-MEK-ERK activation, to regulating the MAPK pathway and to providing a further mechanism underlying GCH immunosuppression.

    Molecular and cellular biology 2002;22;22;7929-41

  • Activation of the mitogen activated protein kinase extracellular signal-regulated kinase 1 and 2 by the nitric oxide-cGMP-cGMP-dependent protein kinase axis regulates the expression of matrix metalloproteinase 13 in vascular endothelial cells.

    Zaragoza C, Soria E, López E, Browning D, Balbín M, López-Otín C and Lamas S

    Centro de Investigaciones Biológicas, Instituto Reina Sofía de Investigaciones Nefrológicas, Consejo Superior de Investigaciones Científicas, and Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain.

    Matrix metalloproteinases (MMPs) are synthesized in response to diverse stimuli, including cytokines, growth factors, hormones, and oxidative stress. Here we show that the nitric oxide (NO) donor 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA-NO) and NO from murine macrophages transcriptionally regulate MMP-13 expression in vascular endothelial cells (BAEC). The cGMP analog, 8-bromo-cGMP (8-Br-cGMP) mimicked the effect of NO, whereas incubation with the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, or the cGMP-dependent protein kinase (PKG) inhibitor phenyl-1,N (2)- etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rp-isomer (PET) reduced the stimulatory effect of DEA-NO on the activation of the MMP-13 promoter. Overexpression of the catalytic subunit of PKG1-alpha resulted in a 5- to 6-fold increase of the MMP-13 regulatory region over control cells. On the other hand, incubation with the mitogen-activated protein/extracellular signal-regulated kinase inhibitor 2'-amino-3'-methoxyflavone (PD98059) significantly reduced DEA-NO and 8-Br-cGMP promoter activation and mRNA expression of MMP-13 in transfected BAEC. Moreover, a complex between PKG1-alpha and the G-protein Raf-1, an upstream activator of the extracellular signal-regulated kinase signaling pathway, was detected in cells overexpressing PKG1-alpha or treated either with DEA-NO or 8-Br-cGMP. Thus, we propose that the NO-cGMP-PKG pathway enhances MMP-13 expression by the activation of ERK 1,2. This effect of NO may be important in the context of pathophysiological conditions such as inflammation or atherogenesis [corrected].

    Molecular pharmacology 2002;62;4;927-35

  • Inhibition of phosphorylation of BAD and Raf-1 by Akt sensitizes human ovarian cancer cells to paclitaxel.

    Mabuchi S, Ohmichi M, Kimura A, Hisamoto K, Hayakawa J, Nishio Y, Adachi K, Takahashi K, Arimoto-Ishida E, Nakatsuji Y, Tasaka K and Murata Y

    Department of Obstetrics and Gynecology, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.

    We studied the roles of the phosphatidylinositol 3-kinase (PI-3K)-Akt-BAD cascade, ERK-BAD cascade, and Akt-Raf-1 cascade in the paclitaxel-resistant SW626 human ovarian cancer cell line, which lacks functional p53. Treatment of SW626 cells with paclitaxel activates Akt and ERK with different time frames. Interference with the Akt cascade either by treatment with PI-3K inhibitor (wortmannin or LY294002) or by exogenous expression of a dominant negative Akt in SW626 cells caused decreased cell viability following treatment with paclitaxel. Interference with the ERK cascade by treatment with an MEK inhibitor, PD98059, in SW626 cells also caused decreased cell viability following treatment with paclitaxel. Treatment of cells with paclitaxel also stimulated the phosphorylation of BAD at both the Ser-112 and Ser-136 sites. The phosphorylation of BAD at Ser-136 was blocked by treatment with wortmannin or cotransfection with the dominant negative Akt. On the other hand, the phosphorylation of BAD at Ser-112 was blocked by PD98059. We further examined the role of BAD in the viability following paclitaxel treatment using BAD mutants. Exogenous expression of doubly substituted BAD2SA in SW626 cells caused decreased viability following treatment with paclitaxel. Moreover, because paclitaxel-induced apoptosis is mediated by activated Raf-1 and the region surrounding Ser-259 in Raf-1 conforms to a consensus sequence for phosphorylation by Akt, the regulation of Raf-1 by Akt was examined. We demonstrated an association between Akt and Raf-1 and showed that the phosphorylation of Raf-1 on Ser-259 induced by paclitaxel was blocked by treatment with wortmannin or LY294002. Furthermore, interference with the Akt cascade induced by paclitaxel up-regulated Raf-1 activity, and expression of constitutively active Akt inhibited Raf-1 activity, suggesting that Akt negatively regulates Raf-1. Our findings suggest that paclitaxel induces the phosphorylation of BAD Ser-112 via the ERK cascade, and the phosphorylation of both BAD Ser-136 and Raf-1 Ser-259 via the PI-3K-Akt cascade, and that inhibition of either of these cascades sensitizes ovarian cancer cells to paclitaxel.

    The Journal of biological chemistry 2002;277;36;33490-500

  • Untying the regulation of the Raf-1 kinase.

    Dhillon AS and Kolch W

    The Beatson Institute for Cancer Research, Cancer Research UK Beatson Laboratories Garscube Estate, Glasgow, UK.

    The Raf-1 kinase is the entry point to the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK-1/2) signaling pathway, which controls fundamental cellular functions including proliferation, differentiation, and survival. As such, Raf-1 is regulated by complex mechanisms that are incompletely understood. Recent results have shown that release from repression is an important event that facilitates the interaction of Raf-1 with the Ras activator and its substrate, MAPK/ERK-1/2 kinase. A number of distinct activation steps contribute in a combinatorial fashion to regulate and adjust Raf-1 activity. The efficiency of downstream signal transmission is modulated by protein:protein interactions, and new data consolidate an important role for kinase suppressor of ras (KSR) as a scaffolding protein. KSR is a dynamic scaffold whose function and localization is regulated by phosphorylation.

    Archives of biochemistry and biophysics 2002;404;1;3-9

  • Negative regulation of mitochondrial VDAC channels by C-Raf kinase.

    Le Mellay V, Troppmair J, Benz R and Rapp UR

    Institut für Medizinische Strahlenkunde und Zellforschung (MSZ), Universität Würzburg, 97078 Würzburg, Germany. lemellay@univ-montp2.fr

    Background: Growth of cancer cells results from the disturbance of positive and negative growth control mechanisms and the prolonged survival of these genetically altered cells due to the failure of cellular suicide programs. Genetic and biochemical approaches have identified Raf family serine/threonine kinases B-Raf and C-Raf as major mediators of cell survival. C-Raf cooperates with Bcl-2/Bcl-XL in suppression of apoptosis by a mechanism that involves targeting of C-Raf to the outer mitochondrial membrane and inactivation of the pro-apoptotic protein Bad. However, apoptosis suppression by C-Raf also occurs in cells lacking expression of Bad or Bcl-2.

    Results: Here we show that even in the absence of Bcl-2/Bcl-XL, mitochondria-targeted C-Raf inhibits cytochrome c release and caspase activation induced by growth factor withdrawal. To clarify the mechanism of Bcl-2 independent survival control by C-Raf at the mitochondria a search for novel mitochondrial targets was undertaken that identified voltage-dependent anion channel (VDAC), a mitochondrial protein (porin) involved in exchange of metabolites for oxidative phosphorylation. C-Raf forms a complex with VDAC in vivo and blocks reconstitution of VDAC channels in planar bilayer membranes in vitro.

    Conclusion: We propose that this interaction may be responsible for the Raf-induced inhibition of cytochrome c release from mitochondria in growth factor starved cells. Moreover, C-Raf kinase-induced VDAC inhibition may regulate the metabolic function of mitochondria and mediate the switch to aerobic glycolysis that is common to cancer cells.

    BMC cell biology 2002;3;14

  • Targeting Raf-1 gene expression by a DNA enzyme inhibits juvenile myelomonocytic leukemia cell growth.

    Iversen PO, Emanuel PD and Sioud M

    Institute for Nutrition Research, University of Oslo, and Department of Immunology, Molecular Medicine Group, the Norwegian Radium Hospital, Oslo, Norway. poiversen@hotmail.com

    Juvenile myelomonocytic leukemia (JMML) is an aggressive childhood disorder with few therapeutic options. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha) promote JMML cell growth. A hyperactive function of the ras oncogene is a hallmark of JMML. We therefore targeted the protein kinase Raf-1 downstream of Ras using a DNA enzyme that degrades mRNA-Raf-1. Western blots of JMML cell lysates revealed phosphorylated Raf-1 protein, indicating constitutive activation. Addition of GM-CSF, but not TNF-alpha, increased phosphorylation of both Raf-1 and the mitogen-activated protein kinases (MAPKs) JNK-1 and ERK-1. Depletion of Raf-1 protein markedly impaired activation of MAPKs, induced substantial inhibition of JMML cell colony formation, and virtually abolished GM-CSF hypersensitivity in JMML cells. Exogenous TNF-alpha, but not GM-CSF, restored colony formation of JMML cells pretreated with the enzyme. We could not detect any effect of the enzyme on the proliferation of normal bone marrow cells, indicating its specificity and potential safety. When immunodeficient mice engrafted with JMML cells were treated continuously with the enzyme via a peritoneal osmotic mini-pump for 4 weeks, a profound reduction in the JMML cell numbers in the recipient murine bone marrows was found. We conclude that GM-CSF is a chief regulator of JMML growth and exerts its proleukemic effects primarily via the Ras/Raf-1 signaling cascade. TNF-alpha plays a permissive role, being dependent upon GM-CSF to induce JMML cell proliferation. The DNA enzyme efficiently catabolized mRNA-Raf-1 with subsequent inhibition of JMML cell growth, suggesting its potential as a mechanism-based therapy in this fatal leukemia.

    Funded by: NCI NIH HHS: CA80916

    Blood 2002;99;11;4147-53

  • Cyclic AMP blocks cell growth through Raf-1-dependent and Raf-1-independent mechanisms.

    Dumaz N, Light Y and Marais R

    Cancer Research UK Centre for Cell and Molecular Biology, Institute of Cancer Research, London SW3 6JB, United Kingdom.

    It is widely accepted that cyclic AMP (cAMP) can block cell growth by phosphorylating Raf-1 on serine 43 and inhibiting signaling to extracellular signal-regulated protein kinase. We show that the suppression of Raf-1 by cAMP is considerably more complex than previously reported. When cellular cAMP is elevated, Raf-1 is phosphorylated on three residues (S43, S233, and S259), which work independently to block Raf-1. Both Ras-dependent and Ras-independent processes are disrupted. However, when cAMP-insensitive versions of Raf-1 are expressed in NIH 3T3 cells, their growth is still strongly suppressed when cAMP is elevated. Thus, although Raf-1 appears to be an important cAMP target, other pathways are also targeted by cAMP, providing alternative mechanisms that lead to suppression of cell growth.

    Molecular and cellular biology 2002;22;11;3717-28

  • Cyclic AMP-dependent kinase regulates Raf-1 kinase mainly by phosphorylation of serine 259.

    Dhillon AS, Pollock C, Steen H, Shaw PE, Mischak H and Kolch W

    The Beatson Institute for Cancer Research, Bearsden, Glasgow G61 1BD, United Kingdom.

    The Raf-1 kinase activates the ERK (extracellular-signal-regulated kinase) pathway. The cyclic AMP (cAMP)-dependent protein kinase (PKA) can inhibit Raf-1 by direct phosphorylation. We have mapped all cAMP-induced phosphorylation sites in Raf-1, showing that serines 43, 259, and 621 are phosphorylated by PKA in vitro and induced by cAMP in vivo. Serine 43 phosphorylation decreased the binding to Ras in serum-starved but not in mitogen-stimulated cells. However, the kinase activity of a RafS43A mutant was fully inhibited by PKA. Mutation of serine 259 increased the basal Raf-1 activity and rendered it largely resistant to inhibition by PKA. cAMP increased Raf-1 serine 259 phosphorylation in a PKA-dependent manner with kinetics that correlated with ERK deactivation. PKA also decreased Raf-1 serine 338 phosphorylation of Raf-1, previously shown to be required for Raf-1 activation. Serine 338 phosphorylation of a RafS259A mutant was unaffected by PKA. Using RafS259 mutants we also demonstrate that Raf-1 is the sole target for PKA inhibition of ERK and ERK-induced gene expression, and that Raf-1 inhibition is mediated mainly through serine 259 phosphorylation.

    Molecular and cellular biology 2002;22;10;3237-46

  • Raf-induced cell cycle progression in human TF-1 hematopoietic cells.

    Chang F, Steelman LS and McCubrey JA

    Department of Microbiology and Immunology; Brody School of Medicine at East Carolina University; Greenville, North Carolina 27858, USA. mubreyi@mail.ecu.edu

    Ras/Raf/MEK/ERK is a crucial pathway regulating cell cycle progression, apoptosis, and drug resistance. The Ras oncogene is frequently mutated in human cancer, which can result in the activation of the downstream Raf/MEK/ERK cascade leading to cell cycle progression in the absence of a growth stimulus. Raf-induced proliferation has been observed in hematopoietic cells. However, the mechanisms by which Raf affects cell cycle progression are not well described. To investigate the importance of Raf/MEK/ERK signaling in human hematopoietic cell growth, the effects of three different Raf genes, A-Raf, B-Raf and Raf-1, on cell cycle progression and regulatory gene expression were examined in TF-1 cells transformed to grow in response to beta-estradiol-regulated DeltaRaf:ER genes. Raf activation increased the expression of cyclin A, cyclin D, cyclin E, and p21(Cip1), which are associated with G(1) progression. Activated DeltaRaf-1:ER and DeltaA-Raf:ER but not DeltaB-Raf:ER increased Cdk2 and Cdk4 kinase activity. The regulatory role of p16(Ink4a), a potent Cdk4 kinase inhibitor, on the kinase activity of Cdk2 and Cdk4 was also examined. Raf induced p16(Ink4a) suppressor but this did not eliminate Cdk4 kinase activity. These results indicate that human hematopoietic cells transformed to grow in response to activated Raf can be used to elucidate the mechanisms by which various cell cycle regulatory molecules effect cell cycle progression. Furthermore, the differences that the various Raf isoforms have on Cdk4 activity and other cell cycle regulatory molecules can be determined in these cells.

    Funded by: NCI NIH HHS: R01CA51025

    Cell cycle (Georgetown, Tex.) 2002;1;3;220-6

  • Phosphorylation-dependent interaction of kinesin light chain 2 and the 14-3-3 protein.

    Ichimura T, Wakamiya-Tsuruta A, Itagaki C, Taoka M, Hayano T, Natsume T and Isobe T

    Department of Chemistry, Graduate School of Science, Tokyo Metropolitan University, Hachioji-shi, Tokyo 192-0397, Japan. ichimura@mail.comp.metro-u.ac.jp

    The protein 14-3-3 is a key regulator in a cell signaling pathway mediated by protein phosphorylation. To identify the cellular targets of this protein systematically, we have employed a proteomic approach: protein components pulled down from PC12 cells stably expressing a myc-tagged 14-3-3eta isoform were analyzed by means of SDS-PAGE and mass spectrometry. This procedure allowed us to identify more than 30 proteins that include various known and unknown targets of the 14-3-3 protein. Among them are several proteins in the membrane traffic pathway, such as the heavy and light chains (KHC/KIF5B and KLC2) of conventional kinesin, a heterotetrameric mechanochemical motor involved in the ATP-dependent movement of vesicles and organelles along microtubules. Subsequent analysis showed that 14-3-3 directly binds to kinesin heterodimers through interaction with KLC2 and that this interaction is dependent on the phosphorylation of KLC2. Studies on the interaction between 14-3-3 and KLC2 variants expressed in cultured cells coupled with mass spectrometric analysis proved that Ser575 is the site of phosphorylation in KLC2 that is responsible for the in vivo interaction with the 14-3-3 protein. These data add KLC2 to the growing list of 14-3-3 targets, and suggest a role of 14-3-3 in the phosphorylation-regulated cellular transport of vesicles and organelles.

    Biochemistry 2002;41;17;5566-72

  • PAK1 primes MEK1 for phosphorylation by Raf-1 kinase during cross-cascade activation of the ERK pathway.

    Coles LC and Shaw PE

    School of Biomedical Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham NG7 2UH, UK.

    The serine/threonine kinase Raf-1 acts downstream of Ras in the MAPK pathway leading to ERK activation in response to mitogens. Raf-1 has oncogenic potential, but is normally controlled by a complex interplay of inhibitory and activating mechanisms. Although Raf-1 is phosphorylated in unstimulated cells, mitogens cause its membrane recruitment by Ras and subsequent phosphorylation on additional sites. Some of these events modulate Raf-1 kinase activity while others determine interactions with other proteins. These changes regulate the ability of Raf-1 to phosphorylate its downstream targets MEK1 and MEK2. Rho family small G proteins act synergistically with Raf-1 to stimulate the ERK pathway by a cross-cascade mechanism that enhances MEK phosphorylation by Raf-1. Here we show that both Raf-1 and MEK1 are phosphorylated by PAK1 and that mutations at PAK1 phosphorylation sites in either protein prevent cross-cascade activation. In contrast, MEK1 activation by constitutively-active Raf-1 is refractory to mutations at PAK1 phosphorylation sites. Phosphorylation of MEK1 on serine 298 does not appear to regulate the interaction between Raf-1 and MEK1, but rather the ability of Raf-1 to phosphorylate MEK1 with which it is complexed in vivo. Our findings indicate that PAK1 primes MEK1 for activation by Raf-1 and imply another level of regulation in the ERK cascade.

    Oncogene 2002;21;14;2236-44

  • Involvement of phosphatidylinositol 3-kinase, but not RalGDS, in TC21/R-Ras2-mediated transformation.

    Murphy GA, Graham SM, Morita S, Reks SE, Rogers-Graham K, Vojtek A, Kelley GG and Der CJ

    Department of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7295, USA. gmurphy@med.unc.edu

    Oncogenic Ras and activated forms of the Ras-related protein TC21/R-Ras2 share similar abilities to alter cell proliferation. However, in contrast to Ras, we found previously that TC21 fails to activate the Raf-1 serine/threonine kinase. Thus, TC21 must utilize non-Raf effectors to regulate cell function. In this study, we determined that TC21 interacts strongly with some (RalGDS, RGL, RGL2/Rlf, AF6, and the phosphatidylinositol 3-kinase (PI3K) catalytic subunit p110delta), and weakly with other Ras small middle dotGTP-binding proteins. In addition, library screening identified novel TC21-interacting proteins. We also determined that TC21, similar to Ras, mediates activation of phospholipase Cepsilon. We then examined if RalGDS, a RalA guanine nucleotide exchange factor, or PI3K are effectors for TC21-mediated signaling and cell proliferation in murine fibroblasts. We found that overexpression of full-length RalGDS reduced the focus forming activity of activated TC21. Furthermore, expression of activated Ras, but not TC21, enhanced GTP loading on RalA. In fact, TC21 attenuated insulin-stimulated RalA small middle dotGTP formation. In contrast, like Ras, expression of activated TC21 resulted in membrane translocation and an increase in the PI3K-dependent phosphorylation of Akt, and inhibition of PI3K activity interfered with TC21 focus formation. Finally, unlike Ras, TC21 did not activate the Rac small GTPase, indicating that Ras may not activate Rac by PI3K. Taken together, these results suggest that PI3K, but not RalGDS, is an important mediator of cell proliferation by TC21.

    Funded by: NCI NIH HHS: CA42978, CA55008; NIDDK NIH HHS: DK56294

    The Journal of biological chemistry 2002;277;12;9966-75

  • The role of Hsp90N, a new member of the Hsp90 family, in signal transduction and neoplastic transformation.

    Grammatikakis N, Vultur A, Ramana CV, Siganou A, Schweinfest CW, Watson DK and Raptis L

    Department of Microbiology and Immunology, Queen's University, Kingston, Ontario K7L 3N6, Canada.ngrammat@earthlink.com

    The 90-kDa heat shock protein (Hsp90), the target of the ansamycin class of anti-cancer drugs, is required for the conformational activation of a specific group of signal transducers, including Raf-1. In this report we have identified a 75-kDa Raf-associated protein as Hsp90N, a novel member of the Hsp90 family. Intriguingly, the ansamycin-binding domain is replaced in Hsp90N by a much shorter, hydrophobic sequence, preceded by a putative myristylation signal. We demonstrate that, although much less abundant, Hsp90N binds Raf with a higher affinity than Hsp90. In sharp contrast to Hsp90, Hsp90N does not associate with p50(cdc37), the Hsp90 kinase cofactor. Hsp90N was found to activate Raf in transiently transfected cells, while Rat F111 fibroblasts stably transfected with Hsp90N exhibited elevated activity of the Raf and downstream ERK kinases. This may be due to Raf binding to myristylated Hsp90N, followed by Raf translocation to the membrane. To examine whether Hsp90N could therefore substitute for Ras in Raf recruitment to the cell membrane, Hsp90N was transfected in c-Ras-deficient, 10T1/2-derived preadipocytes. Our results indicate that, as shown before for activated Ras or Raf, the introduction of even low levels of Hsp90N through transfection in c-Ras-deficient preadipocytes causes a dramatic block of differentiation. Higher levels of Hsp90N expression resulted in neoplastic transformation, including interruption of gap junctional, intercellular communication, and anchorage-independent proliferation. These results indicate that the observed activation of Raf by Hsp90N has a profound biological effect, which is largely c-Ras-independent. With the recent finding that p50(cdc37) is tumorigenic in transgenic mice, these results reinforce the intriguing observation that the family of heat shock proteins represents a novel class of molecules with oncogenic potential.

    The Journal of biological chemistry 2002;277;10;8312-20

  • Dephosphorylation of Ser-259 regulates Raf-1 membrane association.

    Kubicek M, Pacher M, Abraham D, Podar K, Eulitz M and Baccarini M

    Department of Cell Biology and Microbiology, Institute of Microbiology and Genetics, Vienna Biocenter, Dr. Bohr Gasse 9, 1030 Vienna, Austria.

    Mitogenic stimulation of Raf-1 is a complex yet incompletely understood process involving membrane relocalization and phosphorylation of activating residues. We recently reported that Raf-1-associated protein phosphatase 2A contributes to kinase activation, an effect mediated via Ser-259 of Raf-1. Here, we show that mitogens stimulate Ser-259 dephosphorylation and Raf-1/protein phosphatase 2A association concomitantly with membrane accumulation and activation of Raf-1. Blocking Ser-259 dephosphorylation inhibits the two latter events, but it does not prevent activation of a S259A Raf-1 mutant, which is preferentially localized at the membrane independently of mitogenic stimulation. Inhibition of Ser-259 dephosphorylation has no effect on the activation of membrane-tethered Raf-1 (Raf-1CAAX). These data show that Ser-259 dephosphorylation contributes to Raf-1 activation by supporting its membrane accumulation rather than by increasing the specific activity of the kinase and provide a mechanistic basis for the support of kinase activation by Raf-1-associated protein phosphatase 2A.

    The Journal of biological chemistry 2002;277;10;7913-9

  • Interaction between active Pak1 and Raf-1 is necessary for phosphorylation and activation of Raf-1.

    Zang M, Hayne C and Luo Z

    Diabetes and Metabolism Research Unit, Endocrinology Section, Evans Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118, USA.

    Activation of Raf-1 is a complex process in which phosphorylation of Ser(338)-Tyr(341) is a critical step. Previous studies have shown that Pak1/2 is implicated in both Ras-dependent and -independent activation of Raf-1 by phosphorylating Raf Ser(338). The present study explores the structural basis of Raf-1 phosphorylation by Pak1. We found that Pak directly associates with Raf-1 under both physiological and overexpressed conditions. The association is greatly stimulated by 4beta-12-O-tetradecanoylphorbol-13-acetate and nocodazole and by expression of the active mutants of Rac and Ras. The active forms of Pak generated by mutation of Thr(423) to Glu or truncation of the amino-terminal moiety exhibit a greater binding to Raf than the wild type, whereas the kinase-dead mutant Pak barely binds Raf. The extent of binding to Raf-1 is correlated with the ability of Pak to phosphorylate Raf and induce mitogen-activated protein kinase activation. Furthermore, the Raf-1 binding site is defined to the carboxyl terminus of the Pak catalytic domain. In addition, our results suggest that the amino-terminal regulatory region of Raf inhibits the interaction. Taken together, the results indicate that the interaction depends on the active conformations of Pak and Raf. They also argue that Pak1 is a physiological candidate for phosphorylation of Raf Ser(338) during the course of Raf activation.

    Funded by: NIGMS NIH HHS: GM 57959

    The Journal of biological chemistry 2002;277;6;4395-405

  • The RAS effector RIN1 directly competes with RAF and is regulated by 14-3-3 proteins.

    Wang Y, Waldron RT, Dhaka A, Patel A, Riley MM, Rozengurt E and Colicelli J

    Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, California 90095, USA.

    Activation of RAS proteins can lead to multiple outcomes by virtue of regulated signal traffic through alternate effector pathways. We demonstrate that the RAS effector protein RIN1 binds to activated RAS with an affinity (K(d), 22 nM) similar to that observed for RAF1. At concentrations close to their equilibrium dissociation constant values, RIN1 and RAF1 compete directly for RAS binding. RIN1 was also observed to inhibit cellular transformation by activated mutant RAS. This distinguishes RIN1 from other RAS effectors, which are transformation enhancing. Blockade of transformation was mediated by the RAS binding domain but required membrane localization. RIN1 recognizes endogenous RAS following transient activation by epidermal growth factor, and a portion of RIN1 fractionates to the cell membrane in a manner consistent with a reversible interaction. RIN1 also binds to 14-3-3 proteins through a sequence including serine 351. Mutation of this residue abolished the 14-3-3 binding capacity of RIN1 and led to more efficient blockade of RAS-mediated transformation. The mutant protein, RIN1(S351A), showed a shift in localization to the plasma membrane. Serine 351 is a substrate for protein kinase D (PKD [also known as PKCmu]) in vitro and in vivo. These data suggest that the normal localization and function of RIN1, as well as its ability to compete with RAF, are regulated in part by 14-3-3 binding, which in turn is controlled by PKD phosphorylation.

    Funded by: NCI NIH HHS: CA 56301, R01 CA056301; NIDDK NIH HHS: DK 55003, KO1 DK 02834, R01 DK055003

    Molecular and cellular biology 2002;22;3;916-26

  • Decreased expression of hippocampal cholinergic neurostimulating peptide precursor protein mRNA in the hippocampus in Alzheimer disease.

    Maki M, Matsukawa N, Yuasa H, Otsuka Y, Yamamoto T, Akatsu H, Okamoto T, Ueda R and Ojika K

    Second Department of Internal Medicine, Medical School, Nagoya City University, Japan.

    Hippocampal cholinergic neurostimulating peptide (HCNP) is involved in the phenotype development of the septo-hippocampal system. HCNP precursor protein (HCNP-pp) is known to interact with other molecules including phosphatidylethanolamine and Raf-1 kinase, and is also known as phosphatidylethanolamine-binding protein and raf kinase-inhibitory protein. To assess whether HCNP-pp is involved in the pathogenesis of Alzheimer disease (AD), the expression levels of its mRNA in the hippocampus of autopsy brains from patients with dementia (including AD and ischemic vascular dementia) were compared with those of non-demented control subjects. The in situ hybridization analysis revealed that the expression of HCNP-pp mRNA in patients with clinically late-onset AD was decreased in the hippocampal CA1 field, but not in the CA3 field or the dentate gyrus. The early-onset AD patients showed a wide range of expression levels in the hippocampal sub-regions. Northern blot analysis of HCNP-pp mRNA in brain tissue supported these observations. Since HCNP is known to stimulate the enzymatic activity of choline acetyltransferase in neurons, its low expression in the CAI field of AD patients may explain the downregulation of cholinergic neurons seen in these patients and may thus contribute to the pathogenic processes underlying AD.

