G2Cdb::Gene report

Gene id
G00001422
Gene symbol
MAP2K1 (HGNC)
Species
Homo sapiens
Description
mitogen-activated protein kinase kinase 1
Orthologue
G00000173 (Mus musculus)

Databases (8)

Gene
ENSG00000169032 (Ensembl human gene)
5604 (Entrez Gene)
78 (G2Cdb plasticity & disease)
MAP2K1 (GeneCards)
Literature
176872 (OMIM)
Marker Symbol
HGNC:6840 (HGNC)
Protein Expression
3834 (human protein atlas)
Protein Sequence
Q02750 (UniProt)

Synonyms (2)

  • MAPKK1
  • MEK1

Diseases (1)

Disease Nervous effect Mutations Found Literature Mutations Type Genetic association?
D00000303: Cardiofaciocutaneous syndrome Y Y (16439621) Microinsertion (MI) Y

References

  • Germline mutations in genes within the MAPK pathway cause cardio-facio-cutaneous syndrome.

    Rodriguez-Viciana P, Tetsu O, Tidyman WE, Estep AL, Conger BA, Cruz MS, McCormick F and Rauen KA

    Comprehensive Cancer Center and Cancer Research Institute, University of California, San Francisco, CA 94115, USA.

    Cardio-facio-cutaneous (CFC) syndrome is a sporadic developmental disorder involving characteristic craniofacial features, cardiac defects, ectodermal abnormalities, and developmental delay. We demonstrate that heterogeneous de novo missense mutations in three genes within the mitogen-activated protein kinase (MAPK) pathway cause CFC syndrome. The majority of cases (18 out of 23) are caused by mutations in BRAF, a gene frequently mutated in cancer. Of the 11 mutations identified, two result in amino acid substitutions that occur in tumors, but most are unique and suggest previously unknown mechanisms of B-Raf activation. Furthermore, three of five individuals without BRAF mutations had missense mutations in either MEK1 or MEK2, downstream effectors of B-Raf. Our findings highlight the involvement of the MAPK pathway in human development and will provide a molecular diagnosis of CFC syndrome.

    Funded by: NICHD NIH HHS: HD048502

    Science (New York, N.Y.) 2006;311;5765;1287-90

Literature (189)

Pubmed - other

  • 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.

  • p38mapk and MEK1/2 inhibition contribute to cellular oxidant injury after hypoxia.

    Powell CS and Wright MM

    Research Service, VAMC (151 1201 NW 16(th) St., Miami, FL 33125-1693, USA.

    Lung epithelial cells produce increased reactive oxygen species (ROS) after hypoxia exposure, and they are more susceptible after hypoxia to injury by agents that generate superoxide [O2-; e.g., 2,3-dimethoxy-1,4-naphthoquinone (DMNQ)]. Cellular GSH and MnSOD both decrease in hypoxic lung epithelial cells, altering the redox state. Because ROS participate in signaling pathways involved in cell death or survival, we tested the hypothesis that mitogen-activated protein kinases (MAPK) were involved in a protective response against cellular injury during reoxygenation. Human lung epithelial A549 cells were incubated in hypoxia (<1% O2 for 24 h) and then reoxygenated by return to air. p38mapk and MKK3 phosphorylation both decreased after hypoxia. During reoxygenation, cells were incubated with DMNQ (0-50 microM), a redox cycling quinone that produces O2-. Hypoxia preexposure significantly increased epithelial cell lysis resulting from DMNQ. Addition of the p38mapk inhibitors SB-202190 or SB-203580 markedly increased cytotoxicity, as did the mitogen/extracellular signal-regulated kinase (MEK) 1/2 inhibitor PD-98059 (all 10 microM), suggesting a protective effect of downstream molecules activated by the kinases. Transfection of A549 cells with a dominant active MKK3 plasmid (MKK3[Glu]) partially inhibited cytolysis resulting from DMNQ, whereas the inactive MKK3 plasmid (MKK3[Ala]) had less evident protective effects. Stress-related signaling pathways in epithelial cells are modulated by hypoxia and confer protection from reoxygenation, since hypoxia and chemical inhibition of p38mapk and MEK1/2 similarly increase cytolysis resulting from O2-.

    Funded by: NHLBI NIH HHS: HL-57801

  • RNAi-mediated MEK1 knock-down prevents ERK1/2 activation and abolishes human hepatocarcinoma growth in vitro and in vivo.

    Gailhouste L, Ezan F, Bessard A, Frémin C, Rageul J, Langouët S and Baffet G

    EA 4427-SeRAIC, IFR 140, Université de Rennes 1, F-35043 Rennes, France.