    Journal of neuropathology and experimental neurology 2002;61;2;176-85

  • Identification of a C-terminal region that regulates mitogen-activated protein kinase kinase-1 cytoplasmic localization and ERK activation.

    Cha H, Lee EK and Shapiro P

    Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201, USA.

    The C-terminal region of mitogen-activated protein kinase kinase-1 and 2 (MKK1 and MKK2) may function in regulating interactions with upstream kinases or the magnitude and duration of ERK mitogen-activated protein kinase activity. The MKK C-terminal region contains a proline-rich region that reportedly functions in regulating interactions with the Raf-1 kinase and ERK activity. In addition, phosphorylation sites in the C terminus of MKK1 have been suggested to either sustain or attenuate MKK1 activity. To further understand how phosphorylation at the C terminus of MKK1 and protein interactions regulate MKK1 function, we have generated several MKK1 C-terminal deletion mutants and examined their function in regulating MKK1 localization, ERK protein activation, and cell growth. A deletion of C-terminal amino acids encompassing two putative alpha-helices between residues 330 and 379 caused a re-distribution of mutant MKK1 proteins to membrane compartments. Immunofluorescence analysis of MKK1 mutants revealed a loss of homogenous cytosolic distribution that is typically observed with MKK1 wild type, suggesting this region regulates MKK1 cellular localization. In contrast, MKK1 C-terminal deletion mutants localized to various sized punctate regions that overlapped with lysosome compartments. ERK activation in response to constitutively active Raf-1 or growth factor stimulus was attenuated in cells expressing MKK1 C-terminal deletion mutants. This could be partly explained by the inability of Raf-1 to phosphorylate MKK1 C-terminal deletion mutants even though the phosphorylation sites were intact in these mutants. Finally, we show that cells expressing MKK1 C-terminal deletion mutants displayed characteristic patterns of apoptotic cell death and reduced cell proliferation. These findings identify a novel C-terminal region between amino acid residues 330 and 379 on MKK1 that is necessary for regulating the cytoplasmic distribution and subsequent ERK protein activation necessary for cell survival and viability.

    The Journal of biological chemistry 2001;276;51;48494-501

  • Downregulation of the Ras-mitogen-activated protein kinase pathway by the EphB2 receptor tyrosine kinase is required for ephrin-induced neurite retraction.

    Elowe S, Holland SJ, Kulkarni S and Pawson T

    Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada.

    Activation of the EphB2 receptor tyrosine kinase by clustered ephrin-B1 induces growth cone collapse and neurite retraction in differentiated NG108 neuronal cells. We have investigated the cytoplasmic signaling events associated with EphB2-induced cytoskeletal reorganization in these neuronal cells. We find that unlike other receptor tyrosine kinases, EphB2 induces a pronounced downregulation of GTP-bound Ras and consequently of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. A similar inhibition of the Ras-MAPK pathway was observed on stimulation of endogenous EphB2 in COS-1 cells. Inactivation of Ras, induced by ephrin B1 stimulation of NG108 neuronal cells, requires EphB2 tyrosine kinase activity and is blocked by a truncated form of p120-Ras GTPase-activating protein (p120-RasGAP), suggesting that EphB2 signals through the SH2 domain protein p120-RasGAP to inhibit the Ras-MAPK pathway. Suppression of Ras activity appears functionally important, since expression of a constitutively active variant of Ras impaired the ability of EphB2 to induce neurite retraction. In addition, EphB2 attenuated the elevation in ERK activation induced by attachment of NG108 cells to fibronectin, indicating that the EphB2 receptor can modulate integrin signaling to the Ras GTPase. These results suggest that a primary function of EphB2, a member of the most populous family of receptor tyrosine kinases, is to inactivate the Ras-MAPK pathway in a fashion that contributes to cytoskeletal reorganization and adhesion responses in neuronal growth cones.

    Molecular and cellular biology 2001;21;21;7429-41

  • Cooperation of a ubiquitin domain protein and an E3 ubiquitin ligase during chaperone/proteasome coupling.

    Demand J, Alberti S, Patterson C and Höhfeld J

    Abteilung für Molekulare Zellbiologie, Max-Planck-Institut für Biochemie, D-82152, Martinsried, Germany.

    Background: Molecular chaperones recognize nonnative proteins and orchestrate cellular folding processes in conjunction with regulatory cofactors. However, not every attempt to fold a protein is successful, and misfolded proteins can be directed to the cellular degradation machinery for destruction. Molecular mechanisms underlying the cooperation of molecular chaperones with the degradation machinery remain largely enigmatic so far.

    Results: By characterizing the chaperone cofactors BAG-1 and CHIP, we gained insight into the cooperation of the molecular chaperones Hsc70 and Hsp70 with the ubiquitin/proteasome system, a major system for protein degradation in eukaryotic cells. The cofactor CHIP acts as a ubiquitin ligase in the ubiquitination of chaperone substrates such as the raf-1 protein kinase and the glucocorticoid hormone receptor. During targeting of signaling molecules to the proteasome, CHIP may cooperate with BAG-1, a ubiquitin domain protein previously shown to act as a coupling factor between Hsc/Hsp70 and the proteasome. BAG-1 directly interacts with CHIP; it accepts substrates from Hsc/Hsp70 and presents associated proteins to the CHIP ubiquitin conjugation machinery. Consequently, BAG-1 promotes CHIP-induced degradation of the glucocorticoid hormone receptor in vivo.

    Conclusions: The ubiquitin domain protein BAG-1 and the CHIP ubiquitin ligase can cooperate to shift the activity of the Hsc/Hsp70 chaperone system from protein folding to degradation. The chaperone cofactors thus act as key regulators to influence protein quality control.

    Current biology : CB 2001;11;20;1569-77

  • Positive and negative regulation of Raf kinase activity and function by phosphorylation.

    Chong H, Lee J and Guan KL

    Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109-0606, USA.

    Activating and inhibitory phosphorylation mechanisms play an essential role in regulating Raf kinase activity. Here we demonstrate that phosphorylation of C-Raf in the kinase activation loop (residues T491 and S494) is necessary, but not sufficient, for activation. C-Raf has additional activating phosphorylation sites at S338 and Y341. Mutating all four of these residues to acidic residues, S338D/Y341D/T491E/S494D (DDED), in C-Raf results in constitutive activity. However, acidic residue substitutions at the corresponding activation loop sites in B-Raf are sufficient to confer constitutive activity. B-Raf and C-Raf also utilize similar inhibitory phosphorylation mechanisms to regulate kinase activity. B-Raf has multiple inhibitory phosphorylation sites necessary for full kinase inhibition where C-Raf requires only one. We examined the functional significance of these inhibitory and activating phosphorylations in Caenorhabditis elegans lin-45 Raf. Eliminating the inhibitory phosphorylation or mimicking activating phosphorylation sites is sufficient to confer constitutive activity upon lin-45 Raf and induce multi-vulva phenotypes in C.elegans. Our results demonstrate that different members of the Raf family kinases have both common and distinct phosphorylation mechanisms to regulate kinase activity and biological function.

    Funded by: NIGMS NIH HHS: 5-T32-GM07544

    The EMBO journal 2001;20;14;3716-27

  • Raf-1 promotes cell survival by antagonizing apoptosis signal-regulating kinase 1 through a MEK-ERK independent mechanism.

    Chen J, Fujii K, Zhang L, Roberts T and Fu H

    Department of Pharmacology, Emory University School of Medicine, Atlanta, GA 30322, USA.

    The Ser/Thr kinase Raf-1 is a protooncogene product that is a central component in many signaling pathways involved in normal cell growth and oncogenic transformation. Upon activation, Raf-1 phosphorylates mitogen-activated protein kinase kinase (MEK), which in turn activates mitogen-activated protein kinase/extracellular signal-regulated kinases (MAPK/ERKs), leading to the propagation of signals. Depending on specific stimuli and cellular environment, the Raf-1--MEK--ERK cascade regulates diverse cellular processes such as proliferation, differentiation, and apoptosis. Here, we describe a MEK--ERK-independent prosurvival function of Raf-1. We found that Raf-1 interacts with the proapoptotic, stress-activated protein kinase ASK1 (apoptosis signal-regulating kinase 1) in vitro and in vivo. Deletion analysis localized the Raf-1 binding site to the N-terminal regulatory fragment of ASK1. This interaction allows Raf-1 to act independently of the MEK--ERK pathway to inhibit apoptosis. Furthermore, catalytically inactive forms of Raf-1 can mimic the wild-type effect, raising the possibility of a kinase-independent function of Raf-1. Thus, Raf-1 may promote cell survival through its protein-protein interactions in addition to its established MEK kinase function.

    Funded by: NIGMS NIH HHS: GM53165, GM60033

    Proceedings of the National Academy of Sciences of the United States of America 2001;98;14;7783-8

  • Structure--function relationships in HIV-1 Nef.

    Geyer M, Fackler OT and Peterlin BM

    Howard Hughes Medical Institute, University of California, San Francisco 94143-0703, CA, USA. mgeyer@cc.ucsf.edu

    The accessory Nef protein of HIV and SIV is essential for viral pathogenesis, yet it is perplexing in its multitude of molecular functions. In this review we analyse the structure-function relationships of motifs recently proposed to play roles in aspects of Nef modification, signalling and trafficking, and thereby to impinge on the ability of the virus to survive in, and to manipulate, its cellular host. Based on the full-length structure assembly of HIV Nef, we correlate surface accessibility with secondary structure elements and sequence conservation. Motifs involved in Nef-mediated CD4 and MHC I downregulation are located in flexible regions of Nef, suggesting that the formation of the transient trafficking complexes involved in these processes depends on the recognition of primary sequences. In contrast, the interaction sites for signalling molecules that contain SH3 domains or the p21-activated kinases are associated with the well folded core domain, suggesting the recognition of highly structured protein surfaces.

    EMBO reports 2001;2;7;580-5

  • Cloning and characterization of MST4, a novel Ste20-like kinase.

    Qian Z, Lin C, Espinosa R, LeBeau M and Rosner MR

    Ben May Institute for Cancer Research and Department of Medicine, University of Chicago, Chicago, Illinois 60637, USA.

    MST4, a novel member of the germinal center kinase subfamily of human Ste20-like kinases, was cloned and characterized. Composed of a C-terminal regulatory domain and an N-terminal kinase domain, MST4 is most closely related to mammalian Ste20 kinase family member MST3. Both the kinase and C-terminal regulatory domains of MST4 are required for full activation of the kinase. Northern blot analysis indicates that MST4 is ubiquitously distributed, and the MST4 gene is localized to chromosome Xq26, a disease-rich region, by fluorescence in situ hybridization. Although some members of the MST4 family function as upstream regulators of mitogen-activated protein kinase cascades, expression of MST4 in 293 cells was not sufficient to activate or potentiate extracellular signal-regulated kinase, c-Jun N-terminal kinase, or p38 kinase. An alternatively spliced isoform of MST4 (MST4a) was isolated by yeast two-hybrid interaction with the catalytic domain of Raf from a human fetal brain cDNA library and also found in a variety of human fetal and adult tissues. MST4a lacks an exon encoding kinase subdomains IX-XI that stabilizes substrate binding. The existence of both MST4 isoforms suggests that the MST4 kinase activity is highly regulated, and MST4a may function as a dominant-negative regulator of the MST4 kinase.

    Funded by: NCI NIH HHS: CA40046; NIGMS NIH HHS: T32 GM07151; NINDS NIH HHS: NS 33858

    The Journal of biological chemistry 2001;276;25;22439-45

  • Inhibitory actions of ceramide upon PKC-epsilon/ERK interactions.

    Bourbon NA, Yun J, Berkey D, Wang Y and Kester M

    Department of Pharmacology, College of Medicine, Pennsylvania State University, Hershey, PA 17033, USA.

    We have previously shown that interleukin-1 receptor-generated ceramide induces growth arrest in smooth muscle pericytes by inhibiting an upstream kinase in the extracellular signal-regulated kinase (ERK) cascade. Here, we now report the mechanism by which ceramide inhibits ERK activity. Ceramide renders the human embryonic kidney 293 cells (HEK 293) resistant to the mitogenic actions of growth factors and activators of protein kinase C (PKC). A role for PKC to mediate ceramide inhibition of growth factor-induced ERK activity and mitogenesis is suggested, as exogenous ceramide directly inhibits both immunoprecipitated and recombinant PKC-epsilon activities. To confirm that PKC-epsilon is necessary for ceramide-inhibited ERK activity, HEK 293 cells were transfected with a dominant-negative mutant of PKC-epsilon (DeltaPKC-epsilon). These transfected cells respond to insulin-like growth factor I (IGF-I) with a significantly decreased ERK activity that is not further reduced by ceramide treatment. Coimmunoprecipitation studies reveal that the treatment with IGF-I induces the association of ERK with PKC-epsilon but not with PKC-zeta. Ceramide treatment significantly inhibits the IGF-I-induced PKC-epsilon interaction with bioactive phosphorylated ERK. Ceramide also inhibits IGF-I-induced PKC-epsilon association with Raf-1, an upstream kinase of ERK. Together, these studies demonstrate that ceramide exerts anti-mitogenic actions by limiting the ability of PKC-epsilon to form a signaling complex with Raf-1 and ERK.

    Funded by: NIDDK NIH HHS: DK-53715

    American journal of physiology. Cell physiology 2001;280;6;C1403-11

  • Active Ras induces heterodimerization of cRaf and BRaf.

    Weber CK, Slupsky JR, Kalmes HA and Rapp UR

    Abteilung für Naturheilkunde und Klinische Pharmakologie and Abteilung Innere Medizin I, Universität Ulm, D-89081 Ulm, Germany.

    Growth factor-induced signalling leads to activation of members of the Ras family and subsequent stimulation of different Raf isoforms. Within the mechanism of Raf activation, two isoforms of Raf, cRaf and BRaf, may cooperate. We investigated the relationship between cRaf and BRaf and found that active Ras induced heterodimerization of cRaf and BRaf, an effect that was dependent on the serine residue at position 621 of cRAF: Moreover, we also found that cRaf COOH-terminus constitutively associated with BRaf, whereas the NH(2) terminus did not, even in the presence of active RAS: These data suggest that Ras induces the cRaf-BRaf complex formation through the exposure of 14-3-3 binding sites in the COOH-terminus of cRAF: Thus, Ras-induced cRaf-Braf heterodimerization may explain the observed cooperativity of cRaf and BRaf in cells responding to growth factor signals.

    Cancer research 2001;61;9;3595-8

  • Kinase suppressor of Ras signals through Thr269 of c-Raf-1.

    Xing HR and Kolesnick R

    Laboratory of Signal Transduction, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.

    We recently established a two-stage in vitro assay for KSR kinase activity in which KSR never comes in contact with any recombinant kinase other than c-Raf-1 and defined the epidermal growth factor (EGF) as a potent activator of KSR kinase activity (Xing, H. R., Lozano, J., and Kolesnick, R. (2000) J. Biol. Chem. 275, 17276-17280). That study, however, did not address the mechanism of c-Raf-1 stimulation by activated KSR. Here we show that phosphorylation of c-Raf-1 on Thr(269) by KSR is necessary for optimal activation in response to EGF stimulation. In vitro, KSR specifically phosphorylated c-Raf-1 on threonine residues during the first stage of the two-stage kinase assay. Using purified wild-type and mutant c-Raf-1 proteins, we demonstrate that Thr(269) is the major c-Raf-1 site phosphorylated by KSR in vitro and that phosphorylation of this site is essential for c-Raf-1 activation by KSR. KSR acts via transphosphorylation, not by increasing c-Raf-1 autophosphorylation, as kinase-inactive c-Raf-1(K375M) served as an equally effective KSR substrate. In vivo, low physiologic doses of EGF (0.001-0.1 ng/ml) stimulated KSR activation and induced Thr(269) phosphorylation and activation of c-Raf-1. Low dose EGF did not induce serine or tyrosine phosphorylation of c-Raf-1. High dose EGF (10-100 ng/ml) induced no additional Thr(269) phosphorylation, but rather increased c-Raf-1 phosphorylation on serine residues and Tyr(340)/Tyr(341). A Raf-1 mutant with valine substituted for Thr(269) was unresponsive to low dose EGF, but was serine- and Tyr(340)/Tyr(341)-phosphorylated and partially activated at high dose EGF. This study shows that Thr(269) is the major c-Raf-1 site phosphorylated by KSR. Furthermore, phosphorylation of this site is essential for c-Raf-1 activation by KSR in vitro and for optimal c-Raf-1 activation in response to physiologic EGF stimulation in vivo.

    Funded by: NCI NIH HHS: CA42385

    The Journal of biological chemistry 2001;276;13;9733-41

  • Activation and targeting of extracellular signal-regulated kinases by beta-arrestin scaffolds.

    Luttrell LM, Roudabush FL, Choy EW, Miller WE, Field ME, Pierce KL and Lefkowitz RJ

    The Geriatrics Research, Education and Clinical Center, Durham Veterans Affairs Medical Center, Durham, NC 27705, USA. luttrell@receptor-biol.duke.edu

    Using both confocal immunofluorescence microscopy and biochemical approaches, we have examined the role of beta-arrestins in the activation and targeting of extracellular signal-regulated kinase 2 (ERK2) following stimulation of angiotensin II type 1a receptors (AT1aR). In HEK-293 cells expressing hemagglutinin-tagged AT1aR, angiotensin stimulation triggered beta-arrestin-2 binding to the receptor and internalization of AT1aR-beta-arrestin complexes. Using red fluorescent protein-tagged ERK2 to track the subcellular distribution of ERK2, we found that angiotensin treatment caused the redistribution of activated ERK2 into endosomal vesicles that also contained AT1aR-beta-arrestin complexes. This targeting of ERK2 reflects the formation of multiprotein complexes containing AT1aR, beta-arrestin-2, and the component kinases of the ERK cascade, cRaf-1, MEK1, and ERK2. Myc-tagged cRaf-1, MEK1, and green fluorescent protein-tagged ERK2 coprecipitated with Flag-tagged beta-arrestin-2 from transfected COS-7 cells. Coprecipitation of cRaf-1 with beta-arrestin-2 was independent of MEK1 and ERK2, whereas the coprecipitation of MEK1 and ERK2 with beta-arrestin-2 was significantly enhanced in the presence of overexpressed cRaf-1, suggesting that binding of cRaf-1 to beta-arrestin facilitates the assembly of a cRaf-1, MEK1, ERK2 complex. The phosphorylation of ERK2 in beta-arrestin complexes was markedly enhanced by coexpression of cRaf-1, and this effect is blocked by expression of a catalytically inactive dominant inhibitory mutant of MEK1. Stimulation with angiotensin increased the binding of both cRaf-1 and ERK2 to beta-arrestin-2, and the association of beta-arrestin-2, cRaf-1, and ERK2 with AT1aR. These data suggest that beta-arrestins function both as scaffolds to enhance cRaf-1 and MEK-dependent activation of ERK2, and as targeting proteins that direct activated ERK to specific subcellular locations.

    Funded by: NHLBI NIH HHS: HL16037, R01 HL016037; NIDDK NIH HHS: DK55524, R01 DK055524, R56 DK055524

    Proceedings of the National Academy of Sciences of the United States of America 2001;98;5;2449-54

  • Vascular endothelial growth factor-induced prostacyclin production is mediated by a protein kinase C (PKC)-dependent activation of extracellular signal-regulated protein kinases 1 and 2 involving PKC-delta and by mobilization of intracellular Ca2+.

    Gliki G, Abu-Ghazaleh R, Jezequel S, Wheeler-Jones C and Zachary I

    Department of Medicine, University College London, 5 University Street, London WC1E 6JJ, UK.

    We reported previously that vascular endothelial growth factor (VEGF) stimulates prostacyclin (PGI(2)) production via activation of the extracellular signal-regulated kinase (ERK) cascade. In this paper, we examined the role of protein kinase C (PKC) in this pathway. VEGF-induced PGI(2) generation and arachidonic acid release in human umbilical vein endothelial cells were inhibited by the PKC inhibitors GF109203X and calphostin C. VEGF increased PKC activity and immunoreactivity of the PKCdelta, alpha and epsilon isoforms in particulate fractions of cells. PKC inhibitors blocked VEGF-induced activation of ERK, MEK (mitogen-activated protein kinase kinase) and the cytosolic phospholipase A(2), but had little effect on ERK activation induced by basic fibroblast growth factor. GF109203X, calphostin C and the PKCdelta-selective inhibitor, rottlerin, did not inhibit activation of the KDR receptor for VEGF. Inhibition of Ca(2+) fluxes using BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)] blocked VEGF-induced PGI(2) production but did not inhibit ERK activation. Neither activation nor inhibition of the NO/cGMP pathway had any effect on VEGF induction of ERK activity and PGI(2) synthesis. Wortmannin partially inhibited VEGF stimulation of PGI(2) production, but did not inhibit VEGF-induced ERK activity. VEGF-induced ERK activation and PGI(2) production were blocked by rottlerin, and VEGF increased association of PKCdelta with Raf-1, the upstream activator of MEK. The PKC-selective inhibitor Go6976 did not inhibit ERK activation and had only a partial effect on PGI(2) production. These findings indicate that activation of PKC plays a crucial role in VEGF signalling via the ERK cascade leading to PGI(2) synthesis and suggest that the PKCdelta isoform may be a key mediator of VEGF-induced activation of the ERK pathway via increased association with Raf-1.

    The Biochemical journal 2001;353;Pt 3;503-12

  • Nerve growth factor signaling, neuroprotection, and neural repair.

    Sofroniew MV, Howe CL and Mobley WC

    Department of Neurobiology and Brain Research Institute, University of California Los Angeles, Los Angeles, California 90095-1763, USA. sofroniew@mednet.ucla.edu

    Nerve growth factor (NGF) was discovered 50 years ago as a molecule that promoted the survival and differentiation of sensory and sympathetic neurons. Its roles in neural development have been characterized extensively, but recent findings point to an unexpected diversity of NGF actions and indicate that developmental effects are only one aspect of the biology of NGF. This article considers expanded roles for NGF that are associated with the dynamically regulated production of NGF and its receptors that begins in development, extends throughout adult life and aging, and involves a surprising variety of neurons, glia, and nonneural cells. Particular attention is given to a growing body of evidence that suggests that among other roles, endogenous NGF signaling subserves neuroprotective and repair functions. The analysis points to many interesting unanswered questions and to the potential for continuing research on NGF to substantially enhance our understanding of the mechanisms and treatment of neurological disorders.

    Funded by: NINDS NIH HHS: NS24054

    Annual review of neuroscience 2001;24;1217-81

  • A scaffold protein in the c-Jun NH2-terminal kinase signaling pathways suppresses the extracellular signal-regulated kinase signaling pathways.

    Kuboki Y, Ito M, Takamatsu N, Yamamoto KI, Shiba T and Yoshioka K

    Department of Biosciences, School of Science, Kitasato University, Kanagawa 228-8555, Japan.

    We previously reported that c-Jun NH(2)-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1) functions as a putative scaffold factor in the JNK mitogen-activated protein kinase (MAPK) cascades. In that study we also found MEK1 and Raf-1, which are involved in the extracellular signal-regulated kinase (ERK) MAPK cascades, bind to JSAP1. Here we have defined the regions of JSAP1 responsible for the interactions with MEK1 and Raf-1. Both of the binding regions were mapped to the COOH-terminal region (residues 1054-1305) of JSAP1. We next examined the effect of overexpressing JSAP1 on the activation of ERK by phorbol 12-myristate 13-acetate in transfected COS-7 cells and found that JSAP1 inhibits ERK's activation and that the COOH-terminal region of JSAP1 was required for the inhibition. Finally, we investigated the molecular mechanism of JSAP1's inhibitory function and showed that JSAP1 prevents MEK1 phosphorylation and activation by Raf-1, resulting in the suppression of the activation of ERK. Taken together, these results suggest that JSAP1 is involved both in the JNK cascades, as a scaffolding factor, and the ERK cascades, as a suppressor.

    The Journal of biological chemistry 2000;275;51;39815-8

  • MEKK1 binds raf-1 and the ERK2 cascade components.

    Karandikar M, Xu S and Cobb MH

    Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9041, USA.

    Mitogen-activated protein (MAP) kinase cascades are involved in transmitting signals that are generated at the cell surface into the cytosol and nucleus and consist of three sequentially acting enzymes: a MAP kinase, an upstream MAP/extracellular signal-regulated protein kinase (ERK) kinase (MEK), and a MEK kinase (MEKK). Protein-protein interactions within these cascades provide a mechanism to control the localization and function of the proteins. MEKK1 is implicated in activation of the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and ERK1/2 MAP kinase pathways. We showed previously that MEKK1 binds directly to JNK/SAPK. In this study we demonstrate that endogenous MEKK1 binds to endogenous ERK2, MEK1, and another MEKK level kinase, Raf-1, suggesting that it can assemble all three proteins of the ERK2 MAP kinase module.

    Funded by: NIDDK NIH HHS: DK34128; NIGMS NIH HHS: GM56498

    The Journal of biological chemistry 2000;275;51;40120-7

  • Glucocorticoid receptor interaction with 14-3-3 and Raf-1, a proposed mechanism for cross-talk of two signal transduction pathways.

    Widén C, Zilliacus J, Gustafsson JA and Wikström AC

    Department of Medical Nutrition, Karolinska Institutet, NOVUM, S-141 86 Huddinge, Sweden.

    The glucocorticoid receptor (GR) functions as a ligand-dependent transcription factor. In the present study we describe a specific immunoaffinity chromatography purification of GR from liver cytosol from adrenalectomized rats that may be used to identify hitherto unknown cytosolic GR interacting proteins. We have identified the ubiquitously expressed 14-3-3 as well as Raf-1, a downstream effector of Ras, as GR co-purifying proteins. In our semi-quantitative analysis liganded/activated GR showed the strongest interaction with 14-3-3 and Raf-1, but 14-3-3 was also found to co-purify with GR in a nonliganded/nonactivated state. By extensive salt washes we were also able to demonstrate that the glucocorticoid induced interaction between GR, 14-3-3, and Raf-1, respectively, is remarkably stable and withstood 2.4 m salt. The interaction between GR and 14-3-3 was also verified by 14-3-3 co-immunoprecipitation studies. Our observations that GR and Raf-1 are found within the same protein complex ("receptosome") in the cytoplasm of rat liver cells could provide a mechanistic explanation for glucocorticoid effects on the Raf-1-Ras signaling pathway.

    The Journal of biological chemistry 2000;275;50;39296-301

  • Complexes formation between insulin receptor and extracellular signal-regulated kinases ERKs.

    Lin YL, Mettling C and Chou CK

    Institut de Génétique Humaine, Centre National de la Recherche Scientifique, 141 rue de la Cardonille, Montpellier Cedex 5, 34396, France

    A property of signal transduction pathways that might explain their efficiency and specificity is the formation of signaling complexes. The recent demonstration that adaptor proteins can interact with many components of the extracellular signal-regulated kinases (ERKs) signaling cascade leads us to investigate whether such complexes may include the transmembrane receptor. The present work shows that in human hepatoma Hep3B cells, insulin receptor (IR) can be coimmunoprecipitated with other components of the ERKs cascade: insulin receptor substrate (IRS), Raf-1, and ERKs. Furthermore, these complexes formed near the cytoplasmic membrane even prior to insulin stimulation.