    The mitogen-activated protein kinases MEK/ERK pathway regulates fundamental processes in malignant cells and represents an attractive target in the development of new cancer treatments especially for human hepatocarcinoma highly resistant to chemotherapy. Although gene extinction experiments have suggested distinct roles for these proteins, the MEK/ERK cascade remains widely considered as exhibiting an overlap of functions. To investigate the functionality of each kinase in tumorigenesis, we have generated stably knock-down clones for MEK1/2 and ERK1/2 isoforms in the human hepatocellular carcinoma line HuH7. Our results have shown that RNAi strategy allows a specific disruption of the targeted kinases and argued for the critical function of MEK1 in liver tumor growth. Transient and stable extinction experiments demonstrated that MEK1 isoform acts as a major element in the signal transduction by phosphorylating ERK1 and ERK2 after growth factors stimulation, whereas oncogenic level of ERK1/2 phosphorylation appears to be MEK1 and MEK2 dependent in basal condition. In addition, silencing of MEK1 or ERK2 abolished cell proliferation and DNA replication in vitro as well as tumor growth in vivo after injection in rodent. In contrast, targeting MEK2 or ERK1 had no effect on hepatocarcinoma progression. These results strongly corroborate the relevance of targeting the MEK cascade as attested by pharmacologic drugs and support the potential application of RNAi in future development of more effective cancer therapies. Our study emphasizes the importance of the MEK/ERK pathway in human hepatocarcinoma cell growth and argues for a crucial role of MEK1 and ERK2 in this regulation.

    International journal of cancer 2010;126;6;1367-77

  • 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

  • Helicobacter pylori lipopolysaccharides upregulate toll-like receptor 4 expression and proliferation of gastric epithelial cells via the MEK1/2-ERK1/2 mitogen-activated protein kinase pathway.

    Yokota S, Okabayashi T, Rehli M, Fujii N and Amano K

    Department of Microbiology, Sapporo Medical University School of Medicine, South-1, West-17, Chuo-ku, Sapporo 060-8556, Japan. syokota@sapmed.ac.jp

    Helicobacter pylori is recognized as an etiological agent of gastroduodenal diseases. H. pylori produces various toxic substances, including lipopolysaccharide (LPS). However, H. pylori LPS exhibits extremely weakly endotoxic activity compared to the typical LPS, such as that produced by Escherichia coli, which acts through Toll-like receptor 4 (TLR4) to induce inflammatory molecules. The gastric epithelial cell lines MKN28 and MKN45 express TLR4 at very low levels, so they show very weak interleukin-8 (IL-8) production in response to E. coli LPS, but pretreatment with H. pylori LPS markedly enhanced IL-8 production induced by E. coli LPS by upregulating TLR4 via TLR2 and the MEK1/2-ERK1/2 pathway. The transcription factor NF-Y was activated by this signal and promoted transcription of the tlr4 gene. These MEK1/2-ERK1/2 signal-mediated activities were more potently activated by LPS carrying a weakly antigenic epitope, which is frequently found in gastric cancers, than by LPS carrying a highly antigenic epitope, which is associated with chronic gastritis. H. pylori LPS also augmented the proliferation rate of gastric epithelial cells via the MEK1/2-ERK1/2 pathway. H. pylori LPS may be a pathogenic factor causing gastric tumors by enhancing cell proliferation and inflammation via the MEK1/2-ERK1/2 mitogen-activated protein kinase cascade in gastric epithelial cells.

    Infection and immunity 2010;78;1;468-76

  • High-density association study of 383 candidate genes for volumetric BMD at the femoral neck and lumbar spine among older men.

    Yerges LM, Klei L, Cauley JA, Roeder K, Kammerer CM, Moffett SP, Ensrud KE, Nestlerode CS, Marshall LM, Hoffman AR, Lewis C, Lang TF, Barrett-Connor E, Ferrell RE, Orwoll ES, Zmuda JM and MrOS Research Group

    Department of Epidemiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.