    Molecular cell biology research communications : MCBRC 2000;4;4;234-8

  • Calyculin A-induced vimentin phosphorylation sequesters 14-3-3 and displaces other 14-3-3 partners in vivo.

    Tzivion G, Luo ZJ and Avruch J

    Diabetes Unit, the Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.

    14-3-3 proteins bind their targets through a specific serine/threonine-phosphorylated motif present on the target protein. This binding is a crucial step in the phosphorylation-dependent regulation of various key proteins involved in signal transduction and cell cycle control. We report that treatment of COS-7 cells with the phosphatase inhibitor calyculin A induces association of 14-3-3 with a 55-kDa protein, identified as the intermediate filament protein vimentin. Association of vimentin with 14-3-3 depends on vimentin phosphorylation and requires the phosphopeptide-binding domain of 14-3-3. The region necessary for binding to 14-3-3 is confined to the vimentin amino-terminal head domain (amino acids 1-96). Monomeric forms of 14-3-3 do not bind vimentin in vivo or in vitro, indicating that a stable complex requires the binding of a 14-3-3 dimer to two sites on a single vimentin polypeptide. The calyculin A-induced association of vimentin with 14-3-3 in vivo results in the displacement of most other 14-3-3 partners, including the protooncogene Raf, which nevertheless remain capable of binding 14-3-3 in vitro. Concomitant with 14-3-3 displacement, calyculin A treatment blocks Raf activation by EGF; however, this inhibition is completely overcome by 14-3-3 overexpression in vivo or by the addition of prokaryotic recombinant 14-3-3 in vitro. Thus, phosphovimentin, by sequestering 14-3-3 and limiting its availability to other target proteins can affect intracellular signaling processes that require 14-3-3.

    The Journal of biological chemistry 2000;275;38;29772-8

  • Stimulation of Ras guanine nucleotide exchange activity of Ras-GRF1/CDC25(Mm) upon tyrosine phosphorylation by the Cdc42-regulated kinase ACK1.

    Kiyono M, Kato J, Kataoka T, Kaziro Y and Satoh T

    Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan.

    Ras-GRF1 is a brain-specific guanine nucleotide exchange factor (GEF) for Ras, whose activity is regulated in response to Ca(2+) influx and G protein-coupled receptor signals. In addition, Ras-GRF1 acts as a GEF for Rac when tyrosine-phosphorylated following G protein-coupled receptor stimulation. However, the mechanisms underlying the regulation of Ras-GRF1 functions remain incompletely understood. We show here that activated ACK1, a nonreceptor tyrosine kinase that belongs to the focal adhesion kinase family, causes tyrosine phosphorylation of Ras-GRF1. On the other hand, kinase-deficient ACK1 exerted no effect. GEF activity of Ras-GRF1 toward Ha-Ras, as defined by in vitro GDP binding and release assays, was augmented after tyrosine phosphorylation by ACK1. In contrast, GEF activity toward Rac1 remained latent, implying that ACK1 does not represent a tyrosine kinase that acts downstream of G protein-coupled receptors. Consistent with enhanced Ras-GEF activity, accumulation of the GTP-bound form of Ras within the cell was shown through the use of Ras-binding domain pull-down assays. Furthermore, Ras-dependent activation of ERK2 by Ras-GRF1 was enhanced following co-expression of activated ACK1. These results implicate ACK1 as an upstream modulator of Ras-GRF1 and suggest a signaling cascade consisting of Cdc42, ACK1, Ras-GRF1, and Ras in neuronal cells.

    The Journal of biological chemistry 2000;275;38;29788-93

  • The junctional multidomain protein AF-6 is a binding partner of the Rap1A GTPase and associates with the actin cytoskeletal regulator profilin.

    Boettner B, Govek EE, Cross J and Van Aelst L

    Cold Spring Harbor Laboratories, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA.

    The AF-6 protein is a multidomain protein that contains two potential Ras-binding domains within its N terminus. Because of this feature, AF-6 has been isolated in both two-hybrid and biochemical approaches and is postulated to be a potential Ras-effector protein. Herein, we show that it is specifically the first Ras-binding domain of AF-6 that mediates this interaction and that the Ras-related Rap1A protein can associate with this motif even more efficiently than the oncogenic Ha-, K-, and N-Ras GTPases. We further demonstrate that both Ras and Rap1 interact with full-length AF-6 in vivo in mammalian cells and that a fraction of Rap1 colocalizes with AF-6 at the membrane. Dominant active Rap1A, in contrast to Ras, when introduced into epithelial MDCK and MCF-7 cells, does not perturb AF-6-specific residency in cell-cell adhesion complexes. In a pursuit to gain further understanding of the role of AF-6 in junctions, we identified profilin as an AF-6-binding protein. Profilin activates monomeric actin units for subsequent polymerization steps at barbed ends of actin filaments and has been shown to participate in cortical actin assembly. To our knowledge, AF-6 is the only integral component in cell-cell junctions discovered thus far that interacts with profilin and thus could modulate actin modeling proximal to adhesion complexes.

    Proceedings of the National Academy of Sciences of the United States of America 2000;97;16;9064-9

  • Isoform-specific localization of A-RAF in mitochondria.

    Yuryev A, Ono M, Goff SA, Macaluso F and Wennogle LP

    Novartis Institute for Biomolecular Research, Summit, NJ 07901, USA.

    RAF kinase is a family of isoforms including A-RAF, B-RAF, and C-RAF. Despite the important role of RAF in cell growth and proliferation, little evidence exists for isoform-specific function of RAF family members. Using Western analysis and immunogold labeling, A-RAF was selectively localized in highly purified rat liver mitochondria. Two novel human proteins, which interact specifically with A-RAF, were identified, and the full-length sequences are reported. These proteins, referred to as hTOM and hTIM, are similar to components of mitochondrial outer and inner membrane protein-import receptors from lower organisms, implicating their involvement in the mitochondrial transport of A-RAF. hTOM contains multiple tetratricopeptide repeat (TPR) domains, which function in protein-protein interactions. TPR domains are frequently present in proteins involved in cellular transport systems. In contrast, protein 14-3-3, an abundant cytosolic protein that participates in many facets of signal transduction, was found to interact with C-RAF but not with A-RAF N-terminal domain. This information is discussed in view of the important role of mitochondria in cellular functions involving energy balance, proliferation, and apoptosis and the potential role of A-RAF in regulating these systems.

    Molecular and cellular biology 2000;20;13;4870-8

  • Rac-1 and Raf-1 kinases, components of distinct signaling pathways, activate myotonic dystrophy protein kinase.

    Shimizu M, Wang W, Walch ET, Dunne PW and Epstein HF

    Department of Neurology, NS B302, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.

    Myotonic dystrophy protein kinase (DMPK) is a serine-threonine protein kinase encoded by the myotonic dystrophy (DM) locus on human chromosome 19q13.3. It is a close relative of other kinases that interact with members of the Rho family of small GTPases. We show here that the actin cytoskeleton-linked GTPase Rac-1 binds to DMPK, and coexpression of Rac-1 and DMPK activates its transphosphorylation activity in a GTP-sensitive manner. DMPK can also bind Raf-1 kinase, the Ras-activated molecule of the MAP kinase pathway. Purified Raf-1 kinase phosphorylates and activates DMPK. The interaction of DMPK with these distinct signals suggests that it may play a role as a nexus for cross-talk between their respective pathways and may partially explain the remarkable pleiotropy of DM.

    FEBS letters 2000;475;3;273-7

  • The caspase-8 inhibitor FLIP promotes activation of NF-kappaB and Erk signaling pathways.

    Kataoka T, Budd RC, Holler N, Thome M, Martinon F, Irmler M, Burns K, Hahne M, Kennedy N, Kovacsovics M and Tschopp J

    Institute of Biochemistry, University of Lausanne, BIL Biomedical Research Center, Epalinges, CH-1066, Switzerland.

    Background: Activation of Fas (CD95) by its ligand (FasL) rapidly induces cell death through recruitment and activation of caspase-8 via the adaptor protein Fas-associated death domain protein (FADD). However, Fas signals do not always result in apoptosis but can also trigger a pathway that leads to proliferation. We investigated the level at which the two conflicting Fas signals diverge and the protein(s) that are implicated in switching the response.

    Results: Under conditions in which proliferation of CD3-activated human T lymphocytes is increased by recombinant FasL, there was activation of the transcription factors NF-kappaB and AP-1 and recruitment of the caspase-8 inhibitor and FADD-interacting protein FLIP (FLICE-like inhibitory protein). Fas-recruited FLIP interacts with TNF-receptor associated factors 1 and 2, as well as with the kinases RIP and Raf-1, resulting in the activation of the NF-kappaB and extracellular signal regulated kinase (Erk) signaling pathways. In T cells these two signal pathways are critical for interleukin-2 production. Increased expression of FLIP in T cells resulted in increased production of interleukin-2.

    Conclusions: We provide evidence that FLIP is not simply an inhibitor of death-receptor-induced apoptosis but that it also mediates the activation of NF-kappaB and Erk by virtue of its capacity to recruit adaptor proteins involved in these signaling pathways.

    Funded by: FIC NIH HHS: F06 TW02294; NIAID NIH HHS: AI36333

    Current biology : CB 2000;10;11;640-8

  • Mechanism of suppression of the Raf/MEK/extracellular signal-regulated kinase pathway by the raf kinase inhibitor protein.

    Yeung K, Janosch P, McFerran B, Rose DW, Mischak H, Sedivy JM and Kolch W

    Department of Molecular Biology, Brown University, Providence, Rhode Island 02912, USA.

    We have recently identified the Raf kinase inhibitor protein (RKIP) as a physiological endogenous inhibitor of the Raf-1/MEK/extracellular signal-regulated kinase (ERK) pathway. RKIP interfered with MEK phosphorylation and activation by Raf-1, resulting in the suppression of both Raf-1-induced transformation and AP-1-dependent transcription. Here we report the molecular mechanism of RKIP's inhibitory function. RKIP can form ternary complexes with Raf-1, MEK, and ERK. However, whereas MEK and ERK can simultaneously associate with RKIP, Raf-1 binding to RKIP and that of MEK are mutually exclusive. RKIP is able to dissociate a Raf-1-MEK complex and behaves as a competitive inhibitor of MEK phosphorylation. Mapping of the binding domains showed that MEK and Raf-1 bind to overlapping sites in RKIP, whereas MEK and RKIP associate with different domains in Raf-1, and Raf-1 and RKIP bind to different sites in MEK. Both the Raf-1 and the MEK binding sites in RKIP need to be destroyed in order to relieve RKIP-mediated suppression of the Raf-1/MEK/ERK pathway, indicating that binding of either Raf-1 or MEK is sufficient for inhibition. The properties of RKIP reveal the specific sequestration of interacting components as a novel motif in the cell's repertoire for the regulation of signaling pathways.

    Funded by: NIGMS NIH HHS: R01 GM55435

    Molecular and cellular biology 2000;20;9;3079-85

  • The leucine-rich repeat protein SUR-8 enhances MAP kinase activation and forms a complex with Ras and Raf.

    Li W, Han M and Guan KL

    Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109 USA.

    Caenorhabditis elegans sur-8 encodes a positive regulator of Ras signaling. We investigated the mechanism by which the human Sur-8 homolog can positively regulate Ras-MAP kinase signaling in mammalian cells. Sur-8 expression enhances Ras- or EGF-induced Raf and ERK activation but has no effect on ERK activation induced by active Raf or MEK. Furthermore, Sur-8 expression does not increase AKT or JNK activation. Sur-8 interacts with Ras and Raf and is able to form a ternary complex with the two proteins. Thus, Sur-8 may function as a scaffold that enhances Ras-MAP kinase signal transduction by facilitating the interaction between Ras and Raf.

    Genes & development 2000;14;8;895-900

  • The Ras branch of small GTPases: Ras family members don't fall far from the tree.

    Reuther GW and Der CJ

    Department of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, North Carolina 27599-7295, USA. greuther@med.unc.edu

    The Ras branch of the Ras superfamily consists of small GTPases most closely related to Ras and include the R-Ras, Rap, Ral, Rheb, Rin and Rit proteins. Although our understanding of Ras signaling and biology is now considerable, recent observations suggest that Ras function is more complex than previously believed. First, the three Ras proteins may not be functionally identical. Second, Ras function involves functional cross-talk with their close relatives.

    Funded by: NCI NIH HHS: CA42978, CA55008, CA63071

    Current opinion in cell biology 2000;12;2;157-65

  • 14-3-3 isotypes facilitate coupling of protein kinase C-zeta to Raf-1: negative regulation by 14-3-3 phosphorylation.

    Van Der Hoeven PC, Van Der Wal JC, Ruurs P, Van Dijk MC and Van Blitterswijk J

    Division of Cellular Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

    14-3-3 Proteins may function as adapters or scaffold in signal-transduction pathways. We found previously that protein kinase C-zeta (PKC-zeta) can phosphorylate and activate Raf-1 in a signalling complex [van Dijk, Hilkmann and van Blitterswijk (1997) Biochem. J. 325, 303-307]. We report now that PKC-zeta-Raf-1 interaction is mediated by 14-3-3 proteins in vitro and in vivo. Co-immunoprecipitation experiments in COS cells revealed that complex formation between PKC-zeta and Raf-1 is mediated strongly by the 14-3-3beta and -theta; isotypes, but not by 14-3-3zeta. Far-Western blotting revealed that 14-3-3 binds PKC-zeta directly at its regulatory domain, where a S186A mutation in a putative 14-3-3-binding domain strongly reduced the binding and the complex formation with 14-3-3beta and Raf-1. Treatment of PKC-zeta with lambda protein phosphatase also reduced its binding to 14-3-3beta in vitro. Preincubation of an immobilized Raf-1 construct with 14-3-3beta facilitated PKC-zeta binding. Together, the results suggest that 14-3-3 binds both PKC-zeta (at phospho-Ser-186) and Raf-1 in a ternary complex. Complex formation was much stronger with a kinase-inactive PKC-zeta mutant than with wild-type PKC-zeta, supporting the idea that kinase activity leads to complex dissociation. 14-3-3beta and -&theta; were substrates for PKC-zeta, whereas 14-3-3zeta was not. Phosphorylation of 14-3-3beta by PKC-zeta negatively regulated their physical association. 14-3-3beta with its putative PKC-zeta phosphorylation sites mutated enhanced co-precipitation between PKC-zeta and Raf-1, suggesting that phosphorylation of 14-3-3 by PKC-zeta weakens the complex in vivo. We conclude that 14-3-3 facilitates coupling of PKC-zeta to Raf-1 in an isotype-specific and phosphorylation-dependent manner. We suggest that 14-3-3 is a transient mediator of Raf-1 phosphorylation and activation by PKC-zeta.

    The Biochemical journal 2000;345 Pt 2;297-306

  • Integration of calcium and cyclic AMP signaling pathways by 14-3-3.

    Chow CW and Davis RJ

    Howard Hughes Medical Institute, Program in Molecular Medicine, Department of Biochemistry, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.

    Calcium-stimulated nuclear factor of activated T cells (NFAT) transcription activity at the interleukin-2 promoter is negatively regulated by cyclic AMP (cAMP). This effect of cAMP is mediated, in part, by protein kinase A phosphorylation of NFAT. The mechanism of regulation involves the creation of a phosphorylation-dependent binding site for 14-3-3. Decreased NFAT phosphorylation caused by the calcium-stimulated phosphatase calcineurin, or mutation of the PKA phosphorylation sites, disrupted 14-3-3 binding and increased NFAT transcription activity. In contrast, NFAT phosphorylation caused by cAMP increased 14-3-3 binding and reduced NFAT transcription activity. The regulated interaction between NFAT and 14-3-3 provides a mechanism for the integration of calcium and cAMP signaling pathways.

    Funded by: NCI NIH HHS: CA65861, CA72009

    Molecular and cellular biology 2000;20;2;702-12

  • Localization of endogenous Grb10 to the mitochondria and its interaction with the mitochondrial-associated Raf-1 pool.

    Nantel A, Huber M and Thomas DY

    Eukaryotic Genetics Group, Biotechnology Research Institute, National Research Council, Department of Anatomy, Montreal, H4P 2R2 Quebec, Canada. andre.nantel@nrc.ca

    Grb10 belongs to a small family of adapter proteins that are known to interact with a number of receptor tyrosine kinases and signaling molecules. We have recently demonstrated that the Grb10 SH2 domain interacts with both the Raf-1 and MEK1 kinases. Overexpression of Grb10 genes with mutations in their SH2 domains promotes apoptosis in cultured cells, a phenotype that is reversed by concomitant overexpression of the wild type gene. Using immunofluorescence microscopy and subcellular fractionation we now show that most of the Grb10 molecules are peripherally associated with mitochondria. Following insulin-like growth factor I or serum treatment, small pools of Grb10 can also be found at the plasma membrane and in actin-rich membrane ruffles, whereas overexpression of Grb10 leads to its mislocalization to the cytosol. Two-hybrid analysis shows that the Grb10-binding site on Raf-1 co-localizes with its Ras-binding domain. Finally, we show that the endogenous Grb10 and Raf-1 proteins can be co-immunoprecipitated from a partially purified mitochondrial extract, an interaction that is enhanced following the activation of Raf-1 by ultraviolet radiation. Thus, we infer that Grb10 may regulate signaling between plasma membrane receptors and the apoptosis-inducing machinery on the mitochondrial outer membrane by modulating the anti-apoptotic activity of mitochondrial Raf-1.

    The Journal of biological chemistry 1999;274;50;35719-24

  • Phosphorylation and regulation of Raf by Akt (protein kinase B).

    Zimmermann S and Moelling K

    Institute of Medical Virology, University of Zurich, Gloriastrasse 30/32, CH-8028 Zurich, Switzerland.

    Activation of the protein kinase Raf can lead to opposing cellular responses such as proliferation, growth arrest, apoptosis, or differentiation. Akt (protein kinase B), a member of a different signaling pathway that also regulates these responses, interacted with Raf and phosphorylated this protein at a highly conserved serine residue in its regulatory domain in vivo. This phosphorylation of Raf by Akt inhibited activation of the Raf-MEK-ERK signaling pathway and shifted the cellular response in a human breast cancer cell line from cell cycle arrest to proliferation. These observations provide a molecular basis for cross talk between two signaling pathways at the level of Raf and Akt.

    Science (New York, N.Y.) 1999;286;5445;1741-4

  • JSAP1, a novel jun N-terminal protein kinase (JNK)-binding protein that functions as a Scaffold factor in the JNK signaling pathway.

    Ito M, Yoshioka K, Akechi M, Yamashita S, Takamatsu N, Sugiyama K, Hibi M, Nakabeppu Y, Shiba T and Yamamoto KI

    Department of Biosciences, School of Science, Kitasato University, Kanagawa 228, Japan.

    The major components of the mitogen-activated protein kinase (MAPK) cascades are MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). Recent rapid progress in identifying members of MAPK cascades suggests that a number of such signaling pathways exist in cells. To date, however, how the specificity and efficiency of the MAPK cascades is maintained is poorly understood. Here, we have identified a novel mouse protein, termed Jun N-terminal protein kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), by a yeast two-hybrid screen, using JNK3 MAPK as the bait. Of the mammalian MAPKs tested (JNK1, JNK2, JNK3, ERK2, and p38alpha), JSAP1 preferentially coprecipitated with the JNKs in cotransfected COS-7 cells. JNK3 showed a higher binding affinity for JSAP1, compared with JNK1 and JNK2. In similar cotransfection studies, JSAP1 also interacted with SEK1 MAPKK and MEKK1 MAPKKK, which are involved in the JNK cascades. The regions of JSAP1 that bound JNK, SEK1, and MEKK1 were distinct from one another. JNK and MEKK1 also bound JSAP1 in vitro, suggesting that these interactions are direct. In contrast, only the activated form of SEK1 associated with JSAP1 in cotransfected COS-7 cells. The unstimulated SEK1 bound to MEKK1; thus, SEK1 might indirectly associate with JSAP1 through MEKK1. Although JSAP1 coprecipitated with MEK1 MAPKK and Raf-1 MAPKKK, and not MKK6 or MKK7 MAPKK, in cotransfected COS-7 cells, MEK1 and Raf-1 do not interfere with the binding of SEK1 and MEKK1 to JSAP1, respectively. Overexpression of full-length JSAP1 in COS-7 cells led to a considerable enhancement of JNK3 activation, and modest enhancement of JNK1 and JNK2 activation, by the MEKK1-SEK1 pathway. Deletion of the JNK- or MEKK1-binding regions resulted in a significant reduction in the enhancement of the JNK3 activation in COS-7 cells. These results suggest that JSAP1 functions as a scaffold protein in the JNK3 cascade. We also discuss a scaffolding role for JSAP1 in the JNK1 and JNK2 cascades.

    Molecular and cellular biology 1999;19;11;7539-48

  • Rb and prohibitin target distinct regions of E2F1 for repression and respond to different upstream signals.

    Wang S, Nath N, Fusaro G and Chellappan S

    Department of Pathology, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.

    E2F transcription factor is subject to stringent regulation by a variety of molecules. We recently observed that prohibitin, a potential tumor suppressor protein, binds to the retinoblastoma (Rb) protein and represses E2F transcriptional activity. Here we demonstrate that prohibitin requires the marked box region of E2F for repression; further, prohibitin can effectively inhibit colony formation induced by overexpression of E2F1 in T47D cells. Prohibitin was also found to interact with the signaling kinase c-Raf-1, and Raf-1 could effectively reverse prohibitin-mediated repression of E2F activity. Agents such as E1A, p38 kinase, and cyclins D and E had no effect on prohibitin-mediated repression of E2F1, but all of these molecules could reverse Rb function. Similarly, stimulation of the immunoglobulin M signaling pathway in Ramos cells could inactivate prohibitin, but this had no effect on Rb function. Serum stimulation of quiescent Ramos cells inactivated Rb and prohibitin with different kinetics; further, while the serum-dependent inactivation of Rb was dependent on cyclin-dependent kinase activity, the inactivation of prohibitin was not. We believe that prohibitin is a novel regulator of E2F function which channels specific signaling cascades to the cell cycle regulatory machinery.

    Funded by: NCI NIH HHS: R0-1 CA63136, R01 CA063136

    Molecular and cellular biology 1999;19;11;7447-60

  • Contribution of the ERK5/MEK5 pathway to Ras/Raf signaling and growth control.

    English JM, Pearson G, Hockenberry T, Shivakumar L, White MA and Cobb MH

    Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041, USA.

    The activity of the catalytic domain of the orphan MAP kinase ERK5 is increased by Ras but not Raf-1 in cells, which suggests that ERK5 might mediate Raf-independent signaling by Ras. We found that Raf-1 does contribute to Ras activation of ERK5 but in a manner that does not correlate with Raf-1 catalytic activity. A clue to the mechanism of action of Raf-1 on ERK5 comes from the observation that endogenous Raf-1 binds to endogenous ERK5, suggesting the involvement of regulatory protein-protein interactions. This interaction is specific because Raf-1 binds only to ERK5 and not ERK2 or SAPK. Finally, we demonstrate the ERK5/MEK5 pathway is required for Raf-dependent cellular transformation and that a constitutively active form of MEK5, MEK5DD, synergizes with Raf to transform NIH 3T3 cells. These observations suggest that ERK5 plays a large role in Raf-1-mediated signal transduction.

    Funded by: NCI NIH HHS: CA71443, T32-CA66187; NIDDK NIH HHS: DK34128

    The Journal of biological chemistry 1999;274;44;31588-92

  • Signal transduction elements of TC21, an oncogenic member of the R-Ras subfamily of GTP-binding proteins.

    Movilla N, Crespo P and Bustelo XR

    Department of Pathology, State University of New York at Stony Brook, University Hospital, Level 2, Room 718-B, Stony Brook, New York, NY 11794-7025, USA.

    TC21 is a Ras-like GTPase with high oncogenic potential that is found mutated in some human tumors and overexpressed in breast cancer cell lines. We have conducted cellular and biochemical studies in order to understand the role of this protein in signal transduction and to unveil the signaling elements that participate in the TC21 pathway. Using gene transfer experiments, we demonstrate here that the TC21 oncogene can induce both cellular transformation in mouse fibroblasts and neuronal-like differentiation in rat PC12 cells. Interestingly, the proto-oncogenic version of TC21 shows also a lower, but significant, activity in both biological processes. We also demonstrate that the similarity of the cellular responses induced by TC21 and Ras derive from the utilization of overlapping pathways. Thus, the exchange of guanosine nucleotides in wild type TC21 is catalyzed by Ras exchange factors. Moreover, TC21 binds physically to c-Raf-1 in a GTP-dependent manner. Finally, overexpression of TC21G23V in NIH3T3 cells results in the activation of c-Raf-1 and the MAPK and the JNK branches of serine/threonine cascades. From these results, we conclude that TC21 promotes Ras-like responses in diverse cell types due to the use of overlapping, if not identical, signaling elements of the Ras oncogenic pathway.

    Funded by: NCI NIH HHS: CA7373501

    Oncogene 1999;18;43;5860-9

  • M-Ras, a widely expressed 29-kD homologue of p21 Ras: expression of a constitutively active mutant results in factor-independent growth of an interleukin-3-dependent cell line.

    Ehrhardt GR, Leslie KB, Lee F, Wieler JS and Schrader JW

    The Biomedical Research Centre, University of British Columbia, Vancouver, BC, Canada.

    M-Ras, a recently identified homologue of p21 Ras, is widely expressed, with levels of the 29-kD protein in spleen, thymus, and NIH 3T3 fibroblasts equaling or exceeding those of p21 Ras. A G22V mutant of M-Ras was constitutively active and its expression in an interleukin-3 (IL-3)-dependent mast cell/megakaryocyte cell line resulted in increased survival in the absence of IL-3, increased growth in IL-4, and, at high expression levels, in factor-independent growth. Expression of M-Ras G22V, however, had a negative effect on growth in the presence of IL-3, suggesting that M-Ras has both positive and negative effects on growth. Expression of M-Ras G22V in NIH-3T3 fibroblasts resulted in morphological transformation and growth to higher cell densities. M-Ras G22V induced activation of the c-fos promoter, and bound weakly to the Ras-binding domains of Raf-1 and RalGDS. Expression of a mutant of M-Ras G22V that was no longer membrane-bound partially inhibited (40%) activation of the c-fos promoter by N-Ras Q61K, suggesting that M-Ras shared some, but not all, of the effectors of N-Ras. An S27N mutant of M-Ras, like the analogous H-Ras S17N mutant, was a dominant inhibitor of activation of the c-fos promoter by constitutively active Src Y527F, suggesting that M-Ras and p21 Ras shared guanine nucleotide exchange factors and are likely to be activated in parallel. Moreover, M-Ras was recognized by the monoclonal anti-Ras antibody Y13-259, commonly used to study the function and activity of p21 Ras. Mammalian M-Ras and a Caenorhabditis elegans orthologue exhibit conserved structural features, and these are likely to mediate activation of distinctive signaling paths that function in parallel to those downstream of p21 Ras.

    Blood 1999;94;7;2433-44

  • Direct interaction of p21 cyclin-dependent kinase inhibitor with the retinoblastoma tumor suppressor protein.

    Nakanishi M, Kaneko Y, Matsushime H and Ikeda K

    Department of Geriatric Research, National Institute for Longevity Sciences, 36-3 Gengo, Morioka-cho, Obu, Aichi, 474, Japan. mkt-naka@med.nagoya-cu.ac.jp

    The p21CKI forms a physical complex with the retinoblastoma protein (pRb) both in vitro and in vivo. The A/B pocket region of pRb and the N-terminal region of p21 were indispensable for this interaction. Among p21 family members, p57, but not p27, associated with pRb. Overexpression of cyclin D1, Cdk4, and E2F1 in the cells expressing pRb and p21 did not perturb the interaction between p21 and pRb. Coexpression of p21 in cells expressing pRb, cyclin D1, and Cdk4 prevented pRb hyperphosphorylation by cyclin D1/Cdk4. On the other hand, hyperphosphorylation of pRb by an excess amount of cyclin/Cdk disrupted pRb/p21 complex formation in vitro. These findings suggest that pRb may be dynamically regulated by the relative binding and activities of p21 and Cdks.

    Biochemical and biophysical research communications 1999;263;1;35-40

  • The strength of interaction at the Raf cysteine-rich domain is a critical determinant of response of Raf to Ras family small GTPases.

    Okada T, Hu CD, Jin TG, Kariya K, Yamawaki-Kataoka Y and Kataoka T

    Department of Physiology II, Kobe University School of Medicine, Chuo-ku, Kobe 650-0017, Japan.

    To be fully activated at the plasma membrane, Raf-1 must establish two distinct modes of interactions with Ras, one through its Ras-binding domain and the other through its cysteine-rich domain (CRD). The Ras homologue Rap1A is incapable of activating Raf-1 and even antagonizes Ras-dependent activation of Raf-1. We proposed previously that this property of Rap1A may be attributable to its greatly enhanced interaction with Raf-1 CRD compared to Ras. On the other hand, B-Raf, another Raf family member, is activatable by both Ras and Rap1A. When interactions with Ras and Rap1A were measured, B-Raf CRD did not exhibit the enhanced interaction with Rap1A, suggesting that the strength of interaction at CRDs may account for the differential action of Rap1A on Raf-1 and B-Raf. The importance of the interaction at the CRD is further supported by a domain-shuffling experiment between Raf-1 and B-Raf, which clearly indicated that the nature of CRD determines the specificity of response to Rap1A: Raf-1, whose CRD is replaced by B-Raf CRD, became activatable by Rap1A, whereas B-Raf, whose CRD is replaced by Raf-1 CRD, lost its response to Rap1A. Finally, a B-Raf CRD mutant whose interaction with Rap1A is selectively enhanced was isolated and found to possess the double mutation K252E/M278T. B-Raf carrying this mutation was not activated by Rap1A but retained its response to Ras. These results indicate that the strength of interaction with Ras and Rap1A at its CRD may be a critical determinant of regulation of the Raf kinase activity by the Ras family small GTPases.

    Molecular and cellular biology 1999;19;9;6057-64

  • Mutational analysis of Raf-1 cysteine rich domain: requirement for a cluster of basic aminoacids for interaction with phosphatidylserine.

    Improta-Brears T, Ghosh S and Bell RM

    Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA.

    Activation of Raf-1 kinase is preceded by a translocation of Raf-1 to the plasma membrane in response to external stimuli. The membrane localization of Raf-1 is facilitated through its interaction with activated Ras and with membrane phospholipids. Previous evidence suggests that the interaction of Raf-1 with Ras is mediated by two distinct domains within the N-terminal region of Raf-1 comprising amino acid residues 51-131 and residues 139-184, the latter of which codes for a zinc containing cysteine-rich domain. The cysteine-rich domain of Raf-1 is also reported to associate with other proteins, such as 14-3-3, and for selectively binding acidic phospholipids, particularly phosphatidylserine (PS). In the present study, we have investigated the consequences of progressive deletions and point mutations within the cysteine-rich domain of Raf-1 on its ability to bind PS. A reduced interaction with PS was observed in vitro for all deletion mutants of Raf-1 expressed either as full-length proteins or as fragments containing the isolated cysteine-rich domain. In particular, the cluster of basic amino acids R143, K144, and K148 appeared to be critical for interaction with PS, since substitution of all three residues to alanine resulted in a protein that failed to interact with liposomes enriched for PS. Expression of Raf-1 in vivo, containing point mutations in the cysteine-rich domain resulted in a truncated polypeptide that lacked both the Ras and PS binding sites and could no longer translocate to the plasma membrane upon serum stimulation. These results indicate that the basic residues 143, 144 and 148 in the anterior half of Raf-1 cysteine-rich domain play a role in the association with the lipid bilayer and possibly in protein stability, therefore they might contribute to Raf-1 localization and subsequent activation.

    Molecular and cellular biochemistry 1999;198;1-2;171-8

  • 14-3-3Gamma interacts with and is phosphorylated by multiple protein kinase C isoforms in PDGF-stimulated human vascular smooth muscle cells.

    Autieri MV and Carbone CJ

    Department of Cardiology and Physiology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA. mautieri@unix.temple.edu

    It has recently been demonstrated that some members of the 14-3-3 protein family play an important role in signal transduction leading to cellular proliferation. We have previously shown that expression of 14-3-3gamma is induced by growth factors in human vascular smooth muscle cells (VSMC). In this study, we cloned the human homolog of 14-3-3gamma and observed many potential phosphorylation sites, suggesting the potential for post-translational modification. In VSMC treated with platelet-derived growth factor (PDGF), 14-3-3gamma protein was expressed and phosphorylated in an activation-dependent manner. Platelet-derived growth factor-induced phosphorylation could be inhibited by phosphokinase C (PKC) inhibitory compounds, and 14-3-3gamma could be phosphorylated in the absence of PDGF by compounds that activate PKC. We also demonstrated interaction between 14-3-3gamma and several PKC isoforms (alpha, beta, gamma, theta, and delta), implicating these PKC family isoforms as the kinases responsible for PDGF-induced 14-3-3gamma phosphorylation. We found that 14-3-3gamma interacted with the signal transduction protein Raf-1, suggesting that 14-3-3gamma provides a link between this protein and PKC. Thus, 14-3-3gamma may represent a signal transduction protein that is regulated transcriptionally and post-transcriptionally by growth factors.

    DNA and cell biology 1999;18;7;555-64

  • Tyrosine phosphorylation of the proto-oncoprotein Raf-1 is regulated by Raf-1 itself and the phosphatase Cdc25A.

    Xia K, Lee RS, Narsimhan RP, Mukhopadhyay NK, Neel BG and Roberts TM

    Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.

    There is a growing body of evidence demonstrating that Raf-1 is phosphorylated on tyrosines upon stimulation of a variety of receptors. Although detection of Raf-1 tyrosine phosphorylation has remained elusive, genetic analyses have demonstrated it to be important for Raf-1 activation. Here we report new findings which indicate that Raf-1 tyrosine phosphorylation is regulated in vivo. In both a mammalian and baculovirus expression system, a kinase-inactive allele of Raf-1 was found to be tyrosine phosphorylated at levels much greater than that of wild-type Raf-1. The level of tyrosine phosphate on Raf-1 was markedly increased upon treatment with phosphatase inhibitors either before or after cell lysis. Cdc25A was found to dephosphorylate Raf-1 on tyrosines that resulted in a significant decrease in Raf-1 kinase activity. In NIH 3T3 cells, coexpression of wild-type Raf-1 and phosphatase-inactive Cdc25A led to a marked increase in Raf-1 tyrosine phosphorylation in response to platelet-derived growth factor. These data suggest that the tyrosine phosphorylation of Raf-1 is regulated not only by itself but also by Cdc25A.

    Funded by: NCI NIH HHS: 2RO1CA43803-11A1, CA49152

    Molecular and cellular biology 1999;19;7;4819-24

  • Aiolos transcription factor controls cell death in T cells by regulating Bcl-2 expression and its cellular localization.

    Romero F, Martínez-A C, Camonis J and Rebollo A

    Laboratoire d'Oncologie Cellulaire et Moleculaire, INSERM U363, Hôpital Cochin, 27 rue du Faubourg Saint-Jacques, F-75014 Paris, France.

    We searched for proteins that interact with Ras in interleukin (IL)-2-stimulated or IL-2-deprived cells, and found that the transcription factor Aiolos interacts with Ras. The Ras-Aiolos interaction was confirmed in vitro and in vivo by co-immunoprecipitation. Indirect immunofluorescence shows that IL-2 controls the cellular distribution of Aiolos and induces its tyrosine phosphorylation, required for dissociation from Ras. We also identified functional Aiolos-binding sites in the Bcl-2 promoter, which are able to activate the luciferase reporter gene. Mutation of Aiolos-binding sites within the Bcl-2 promoter inhibits transactivation of the reporter gene luciferase, suggesting direct control of Bcl-2 expression by Aiolos. Co-transfection experiments confirm that Aiolos induces Bcl-2 expression and prevents apoptosis in IL-2-deprived cells. We propose a model for the regulation of Bcl-2 expression via Aiolos.

    The EMBO journal 1999;18;12;3419-30

  • The ankyrin repeat-containing adaptor protein Tvl-1 is a novel substrate and regulator of Raf-1.

    Lin JH, Makris A, McMahon C, Bear SE, Patriotis C, Prasad VR, Brent R, Golemis EA and Tsichlis PN

    Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.

    Tvl-1 is a 269-amino acid ankyrin repeat protein expressed primarily in thymus, lung, and testes that was identified by screening a murine T-cell two-hybrid cDNA library for proteins that associate with the serine-threonine kinase Raf-1. The interaction of Tvl-1 with Raf-1 was confirmed by co-immunoprecipitation of the two proteins from COS-1 cells transiently transfected with Tvl-1 and Raf-1 expression constructs as well as by co-immunoprecipitation of the endogenous proteins from CV-1 and NB2 cells. Tvl-1 interacts with Raf-1 via its carboxyl-terminal ankyrin repeat domain. The same domain also mediates Tvl-1 homodimerization. Tvl-1 was detected by immunofluorescence in both the cytoplasm and the nucleus suggesting that in addition to Raf-1 it may also interact with nuclear proteins. Activated Raf-1 phosphorylates Tvl-1 both in vitro and in vivo. In baculovirus-infected Sf9 insect cells, Tvl-1 potentiates the activation of Raf-1 by Src and Ras while in COS-1 cells it potentiates the activation of Raf-1 by EGF. These data suggest that Tvl-1 is both a target as well as a regulator of Raf-1. The human homologue of Tvl-1 maps to chromosome 19p12, upstream of MEF2B with the two genes in a head to head arrangement.

    Funded by: NCI NIH HHS: CA06927, R01-CA38147, T32-CA09683

    The Journal of biological chemistry 1999;274;21;14706-15

  • Serine and tyrosine phosphorylations cooperate in Raf-1, but not B-Raf activation.

    Mason CS, Springer CJ, Cooper RG, Superti-Furga G, Marshall CJ and Marais R

    CRC Centre for Cell and Molecular Biology, Institute of Cancer Research, 237 Fulham Road, London SW3 6JB.

    The Raf family of serine/threonine protein kinases couple growth factor receptor stimulation to mitogen activated protein kinase activation, but their own regulation is poorly understood. Using phospho-specific antisera, we show that activated Raf-1 is phosphorylated on S338 and Y341. Expression of Raf-1 with oncogenic Ras gives predominantly S338 phosphorylation, whereas activated Src gives predominantly Y341 phosphorylation. Phosphorylation at both sites is maximal only when both oncogenic Ras and activated Src are present. Raf-1 that cannot interact with Ras-GTP is not phosphorylated, showing that phosphorylation is Ras dependent, presumably occurring at the plasma membrane. Mutations which prevent phosphorylation at either site block Raf-1 activation and maximal activity is seen only when both are phosphorylated. Mutations at S339 or Y340 do not block Raf-1 activation. While B-Raf lacks a tyrosine phosphorylation site equivalent to Y341 of Raf-1, S445 of B-Raf is equivalent to S338 of Raf-1. Phosphorylation of S445 is constitutive and is not stimulated by oncogenic Ras. However, S445 phosphorylation still contributes to B-Raf activation by elevating basal and consequently Ras-stimulated activity. Thus, there are considerable differences between the activation of the Raf proteins; Ras-GTP mediates two phosphorylation events required for Raf-1 activation but does not regulate such events for B-Raf.

    The EMBO journal 1999;18;8;2137-48

  • Activation of the Raf/MAP kinase cascade by the Ras-related protein TC21 is required for the TC21-mediated transformation of NIH 3T3 cells.

    Rosário M, Paterson HF and Marshall CJ

    CRC Centre for Cell and Molecular Biology, Chester Beatty Laboratories, Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK.

    TC21 is a member of the Ras superfamily of small GTP-binding proteins and, like Ras, has been implicated in the regulation of growth-stimulating pathways. Point mutations introduced into TC21 based on equivalent H-Ras oncogenic mutations are transforming in cultured cells, and oncogenic mutations in TC21 have been isolated from several human tumours. The mechanism of TC21 signalling in transformation is poorly understood. While activation of the serine/threonine kinases Raf-1 and B-Raf has been implicated in signalling pathways leading to transformation by H-Ras, it has been argued that TC21 does not activate Raf-1 or B-Raf. Since the Raf-signalling pathway is important in transformation by other Ras proteins, we assessed whether the Raf pathway is important to transformation by TC21. Raf-1 and B-Raf are constitutively active in TC21-transformed cells and the ERK/MAPK cascade is required for the maintenance of the transformed state. We demonstrate that oncogenic V23 TC21, like Ras, interacts with Raf-1 and B-Raf (but not with A-Raf), resulting in the translocation of the Raf proteins to the plasma membrane and in their activation. Furthermore, using point mutations in the effector loop of TC21, we show that the interaction of TC21 with Raf-1 is crucial for transformation.

    Funded by: Wellcome Trust

    The EMBO journal 1999;18;5;1270-9

  • Effect of phosphorylation on activities of Rap1A to interact with Raf-1 and to suppress Ras-dependent Raf-1 activation.

    Hu CD, Kariya K, Okada T, Qi X, Song C and Kataoka T

    Department of Physiology II, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.

    Rap1A is phosphorylated by cAMP-dependent protein kinase (PKA), and this phosphorylation has been shown to modulate its interaction with other proteins. However, it is not known whether Rap1A phosphorylation is involved in regulation of its cellular functions, including suppression of Ras-dependent Raf-1 activation. We have previously shown that this suppressive activity of Rap1A is attributable to its greatly enhanced ability to bind to the cysteine-rich region (CRR, residues 152-184) of Raf-1 compared with that of Ras. Here, we show that phosphorylation of Rap1A by PKA abolished its binding activity to CRR. Furthermore, a mutant Rap1A(S180E), whose sole PKA phosphorylation residue, Ser-180, was substituted by an acidic residue, Glu, to mimic its phosphorylated form, failed to suppress Ras-dependent Raf-1 activation in COS-7 cells. These results indicate that the CRR binding activity and the Ras-suppressive function of Rap1A can be modulated through phosphorylation and suggest that Rap1A may function as a PKA-dependent regulator of Raf-1 activation, not merely as a suppressor.

    The Journal of biological chemistry 1999;274;1;48-51

  • Raf-1 physically interacts with Rb and regulates its function: a link between mitogenic signaling and cell cycle regulation.

    Wang S, Ghosh RN and Chellappan SP

    Department of Pathology, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA.

    Cells initiate proliferation in response to growth factor stimulation, but the biochemical mechanisms linking signals received at the cell surface receptors to the cell cycle regulatory molecules are not yet clear. In this study, we show that the signaling molecule Raf-1 can physically interact with Rb and p130 proteins in vitro and in vivo and that this interaction can be detected in mammalian cells without overexpressing any component. The binding of Raf-1 to Rb occurs subsequent to mitogen stimulation, and this interaction can be detected only in proliferating cells. Raf-1 can inactivate Rb function and can reverse Rb-mediated repression of E2F1 transcription and cell proliferation efficiently. The region of Raf-1 involved in Rb binding spanned residues 1 to 28 at the N terminus, and functional inactivation of Rb required a direct interaction. Serum stimulation of quiescent human fibroblast HSF8 cells led to a partial translocation of Raf-1 into the nucleus, where it colocalized with Rb. Further, Raf-1 was able to phosphorylate Rb in vitro quite efficiently. We believe that the physical interaction of Raf-1 with Rb is a vital step in the growth factor-mediated induction of cell proliferation and that Raf-1 acts as a direct link between cell surface signaling cascades and the cell cycle machinery.

    Funded by: NCI NIH HHS: CA63136

    Molecular and cellular biology 1998;18;12;7487-98

  • The protein kinase Pak3 positively regulates Raf-1 activity through phosphorylation of serine 338.

    King AJ, Sun H, Diaz B, Barnard D, Miao W, Bagrodia S and Marshall MS

    Department of Medicine, Indiana University School of Medicine, The Walther Oncology Center, Indianapolis 46202, USA.

    The pathway involving the signalling protein p21Ras propagates a range of extracellular signals from receptors on the cell membrane to the cytoplasm and nucleus. The Ras proteins regulate many effectors, including members of the Raf family of protein kinases. Ras-dependent activation of Raf-1 at the plasma membrane involves phosphorylation events, protein-protein interactions and structural changes. Phosphorylation of serine residues 338 or 339 in the catalytic domain of Raf-1 regulates its activation in response to Ras, Src and epidermal growth factor. Here we show that the p21-activated protein kinase Pak3 phosphorylates Raf-1 on serine 338 in vitro and in vivo. The p21-activated protein kinases are regulated by the Rho-family GTPases Rac and Cdc42. Our results indicate that signal transduction through Raf-1 depends on both Ras and the activation of the Pak pathway. As guanine-nucleotide-exchange activity on Rac can be stimulated by a Ras-dependent phosphatidylinositol-3-OH kinase, a mechanism could exist through which one Ras effector pathway can be influenced by another.

    Nature 1998;396;6707;180-3

  • JNKK1 organizes a MAP kinase module through specific and sequential interactions with upstream and downstream components mediated by its amino-terminal extension.

    Xia Y, Wu Z, Su B, Murray B and Karin M

    Laboratory of Gene Regulation and Signal Transduction, Department of Pharmacology, University of California San Diego, La Jolla, California 92093-0636 USA.

    MAP kinase (MAPK) cascades are composed of a MAPK, MAPK kinase (MAPKK), and a MAPKK kinase (MAPKKK). Despite the existence of numerous components and ample opportunities for crosstalk, most MAPKs are specifically and distinctly activated. We investigated the basis for specific activation of the JNK subgroup of MAPKs. The specificity of JNK activation is determined by the MAPKK JNKK1, which interacts with the MAPKKK MEKK1 and JNK through its amino-terminal extension. Inactive JNKK1 mutants can disrupt JNK activation by MEKK1 or tumor necrosis factor (TNF) in intact cells only if they contain an intact amino-terminal extension. Mutations in this region interfere with the ability of JNKK1 to respond to TNF but do not affect its activation by physical stressors. As JNK and MEKK1 compete for binding to JNKK1 and activation of JNKK1 prevents its binding to MEKK1, activation of this module is likely to occur through sequential MEKK1:JNKK1 and JNKK1:JNK interactions. These results underscore a role for the amino-terminal extension of MAPKKs in determination of response specificity.

    Funded by: NHLBI NIH HHS: HL 35018

    Genes & development 1998;12;21;3369-81

  • Premature senescence involving p53 and p16 is activated in response to constitutive MEK/MAPK mitogenic signaling.

    Lin AW, Barradas M, Stone JC, van Aelst L, Serrano M and Lowe SW

    Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.

    Oncogenic Ras transforms immortal rodent cells to a tumorigenic state, in part, by constitutively transmitting mitogenic signals through the mitogen-activated protein kinase (MAPK) cascade. In primary cells, Ras is initially mitogenic but eventually induces premature senescence involving the p53 and p16(INK4a) tumor suppressors. Constitutive activation of MEK (a component of the MAPK cascade) induces both p53 and p16, and is required for Ras-induced senescence of normal human fibroblasts. Furthermore, activated MEK permanently arrests primary murine fibroblasts but forces uncontrolled mitogenesis and transformation in cells lacking either p53 or INK4a. The precisely opposite response of normal and immortalized cells to constitutive activation of the MAPK cascade implies that premature senescence acts as a fail-safe mechanism to limit the transforming potential of excessive Ras mitogenic signaling. Consequently, constitutive MAPK signaling activates p53 and p16 as tumor suppressors.

    Funded by: NCI NIH HHS: CA13106, CA72982, P01 CA013106

    Genes & development 1998;12;19;3008-19

  • 14-3-3 proteins are required for maintenance of Raf-1 phosphorylation and kinase activity.

    Thorson JA, Yu LW, Hsu AL, Shih NY, Graves PR, Tanner JW, Allen PM, Piwnica-Worms H and Shaw AS

    Center for Immunology and Department of Pathology, Washington University School of Medicine, St. Louis, Missouri, USA.

    By binding to serine-phosphorylated proteins, 14-3-3 proteins function as effectors of serine phosphorylation. The exact mechanism of their action is, however, still largely unknown. Here we demonstrate a requirement for 14-3-3 for Raf-1 kinase activity and phosphorylation. Expression of dominant negative forms of 14-3-3 resulted in the loss of a critical Raf-1 phosphorylation, while overexpression of 14-3-3 resulted in enhanced phosphorylation of this site. 14-3-3 levels, therefore, regulate the stoichiometry of Raf-1 phosphorylation and its potential activity in the cell. Phosphorylation of Raf-1, however, was insufficient by itself for kinase activity. Removal of 14-3-3 from phosphorylated Raf abrogated kinase activity, whereas addition of 14-3-3 restored it. This supports a paradigm in which the effects of phosphorylation on serine as well as tyrosine residues are mediated by inducible protein-protein interactions.

    Funded by: NIAID NIH HHS: AI54094; NIGMS NIH HHS: F32 GM018428, GM18428, GM47017, R01 GM047017

    Molecular and cellular biology 1998;18;9;5229-38

  • Autoregulation of the Raf-1 serine/threonine kinase.

    Cutler RE, Stephens RM, Saracino MR and Morrison DK

    Molecular Basis of Carcinogenesis Laboratory, Advanced BioSciences Laboratories-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702, USA.

    The Raf-1 serine/threonine kinase is a key protein involved in the transmission of many growth and developmental signals. In this report, we show that autoinhibition mediated by the noncatalytic, N-terminal regulatory region of Raf-1 is an important mechanism regulating Raf-1 function. The inhibition of the regulatory region occurs, at least in part, through binding interactions involving the cysteine-rich domain. Events that disrupt this autoinhibition, such as mutation of the cysteine-rich domain or a mutation mimicking an activating phosphorylation event (Y340D), alleviate the repression of the regulatory region and increase Raf-1 activity. Based on the striking similarites between the autoregulation of the serine/threonine kinases protein kinase C, Byr2, and Raf-1, we propose that relief of autorepression and activation at the plasma membrane is an evolutionarily conserved mechanism of kinase regulation.

    Proceedings of the National Academy of Sciences of the United States of America 1998;95;16;9214-9

  • Binding of human immunodeficiency virus type 1 to CD4 and CXCR4 receptors differentially regulates expression of inflammatory genes and activates the MEK/ERK signaling pathway.

    Popik W, Hesselgesser JE and Pitha PM

    Oncology Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, USA.

    We have previously shown that binding of human immunodeficiency virus type 1 (HIV-1) virions to CD4 receptors stimulates association of Lck with Raf-1 and results in the activation of Raf-1 kinase in a Ras-independent manner. In the present study, we demonstrate that HIV-1 envelope glycoproteins of both T-cell-tropic and macrophagetropic strains rapidly activate the ERK/mitogen-activated protein (MAP) kinase pathway and the binding of nuclear transcription factors (AP-1, NF-kappaB, and C/EBP) and stimulate expression of cytokine and chemokine genes. The activation of this signaling pathway requires functional CD4 receptors and is independent of binding to CXCR4. Binding of the natural ligand stromal cell-derived factor 1 (SDF-1) to CXCR4, which inhibits entry of T-cell-tropic HIV-1, activates also the ERK/MAP kinase pathway. However, SDF-1 did not affect the CD4-mediated expression of cytokine and chemokine genes. These results provide firm molecular evidence that binding of HIV-1 envelope glycoproteins to CD4 receptor initiates a signaling pathway(s) independent of the binding to the chemokine receptor that leads to the aberrant expression of inflammatory genes and may contribute significantly to HIV-1 replication as well as to deregulation of the immune system.

    Funded by: NIAID NIH HHS: AI26123, AI40838, AI42557

    Journal of virology 1998;72;8;6406-13

  • Activation of the 41/43 kDa mitogen-activated protein kinase signaling pathway is required for hepatocyte growth factor-induced cell scattering.

    Tanimura S, Chatani Y, Hoshino R, Sato M, Watanabe S, Kataoka T, Nakamura T and Kohno M

    Laboratory of Cell Regulation, School of Pharmaceutical Sciences, Nagasaki University, Japan.

    Hepatocyte growth factor (HGF) markedly induced the spreading, dissociation and scattering of Madin-Darby canine kidney epithelial cells (MDCK) and human stomach adenocarcinoma cells (TMK1). Scattering of MDCK and TMK1 cells was induced by 12-O-tetradecanoyl-phorbol-13-acetate (PMA) and epidermal growth factor (EGF), respectively. In all these agent-stimulated cells, rapid activation of Raf-1, MAP kinase/ERK kinase (MEK), 41/43 kDa MAP kinases and p90rsk was commonly observed. In contrast, PMA neither induced the scattering nor activation of all these kinases in TMK1 cells. Pretreatment of MDCK and TMK1 cells with 2-(2-amino-3-methoxyphenyl) choromone (AMPC), a specific inhibitor of MEK, selectively inhibited the HGF-, PMA- and EGF-stimulated activities of MEK, 41/43 kDa MAP kinases and p90rsk in a dose dependent manner. AMPC-pretreatment, however, did not affect HGF-, PMA- or EGF-induced activation of Raf-1, nor HGF-induced activation of phosphatidylinositol 3-kinase in these cells. Importantly, HGF-, PMA- and EGF-induced scattering of MDCK and TMK1 cells was inhibited at doses of AMPC similar to those that gave comparable levels of inhibition of the activities of MEK, 41/43 kDa MAP kinases and p90rsk. These results suggest that activation of the 41/43 kDa MAP kinase signaling pathway is required for the motility response of MDCK and TMK1 cells induced by agents such as HGF, PMA and EGF.

    Oncogene 1998;17;1;57-65

  • Binding of c-Raf1 kinase to a conserved acidic sequence within the carboxyl-terminal region of the HIV-1 Nef protein.

    Hodge DR, Dunn KJ, Pei GK, Chakrabarty MK, Heidecker G, Lautenberger JA and Samuel KP

    Laboratory of Leukocyte Biology, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702, USA.