    Genetics is a well-established but poorly understood determinant of BMD. Whereas some genetic variants may influence BMD throughout the body, others may be skeletal site specific. We initially screened for associations between 4608 tagging and potentially functional single nucleotide polymorphisms (SNPs) in 383 candidate genes and femoral neck and lumbar spine volumetric BMD (vBMD) measured from QCT scans among 862 community-dwelling white men >or=65 yr of age in the Osteoporotic Fractures in Men Study (MrOS). The most promising SNP associations (p < 0.01) were validated by genotyping an additional 1156 white men from MrOS. This analysis identified 8 SNPs in 6 genes (APC, DMP1, FGFR2, FLT1, HOXA, and PTN) that were associated with femoral neck vBMD and 13 SNPs in 7 genes (APC, BMPR1B, FOXC2, HOXA, IGFBP2, NFATC1, and SOST) that were associated with lumbar spine vBMD in both genotyping samples (p < 0.05). Although most associations were specific to one skeletal site, SNPs in the APC and HOXA gene regions were associated with both femoral neck and lumbar spine BMD. This analysis identifies several novel and robust genetic associations for volumetric BMD, and these findings in combination with other data suggest the presence of genetic loci for volumetric BMD that are at least to some extent skeletal-site specific.

    Funded by: NCRR NIH HHS: UL1 RR024140, UL1 RR024153; NIA NIH HHS: T32 AG000181, T32-AG00181, U01 AG018197, U01 AG027810, U01 AG042140, U01 AG18197, U01-AG027810; NIAMS NIH HHS: R01 AR051124, R01-AR051124, U01 AR045580, U01 AR045583, U01 AR045614, U01 AR045632, U01 AR045647, U01 AR045654, U01 AR45580, U01 AR45583, U01 AR45614, U01 AR45632, U01 AR45647, U01 AR45654

    Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 2009;24;12;2039-49

  • RAS signaling dysregulation in human embryonal Rhabdomyosarcoma.

    Martinelli S, McDowell HP, Vigne SD, Kokai G, Uccini S, Tartaglia M and Dominici C

    Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy. simone.martinelli@iss.it

    Rhabdomyosarcoma (RMS) is a common childhood solid tumor, resulting from dysregulation of the skeletal myogenesis program. Two major histological subtypes occur in childhood RMS, embryonal and alveolar. While chromosomal rearrangements account for the majority of alveolar tumors, the genetic defects underlying the pathogenesis of embryonal RMS remain largely undetermined. A few studies performed on small series of embryonal tumors suggest that dysregulation of RAS function may be relevant to disease pathogenesis. To explore further the biological and clinical relevance of mutations with perturbing consequences on RAS signaling in embryonal RMS, we investigated the prevalence of PTPN11, HRAS, KRAS, NRAS, BRAF, MEK1, and MEK2 mutations in a relatively large cohort of primary tumors. While HRAS and KRAS were found to be rarely mutated, we identified somatic NRAS lesions in 20% of cases. All mutations were missense and affected codon 61, with the introduction of a positive charged amino acid residue representing the most common event. PTPN11 was found mutated in one tumor specimen, confirming that somatic defects in this gene are relatively uncommon in RMS, while no mutation was observed in BRAF and MEK genes. Although no clear association of mutations with any clinical variable was observed, comparison of the outcome between mutation-positive and mutation-negative cases indicated a trend for a higher percentage of patients exhibiting a better outcome in the former. Our findings provide evidence that dysregulation of RAS signaling is a major event contributing to embryonal RMS pathogenesis.

    Funded by: Telethon: GGP07115

    Genes, chromosomes & cancer 2009;48;11;975-82

  • Distinct genetic alterations in the mitogen-activated protein kinase pathway dictate sensitivity of thyroid cancer cells to mitogen-activated protein kinase kinase 1/2 inhibition.

    Schweppe RE, Kerege AA, Sharma V, Poczobutt JM, Gutierrez-Hartmann A, Grzywa RL and Haugen BR

    University of Colorado Denver, Aurora, 80045, USA. rebecca.schweppe@ucdenver.edu

    Background: The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway plays an important role in papillary and anap 1ce lastic thyroid cancer (PTC and ATC) due to activating mutations in BRAF, RAS, or rearrangements in RET/PTC1. The objective of this study was to thoroughly test whether the BRAF V600E mutation predicts response to mitogen-activated protein kinase kinase 1/2 (MKK1/2) inhibition, as shown in other tumor types, using an authenticated panel of thyroid cancer cell lines.

    Methods: PTC and ATC 1d6a cells harboring distinct mutations in the MAPK pathway were treated with two different inhibitors selective for MKK1/2 (CI-1040 or U0126). The consequences of MKK1/2 inhibition on cell growth, survival, invasion, and MAPK signaling was determined.