    Nef is a membrane-associated cytoplasmic phosphoprotein that is well conserved among the different human (HIV-1 and HIV-2) and simian immunodeficiency viruses and has important roles in down-regulating the CD4 receptor and modulating T-cell signaling pathways. The ability to modulate T-cell signaling pathways suggests that Nef may physically interact with T-cell signaling proteins. In order to identify Nef binding proteins and map their site(s) of interaction, we targeted a highly conserved acidic sequence at the carboxyl-terminal region of Nef sharing striking similarity with an acidic sequence at the c-Raf1-binding site within the Ras effector region. Here, we used deletion and site-specific mutagenesis to generate mutant Nef proteins fused to bacterial glutathione S-transferase in in vitro precipitation assays and immunoblot analysis to map the specific interaction between the HIV-1LAI Nef and c-Raf1 to a conserved acidic sequence motif containing the core sequence Asp-Asp-X-X-X-Glu (position 174-179). Significantly, we demonstrate that substitution of the nonpolar glycine residue for either or both of the conserved negatively charged aspartic acid residues at positions 174 and 175 in the full-length recombinant Nef protein background completely abrogated binding of c-Raf1 in vitro. In addition, lysates from a permanent CEM T-cell line constitutively expressing the native HIV-1 Nef protein was used to coimmunoprecipitate a stable Nef-c-Raf1 complex, suggesting that molecular interactions between Nef and c-Raf1, an important downstream transducer of cell signaling through the c-Raf1-MAP kinase pathway, occur in vivo. This interaction may account for the Nef-induced perturbations of T-cell signaling and activation pathways in vitro and in vivo.

    Funded by: NCRR NIH HHS: 2P20RR011606-02

    The Journal of biological chemistry 1998;273;25;15727-33

  • Interaction of the Grb10 adapter protein with the Raf1 and MEK1 kinases.

    Nantel A, Mohammad-Ali K, Sherk J, Posner BI and Thomas DY

    Eukaryotic Genetics Group, Biotechnology Research Institute, National Research Council, 6100 Royalmount, Montreal, Quebec H4P 2R2, Canada. andre.nantel@bri.nrc.ca

    Grb10 and its close homologues Grb7 and Grb14, belong to a family of adapter proteins characterized by a proline-rich region, a central PH domain, and a carboxyl-terminal Src homology 2 (SH2) domain. Their interaction with a variety of activated tyrosine kinase receptors is well documented, but their actual function remains a mystery. The Grb10 SH2 domain was isolated from a two-hybrid screen using the MEK1 kinase as a bait. We show that this unusual SH2 domain interacts, in a phosphotyrosine-independent manner, with both the Raf1 and MEK1 kinases. Mutation of the MEK1 Thr-386 residue, which is phosphorylated by mitogen-activated protein kinase in vitro, reduces binding to Grb10 in a two-hybrid assay. Interaction of Grb10 with Raf1 is constitutive, while interaction between Grb10 and MEK1 needs insulin treatment of the cells and follows mitogen-activated protein kinase activation. Random mutagenesis of the SH2 domain demonstrated that the Arg-betaB5 and Asp-EF2 residues are necessary for binding to the epidermal growth factor and insulin receptors as well as to the two kinases. In addition, we show that a mutation in Ser-betaB7 affects binding only to the receptors, while a mutation in Thr-betaC5 abrogates binding only to MEK1. Finally, transfection of Grb10 genes with specific mutations in their SH2 domains induces apoptosis in HTC-IR and COS-7 cells. These effects can be competed by co-expression of the wild type protein, suggesting that these mutants act by sequestering necessary signaling components.

    The Journal of biological chemistry 1998;273;17;10475-84

  • Identification of Nore1 as a potential Ras effector.

    Vavvas D, Li X, Avruch J and Zhang XF

    The Diabetes Unit and Medical Services and the Department of Medicine, Harvard Medical School, Massachusetts General Hospital East, Charlestown, Massachusetts 02129, USA.

    The small GTP-binding protein Ras is pivotal in transmitting growth and differentiation signals downstream of cell surface receptors. Many observations have indicated that Ras transmits signals from cell surface receptors into multiple pathways via direct interaction with different effectors in mammalian cells. We have identified a novel potential Ras effector or target named Nore1. Nore1 has no significant sequence similarity to known mammalian proteins and lacks an identifiable catalytic domain, but contains sequence motifs that predict DAG_PE binding and SH3 domain binding. We show that Nore1 directly interacts with Ras in vitro in a GTP-dependent manner, and the interaction requires an intact Ras effector domain. Nore1 becomes associated with Ras in situ following activation of epidermal growth factor receptor in COS-7 and in KB cells.

    Funded by: NIGMS NIH HHS: GM51281

    The Journal of biological chemistry 1998;273;10;5439-42

  • Identification of a specific effector of the small GTP-binding protein Rap2.

    Janoueix-Lerosey I, Pasheva E, de Tand MF, Tavitian A and de Gunzburg J

    Unité INSERM 248, Institut Curie, Section de Recherche, Paris, France.

    Rap2 is a small GTP-binding protein that belongs to the Ras superfamily and whose function is still unknown. To elucidate Rap2 function, we searched for potential effectors by screening a mouse brain cDNA library in a yeast two-hybrid system using as a bait a Rap2A protein bearing a mutation of Gly to Val at position 12. This strategy lead to the identification of a protein that interacts specifically with Rap2A complexed with GTP, and requires an intact effector domain of Rap2A for interaction; we designated this protein Rap2-interacting protein 8 (RPIP8). Biochemical data obtained from in vitro studies with purified proteins confirmed the genetic results. Mouse RPIP8 consists of 446 amino acids, bears a coiled-coil domain between residues 265 and 313, and is expressed principally in brain. Its human counterpart, of 400 amino acids, exhibits 93.7% identity in their common region. A search for similar sequences in expressed-sequence-tags databanks revealed the presence in human and rodents of mRNAs encoding the 400-residue and 446-residue forms of RPIP8. Furthermore a doublet of 45-50 kDa, corresponding to the 400-residue and 446-residue forms of the protein, was detected by western blotting of mouse brain extracts and lysates from pheochromocytoma PC12 cells and the pancreatic beta-cell lines HIT-T15 and RIN-m5F. Using transient transfections of HIT-T15 cells it was possible to demonstrate that [Val12]Rap2 and wild-type Rap2 could be immunoprecipitated with RPIP8. These data therefore argue for RPIP8 being a specific effector of the Rap2 protein in cells exhibiting neuronal properties.

    European journal of biochemistry 1998;252;2;290-8

  • Interferon gamma activation of Raf-1 is Jak1-dependent and p21ras-independent.

    Sakatsume M, Stancato LF, David M, Silvennoinen O, Saharinen P, Pierce J, Larner AC and Finbloom DS

    Division of Cytokine Biology, Center for Biologics Research and Evaluation, Food and Drug Administration, Bethesda, Maryland 20892, USA.

    Signal transduction through the interferongamma (IFNgamma) receptor involves the formation of a ligand-dependent multimolecular association of receptor chains (alpha and beta), Janus tyrosine kinases (Jak1 and Jak2), and the transcription factor (signal transducers and activators of transcription 1alpha (STAT1alpha)) in addition to activation of mitogen-activated protein kinases (MAPK). Interactions between components of the Jak/STAT cascade and the p21(ras)/Raf-1/MAPK cascade are unexplored. Treatment of HeLa cells with IFNgamma resulted in the rapid and transient activation of Raf-1 and MAPK. Parallel activation of cells resulted in essentially no enhancement of p21(ras) activation despite marked enhancement after treatment with epidermal growth factor. In HeLa (E1C3) and fibrosarcoma (U4A) cell lines, both of which are deficient in Jak1 kinase, Raf-1 activation by IFNgamma was absent. Reconstitution of Raf-1 activity was observed only with kinase active Jak1 in both cell lines. In COS cells, transient expression of wild type or kinase-inactive Jak1 coimmunoprecipitated with Raf-1, but activation of Raf-1 activity was only observed in cells expressing kinase-active Jak1. These observations suggest that a kinase-active Jak1 is required for IFNgamma activation of Raf-1 that is p21(ras)-independent.

    The Journal of biological chemistry 1998;273;5;3021-6

  • Construction and characterization of a full length-enriched and a 5'-end-enriched cDNA library.

    Suzuki Y, Yoshitomo-Nakagawa K, Maruyama K, Suyama A and Sugano S

    International and Interdisciplinary Studies, The University of Tokyo, Japan.

    Using 'oligo-capped' mRNA [Maruyama, K., Sugano, S., 1994. Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides. Gene 138, 171-174], whose cap structure was replaced by a synthetic oligonucleotide, we constructed two types of cDNA library. One is a 'full length-enriched cDNA library' which has a high content of full-length cDNA clones and the other is a '5'-end-enriched cDNA library', which has a high content of cDNA clones with their mRNA start sites. The 5'-end-enriched library was constructed especially for isolating the mRNA start sites of long mRNAs. In order to characterize these libraries, we performed one-pass sequencing of randomly selected cDNA clones from both libraries (84 clones for the full length-enriched cDNA library and 159 clones for the 5'-end-enriched cDNA library). The cDNA clones of the polypeptide chain elongation factor 1 alpha were most frequently (nine clones) isolated, and more than 80% of them (eight clones) contained the mRNA start site of the gene. Furthermore, about 80% of the cDNA clones of both libraries whose sequence matched with known genes had the known 5' ends or sequences upstream of the known 5' ends (28 out of 35 for the full length-enriched library and 51 out of 62 for the 5'-end-enriched library). The longest full-length clone of the full length-enriched cDNA library was about 3300 bp (among 28 clones). In contrast, seven clones (out of the 51 clones with the mRNA start sites) from the 5'-end-enriched cDNA library came from mRNAs whose length is more than 3500 bp. These cDNA libraries may be useful for generating 5' ESTs with the information of the mRNA start sites that are now scarce in the EST database.

    Gene 1997;200;1-2;149-56

  • c-Raf kinase binds to N-terminal domain of c-Myc.

    Alexandrov I, Shlyakhova L, Vartanian A, Zajac-Kaye M and Alexandrova N

    Mental Health Research Center, Russian Academy of Medical Sciences, Moscow.

    We have demonstrated that the 50 N-terminal amino acids of c-Myc bind a kinase activity, which phosphorylates Myc in vitro predominantly on Thr8. We also have shown that c-Raf, a widely known Ser/Thr kinase, involved in the Ras signaling pathway, binds to the same portion of c-Myc in vitro. In addition we were able to precipitate native c-Myc/Raf complex from various cell lysates. Physical interaction of Myc and Raf may potentially be a part of their well-known functional cooperation.

    FEBS letters 1997;414;2;465-70

  • 14-3-3 zeta negatively regulates raf-1 activity by interactions with the Raf-1 cysteine-rich domain.

    Clark GJ, Drugan JK, Rossman KL, Carpenter JW, Rogers-Graham K, Fu H, Der CJ and Campbell SL

    Department of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA.

    Although Raf-1 is a critical effector of Ras signaling and transformation, the mechanism by which Ras promotes Raf-1 activation is complex and remains poorly understood. We recently reported that Ras interaction with the Raf-1 cysteine-rich domain (Raf-CRD, residues 139-184) may be required for Raf-1 activation. The Raf-CRD is located in the NH2-terminal negative regulatory domain of Raf-1 and is highly homologous to cysteine-rich domains found in protein kinase C family members. Recent studies indicate that the structural integrity of the Raf-CRD is also critical for Raf-1 interaction with 14-3-3 proteins. However, whether 14-3-3 proteins interact directly with the Raf-CRD and how this interaction may mediate Raf-1 function has not been determined. In the present study, we demonstrate that 14-3-3 zeta binds directly to the isolated Raf-CRD. Moreover, mutation of Raf-1 residues 143-145 impairs binding of 14-3-3, but not Ras, to the Raf-CRD. Introduction of mutations that impair 14-3-3 binding resulted in full-length Raf-1 mutants with enhanced transforming activity. Thus, 14-3-3 interaction with the Raf-CRD may serve in negative regulation of Raf-1 function by facilitating dissociation of 14-3-3 from the NH2 terminus of Raf-1 to promote subsequent events necessary for full activation of Raf-1.

    Funded by: NCI NIH HHS: CA42978, CA64569, CA70308; ...

    The Journal of biological chemistry 1997;272;34;20990-3

  • Phosphorylation of Raf-1 serine 338-serine 339 is an essential regulatory event for Ras-dependent activation and biological signaling.

    Diaz B, Barnard D, Filson A, MacDonald S, King A and Marshall M

    Department of Medicine, Walther Oncology Center, Indiana University School of Medicine, Indianapolis 46202, USA.

    Activation of the Raf serine/threonine protein kinases is tightly regulated by multiple phosphorylation events. Phosphorylation of either tyrosine 340 or 341 in the catalytic domain of Raf-1 has been previously shown to induce the ability of the protein kinase to phosphorylate MEK. By using a combination of mitogenic and enzymatic assays, we found that phosphorylation of the adjacent residue, serine 338, and, to a lesser extent, serine 339 is essential for the biological and enzymatic activities of Raf-1. Replacement of S338 with alanine blocked the ability of prenylated Raf-CX to transform Rat-1 fibroblasts. Similarly, the loss of S338-S339 in Raf-1 prevented protein kinase activation in COS-7 cells by either oncogenic Ras[V12] or v-Src. Consistent with phosphorylation of S338-S339, acidic amino acid substitutions of these residues partially restored transforming activity to Raf-CX, as well as kinase activation of Raf-1 by Ras[V12] or v-Src. Two-dimensional phosphopeptide mapping of wild-type Raf-CX and Raf-CX[A338A339] confirmed the presence of a phosphoserine-containing peptide with the predicted mobility in the wild-type protein which was absent from the mutant. This peptide could be quantitatively precipitated by an antipeptide antibody specific for the 18-residue tryptic peptide containing S338-S339 and was demonstrated to contain only phosphoserine. Phosphorylation of this peptide in Raf-1 was significantly increased by coexpression with Ras[V12]. These data demonstrate that Raf-1 residues 338 to 341 constitute a unique phosphoregulatory site in which the phosphorylation of serine and tyrosine residues contributes to the regulation of Raf by Ras, Src, and Ras-independent membrane localization.

    Molecular and cellular biology 1997;17;8;4509-16

  • Activation of CDC 25 phosphatase and CDC 2 kinase involved in GL331-induced apoptosis.

    Huang TS, Shu CH, Yang WK and Whang-Peng J

    Cancer Research Division, National Health Research Institutes, Veterans General Hospital-Taipei, Taiwan, Republic of China. tshuang@gate.sinica.edu.tw

    CDC 25 is a dual phosphatase responsible for dephosphorylation and, thus, activation of CDC 2 kinase in G2. Abnormal activation of cyclin B-associated CDC 2 kinase has been implicated in apoptosis induced by cancer chemotherapeutic agents such as paclitaxel (Taxol) and etoposide (VP-16). In this study, we found that the CDC 2 kinase could be transiently activated when nasopharyngeal carcinoma NPC-TW01 cells were treated for 3 h with a new anticancer agent, GL331. GL331 treatment also induced a concomitant increase in CDC 25A phosphatase activity and a reduced level of Tyr-15-phosphorylated CDC 2 in NPC-TW01 cells. Furthermore, subsequent apoptotic DNA fragmentation induced by GL331 could be interrupted by treatment of the cells with the cyclin B1-specific antisense oligonucleotides, suggesting that abnormal activation of cyclin B1-associated CDC 2 kinase and CDC 25A phosphatase was involved in GL331-induced apoptosis. Raf-1 has been shown to associate with CDC 25A and, thus, to stimulate its phosphatase activity. Our results revealed that GL331 could facilitate the association of CDC 25A with Raf-1, resulting in the cascade of CDC 25A phosphatase activation and CDC 2 kinase activation, as well as related signaling pathways, and ultimately causing apoptosis in cancer cells.

    Cancer research 1997;57;14;2974-8

  • Interaction of activated Ras with Raf-1 alone may be sufficient for transformation of rat2 cells.

    Stang S, Bottorff D and Stone JC

    Department of Biochemistry, University of Alberta, Edmonton, Canada.

    v-H-ras effector mutants have been assessed for transforming activity and for the ability of the encoded proteins to interact with Raf-1-, B-Raf-, byr2-, ralGDS-, and CDC25-encoded proteins in the yeast two-hybrid system. Transformation was assessed in rat2 cells as well as in a mutant cell line, rv68BUR, that affords a more sensitive transformation assay. Selected mutant Ras proteins were also examined for their ability to interact with an amino-terminal fragment of Raf-1 in vitro. Finally, possible cooperation between different v-H-ras effector mutants and between effector mutants and overexpressed Raf-1 was assessed. Ras transforming activity was shown to correlate best with the ability of the encoded protein to interact with Raf-1. No evidence for cooperation between v-H-ras effector mutants was found. Signaling through the Raf1-MEK-mitogen-activated protein kinase cascade may be the only effector pathway contributing to RAS transformation in these cells.

    Molecular and cellular biology 1997;17;6;3047-55

  • Raf-1 kinase and exoenzyme S interact with 14-3-3zeta through a common site involving lysine 49.

    Zhang L, Wang H, Liu D, Liddington R and Fu H

    Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.

    14-3-3 proteins are a family of conserved dimeric molecules that bind to a range of cellular proteins involved in signal transduction and oncogenesis. Our solution of the crystal structure of 14-3-3zeta revealed a conserved amphipathic groove that may allow the association of 14-3-3 with diverse ligands (Liu, D., Bienkowska, J., Petosa, C., Collier, R. J., Fu, H., and Liddington, R. (1995) Nature 376, 191-194). Here, the contributions of three positively charged residues (Lys-49, Arg-56, and Arg-60) that lie in this Raf-binding groove were investigated. Two of the charge-reversal mutations greatly (K49E) or partially (R56E) decreased the interaction of 14-3-3zeta with Raf-1 kinase, whereas R60E showed only subtle effects on the binding. Interestingly, these mutations exhibited similar effects on the functional interaction of 14-3-3zeta with another target protein, exoenzyme S (ExoS), an ADP-ribosyltransferase from Pseudomonas aeruginosa. The EC50 values of 14-3-3zeta required for ExoS activation increased by approximately 110-, 5-, and 2-fold for the K49E, R56E, and R60E mutants, respectively. The drastic reduction of 14-3-3zeta/ligand affinity by the K49E mutation is due to a local electrostatic effect, rather than the result of a gross structural alteration, as evidenced by partial proteolysis and circular dichroism analysis. This work identifies the first point mutation (K49E) that dramatically disrupts 14-3-3zeta/ligand interactions. The parallel effects of this single point mutation on both Raf-1 binding and ExoS activation strongly suggest that diverse associated proteins share a common structural binding determinant on 14-3-3zeta.

    Funded by: NIGMS NIH HHS: GM53165

    The Journal of biological chemistry 1997;272;21;13717-24

  • Protein binding and signaling properties of RIN1 suggest a unique effector function.

    Han L, Wong D, Dhaka A, Afar D, White M, Xie W, Herschman H, Witte O and Colicelli J

    Department of Biological Chemistry, Molecular Genetics, and Immunology, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90095, USA.

    Human RIN1 was first characterized as a RAS binding protein based on the properties of its carboxyl-terminal domain. We now show that full-length RIN1 interacts with activated RAS in mammalian cells and defines a minimum region of 434 aa required for efficient RAS binding. RIN1 interacts with the "effector domain" of RAS and employs some RAS determinants that are common to, and others that are distinct from, those required for the binding of RAF1, a known RAS effector. The same domain of RIN1 that binds RAS also interacts with 14-3-3 proteins, extending the similarity between RIN1 and other RAS effectors. When expressed in mammalian cells, the RAS binding domain of RIN1 can act as a dominant negative signal transduction blocker. The amino-terminal domain of RIN1 contains a proline-rich sequence similar to consensus Src homology 3 (SH3) binding regions. This RIN1 sequence shows preferential binding to the ABL-SH3 domain in vitro. Moreover, the amino-terminal domain of RIN1 directly associates with, and is tyrosine phosphorylated by, c-ABL. In addition, RIN1 encodes a functional SH2 domain that has the potential to activate downstream signals. These data suggest that RIN1 is able to mediate multiple signals. A differential pattern of expression and alternate splicing indicate several levels of RIN1 regulation.

    Funded by: NCI NIH HHS: CA53867, CA56301, R01 CA056301, R01 CA071443; NIGMS NIH HHS: GM24787

    Proceedings of the National Academy of Sciences of the United States of America 1997;94;10;4954-9

  • Coassociation of Rap1A and Ha-Ras with Raf-1 N-terminal region interferes with ras-dependent activation of Raf-1.

    Hu CD, Kariya Ki, Kotani G, Shirouzu M, Yokoyama S and Kataoka T

    Department of Physiology II, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650, Japan.

    Raf-1 is a major downstream effector of mammalian Ras. Binding of the effector domain of Ras to the Ras-binding domain of Raf-1 is essential for Ras-dependent Raf-1 activation. However, Rap1A, which has an identical effector domain to that of Ras, cannot activate Raf-1 and even antagonizes several Ras functions in vivo. Recently, we identified the cysteine-rich region (CRR) of Raf-1 as another Ras-binding domain. Ha-Ras proteins carrying mutations N26G and V45E, which failed to bind to CRR, also failed to activate Raf-1. Since these mutations replace Ras residues with those of Rap1A, we examined if Rap1A lacks the ability to bind to CRR. Contrary to the expectation, Rap1A exhibited a greatly enhanced binding to CRR compared with Ha-Ras. Enhanced CRR binding was also found with Ha-Ras carrying another Rap1A-type mutation E31K. Both Rap1A and Ha-Ras(E31K) mutant failed to activate Raf-1 and interfered with Ha-Ras-dependent activation of Raf-1 in Sf9 cells. Enhanced binding of Rap1A to CRR led to co-association of Rap1A and Ha-Ras with Raf-1 N-terminal region through binding to CRR and Ras-binding domain, respectively. These results suggest that Rap1A interferes with Ras-dependent Raf-1 activation by inhibiting binding of Ras to Raf-1 CRR.

    The Journal of biological chemistry 1997;272;18;11702-5

  • Role of phosphoinositide 3-OH kinase in cell transformation and control of the actin cytoskeleton by Ras.

    Rodriguez-Viciana P, Warne PH, Khwaja A, Marte BM, Pappin D, Das P, Waterfield MD, Ridley A and Downward J

    Imperial Cancer Research Fund, London, United Kingdom.

    The pathways by which mammalian Ras proteins induce cortical actin rearrangement and cause cellular transformation are investigated using partial loss of function mutants of Ras and activated and inhibitory forms of various postulated target enzymes for Ras. Efficient transformation by Ras requires activation of other direct effectors in addition to the MAP kinase kinase kinase Raf and is inhibited by inactivation of the PI 3-kinase pathway. Actin rearrangement correlates with the ability of Ras mutants to activate PI 3-kinase. Inhibition of PI 3-kinase activity blocks Ras induction of membrane ruffling, while activated PI 3-kinase is sufficient to induce membrane ruffling, acting through Rac. The ability of activated Ras to stimulate PI 3-kinase in addition to Raf is therefore important in Ras transformation of mammalian cells and essential in Ras-induced cytoskeletal reorganization.

    Cell 1997;89;3;457-67

  • Physical association with ras enhances activation of membrane-bound raf (RafCAAX).

    Mineo C, Anderson RG and White MA

    Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9039, USA.

    The transforming activity of artificially membrane-targeted Raf1 suggests that Ras-mediated recruitment of Raf1 to the plasma membrane is an important step in Raf1 activation. Cellular Ras is concentrated in the caveolae, a microdomain of the plasma membrane that is highly enriched in caveolin, glycosylphosphatidylinositol-anchored proteins, and signal transduction molecules. Growth factor stimulation recruits Raf1 to this membrane domain. Whether Ras simply promotes Raf1 association with caveolae membranes or also modulates subsequent activation events is presently unclear. We have identified a ras variant, ras12V,37G, that does not interact with Raf1 but does interact with a mutant raf1, raf1(257L). To examine the role of Ras in the activation of membrane-bound Raf1, raf1CAAX, and raf1(257L)CAAX, membrane-targeted variants of Raf1 and raf1(257L), respectively, were expressed in fibroblasts with or without coexpression of ras12V, 37G. Cell fractionation localized both raf1CAAX and raf1(257L)CAAX to caveolae membranes independent of ras12V,37G expression; however, coexpression of ras12V,37G enhanced the activation of raf(257L)CAAX, but not raf1CAAX, as monitored by induction of cellular transformation, increased Raf kinase activity, and induction of activated MAP kinase. These results suggest that the Ras/Raf1 interaction plays a role in Raf1 activation that is distinct from membrane recruitment.

    Funded by: NCI NIH HHS: CA71443; NHLBI NIH HHS: HL20948; NIGMS NIH HHS: GM43169

    The Journal of biological chemistry 1997;272;16;10345-8

  • The complexity of Raf-1 regulation.

    Morrison DK and Cutler RE

    Molecular Basis of Carcinogenesis Laboratory, ABL-Basic Research Program, National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, MD 21702, USA.

    The activation of the serine/threonine kinase Raf-1 is proving to be an intricate multistep process. Recent advances in elucidating how Raf-1 becomes activated in response to signaling events have emphasized the role of phosphorylation and protein interactions in Raf-1 regulation. The picture clearly emerging is that Raf-1 activity can be regulated by multiple mechanisms.

    Current opinion in cell biology 1997;9;2;174-9

  • Signal transduction by the neurotrophin receptors.

    Kaplan DR and Miller FD

    Brain Tumor Research Centre, Montreal Neurological Institute, 3801 University Street, Montreal, PQ, Canada, H3A 2B4. mcdv@musica.mcgill.ca

    The neurotrophins signal cell survival, differentiation, growth cessation, and apoptosis through two cell surface receptors, the Trks and p75NTR (p75 neurotrophin receptor). Recent advances indicate that the particular events that are mediated by neurotrophins are dependent upon the cell type and the expression pattern of each neurotrophin receptor. For example, TrkA activation induces cell death of neural tumor cells, and survival and differentiation of neurons. Likewise, p75NTR, when activated in the absence of a strong Trk signal, induces apoptosis of neurons, while in the presence of Trk it enhances responses to neurotrophin. These differing responses point to a complex interplay between neurotrophin-stimulated survival, differentiation, and apoptosis pathways.

    Current opinion in cell biology 1997;9;2;213-21

  • Identification of Raf-1 Ser621 kinase activity from NIH 3T3 cells as AMP-activated protein kinase.

    Sprenkle AB, Davies SP, Carling D, Hardie DG and Sturgill TW

    Howard Hughes Medical Institute, Department of Medicine, University of Virginia, Charlottesville 22908, USA.

    Raf-1 is extensively phosphorylated on Ser621 in both quiescent and mitogen-stimulated cells. To identify the responsible kinase(s), cytosolic fractions of NIH 3T3 cells were analyzed for Ser621 peptide kinase activity. One major peak of activity was detected and identified as AMP-activated protein kinase (AMPK) by immunodepletion experiments. AMPK phosphorylated the catalytic domain of Raf-1, expressed in Escherichia coli as a soluble GST fusion protein, to generate a single tryptic [32P]phosphopeptide containing exclusively phospho-Ser621. AMPK also phosphorylated full-length, kinase-defective Raf-1 (K375M) to generate two [32P]phosphopeptides, one co-migrating with synthetic tryptic peptide containing phospho-Ser621 and the other with phospho-Ser259.

    Funded by: Wellcome Trust

    FEBS letters 1997;403;3;254-8

  • Rheb interacts with Raf-1 kinase and may function to integrate growth factor- and protein kinase A-dependent signals.