    Results: Inhibition of MKK1/2 using CI-1040 or U0126 differentially inhibits the growth of a panel of PTC and ATC cell lines in two-dimensional culture, with those harboring the BRAF V600E mutation (SW1736) or BRAF-V600E/PI3K-E542K mutations (K1) being the most sensitive, the RET/PTC1 rearrangement (TPC1) and BRAF V600E mutant (BCPAP), intermediate, and the HRAS-G13R mutant (C643), the least sensitive. Growth of these cells is more sensitive to MKK1/2 inhibition when grown in 2% versus 10% serum. Baseline levels of phospho-ERK1/2 were similar in all of the cell lines, and inhibition phospho-ERK1/2 did not predict sensitivity to MKK1/2 inhibition. When cells are grown in three-dimensional culture, MKK1/2 inhibition of growth correlates with mutational status (BRAF > RET/PTC1 > RAS). Finally, PTC and ATC invasiveness is differentially inhibited by CI-1040, which is independent of tumor type or mutation present.

    Conclusions: Different mutations in the MAPK pathway play distinct roles in the growth and invasion of thyroid cancer cells. These results indicate that MKK1/2 inhibitors have the potential to inhibit thyroid cancer growth and invasion, but that responses differ based on mutation status and growth conditions.

    Funded by: NCI NIH HHS: CA100560, K12 CA086913, P30 CA 046934

    Thyroid : official journal of the American Thyroid Association 2009;19;8;825-35

  • In vivo antitumor activity of MEK and phosphatidylinositol 3-kinase inhibitors in basal-like breast cancer models.

    Hoeflich KP, O'Brien C, Boyd Z, Cavet G, Guerrero S, Jung K, Januario T, Savage H, Punnoose E, Truong T, Zhou W, Berry L, Murray L, Amler L, Belvin M, Friedman LS and Lackner MR

    Translational Oncology, Genentech, Inc., South San Francisco, California 94080, USA.

    Purpose: The pathways underlying basal-like breast cancer are poorly understood, and as yet, there is no approved targeted therapy for this disease. We investigated the role of mitogen-activated protein kinase kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) inhibitors as targeted therapies for basal-like breast cancer.

    We used pharmacogenomic analysis of a large panel of breast cancer cell lines with detailed accompanying molecular information to identify molecular predictors of response to a potent and selective inhibitor of MEK and also to define molecular mechanisms underlying combined MEK and PI3K targeting in basal-like breast cancer. Hypotheses were confirmed by testing in multiple tumor xenograft models.

    Results: We found that basal-like breast cancer models have an activated RAS-like transcriptional program and show greater sensitivity to a selective inhibitor of MEK compared with models representative of other breast cancer subtypes. We also showed that loss of PTEN is a negative predictor of response to MEK inhibition, that treatment with a selective MEK inhibitor caused up-regulation of PI3K pathway signaling, and that dual blockade of both PI3K and MEK/extracellular signal-regulated kinase signaling synergized to potently impair the growth of basal-like breast cancer models in vitro and in vivo.

    Conclusions: Our studies suggest that single-agent MEK inhibition is a promising therapeutic modality for basal-like breast cancers with intact PTEN, and also provide a basis for rational combination of MEK and PI3K inhibitors in basal-like cancers with both intact and deleted PTEN.

    Clinical cancer research : an official journal of the American Association for Cancer Research 2009;15;14;4649-64

  • MEK1 mutations, but not ERK2 mutations, occur in melanomas and colon carcinomas, but none in thyroid carcinomas.

    Murugan AK, Dong J, Xie J and Xing M

    Mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in the pathogenesis of melanoma, colon cancer and thyroid cancer, which commonly harbor RAS and BRAF mutations. However, mutations in exon 2 of MEK1 and exon 7 of ERK2 have not been investigated in these cancers although they are occasionally found in some other cancers or cell lines. In this study, we performed mutational analysis to search for these mutations in 185 samples, including 167 tumor samples and 18 cell lines of melanoma, colon cancer and thyroid cancer. We found one MEK1 mutation in 1 of 37 (3%) melanoma tumor samples and another MEK1 mutation in 1 of 45 (2.2%) colon cancer samples. We did not find any MEK1 mutation in 99 thyroid tumor samples and 12 thyroid cancer cell lines. We also did not find any ERK2 mutation in melanoma, colon cancer and thyroid cancer. We thus report for the first time a low prevalence of MEK1 mutations in melanoma and colon cancer. Both of the two mutants have been demonstrated to be activating in the MAPK signaling pathway and may therefore provide potential target for effective therapy in cases of melanomas and colon cancer harboring these mutations.

    Cell cycle (Georgetown, Tex.) 2009;8;13;2122-4

  • Protein kinase SGK1 enhances MEK/ERK complex formation through the phosphorylation of ERK2: implication for the positive regulatory role of SGK1 on the ERK function during liver regeneration.