    Yee WM and Worley PF

    Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

    Rheb is a recently described member of the Ras family that was originally identified as an immediate-early gene in brain but is also widely expressed in other tissues. Here we demonstrate that Rheb interacts with and appears to regulate Raf-1 kinase, an essential component of the H-Ras signaling pathway. In direct contrast to H-Ras, however, the interaction of Rheb with Raf-1 is potentiated by growth factors in combination with agents that increase cyclic AMP (cAMP) levels. Protein kinase A-dependent phosphorylation of serine 43 within the regulatory domain of Raf-1 reciprocally potentiates its interaction with Rheb and decreases its interaction with H-Ras. A single amino acid in the G2 effector domain is critical for the differential properties of Rheb. Since Rheb is an immediate-early gene, our studies suggest that Rheb functions in concert with H-Ras to dynamically integrate cAMP and growth factor signaling.

    Funded by: NIMH NIH HHS: KO2 MH1152, NRSA F31 MH10847, R01 MH53608

    Molecular and cellular biology 1997;17;2;921-33

  • Activation of T cell Raf-1 at mitosis requires the protein-tyrosine kinase Lck.

    Pathan NI, Ashendel CL, Geahlen RL and Harrison ML

    Department of Biology, Purdue University, West Lafayette, Indiana 47907, USA.

    The serine/threonine protein kinase Raf-1 is activated in response to a variety of growth factors in fibroblasts and hematopoietic cells. In T cells, Raf-1 is activated in response to stimulation through the T cell antigen receptor, the interleukin-2 receptor, and by stimulation of protein kinase C. We demonstrate here that in T cells, Raf-1 is also activated during mitosis. The mitotic activation of Raf-1 was not observed in the Lck-deficient cell line, J.CaM.1. During mitosis, Raf-1 was found to interact selectively with a mitotic form of Lck that migrated with a reduced electrophoretic mobility on SDS-polyacrylamide gels. We conclude that Raf-1 is activated during mitosis in T cells and that this mitotic activation of Raf-1 is dependent on the presence of Lck.

    Funded by: NIGMS NIH HHS: GM48099

    The Journal of biological chemistry 1996;271;48;30315-7

  • Bcl-2 targets the protein kinase Raf-1 to mitochondria.

    Wang HG, Rapp UR and Reed JC

    The Burnham Institute, Program on Apoptosis and Cell Death Research, La Jolla, California 92037, USA.

    A green fluorescent protein (GFP)-Raf-1 fusion protein was used to show that Bcl-2 can target this kinase to mitochondria. Active Raf-1 fused with targeting sequences from an outer mitochondrial membrane protein protected cells from apoptosis and resulted in phosphorylation of BAD, a proapoptotic Bcl-2 homolog. Plasma membrane-targeted Raf-1 did not protect from apoptosis and resulted in phosphorylation of ERK-1 and ERK-2. Untargeted active Raf-1 improved Bcl-2-mediated resistance to apoptosis, whereas a kinase-inactive Raf-1 mutant abrogated apoptosis suppression by Bcl-2. Bcl-2 can therefore target Raf-1 to mitochondrial membranes, allowing this kinase to phosphorylate BAD or possibly other protein substrates involved in apoptosis regulation.

    Funded by: NCI NIH HHS: CA-67329

    Cell 1996;87;4;629-38

  • Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-X(L)

    Zha J, Harada H, Yang E, Jockel J and Korsmeyer SJ

    Department of Medicine, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

    Extracellular survival factors alter a cell's susceptibility to apoptosis, often through posttranslational mechanisms. However, no consistent relationship has been established between such survival signals and the BCL-2 family, where the balance of death agonists versus antagonists determines susceptibility. One distant member, BAD, heterodimerizes with BCL-X(L) or BCL-2, neutralizing their protective effect and promoting cell death. In the presence of survival factor IL-3, cells phosphorylated BAD on two serine residues embedded in 14-3-3 consensus binding sites. Only the nonphosphorylated BAD heterodimerized with BCL-X(L) at membrane sites to promote cell death. Phosphorylated BAD was sequestered in the cytosol bound to 14-3-3. Substitution of serine phosphorylation sites further enhanced BAD's death-promoting activity. The rapid phosphorylation of BAD following IL-3 connects a proximal survival signal with the BCL-2 family, modulating this checkpoint for apoptosis.

    Funded by: NCI NIH HHS: R01 CA50239

    Cell 1996;87;4;619-28

  • Binding of human immunodeficiency virus type 1 to CD4 induces association of Lck and Raf-1 and activates Raf-1 by a Ras-independent pathway.

    Popik W and Pitha PM

    Oncology Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21231, USA.

    We have analyzed CD4-mediated signaling during the early stages of human immunodeficiency virus type 1 (HIV-1) infection. Binding of purified HIV-1 virions or recombinant HIV-1 glycoprotein gp120 to CD4 receptors resulted in association and tyrosine phosphorylation and activation of tyrosine kinase Lck and serine/threonine kinase Raf-1. The association between Lck and Raf-1 was mediated by stimulation of the CD4 receptors, since it was abolished by preincubation of the virus with soluble CD4 and was not detected in CD4-negative A201 T cells. However, the Lck-Raf-1 association was restored in A201 cells permanently transfected with human CD4 cDNA and stimulated with anti-CD4 antibodies. In addition, a catalytically active Lck was required for the association of Lck and Raf-1. Surprisingly, the CD4-mediated signaling, induced by the HIV-1 binding, did not result in stimulation of the Ras GTP-binding activity or its association with Raf-1, indicating that the signaling pathway generated by the HIV-1 binding is not identical to the classical Ras/Raf-1 pathway. Furthermore, overexpression of activated Raf-1 in Jurkat T cells stimulated the HIV long terminal repeat promoter activity and significantly enhanced HIV-1 replication. This suggests that the Lck-Raf-1 pathway, rapidly stimulated by the binding of HIV-1 or gp120 to CD4 receptors, may play an essential role in the transcriptional activation of the integrated HIV-1 provirus as well as in its pathogenicity.

    Funded by: NIAID NIH HHS: AI26123

    Molecular and cellular biology 1996;16;11;6532-41

  • Association of a p95 Vav-containing signaling complex with the FcepsilonRI gamma chain in the RBL-2H3 mast cell line. Evidence for a constitutive in vivo association of Vav with Grb2, Raf-1, and ERK2 in an active complex.

    Song JS, Gomez J, Stancato LF and Rivera J

    Section on Chemical Immunology, NIAMSD, National Institutes of Health, Bethesda, Maryland 20892, USA.

    Aggregation of the high affinity receptor for IgE (FcepsilonRI) on the mucosal mast cell line, RBL-2H3, results in the rapid and persistent tyrosine phosphorylation of Vav. Immunoprecipitation of Vav from activated cells revealed co-immunoprecipitated phosphoproteins of molecular weights identical to the FcepsilonRI beta and gamma chains, and the former was reactive with antibody to the FcepsilonRI beta chain. Conversely, Western blots revealed the presence of p95 Vav in FcepsilonRI immunoprecipitates. The association of Vav and of Grb2 with the receptor was found to be regulated by aggregation of the receptor, and the interaction of Vav with the FcepsilonRI was localized to the gamma chain. To gain insight on the signaling pathway in which Vav participates, we investigated the in vivo associations of Vav with other molecules. A reducible chemical cross-linking agent was used to covalently maintain protein interactions under nonreducing conditions. A fraction of Vav increased in mass to form a complex of >300 kDa in molecular mass. Under reducing conditions the cross-linked Vav immunoprecipitates showed the presence of Grb2, Raf-1, and p42(mapk) (ERK2). In vitro kinase assays of Raf-1 activity associated with Vav revealed that this complex had an activity greater than that of Raf-1 derived from nonactivated cells, and aggregation of the FcepsilonRI did not modulate this activity. In contrast, aggregation of the FcepsilonRI increased the total Raf-1 activity by 2-5-fold. These results demonstrate that Vav associates constitutively with components of the mitogen-activated protein kinase pathway to form an active multimeric signaling complex whose in vivo activity and associations may be directed by aggregation of the FcepsilonRI. The findings of this study may also be relevant to other members of the immune recognition receptor family that share the T-cell antigen receptor zeta/gamma chains.

    The Journal of biological chemistry 1996;271;43;26962-70

  • The cytokine-activated tyrosine kinase JAK2 activates Raf-1 in a p21ras-dependent manner.

    Xia K, Mukhopadhyay NK, Inhorn RC, Barber DL, Rose PE, Lee RS, Narsimhan RP, D'Andrea AD, Griffin JD and Roberts TM

    Dana-Farber Cancer Institute, Boston, MA, USA.

    JAK2, a member of the Janus kinase superfamily was found to interact functionally with Raf-1, a central component of the ras/mitogen-activated protein kinase signal transduction pathway. Interferon-gamma and several other cytokines that are known to activate JAK2 kinase were also found to stimulate Raf-1 kinase activity toward MEK-1 in mammalian cells. In the baculovirus coexpression system, Raf-1 was activated by JAK2 in the presence of p21ras. Under these conditions, a ternary complex of p21ras, JAK2, and Raf-1 was observed. In contrast, in the absence of p21ras, coexpression of JAK2 and Raf-1 resulted in an overall decrease in the Raf-1 kinase activity. In addition, JAK2 phosphorylated Raf-1 at sites different from those phosphorylated by pp60v-src. In mammalian cells treated with either erythropoietin or interferon-gamma, a small fraction of Raf-1 coimmunoprecipitated with JAK2 in lysates of cells in which JAK2 was activated as judged by its state of tyrosine phosphorylation. Taken together, these data suggest that JAK2 and p21ras cooperate to activate Raf-1.

    Funded by: NCI NIH HHS: CA36167, CA43803; NIDDK NIH HHS: DK43889

    Proceedings of the National Academy of Sciences of the United States of America 1996;93;21;11681-6

  • Increased expression of the Ras suppressor Rsu-1 enhances Erk-2 activation and inhibits Jun kinase activation.

    Masuelli L and Cutler ML

    Laboratory of Tumor Immunology and Biology, National Cancer Institute, Bethesda, Maryland 20892, USA.

    Studies were undertaken to determine the effect of the Ras suppressor Rsu-1 on Ras signal transduction pathways in two different cell backgrounds. An expression vector containing the mouse rsu-1 cDNA under the control of a mouse mammary tumor virus promoter was introduced into NIH 3T3 cells and the pheochromocytoma cell line PC12. Cell lines developed in the NIH 3T3 background expressed p33rsu-1 at approximately twice the normal endogenous level. However, PC12 cell clones which expressed p33rsu-1 at an increased level in a regulatable fashion in response to dexamethasone were isolated. Analysis of proteins involved in regulation of Ras and responsive to Ras signal transduction revealed similar changes in the two cell backgrounds in the presence of elevated p33rsu-1. There was an increase in the level of SOS, the guanine nucleotide exchange factor, and an increase in the percentage of GTP-bound Ras. In addition, there was an increase in the amount of p120 Ras-specific GTPase-activating protein (GAP) and GAP-associated p190. However, a decrease in Ras GTPase-activating activity was detected in lysates of the Rsu-1 transfectants, and immunoprecipitated p120 GAP from the Rsu-1 transfectants showed less Ras GTPase-activating activity than GAP from control cells. Activation of Erk-2 kinase by growth factor and tetradecanyol phorbol acetate was greater in the Rsu-1 transfectants than in control cells. However, c-Jun amino-terminal kinase activity (Jun kinase) was not activatable by epidermal growth factor in Rsu-1 PC12 cell transfectants, in contrast to the PC12 vector control cell line. Transient expression of p33rsu-1 in Cos1 cells following cotransfection with either hemagglutinin-tagged Jun kinase or hemagglutinin-tagged Erk-2 revealed that Rsu-1 expression inhibited constitutive Jun kinase activity while enhancing Erk-2 activity. Detection of in vitro binding of Rsu-1 to Raf-1 suggested that in Rsu-1 transfectants, increased activation of the Raf-1 pathway occurred at the expense of activation of signal transduction leading to Jun kinase. These results indicate that inhibition of Jun kinase activation was sufficient to inhibit Ras transformation even in the presence of activated Erk-2.

    Molecular and cellular biology 1996;16;10;5466-76

  • 14-3-3 proteins associate with A20 in an isoform-specific manner and function both as chaperone and adapter molecules.

    Vincenz C and Dixit VM

    Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.

    A20, a novel zinc finger protein, is an inhibitor of tumor necrosis factor-induced apoptosis. The mechanism by which A20 exerts its protective effect is currently unknown. Several isoforms of the 14-3-3 proteins were found to interact with A20 in a yeast two-hybrid screen. A20 bound several 14-3-3 isoforms in vitro. Moreover, transfected A20 was found to preferentially bind the endogenous eta14-3-3 isoform, whereas the beta/zeta isoforms co-immunoprecipitated much less efficiently, and epsilon14-3-3 had an intermediate affinity. Importantly, c-Raf, a previously described 14-3-3-interacting protein, also preferentially bound the eta isoform. The cellular localization and subcellular fractionation of A20 was dramatically altered by co-transfected 14-3-3, providing the first experimental evidence for the notion that 14-3-3 can function as a chaperone. Furthermore, c-Raf and A20 co-immunoprecipitated in a 14-3-3-dependent manner, suggesting that 14-3-3 can function as a bridging or adapter molecule.

    Funded by: NCI NIH HHS: CA61348

    The Journal of biological chemistry 1996;271;33;20029-34

  • The solution structure of the Raf-1 cysteine-rich domain: a novel ras and phospholipid binding site.

    Mott HR, Carpenter JW, Zhong S, Ghosh S, Bell RM and Campbell SL

    Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill 27599, USA.

    The Raf-1 protein kinase is the best-characterized downstream effector of activated Ras. Interaction with Ras leads to Raf-1 activation and results in transduction of cell growth and differentiation signals. The details of Raf-1 activation are unclear, but our characterization of a second Ras-binding site in the cysteine-rich domain (CRD) and the involvement of both Ras-binding sites in effective Raf-1-mediated transformation provides insight into the molecular aspects and consequences of Ras-Raf interactions. The Raf-1 CRD is a member of an emerging family of domains, many of which are found within signal transducing proteins. Several contain binding sites for diacylglycerol (or phorbol esters) and phosphatidylserine and are believed to play a role in membrane translocation and enzyme activation. The CRD from Raf-1 does not bind diacylglycerol but interacts with Ras and phosphatidylserine. To investigate the ligand-binding specificities associated with CRDs, we have determined the solution structure of the Raf-1 CRD using heteronuclear multidimensional NMR. We show that there are differences between this structure and the structures of two related domains from protein kinase C (PKC). The differences are confined to regions of the CRDs involved in binding phorbol ester in the PKC domains. Since phosphatidylserine is a common ligand, we expect its binding site to be located in regions where the structures of the Raf-1 and PKC domains are similar. The structure of the Raf-1 CRD represents an example of this family of domains that does not bind diacylglycerol and provides a framework for investigating its interactions with other molecules.

    Funded by: NCI NIH HHS: R01 CA70308-1

    Proceedings of the National Academy of Sciences of the United States of America 1996;93;16;8312-7

  • Loss of Raf-1-binding activity of v-Ha-Ras by the deletion of amino acid residues 64-72 and 143-151.

    Hiwasa T, Kasama M, Nakadai T, Sawada T and Sakiyama S

    Division of Biochemistry, Chiba Cancer Center Research Institute, Japan.

    In order to elucidate the molecular events in signal transduction, examination of the interaction between Ras and Raf-1 seems crucial. Many Raf-1 mutants have been investigated in terms of their binding activities to Ras, where only a few Ras mutants have been examined thus far. We have investigated the Raf-1-binding activities of v-Ha-Ras and 21 insertion/deletion mutants of this protein. The results show that the mutants have varying levels of Raf-1-binding activity that are related neither to their transforming activity nor to their guanine nucleotide-binding activity. Deletion in the effector domain of Ras did not completely abolish Raf-1-binding, whereas the deletion in amino acid residues 64-72 or 143-151 resulted in complete loss of Raf-1-binding activity.

    Cellular signalling 1996;8;5;393-6

  • Ras/Rap effector specificity determined by charge reversal.

    Nassar N, Horn G, Herrmann C, Block C, Janknecht R and Wittinghofer A

    Max-Planck-Institut für molekulare Physiologie, Abteilung Strukturelle Biologie, Dortmund, Germany.

    Members of the Ras subfamily of small GTP-binding proteins have been shown to be promiscuous towards a variety of putative effector molecules such as the protein kinase c-Raf and the Ral-specific guanine nucleotide exchange factor (Ral-GEF). To address the question of specificity of interactions we have introduced the mutations E30D and K31E into Rap and show biochemically, by X-ray structure analysis and by transfection in vivo that the identical core effector region of Ras and Rap (residues 32-40) is responsible for molecular recognition, but that residues outside this region are responsible for the specificity of the interaction. The major determinant for the switch in specificity is the opposite charge of residue 31--Lys in Rap, Glu in Ras--which creates a favourable complementary interface for the Ras-Raf interaction.

    Nature structural biology 1996;3;8;723-9

  • Bcl-2 interacting protein, BAG-1, binds to and activates the kinase Raf-1.

    Wang HG, Takayama S, Rapp UR and Reed JC

    The Burnham Institute, La Jolla, CA 92037, USA.

    The Bcl-2 protein blocks programmed cell death (apoptosis) through an unknown mechanism. Previously we identified a Bcl-2 interacting protein BAG-1 that enhances the anti-apoptotic effects of Bcl-2. Like BAG-1, the serine/threonine protein kinase Raf-1 also can functionally cooperate with Bcl-2 in suppressing apoptosis. Here we show that Raf-1 and BAG-1 specifically interact in vitro and in yeast two-hybrid assays. Raf-1 and BAG-1 can also be coimmunoprecipitated from mammalian cells and from insect cells infected with recombinant baculoviruses encoding these proteins. Furthermore, bacterially-produced BAG-1 protein can increase the kinase activity of Raf-1 in vitro. BAG-1 also activates this mammalian kinase in yeast. These observations suggest that the Bcl-2 binding protein BAG-1 joins Ras and 14-3-3 proteins as potential activators of the kinase Raf-1.

    Funded by: NCI NIH HHS: CA67329

    Proceedings of the National Academy of Sciences of the United States of America 1996;93;14;7063-8

  • Regulation of neuromodulatory actions of angiotensin II in the brain neurons by the Ras-dependent mitogen-activated protein kinase pathway.

    Yang H, Lu D, Yu K and Raizada MK

    Department of Physiology, University of Florida, College of Medicine, Gainesville, Florida 32610, USA.

    Angiotensin II (Ang II) stimulates norepinephrine transporter (NET) and tyrosine hydroxylase (TH) in the neurons, but the signal transduction mechanism of this neuromodulation is not understood. Treatment of neuronal cultures of hypothalamus-brainstem with Ang II resulted in a time- and dose-dependent activation of Ras, Raf-1, and mitogen-activated protein kinase. This activation was mediated by the interaction of Ang II with the AT1, receptor subtype and was associated with the redistribution of AT1 receptor with Ras and Raf-1 on the neuronal membrane. Treatment with antisense oligonucleotide (AON) to mitogen-activated protein kinase decreased mitogen-activated protein kinase immunoreactivity by 70% and attenuated Ang II stimulation of c-fos, NET, and TH mRNA levels. This demonstrates that induction of these genes requires mitogen-activated protein kinase activation by Ang II. In contrast, AON to mitogen-activated protein kinase failed to inhibit Ang II stimulation of plasminogen activator inhibitor-1 mRNA levels. These results suggest that AT1 receptors are coupled to a Ras-Raf-1 mitogen-activated protein kinase signal transduction pathway that is responsible for stimulation of NET and TH, two neuro-modulatory actions of Ang II in the brain.

    Funded by: PHS HHS: 33610 (NIH)

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1996;16;13;4047-58

  • Activation-modulated association of 14-3-3 proteins with Cbl in T cells.

    Liu YC, Elly C, Yoshida H, Bonnefoy-Berard N and Altman A

    Division of Immunobiology, La Jolla Institute for Allergy and Immunology, La Jolla, California 92037, USA.

    14-3-3 proteins have recently been implicated in the regulation of intracellular signaling pathways via their interaction with several oncogene and protooncogene products. We found recently that 14-3-3 associates with several tyrosine-phosphorylated proteins and phosphatidylinositol 3-kinase (PI3-K) in T cells. We report here the identification of the 120-kDa 14-3-3tau-binding phosphoprotein present in activated T cell lysates as Cbl, a protooncogene product of unknown function which was found recently to be a major protein-tyrosine kinase (PTK) substrate, and to interact with several signaling molecules including PI3-K, in T lymphocytes. The association between 14-3-3tau and Cbl was detected both in vitro and in intact T cells and, in contrast to Raf-1, was markedly increased following T cell activation. The use of truncated 14-3-3tau fusion proteins demonstrated that the 15 C-terminal residues are required for the association between 14-3-3 and three of its target proteins, namely, Cbl, Raf-1, and PI3-K. The findings that 14-3-3tau binds both PI3-K and Cbl, together with recent reports of an association between Cbl and PI3-K, suggest that 14-3-3 dimers play a critical role in signal transduction processes by promoting and coordinating protein-protein interactions of signaling proteins.

    Funded by: NCI NIH HHS: CA35299; NIGMS NIH HHS: GM50819

    The Journal of biological chemistry 1996;271;24;14591-5

  • Identification of neurofibromin mutants that exhibit allele specificity or increased Ras affinity resulting in suppression of activated ras alleles.

    Morcos P, Thapar N, Tusneem N, Stacey D and Tamanoi F

    Department of Microbiology and Molecular Genetics, University of California, Los Angeles 90095-1489, USA.

    Neurofibromin plays a critical role in the downregulation of Ras proteins in neurons and Schwann cells. Thus, the ability of neurofibromin to interact with Ras is crucial for its function, as mutations in NF1 that abolish this interaction fail to maintain function. To investigate the neurofibromin-Ras interaction in a systematic manner, we have carried out a yeast two-hybrid screen using a mutant of H-ras, H-rasD92K, defective for interaction with the GTPase-activated protein-related domain (GRD) of NF1. Two screens of a randomly mutagenized NF1-GRD library led to the identification of seven novel NF1 mutants. Characterization of the NF1-GRD mutants revealed that one class of mutants are allele specific for H-raSD92K. These mutants exhibit increased affinity for H-raSD92K and significantly reduced affinity for wild-type H-ras protein. Furthermore, they do not interact with another H-ras mutant defective for interaction with GTPase-activating proteins. Another class of mutants are high-affinity mutants which exhibit dramatically increased affinity for both wild-type and mutant forms of Ras. They also exhibit a striking ability to suppress the heat shock sensitive traits of activated RAS2G19v in yeast cells. Five mutations cluster within a region encompassing residues 1391 to 1436 (region II). Three NF1 patient mutations have previously been identified in this region. Two mutations that we identified occur in a region encompassing residues 1262 to 1276 (region I). Combining high-affinity mutations from both regions results in even greater affinity for Ras. These results demonstrate that two distinct regions of NF1-GRD are involved in the Ras interaction and that single amino acid changes can affect NF1's affinity for Ras.

    Funded by: NIGMS NIH HHS: GM52271; NINDS NIH HHS: NS30054

    Molecular and cellular biology 1996;16;5;2496-503

  • Selective activation of MEK1 but not MEK2 by A-Raf from epidermal growth factor-stimulated Hela cells.

    Wu X, Noh SJ, Zhou G, Dixon JE and Guan KL

    Department of Biological Chemistry and the Institute of Gerontology, the University of Michigan Medical School, Ann Arbor, Michigan 48109, USA.

    Activation of the mitogen-activated protein kinase cascade is a critical event in mitogenic growth factor signal transduction. Mitogen-activated protein kinase is directly activated by a dual specific kinase, MEK, which itself is activated by serine phosphorylation. The c-Raf kinase has been implicated in mediating the signal transduction from mitogenic growth factor receptors to MEK activation. Recently, the B-Raf kinase was shown to be capable of phosphorylating and activating MEK as a result of growth factor stimulation. In this report, we used the yeast two-hybrid screening to isolate MEK interacting proteins. All three members of the Raf family kinases were identified as positive clones when the mutant MEK1S218/222A, in which the two phosphorylation serine residues were substituted by alanines, was used as a bait, whereas no positive clones were isolated when the wild type MEK1 was used as a bait in a similar screening. These results suggest that elimination of the phosphorylation sites of a target protein (MEK1 in our study) may stabilize the interaction between the kinase (Raf) and its substrate (MEK1), possibly due the formation of a nonproductive complex. These observations seem to suggest a general strategy using mutants to identify the upstream kinase of a phosphoprotein or the downstream targets of a kinase. Although c-Raf and B-Raf have been implicated in growth factor-induced MEK activation, little is known about A-Raf. We observed that stimulation of Hela cells with epidermal growth factor resulted in a rapid and transient activation of A-Raf, which is then capable of phosphorylating and activating MEK1. Interestingly, A-Raf does not activate MEK2, although c-Raf can activate both MEK1 and MEK2. Our data demonstrated that A-Raf is, indeed, a MEK1 activator and may play a role in growth factor signaling.

    The Journal of biological chemistry 1996;271;6;3265-71

  • Structure-function analysis of Bcl-2 family proteins. Regulators of programmed cell death.

    Reed JC, Zha H, Aime-Sempe C, Takayama S and Wang HG

    La Jolla Cancer Research Foundation, California 92037, USA.

    The Bcl-2 protein blocks a distal step in an evolutionarily conserved pathway for programmed cell death and apoptosis. To gain better understanding of how this protein functions, we have undertaken a structure-function analysis of this protein, focusing on domains within Bcl-2 that are required for function and for interactions with other proteins. Four conserved domains are present in Bcl-2 and several of its homologs: BH1 (residues 136-155), BH2 (187-202), BH3 (93-107) and BH4 (10-30). Deletion of the BH1, BH2, or BH4 domains of Bcl-2 abolishes its ability to suppress cell death in mammalian cells and prevents homodimerization of these mutant proteins, though these mutants can still bind to the wild-type Bcl-2 protein. These mutants also fail to bind to BAG-1 and Raf-1, two proteins that we have shown can associate with protein complexes containing Bcl-2 and which cooperate with Bcl-2 to suppress cell death. Deletion of either BH1 or BH2 nullifies the ability of Bcl-2 to: (a) suppress death in mammalian cells: (b) block Bax-induced lethality in yeast; and (c) heterodimerize with Bax. In contrast, deletion of the BH4 domain of Bcl-2 nullifies anti-apoptotic function and homodimerization, but does not impair binding to the pro-apoptotic protein Bax. Taken together, the data suggest the possibility that both Bcl-2/Bcl-2 homodimerization and Bcl-2/Bax heterodimerization are necessary but insufficient for the anti-apoptotic function of the Bcl-2 protein. Homodimerization of Bcl-2 with itself involves a head-to-tail interaction, in which an N-terminal domain where BH4 resides interacts with the more distal region of Bcl-2 where BH1, BH2, and BH3 are located. In contrast, Bcl-2/Bax heterodimerization involves a tail-to-tail interaction, that requires the portion of Bcl-2 where BH1, BH2, and BH3 reside and a central region in Bax where the BH3 domain is located. The BH3 domain of Bax is also required for Bax/Bax homodimerization and pro-apoptotic function in both yeast and mammalian cells. Thus, Bcl-2 may suppress cell death at least in part by binding to Bax via the BH3 domain and thereby preventing formation of Bax/Bax homodimers. Further studies however are required to delineate the full significance of Bcl-2/Bcl-2, Bcl-2/Bax, and Bax/Bax dimers and the biochemical mechanisms by which Bcl-2 family proteins ultimately control cell life and death.

    Advances in experimental medicine and biology 1996;406;99-112

  • Cysteine-rich region of Raf-1 interacts with activator domain of post-translationally modified Ha-Ras.

    Hu CD, Kariya K, Tamada M, Akasaka K, Shirouzu M, Yokoyama S and Kataoka T

    Department of Physiology II, Kobe University School of Medicine, Japan.

    The interaction between "switch I/effector domain" of Ha-Ras and the Ras-binding domain (RBD, amino acid 51-131) of Raf-1 is essential for signal transduction. However, the importance of the "activator domain" (approximately corresponding to amino acids 26-28 and 40-49) of Ha-Ras and of the "cysteine-rich region" (CRR, amino acids 152-184) of Raf-1 have also been proposed. Here, we found that Raf-1 CRR interacts directly with Ha-Ras independently of RBD and that participation of CRR is necessary for efficient Ras-Raf binding. Furthermore, Ha-Ras carrying mutations (N26G and V45E) in the activator domain failed to bind CRR, whereas they bound RBD normally. On the contrary, Ha-Ras carrying mutations in the switch I/effector domain exhibited severely reduced ability to bind RBD, whereas their ability to bind CRR was unaffected. Mutants that bound to either RBD or CRR alone failed to activate Raf-1. Ha-Ras without post-translational modifications, which lacks the ability to activate Raf-1, selectively lost the ability to bind CRR. These results suggest that the activator domain of Ha-Ras participates in activation of Raf-1 through interaction with CRR and that post-translational modifications of Ha-Ras are required for this interaction.

    The Journal of biological chemistry 1995;270;51;30274-7

  • Phosphorylation of Raf by ceramide-activated protein kinase.

    Yao B, Zhang Y, Delikat S, Mathias S, Basu S and Kolesnick R

    Laboratory of Signal Transduction, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.

    The sphingomyelin pathway, initiated by hydrolysis of sphingomyelin to ceramide and stimulation of a Ser/Thr ceramide-activated protein (CAP) kinase, mediates tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 beta action. CAP kinase is membrane-bound and proline-directed, recognizing the minimal substrate motif Thr-Leu-Pro. TNF may use the sphingomyelin pathway to signal Raf1 to activate the MAP kinase cascade. Evidence shows that cytoplasmic Raf1 binds to GTP-ras upon cellular stimulation, is recruited to the plasma membrane, and activated. How membrane-bound Raf1 is activated is uncertain, but regulation of its kinase activity may involve its phosphorylation. Specific Raf kinases, however, have not hitherto been identified. Here we report that CAP kinase phosphorylates Raf1 on Thr 269, increasing its activity towards MEK (MAP kinase or ERK kinase). Moreover, in intact HL-60 cells, CAP kinase complexes with Raf1 and, in response to TNF and ceramide analogues, phosphorylates and activates Raf1, implicating CAP kinase as a link between the TNF receptor and Raf1.

    Nature 1995;378;6554;307-10

  • Identification of the 14.3.3 zeta domains important for self-association and Raf binding.

    Luo ZJ, Zhang XF, Rapp U and Avruch J

    Diabetes Research Laboratory, Massachusetts General Hospital, Charlestown 02129, USA.

    The 14.3.3 zeta protein is a ubiquitous and abundant arachidonate-selective acyltransferase and putative phospholipase A2, which self-assembles into dimers and binds to c-Raf-1 and other polypeptides in vitro and in intact cells. The 14.3.3 polypeptides endogenous to Sf9 cells associate in situ with both active and inactive recombinant Raf and copurify at a fairly reproducible molar ratio that is probably 1. Purified baculoviral recombinant Raf, despite its preassociated 14.3.3 polypeptide, binds additional recombinant 14.3.3 zeta polypeptide in vitro, in a saturable and specific reaction, forming a complex that is resistant to 1 M LiCl. A two-hybrid analysis indicates that 14.3.3 zeta binds primarily to Raf noncatalytic sequences distinct from those that bind Ras-GTP, and in vitro 14.3.3 zeta binds to Raf without inhibiting the Ras-Raf association or Raf-catalyzed MEK phosphorylation. Deletion analysis of 14.3.3 zeta (1-245) indicates that the 14.3.3 domain responsible for binding to Raf extends over the carboxyl-terminal 100 amino acids, whereas 14.3.3 dimerization is mediated by amino-terminal sequences. As with Ras, the 14.3.3 zeta polypeptide does not activate purified Raf directly in vitro. Moreover, expression of recombinant 14.3.3 zeta in COS cells beyond the substantial level of endogenous 14.3.3 protein does not alter endogenous Raf kinase, as judged by the activity of a cotransfected Erk-1 reporter. Coexpression of recombinant 14.3.3 with recombinant Myc-tagged Raf in COS cells does increase substantially the Myc-Raf kinase activity achieved during transient expression, which is attributable primarily to an increased level of Myc-Raf polypeptide, without alteration of Myc-Raf specific activity or the activation that occurs in response to epidermal growth factor or 12-O-tetradecanoylphorbol-13-acetate. Nevertheless, evidence that 14.3.3 actively participates in Raf activation in situ is provided by the finding that although full-length 14.3.3 zeta binds active Raf in situ, truncated versions of 14.3.3, some of which bind Raf polypeptide in situ nearly as well as full-length 14.3.3 zeta, are recovered in association only with inactive Raf polypeptides. Thus, 14.3.3 polypeptides bind tightly to one or more sites on c-Raf. Overexpression of 14.3.3 zeta enhances the expression of recombinant Raf, perhaps by stabilizing the Raf polypeptide. In addition, Raf polypeptides bound to truncated 14.3.3 polypeptides are unable to undergo activation in situ, indicating that 14.3.3 participates in the process of Raf activation by mechanisms that remain to be elucidated.

    The Journal of biological chemistry 1995;270;40;23681-7

  • 14-3-3 proteins associate with cdc25 phosphatases.

    Conklin DS, Galaktionov K and Beach D

    Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, NY 11724, USA.

    The cdc25 phosphatases play key roles in cell cycle progression by activating cyclin-dependent kinases. Two members of the 14-3-3 protein family have been isolated in a yeast two-hybrid screen designed to identify proteins that interact with the human cdc25A and cdc25B phosphatases. Genes encoding the human homolog of the 14-3-3 epsilon protein and the previously described 14-3-3 beta protein have been isolated in this screening. 14-3-3 proteins constitute a family of well-conserved eukaryotic proteins that were originally isolated in mammalian brain preparations and that possess diverse biochemical activities related to signal transduction. We present evidence that indicates that cdc25 and 14-3-3 proteins physically interact both in vitro and in vivo. 14-3-3 protein does not, however, affect the phosphatase activity of cdc25A. Raf-1, which is known to bind 14-3-3 proteins, has recently been shown to associate with cdc25A and to stimulate its phosphatase activity. 14-3-3 protein, however, has no effect on the cdc25A-kinase activity of Raf-1. Instead, 14-3-3 may facilitate the association of cdc25 with Raf-1 in vivo, participating in the linkage between mitogenic signaling and the cell cycle machinery.

    Proceedings of the National Academy of Sciences of the United States of America 1995;92;17;7892-6

  • Characterization of the interaction of Raf-1 with ras p21 or 14-3-3 protein in intact cells.

    Koyama S, Williams LT and Kikuchi A

    Department of Biochemistry, Hiroshima University School of Medicine, Japan.

    Several deletion mutants of Raf-1 were expressed with v-ras p21 or 14-3-3 protein in COS-7 cells and Sf9 cells and the interaction of Raf-1 with ras p21 or with 14-3-3 protein in intact cells was examined. Raf(1-135) (residues 1-135) and Raf(1-322) interacted with v-ras p21, but other deletion mutants such as Raf(136-322) or Raf(321-648) did not. Raf(1-322) interacted with 14-3-3 protein much more efficiently than Raf(321-648) did. While Raf(1-135) did not interact with 14-3-3 protein, Raf(136-322) did. These results clearly indicate that Raf-1 simultaneously interacts with both ras p21 and 14-3-3 protein through the distinct binding domains in intact cells.

    FEBS letters 1995;368;2;321-5

  • A direct interaction between G-protein beta gamma subunits and the Raf-1 protein kinase.

    Pumiglia KM, LeVine H, Haske T, Habib T, Jove R and Decker SJ

    Parke-Davis Pharmaceutical Research Division, Department of Signal Transduction, Ann Arbor, Michigan 48106, USA.

    Raf-1 is a serine/threonine protein kinase positioned downstream of Ras in the mitogen-activated protein kinase cascade. Using a yeast two-hybrid strategy to identify other proteins that interact with and potentially regulate Raf-1, we isolated a clone encoding the carboxyl-terminal half of the G beta 2 subunit of heterotrimeric G-proteins. In vitro, purified G beta gamma subunits specifically bound to a GST fusion protein encoding amino acids 1-330 of Raf-1 (Raf/330). Binding assays with truncation mutants of GST-Raf indicate that the region located between amino acids 136 and 239 is a primary determinant for interaction with G beta gamma. In competition experiments, the carboxyl terminus of beta-adrenergic receptor kinase (beta ARK) blocked the binding of G beta gamma to Raf/330; however, the Raf-1-binding proteins, Ras and 14-3-3, had no effect. Scatchard analysis of in vitro binding between Raf/330 and G beta gamma revealed an affinity of interaction (Kd = 163 +/- 36 nM), similar to that seen between G beta gamma and beta ARK (Kd = 87 +/- 24 nM). The formation of native heterotrimeric G alpha beta gamma complexes, as measured by pertussis toxin ADP-ribosylation of G alpha, could be disrupted by increasing amounts of Raf/330, with an EC50 of approximately 200 nM, in close agreement with the estimated binding affinity. In vivo complexes of Raf-1 and G beta gamma were isolated from human embryonic kidney 293-T cells transfected with epitope-tagged G beta 2. The identification and characterization of this novel interaction raises several possibilities for signaling cross-talk between growth factor receptors and those receptors coupled to heterotrimeric G-proteins.

    Funded by: NCI NIH HHS: CA55652

    The Journal of biological chemistry 1995;270;24;14251-4

  • The 2.2 A crystal structure of the Ras-binding domain of the serine/threonine kinase c-Raf1 in complex with Rap1A and a GTP analogue.

    Nassar N, Horn G, Herrmann C, Scherer A, McCormick F and Wittinghofer A

    Max-Planck-Institut für molekulare Physiologie, Abteilung Strukturelle Biologie, Dortmund, Germany.

    The X-ray crystal structure of the complex between the Ras-related protein Rap1A in the GTP-analogue (GppNHp) form and the Ras-binding domain (RBD) of the Ras effector molecule c-Raf1, a Ser/Thr-specific protein kinase, has been solved to a resolution of 2.2 A. It shows that RBD has the ubiquitin superfold and that the structure of Rap1A is very similar to that of Ras. The interaction between the two proteins is mediated by an apparent central antiparallel beta-sheet formed by strands B1-B2 from RBD and strands beta 2-beta 3 from Rap1A. Complex formation is mediated by main-chain and side-chain interactions of the so-called effector residues in the switch I region of Rap1A.

    Nature 1995;375;6532;554-60

  • Solution structure of the Ras-binding domain of c-Raf-1 and identification of its Ras interaction surface.

    Emerson SD, Madison VS, Palermo RE, Waugh DS, Scheffler JE, Tsao KL, Kiefer SE, Liu SP and Fry DC

    Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey 07110, USA.

    The structure of the Ras-binding domain of human c-Raf-1 (residues 55-132) has been determined in solution by nuclear magnetic resonance (NMR) spectroscopy. Following complete assignment of the backbone and side-chain 1H, 15N, and 13C resonances, the structure was calculated using the program CHARMM. Over 1300 NOE-derived constraints were applied, resulting in a detailed structure. The fold of Raf55-132 consists of a five-stranded beta-sheet, a 12-residue alpha-helix, and an additional one-turn helix. It is similar to those of ubiquitin and the IgG-binding domain of protein G, although the three proteins share very little sequence identity. The surface of Raf55-132 that interacts with Ras has been identified by monitoring perturbation of line widths and chemical shifts of 15N-labeled Raf55-132 resonances during titration with unlabeled Ras-GMPPNP. The Ras-binding site is contained within a spatially contiguous patch comprised of the N-terminal beta-hairpin and the C-terminal end of the alpha-helix.

    Biochemistry 1995;34;21;6911-8

  • Raf1 interaction with Cdc25 phosphatase ties mitogenic signal transduction to cell cycle activation.

    Galaktionov K, Jessus C and Beach D

    Howard Hughes Medical Institute, Cold Spring Harbor Laboratory, New York 11724, USA.

    The Ras and Raf1 proto-oncogenes transduce extracellular signals that promote cell growth. Cdc25 phosphatases activate the cell division cycle by dephosphorylation of critical threonine and tyrosine residues within the cyclin-dependent kinases. We show here that Cdc25 phosphatase associates with raf1 in somatic mammalian cells and in meiotic frog oocytes. Furthermore, Cdc25 phosphatase can be activated in vitro in a Raf1-dependent manner. We suggest that activation of the cell cycle by the Ras/Raf1 pathways might be mediated in part by Cdc25.

    Genes & development 1995;9;9;1046-58

  • Two distinct Raf domains mediate interaction with Ras.

    Brtva TR, Drugan JK, Ghosh S, Terrell RS, Campbell-Burk S, Bell RM and Der CJ

    Cirruculum in Genetics and Molecular Biology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill 27599, USA.

    A key event for Ras transformation involves the direct physical association between Ras and the Raf-1 kinase. This interaction promotes both Raf translocation to the plasma membrane and activation of Raf kinase activity. Although substantial experimental evidence has demonstrated that Raf residues 51-131 alone are sufficient for Ras binding, conflicting observations have suggested that the Raf cysteine-rich domain (residues 139-184) may also be important for interaction with Ras. To clarify the role of the Raf cysteine-rich domain in Ras-Raf binding, we have compared the ability of two distinct Raf fragments to interact with Ras using both in vitro Ras binding and in vivo Ras inhibition assays. First, we determined that both Raf sequences 2-140 and 139-186 (designated Raf-Cys) showed preferential binding to active, GTP-bound Ras in vitro. Second, we observed that Raf-Cys antagonized oncogenic Ras(Q61L)-mediated transactivation of Ras-responsive elements and focus-forming activity in NIH 3T3 cells and insulin-induced germinal vesicle breakdown in Xenopus laevis oocytes in vivo. This inhibitory activity suggests that Raf-Cys can interact with Ras in vivo. Taken together, these results suggest that Ras interaction with two distinct domains of Raf-1 may be important in Ras-mediated activation of Raf kinase activity.

    Funded by: NCI NIH HHS: CA42978, CA52072, CA55008; ...

    The Journal of biological chemistry 1995;270;17;9809-12

  • B-Raf protein isoforms interact with and phosphorylate Mek-1 on serine residues 218 and 222.

    Papin C, Eychène A, Brunet A, Pagès G, Pouysségur J, Calothy G and Barnier JV

    Unité Mixte de Recherche 146 du CNRS, Institut Curie, ORSAY, France.

    The B-raf/c-Rmil proto-oncogene belongs to the raf/mil family of serine/threonine protein kinases. It encodes multiple protein isoforms resulting from alternative splicing of two exons located upstream of the kinase domain. Recent studies suggested that B-Raf could be the intermediate molecule between Ras and Mek-1 (MAP Kinase Kinase) in signalling pathways specific of neural cells. However, there has been no evidence for a direct interaction between B-Raf and Mek-1. We report here that different B-Raf isoforms can be co-immunoprecipitated with anti-Mek-1 antisera in COS-1 cells and that the kinase activity of B-Raf is not required for its interaction with Mek-1. We also show that all B-Raf isoforms tested phosphorylate Mek-1 in a time-dependent manner, whereas kinase defective mutants fail to do so. Finally, we demonstrate that the constitutively activated S218D, S222D and S218D/S222D mutants of Mek-1 interact similarly with B-Raf. However, only the S218D and S222D mutants, and not the S218D/S222D double mutant, can be phosphorylated by B-Raf isoforms. Therefore, serine residues 218 and 222, previously shown to regulate Mek-1 activity, appear to be the major phosphorylation sites by B-Raf in vitro.

    Oncogene 1995;10;8;1647-51

  • A human protein selected for interference with Ras function interacts directly with Ras and competes with Raf1.

    Han L and Colicelli J

    Department of Biological Chemistry, UCLA School of Medicine 90024.

    The overexpression of some human proteins can cause interference with the Ras signal transduction pathway in the yeast Saccharomyces cerevisiae. The functional block is located at the level of the effector itself, since these proteins do not suppress activating mutations further downstream in the same pathway. We now demonstrate, with in vivo and in vitro experiments, that the protein encoded by one human cDNA (clone 99) can interact directly with yeast Ras2p and with human H-Ras protein, and we have named this gene rin1 (Ras interaction/interference). The interaction between Ras and Rin1 is enhanced when Ras is bound to GTP. Rin1 is not able to interact with either an effector mutant or a dominant negative mutant of H-Ras. Thus, Rin1 displays a human H-Ras interaction profile that is the same as that seen for Raf1 and yeast adenylyl cyclase, two known effectors of Ras. Moreover, Raf1 directly competes with Rin1 for binding to H-Ras in vitro. Unlike Raf1, however, the Rin1 protein resides primarily at the plasma membrane, where H-Ras is localized. These data are consistent with Rin1 functioning in mammalian cells as an effector or regulator of H-Ras.

    Funded by: NCI NIH HHS: CA-56301

    Molecular and cellular biology 1995;15;3;1318-23

  • RAS and RAF-1 form a signalling complex with MEK-1 but not MEK-2.

    Jelinek T, Catling AD, Reuter CW, Moodie SA, Wolfman A and Weber MJ

    Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville 22908.

    Recent studies have demonstrated the existence of a physical complex containing p21ras (RAS), p74raf-1 (RAF-1), and MEK-1. Although it is clear that formation of this complex depends on the activation state of RAS, it is not known whether this complex is regulated by the activation state of the cell and whether MEK-2 is also present in the complex. To analyze the regulation and specificity of this complex, we utilized immobilized RAS to probe lysates of cultured NIH 3T3 fibroblasts and analyzed the proteins complexing with RAS following serum starvation or stimulation. Complex formation among RAS, RAF-1, and MEK-1 was dependent only on RAS:GMP-PNP and not on cell stimulation. Incubations of lysates with immobilized RAS depleted all RAF-1 from the lysate but bound only a small fraction of cytosolic MEK-1, and further MEK-1 could bind immobilized RAS only if exogenous RAF-1 was added to the lysate. This indicates that binding of MEK-1 to RAS depends on the presence of RAF-1 or an equivalent protein. In contrast to MEK-1, MEK-2 was not detected in the RAS signalling complex. A proline-rich region of MEK-1 containing a phosphorylation site appears to be essential for signalling complex formation. Consistent with the preferential binding of MEK-1 to RAS:RAF-1, the basal activity of MEK-1 in v-ras-transformed cells was found to be elevated sixfold, whereas MEK-2 was elevated only twofold, suggesting that the RAS signalling pathway favors MEK-1 activation.

    Funded by: NCI NIH HHS: CA39076; NIGMS NIH HHS: GM 41220, GM 47332

    Molecular and cellular biology 1994;14;12;8212-8

  • The ubiquitously expressed Syp phosphatase interacts with c-kit and Grb2 in hematopoietic cells.

    Tauchi T, Feng GS, Marshall MS, Shen R, Mantel C, Pawson T and Broxmeyer HE

    Department of Medicine (Hematology/Oncology), Indiana University School of Medicine, Indianapolis 46202.

    The c-kit proto-oncogene encodes a transmembrane tyrosine kinase receptor, which is important for the normal development of hematopoietic cells, melanoblasts, and germ cells. Autophosphorylation of c-kit receptor on tyrosine creates binding sites for cellular src homology 2 (SH2)-containing signaling molecules. The discovery of phosphotyrosine phosphatases that contain SH2 domains suggests roles for these molecules in growth factor signaling pathways. We found that Syp, a phosphotyrosine phosphatase widely expressed in all the tissues in mammals, associates with c-kit receptor after activation with its ligand, steel factor, in the factor-dependent cell line, M07e. Both NH2-terminal and COOH-terminal SH2 domains of Syp, made as glutathione S-transferase fusion proteins, were able to bind to the activated c-kit receptor in vitro. Furthermore, Syp became marginally phosphorylated on tyrosine upon c-kit receptor activation, and tyrosine-phosphorylated Syp was found to be complexed with Grb2 in steel factor-stimulated M07e cells. Direct binding between Syp and Grb2 was also observed in vitro. Last, Ras and Raf interacts in vitro as a result of steel factor-stimulated Ras activation. These results suggest that Syp may be an important signaling component downstream of the c-kit receptor and involved in activation of the Ras signaling pathway in hematopoietic cells.

    Funded by: NCI NIH HHS: R37 CA36464; NHLBI NIH HHS: R01 HL46549, R01 HL49202

    The Journal of biological chemistry 1994;269;40;25206-11

  • Interaction of the protein kinase Raf-1 with 14-3-3 proteins.

    Fu H, Xia K, Pallas DC, Cui C, Conroy K, Narsimhan RP, Mamon H, Collier RJ and Roberts TM

    Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115.

    Members of a family of highly conserved proteins, termed 14-3-3 proteins, were found by several experimental approaches to associate with Raf-1, a central component of a key signal transduction pathway. Optimal complex formation required the amino-terminal regulatory domain of Raf-1. The association of 14-3-3 proteins and Raf-1 was not substantially affected by the activation state of Raf.

    Funded by: NCI NIH HHS: CA57327, R01 CA057327; NIAID NIH HHS: AI22021; NICHD NIH HHS: HD24926

    Science (New York, N.Y.) 1994;266;5182;126-9

  • Human ltk receptor tyrosine kinase binds to PLC-gamma 1, PI3-K, GAP and Raf-1 in vivo.

    Kozutsumi H, Toyoshima H, Hagiwara K, Yazaki Y and Hirai H

    Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan.

    Leukocyte tyrosine kinase (ltk) is a receptor-type tyrosine kinase which is suggested to be expressed in hematopoietic cells and neuronal cells in human. Recently we have cloned a full sized human ltk cDNA which has a 423 amino acid extracellular domain which may bind to unknown ligand(s), and a 415 amino acid cytoplasmic domain which contains a tyrosine kinase domain. To identify the cellular signal transducer proteins binding to the ltk protein, we have analysed the recombinant ltk protein transiently expressed in COS cells. By an in vitro immune complex kinase assay, a major 140 kDa phosphoprotein and other cellular phosphoproteins were co-immunoprecipitated with the 100 kDa ltk protein using anti-ltk monoclonal antibodies. Western blot analysis revealed that the wild-type ltk protein was tyrosine-phosphorylated in vivo and associated with SH2 containing proteins, PLC-gamma 1, p85 subunit of PI3-K and GAP, in vivo. Furthermore, the wild-type ltk protein also binds to a serine/threonine kinase, Raf-1, in vivo. In contrast, none of these signal transducer proteins were associated with a kinase-negative ltk mutant (K544M-ltk) in which methionine at the putative ATP binding site was replaced with lysine. These results suggest that the associations of the ltk protein with those signaling molecules depend on the tyrosine kinase activity of the ltk protein. This is the first detection of cytoplasmic signal transducers that bind to the ltk protein in vivo.

    Oncogene 1994;9;10;2991-8

  • Signaling through transforming G protein-coupled receptors in NIH 3T3 cells involves c-Raf activation. Evidence for a protein kinase C-independent pathway.

    Crespo P, Xu N, Daniotti JL, Troppmair J, Rapp UR and Gutkind JS

    Molecular Signaling Unit, Laboratory of Cellular Development and Oncology, NIDR, National Institutes of Health, Bethesda, Maryland 20892.

    We have studied the role of Raf-1 in mitogenesis and cellular transformation induced by G protein-coupled receptors in NIH 3T3 cells transfected with the human m1 muscarinic receptor. We have observed that in m1-expressing NIH 3T3 cells, the cholinergic agonist carbachol induces a dose- and time-dependent shift in the electrophoretic mobility of p72Raf-1, equivalent to that observed when using phorbol esters or platelet-derived growth factor as stimulants. Phosphoamino acid analysis of slower mobility forms of p72Raf-1 revealed both phosphoserine and phosphothreonine. Carbachol potently induced c-Raf activity as judged by its in vitro phosphorylating activity using MEK as a substrate. However, induction of Raf-1 kinase activity by carbachol occurred much earlier than changes in its electrophoretic mobility. Raf-1 kinase activation followed a kinetic similar to that exhibited by an epitope-tagged ERK2 protein when coexpressed in the same cells. Conventional protein kinase C (PKC) inactivation by means of sustained phorbol ester treatment or by a new nontoxic PKC-specific inhibitor, GF 109203X, abolished p72Raf-1 mobility shift induced by carbachol or by phorbol esters. However, c-Raf and ERK2 enzymatic activity in response to carbachol was at least 50-80% PKC-independent. Furthermore, inhibition of PKC failed to affect DNA synthesis or focus formation induced by carbachol in cells expressing m1 receptors. In contrast, cotransfection of NIH 3T3 cells with the Raf-1 dominant negative mutant Raf-301 (K375W) drastically decreased the transforming ability of m1 receptors. Thus, our findings implicate Raf-1 activation in transformation by G protein-coupled receptors. In addition, our data suggest that activation of p72Raf-1 and ERK2 by G protein-coupled receptors involves PKC-independent pathways.

    The Journal of biological chemistry 1994;269;33;21103-9

  • Critical binding and regulatory interactions between Ras and Raf occur through a small, stable N-terminal domain of Raf and specific Ras effector residues.

    Chuang E, Barnard D, Hettich L, Zhang XF, Avruch J and Marshall MS

    Department of Medicine, Indiana University, Indianapolis 46202.

    Genetic and biochemical evidence suggests that the Ras protooncogene product regulates the activation of the Raf kinase pathway, leading to the proposal that Raf is a direct mitogenic effector of activated Ras. Here we report the use of a novel competition assay to measure in vitro the relative affinity of the c-Raf-1 regulatory region for Ras-GTP, Ras-GDP, and 10 oncogenic and effector mutant Ras proteins. c-Raf-1 associates with normal Ras and the oncogenic V12 and L61 forms of Ras with equal affinity. The moderately transforming mutant Ras[E30K31] also bound to the c-Raf-1 regulatory region with normal affinity. Transformation-defective Ras effector mutants Ras[N33], Ras[S35], and Ras[N38] bound poorly. In contrast, the transformation defective Ras[G26I27] and Ras[E45] mutants bound to the c-Raf-1 regulatory region with nearly wild-type affinity. A stable, high-affinity Ras-binding region of c-Raf-1 was mapped to a 99-amino-acid subfragment of the first 257 residues. The smallest Ras-binding region identified consisted of N-terminal residues 51 to 131, although stable expression of the domain and high-affinity binding were improved by the presence of residues 132 to 149. Deletion of the Raf zinc finger region did not reduce Ras-binding affinity, while removal of the first 50 amino acids greatly increased affinity. Phosphorylation of Raf[1-149] by protein kinase A on serine 43 resulted in significant inhibiton of Ras binding. demonstrating that the mechanism of cyclic AMP downregulation results through structural changes occurring exclusively in this small Ras-binding domain.

    Funded by: NIDDK NIH HHS: DK07519

    Molecular and cellular biology 1994;14;8;5318-25

  • Raf-1 interacts with Fyn and Src in a non-phosphotyrosine-dependent manner.

    Cleghon V and Morrison DK

    Molecular Mechanisms of Carcinogenesis Laboratory, ABL-Basic Research Program National Cancer Institute-Frederick Cancer Research and Development Center, Maryland 21702.

    To identify novel proteins capable of associating with the Raf-1 serine/threonine kinase, we investigated whether Raf-1 could interact with the Src homology 2 (SH2) domains of various signal-transducing molecules. In this report, we demonstrate that Raf-1 associated with the SH2 domain of Fyn (a member of the Src tyrosine kinase family) but not with the SH2 domains of phospholipase C-gamma 1, the p85 alpha subunit of phosphatidylinositol 3-kinase, and SH2-containing protein tyrosine phosphatase 2. Unlike most SH2 domain interactions that require tyrosine-phosphorylated residues, the Raf-1/Fyn SH2 domain association was dependent on the serine phosphorylation of Raf-1. Our results also demonstrate that Raf-1 interacted with the SH2 domain of Src and that this interaction was destabilized by mutation of Arg175 found within the conserved SH2 domain FLVRES sequence. In addition, we show that inclusion of additional Src sequences containing the SH3 domain increased the association of Raf-1 with the Src SH2 domain. Finally, using the baculovirus/Sf9 cell system, we show that coexpression of Raf-1 with full-length Fyn/Src resulted in the coimmunoprecipitation of Raf-1 with Fyn/Src, the tyrosine phosphorylation of Raf-1, and the stimulation of Raf-1 kinase activity. These results suggest that Raf-1 may form a functional complex with Fyn/Src mediated in part by SH2 domains and the serine phosphorylation of Raf-1.

    Funded by: NCI NIH HHS: N01-CO-74101

    The Journal of biological chemistry 1994;269;26;17749-55

  • Identification of the sites in MAP kinase kinase-1 phosphorylated by p74raf-1.

    Alessi DR, Saito Y, Campbell DG, Cohen P, Sithanandam G, Rapp U, Ashworth A, Marshall CJ and Cowley S

    Department of Biochemistry, University of Dundee, Scotland.

    Many growth factors whose receptors are protein tyrosine kinases stimulate the MAP kinase pathway by activating first the GTP-binding protein Ras and then the protein kinase p74raf-1. p74raf-1 phosphorylates and activates MAP kinase kinase (MAPKK). To understand the mechanism of activation of MAPKK, we have identified Ser217 and Ser221 of MAPKK1 as the sites phosphorylated by p74raf-1. This represents the first characterization of sites phosphorylated by this proto-oncogene product. Ser217 and Ser221 lie in a region of the catalytic domain where the activating phosphorylation sites of several other protein kinases are located. Among MAPKK family members, this region is the most conserved, suggesting that all members of the family are activated by the phosphorylation of these sites. A 'kinase-dead' MAPKK1 mutant was phosphorylated at the same residues as the wild-type enzyme, establishing that both sites are phosphorylated directly by p74raf-1, and not by autophosphorylation. Only the diphosphorylated form of MAPKK1 (phosphorylated at both Ser217 and Ser221) was detected, even when the stoichiometry of phosphorylation by p74raf-1 was low, indicating that phosphorylation of one of these sites is rate limiting, phosphorylation of the second then occurring extremely rapidly. Ser217 and Ser221 were both phosphorylated in vivo within minutes when PC12 cells were stimulated with nerve growth factor. Analysis of MAPKK1 mutants in which either Ser217 or Ser221 were changed to glutamic acid, and the finding that inactivation of maximally activated MAPKK1 required the dephosphorylation of both serines, shows that phosphorylation of either residue is sufficient for maximal activation.

    The EMBO journal 1994;13;7;1610-9

  • Interaction of Ras and Raf in intact mammalian cells upon extracellular stimulation.

    Hallberg B, Rayter SI and Downward J

    Imperial Cancer Research Fund, London, United Kingdom.

    It has recently been shown that Ras proteins interact directly with Raf serine/threonine kinases in vitro and in the yeast two-hybrid system, leading to speculation that Raf proteins function as effectors for Ras. Here it is demonstrated that the endogenous Raf-1 protein co-immunoprecipitates with Ras from mammalian cells when the non-neutralizing anti-Ras monoclonal antibody Y13-238 is used. The formation of a Ras-Raf complex is absolutely dependent on prior treatment of the cells with a stimulus that activates Ras: phorbol ester or anti-T cell receptor antibody in the case of human peripheral blood T lymphoblasts, or epidermal growth factor in the case of Rat-1 fibroblasts. Up to 3% of cellular Raf-1 can be found in association with Ras. The association is not competed by addition of exogenous GST-Raf to the cell lysates and is therefore unlikely to be due to Ras-Raf binding after cell lysis. Specific interaction of Ras and Raf therefore occurs in intact mammalian cells in response to stimuli that cause Ras to become GTP-bound.

    The Journal of biological chemistry 1994;269;6;3913-6

  • Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides.

    Maruyama K and Sugano S

    Institute of Medical Science, University of Tokyo, Japan.

    We have devised a method to replace the cap structure of a mRNA with an oligoribonucleotide (r-oligo) to label the 5' end of eukaryotic mRNAs. The method consists of removing the cap with tobacco acid pyrophosphatase (TAP) and ligating r-oligos to decapped mRNAs with T4 RNA ligase. This reaction was made cap-specific by removing 5'-phosphates of non-capped RNAs with alkaline phosphatase prior to TAP treatment. Unlike the conventional methods that label the 5' end of cDNAs, this method specifically labels the capped end of the mRNAs with a synthetic r-oligo prior to first-strand cDNA synthesis. The 5' end of the mRNA was identified quite simply by reverse transcription-polymerase chain reaction (RT-PCR).

    Gene 1994;138;1-2;171-4

  • Raf exists in a native heterocomplex with hsp90 and p50 that can be reconstituted in a cell-free system.

    Stancato LF, Chow YH, Hutchison KA, Perdew GH, Jove R and Pratt WB

    Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109.

    Recently, we have demonstrated that the tyrosine kinase pp60v-src can undergo cell-free assembly into a heterocomplex with rabbit hsp90 and p50 when the immunoadsorbed protein is incubated with rabbit reticulocyte lysate (Hutchison, K. A., Brott, B. K., De Leon, J. H., Perdew, G. H., Jove, R., and Pratt, W. B. (1992) J. Biol. Chem 267, 2902-2908). Using a baculovirus system to express a high level of human c-Raf serine/threonine kinase in Sf9 insect cells, we show here that immunoadsorbed c-Raf undergoes similar lysate-mediated assembly into a heterocomplex with hsp90 and p50. As with pp60v-src and steroid receptors, binding of c-Raf to hsp90 occurs in an ATP-dependent and K(+)-dependent manner and the resulting heterocomplex is stabilized by molybdate. With a very rapid and gentle procedure of Sf9 cell cytosol preparation and c-Raf immunoadsorption, we show coimmunoadsorption of the insect homologue of hsp90. The same procedures permit detection of a native complex of v-Raf with rat hsp90 and p50 in stably transfected rat 3Y1 fibroblasts, and v-Raf is also assembled into a heterocomplex with rabbit hsp90 and p50 by reticulocyte lysate. Using the 22W mutant of c-Raf in which the NH2-terminal half has been deleted, we show that the catalytic domain of the kinase is sufficient for both formation of the native heterocomplex in mouse NIH 3T3 cells and cell-free reconstitution of the heterocomplex by rabbit reticulocyte lysate. Although the native Raf-heat shock protein heterocomplex is less stable than native pp60v-src and glucocorticoid receptor heterocomplexes, by analogy with these proteins its detection may have important implications regarding the mechanism of Raf trafficking through the cytoplasm.

    Funded by: NCI NIH HHS: CA28010, CA55652; NIEHS NIH HHS: ESO4869

    The Journal of biological chemistry 1993;268;29;21711-6

  • Activation of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase by G protein and tyrosine kinase oncoproteins.

    Gardner AM, Vaillancourt RR and Johnson GL

    Division of Basic Sciences, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206.

    Mitogen-activated protein kinases (MAPKs) are rapidly phosphorylated and activated in response to a variety of extracellular stimuli in many different cell types. The kinases that activate MAPK, the MAPK/ERK Kinases (MEKs), are also activated by phosphorylation. We have studied the influence of specific oncogenes on the regulation of MEK activity in NIH3T3 and Rat1a fibroblasts. We show that a similar MEK activity phosphorylates and activates MAPK in both growth factor-stimulated (epidermal growth factor and thrombin) and oncogene (gip2, v-src, and v-raf)-transfected cells. Gip2 and v-Src activated MEK-1 in transfected Rat 1a cells, whereas v-Raf activated MEK-1 in transfected NIH3T3 cells. These cell-selective differences in MEK activation parallel constitutive MAPK activation in these cell lines. Stable expression of the v-ras oncogene resulted in little constitutive MEK activation in either cell line, even though both were highly transformed. The growth factor and oncoprotein regulated MEK activity co-fractionated by Mono S chromatography with the 45-kDa MEK-1 protein. We further demonstrate in NIH3T3 and Rat 1a cells that Raf-1 is activated, as measured by its ability to phosphorylate MEK-1, in response to epidermal growth factor but not thrombin. Thus, the regulatory network of protein kinases that activate MAPK converges at MEK but diverges with the kinases that phosphorylate and activate MEK.

    Funded by: NIDDK NIH HHS: DK37871; NIGMS NIH HHS: GM 30324

    The Journal of biological chemistry 1993;268;24;17896-901

  • Identification of the major phosphorylation sites of the Raf-1 kinase.

    Morrison DK, Heidecker G, Rapp UR and Copeland TD

    ABL-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Maryland 21702.

    Treatment of cells with various growth factors and mitogens results in the rapid hyperphosphorylation and activation of the Raf-1 kinase. To determine if phosphorylation events affect Raf-1 activity, we have initiated experiments to identify the phosphorylation sites of Raf-1. In this report, we find that Ser43, Ser259, and Ser621 are the major sites of Raf-1 which are phosphorylated in mammalian cells and in Sf9 insect cells infected with a recombinant baculovirus encoding human Raf-1. Mutant Raf-1 proteins lacking kinase activity are also phosphorylated on these sites in vivo, indicating that these phosphorylation events are not a consequence of autophosphorylation. Furthermore, we find that Thr268 is the predominant Raf-1 residue phosphorylated in in vitro autokinase assays. In addition, we have examined the biochemical activity of baculovirus-expressed Raf-1 proteins containing mutations at these phosphorylation sites. In in vitro protein kinase assays Ser259 mutant proteins were 2-fold more active than wild-type Raf-1 and Ser621 mutant proteins were inactive as kinases. Analysis of the residues surrounding Ser259 and Ser621 indicates that RSXSXP may be a consensus sequence for the kinase responsible for phosphorylation of Raf-1 at these sites. Interestingly, these RSXSXP sequences are completely conserved throughout evolution in all Raf family members.

    Funded by: NCI NIH HHS: N01-CO-74101

    The Journal of biological chemistry 1993;268;23;17309-16

  • Receptor accessory factor enhances specific DNA binding of androgen and glucocorticoid receptors.

    Kupfer SR, Marschke KB, Wilson EM and French FS

    Department of Pediatrics, University of North Carolina School of Medicine, Chapel Hill 27599.

    Protein-protein interactions are common among transcriptional activators and may have important consequences for gene regulation. Using the mobility shift assay, we have identified a factor that enhances specific DNA binding of truncated rat androgen (AR) and glucocorticoid (GR) receptors by 25- and 6-fold, respectively, through the formation of heteromeric complexes. This factor, designated receptor accessory factor, or RAF, also potentiates DNA binding of full-length human GR. RAF is temperature and trypsin sensitive and is present in a variety of cultured mammalian cells. By gel filtration RAF has a predicted molecular mass of 130 kDa. RAF enhancement of AR-DNA binding is optimal with androgen response element DNA. RAF appears to interact directly with AR because 1) deoxycholate, which interferes with protein-protein but not protein-DNA interactions, prevents RAF.AR.DNA complex formation, 2) RAF activity is recovered from an androgen response element DNA affinity column only in the presence of AR, and 3) RAF increases the size of an AR.DNA complex by gel filtration. Mutagenesis of truncated AR fragments indicates that a region in the NH2-terminal domain is required for RAF to enhance AR-DNA binding. The interaction of RAF with AR and GR suggests that RAF might influence the ability of these nuclear receptors to activate transcription.

    Funded by: NICHD NIH HHS: HD16910, K08-HD00963, P30-HD04466; ...

    The Journal of biological chemistry 1993;268;23;17519-27

  • Identification and characterization of a new mammalian mitogen-activated protein kinase kinase, MKK2.

    Wu J, Harrison JK, Dent P, Lynch KR, Weber MJ and Sturgill TW

    Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville 22908.

    Mitogen-activated protein (MAP) kinases are serine/threonine protein kinases activated by dual phosphorylation on threonine and tyrosine residues. A MAP kinase kinase (MKK1 or MEK1) has been identified as a dual-specificity protein kinase that is sufficient to phosphorylate MAP kinases p42mapk and p44mapk on the regulatory threonine and tyrosine residues. Because of the multiplicity of MAP kinase isoforms and the diverse circumstances and agonists leading to their activation, we thought it unlikely that a single MKK could accommodate this complexity. Indeed, two protein bands with MKK activity have previously been identified after renaturation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We now report the molecular cloning and characterization of a second rat MAP kinase kinase cDNA, MKK2. MKK2 cDNA contains an open reading frame encoding a protein of 400 amino acids, 7 residues longer than MKK1 (MEK1). The amino acid sequence of MKK2 is 81% identical to that of MKK1, but nucleotide sequence differences occur throughout the aligned MKK2 and MKK1 cDNAs, indicating that MKK2 is the product of a distinct gene. MKK1 and MKK2 mRNAs are expressed differently in rat tissues. Both cDNAs when expressed in COS cells displayed the ability to phosphorylate and activate p42mapk and p44mapk, both MKK1 and MKK2 were activated in vivo in response to serum, and both could be phosphorylated and activated by the v-Raf protein in vitro. However, differences between MKK1 and MKK2 in sites of phosphorylation by proline-directed protein kinases predict differences in feedback regulation.

    Funded by: NIDDK NIH HHS: DK38942, DK41077; NIGMS NIH HHS: GM47332

    Molecular and cellular biology 1993;13;8;4539-48

  • Direct interaction of Ras and the amino-terminal region of Raf-1 in vitro.

    Warne PH, Viciana PR and Downward J

    Imperial Cancer Research Fund, London, UK.

    The Ras proteins are key regulators of the growth of eukaryotic cells, but their direct target enzymes, or 'effectors', are unknown. The protein encoded by the c-raf-1 proto-oncogene is thought to function downstream of p21ras because disruption of Raf blocks signalling by Ras in a number of systems. Here we report that the amino-terminal cysteine-rich regulatory region of p74c-raf-1 expressed as a glutathione-S-transferase (GST) fusion protein binds directly to Ras with relatively high affinity (50 nM). The binding is strictly dependent on the Ras protein being in the active GTP-bound conformation rather than the inactive GDP-bound state. Raf-GST interacts with wild-type and oncogenic Ras (Val 12) but fails to interact with a biologically inert effector mutant of Ras (Ala 38) and a dominant negative mutant (Asn 17). A peptide based on the effector region of Ras inhibits the interaction. Raf-GST acts as a potent competitive inhibitor of the GTPase-activating proteins p120GAP and neurofibromin. In addition, Raf itself displays weak GTPase-stimulating activity towards Ras. It is therefore likely that Raf is a direct effector of Ras.

    Nature 1993;364;6435;352-5

  • Normal and oncogenic p21ras proteins bind to the amino-terminal regulatory domain of c-Raf-1.

    Zhang XF, Settleman J, Kyriakis JM, Takeuchi-Suzuki E, Elledge SJ, Marshall MS, Bruder JT, Rapp UR and Avruch J

    Diabetes Unit, Harvard Medical School, Charlestown, Massachusetts.

    In higher eukaryotes, the Ras and Raf-1 proto-oncoproteins transduce growth and differentiation signals initiated by tyrosine kinases. The Ras polypeptide and the amino-terminal regulatory domain of Raf-1 (residues 1-257) are shown to interact, directly in vitro and in a yeast expression system. Raf-1 (1-257) binds GTP-Ras in preference to GDP-Ras, and inhibits Ras-GAP activity. Mutations in and around the Ras effector domain impair Ras binding to Raf-1 (1-257) and Ras transforming activity in parallel.

    Nature 1993;364;6435;308-13

  • Protein kinase C alpha activates RAF-1 by direct phosphorylation.

    Kolch W, Heidecker G, Kochs G, Hummel R, Vahidi H, Mischak H, Finkenzeller G, Marmé D and Rapp UR

    Biological Research, Goedecke AG, Freiburg, Germany.

    The kinase Raf-1 can be activated by treatment of cells with mitogens and by the protein kinase C (PKC) activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) (reviewed in refs 1,2). Activated Raf-1 triggers a protein kinase cascade by direct phosphorylation of MAP kinase kinase, resulting in phosphorylation of ternary complex factor and Jun by MAP kinase. Here we investigate the molecular mechanism and biological consequences of PKC alpha-mediated Raf-1 activation in NIH3T3 fibroblasts. PKC alpha directly phosphorylates and activates Raf-1 both in vitro and in vivo. PKC alpha induces Raf-1 phosphorylation at several sites, including a serine residue at position 499. Mutation of serine at this position or at residue 259 does not abrogate Raf-1 stimulation by a combination of Ras plus the src tyrosine kinase Lck, but severely impedes Raf-1 activation by PKC alpha. Consistent with such a direct interaction is the observation that Raf-1 and PKC alpha cooperate in the transformation of NIH3T3 cells. The Ser499 phosphorylation site is necessary for this synergism.

    Nature 1993;364;6434;249-52

  • Interleukin-2 (IL-2) induces tyrosine kinase-dependent translocation of active raf-1 from the IL-2 receptor into the cytosol.

    Maslinski W, Remillard B, Tsudo M and Strom TB

    Department of Medicine, Beth Israel Hospital, Boston, Massachusetts.

    Stimulation of the interleukin-2 (IL-2) receptor results in phosphorylation and activation of cytosolic Raf-1 serine/threonine kinase. Herein, we report that enzymatically active Raf-1 is physically associated with the IL-2 receptor beta chain (p75) in T-cell blasts. Following stimulation with IL-2, Raf-1 dissociates from the IL-2 receptor complex and translocates to the cytosol. Genistein, a protein tyrosine kinase inhibitor, prevents the dissociation of enzymatically active Raf-1 from the ligand-stimulated IL-2 receptor complex. These data favor a model of IL-2 receptor activation in which an IL-2-activated protein tyrosine kinase phosphorylates the IL-2 receptor and/or receptor-bound Raf-1. Following tyrosine phosphorylation, enzymatically active Raf-1 dissociates from the IL-2 receptor and translocates into the cytosol.

    Funded by: NCI NIH HHS: UO1 CA 48626

    The Journal of biological chemistry 1992;267;22;15281-4

  • A genetic linkage map of 96 loci on the short arm of human chromosome 3.

    Tory K, Latif F, Modi W, Schmidt L, Wei MH, Li H, Cobler P, Orcutt ML, Delisio J, Geil L et al.

    Program Resources Inc., Frederick, Maryland 21701.

    We constructed a genetic map of 96 loci on the short arm of human chromosome 3 (3p) in 59 families provided by the Centre d'Etude du Polymorphisme Humaine (CEPH). Twenty-nine continuously linked loci were placed on the map with likelihood support of at least 1000:1; one locus, D3S213, was placed on the map with likelihood support of 871:1; D3Z1, an alpha satellite centromeric repeat probe, was placed on the map with likelihood support of 159:1; 65 loci were assigned regional locations. The average heterozygosity of the uniquely ordered markers was 49%. The map extends from 3p26, the terminal band of 3p, to the centromere (from D3S211 to D3Z1). Multipoint linkage analysis indicated that the male, female, and sex-averaged maps extend for 102, 147, and 116 cM, respectively. The mean genetic distance between uniquely ordered loci on the sex-averaged map was 4.0 cM. Probe density was greatest for the region of 3p between D3F15S2e and the telomere. The sex-averaged map contained two intervals greater than 10 cM. Seventeen probes were localized by fluorescence in situ hybridization. The loci described in this report will be useful in building an integrated genetic and physical map of this chromosome.

    Genomics 1992;13;2;275-86

  • Localization of the 5' end of the MCF2 oncogene to human chromosome 15q15----q23.

    Galland F, Stefanova M, Lafage M and Birnbaum D

    U.119 INSERM, Marseille, France.

    Oncogenic activation of the MCF.2 cell line-derived transforming sequence gene (MCF2) occurs through substitution of part of its 5' coding region by unrelated nonsyntenic sequences. Analysis of the MCF2 oncogene locus revealed complex recombination events involving four discontinuous human DNA segments. The upstream replacing sequence, named URS, represents the farthest 5' portion of the locus. The URS sequence maps to the D15S93 locus on human chromosome 15q15----q23.

    Cytogenetics and cell genetics 1992;60;2;114-6

  • Raf-1: a kinase currently without a cause but not lacking in effects.

    Li P, Wood K, Mamon H, Haser W and Roberts T

    Division of Cellular and Molecular Biology, Dana-Farber Cancer Institute, Boston, Massachusetts.

    Cell 1991;64;3;479-82

  • Direct activation of the serine/threonine kinase activity of Raf-1 through tyrosine phosphorylation by the PDGF beta-receptor.

    Morrison DK, Kaplan DR, Escobedo JA, Rapp UR, Roberts TM and Williams LT

    Department of Medicine, Howard Hughes Medical Institute, University of California, San Francisco 94143.

    We have examined the interaction between the serine/threonine kinase proto-oncogene product Raf-1 and the tyrosine kinase PDGF beta-receptor. Raf-1 tyrosine phosphorylation and kinase activity were increased by PDGF treatment of 3T3 cells or CHO cells expressing wild-type PDGF receptors but not mutant receptors defective in transmitting mitogenic signals, suggesting that the increase in Raf-1 kinase activity is a significant event in PDGF-induced mitogenesis. Concurrent with these increases, Raf-1 associated with the ligand-activated PDGF receptor. Furthermore, both mammalian Raf-1 and Raf-1 expressed using a recombinant baculoviral vector, associated in vitro with baculoviral-expressed PDGF receptor. This association was markedly decreased by prior phosphatase treatment of the receptor. Following incubation of partially purified baculoviral-expressed PDGF receptor with partially purified Raf-1, Raf-1 became phosphorylated on tyrosine and its serine/threonine kinase activity increased 4- to 6-fold. This is the first demonstration of the direct modulation of a protein activity by a growth factor receptor tyrosine kinase.

    Funded by: NHLBI NIH HHS: R01 HL-32898

    Cell 1989;58;4;649-57

  • The raf oncogene is associated with a radiation-resistant human laryngeal cancer.

    Kasid U, Pfeifer A, Weichselbaum RR, Dritschilo A and Mark GE

    In order to identify the genetic factors associated with the radiation-resistant human laryngeal carcinoma cell line (SQ-20B), tumor cell DNA was transfected into NIH/3T3 cells. A high incidence (six out of six) of raf sequences was found in transfected NIH/3T3 clones and the tumorigenic potential of SQ-20B DNA could be linked to genomic fragments that represent most of the kinase domain of human c-raf-1. An apparently unaltered 3.5-kilobase pair (kb) human c-raf transcript was identified in SQ-20B cells but was not observed in the transfected NIH/3T3 cell clones. Two new transcripts (4.2 kb and 2.6 kb) were found in tumorigenic clones; the large transcript was missing in a very poorly tumorigenic clone. Cytogenetic analysis indicated that the normal autosomes of chromosome 3 were absent in SQ-20B karyotypes and had formed apparently stable marker chromosomes. Unlike the recipient NIH/3T3 cell line, 30 percent of the transformed clone-1 metaphases had minute and double-minute chromosomes representative of amplified DNA sequences. The frequency of the c-raf-1 identification by NIH/3T3 transfection of SQ-20B DNA suggests the presence of some genetic abnormality within this locus.

    Funded by: NCI NIH HHS: CA425969

    Science (New York, N.Y.) 1987;237;4818;1039-41

  • Activation of human c-raf-1 by replacing the N-terminal region with different sequences.

    Tahira T, Ochiai M, Hayashi K, Nagao M and Sugimura T

    Two transformants of NIH 3T3 cells, obtained by the transfection of human colon cancer and normal colon DNAs, contained activated c-raf-1. In both the activated c-raf-1, the 5' half of the c-raf-1 sequence was replaced by sequences other than c-raf-1 as a result of recombinations which occurred at the intron between exons 7 and 8. It was suggested, however, that these recombinations, which conferred the transforming activity on the c-raf-1, occurred during the transfection. In one case analyzed, characteristic sequences were found near the breakpoint and these may be involved in the recombination. It was found, upon analysing the structure of the cDNA derived from one of the activated c-raf-1, that fused mRNA had been transcribed from the recombined gene comprising the non-raf gene and c-raf-1. The mRNA possibly encodes a fused protein. One cDNA clone was derived from alternatively spliced mRNA, although its physiological role is unclear. On comparing the structure of the two human activated c-raf-1 and the rat activated c-raf which we have reported previously, it was revealed that, in these three cases, the sequences joined to the truncated c-raf(-1)1 were different. It was suggested from data which we and others have previously reported that various sequences could be capable of activating c-raf(-1) by replacing its 5' half.

    Nucleic acids research 1987;15;12;4809-20

  • The complete coding sequence of the human raf oncogene and the corresponding structure of the c-raf-1 gene.

    Bonner TI, Oppermann H, Seeburg P, Kerby SB, Gunnell MA, Young AC and Rapp UR

    The complete 648 amino acid sequence of the human raf oncogene was deduced from the 2977 nucleotide sequence of a fetal liver cDNA. The cDNA has been used to obtain clones which extend the human c-raf-1 locus by an additional 18.9 kb at the 5' end and contain all the remaining coding exons.

    Nucleic acids research 1986;14;2;1009-15

  • Structure and biological activity of human homologs of the raf/mil oncogene.

    Bonner TI, Kerby SB, Sutrave P, Gunnell MA, Mark G and Rapp UR

    Two human genes homologous to the raf/mil oncogene have been cloned and sequenced. One, c-raf-2, is a processed pseudogene; the other, c-raf-1, contains nine exons homologous to both raf and mil and two additional exons homologous to mil. A 3' portion of c-raf-1 containing six of the seven amino acid differences relative to murine v-raf can substitute for the 3' portion of v-raf in a transformation assay. Sequence homologies between c-raf-1 and Moloney leukemia virus at both ends of v-raf indicate that the viral gene was acquired by homologous recombination. Although the data are consistent with the traditional model of retroviral transduction, they also raise the possibility that the transduction occurred in a double crossover event between proviral DNA and the murine gene.

    Molecular and cellular biology 1985;5;6;1400-7

Gene lists (3)

Gene List Source Species Name Description Gene count
L00000015 G2C Homo sapiens Human NRC Human orthologues of mouse NRC adapted from Collins et al (2006) 186
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000034 G2C Homo sapiens Pocklington H3 Human orthologues of cluster 3 (mouse) from Pocklington et al (2006) 30
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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