    Won M, Park KA, Byun HS, Kim YR, Choi BL, Hong JH, Park J, Seok JH, Lee YH, Cho CH, Song IS, Kim YK, Shen HM and Hur GM

    Department of Pharmacology, Research Institute for Medical Science, Infection Signaling Network Research Center, Daejeon Regional Cancer Center, College of Medicine, Chungnam National University, 6 Munhwa-dong, Jung-gu, Daejeon 301-131, Republic of Korea.

    Based on the observation of biphasic induction of SGK1 expression in the regenerating liver, we investigated the role of SGK1 in the regulation of MEK/ERK signaling pathway which plays a crucial role in regulating growth and survival signaling.

    Methods: To determine the role of SGK1 in the activation of MEK/ERK signaling cascade, we infected primary hepatocytes with recombinant adenoviral vector encoding SGK1, and assessed its effect on the MEK/ERK signaling pathway.

    Results: Partial hepatectomy resulted in the biphasic transcriptional induction of SGK1 in regenerating liver tissues. Infection of primary hepatocytes with an adenoviral vector encoding SGK1 enhanced the ERK phosphorylation under serum-starved conditions and this was blocked by the expression of kinase-dead SGK1. SGK1 was found to physically interact with ERK1/2 as well as MEK1/2. Furthermore, SGK1 mediated the phosphorylation of ERK2 on Ser(29) in a serum-dependent manner. Replacement of Ser(29) to aspartic acid, which mimics the phosphorylation of Ser(29), enhanced the ERK2 activity as well as the MEK/ERK complexes formation.

    Conclusions: SGK1 expression during liver regeneration is a part of a signaling pathway that is necessary for enhancing ERK signaling activation through modulating the MEK/ERK complex formation.

    Journal of hepatology 2009;51;1;67-76

  • Spectrum of MEK1 and MEK2 gene mutations in cardio-facio-cutaneous syndrome and genotype-phenotype correlations.

    Dentici ML, Sarkozy A, Pantaleoni F, Carta C, Lepri F, Ferese R, Cordeddu V, Martinelli S, Briuglia S, Digilio MC, Zampino G, Tartaglia M and Dallapiccola B

    IRCCS-Casa Sollievo della Sofferenza, San Giovanni Rotondo e Istituto CSS-Mendel, Rome, Italy.

    Cardio-facio-cutaneous syndrome (CFCS) is a rare disease characterized by mental retardation, facial dysmorphisms, ectodermal abnormalities, heart defects and developmental delay. CFCS is genetically heterogeneous and mutations in the KRAS, BRAF, MAP2K1 (MEK1) and MAP2K2 (MEK2) genes, encoding for components of the RAS-mitogen activated protein kinase (MAPK) signaling pathway, have been identified in up to 90% of cases. Here we screened a cohort of 33 individuals with CFCS for MEK1 and MEK2 gene mutations to further explore their molecular spectrum in this disorder, and to analyze genotype-phenotype correlations. Three MEK1 and two MEK2 mutations were detected in six patients. Two missense MEK1 (L42F and Y130H) changes and one in-frame MEK2 (K63_E66del) deletion had not been reported earlier. All mutations were localized within exon 2 or 3. Together with the available records, the present data document that MEK1 mutations are relatively more frequent than those in MEK2, with exons 2 and 3 being mutational hot spots in both genes. Mutational analysis of the affected MEK1 and MEK2 exons did not reveal occurrence of mutations among 75 patients with Noonan syndrome, confirming the low prevalence of MEK gene defects in this disorder. Clinical review of known individuals with MEK1/MEK2 mutations suggests that these patients show dysmorphic features, ectodermal abnormalities and cognitive deficit similar to what was observed in BRAF-mutated patients and in the general CFCS population. Conversely, congenital heart defects, particularly mitral valve and septal defects, and ocular anomalies seem to be less frequent among MEK1/MEK2 mutation-positive patients.

    Funded by: Telethon: GGP07115

    European journal of human genetics : EJHG 2009;17;6;733-40

  • MEK1 binds directly to betaarrestin1, influencing both its phosphorylation by ERK and the timing of its isoprenaline-stimulated internalization.

    Meng D, Lynch MJ, Huston E, Beyermann M, Eichhorst J, Adams DR, Klussmann E, Klusmann E, Houslay MD and Baillie GS

    Neuroscience and Molecular Pharmacology, Faculty of Biomedical and Life Sciences, Wolfson Building, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom.

    betaArrestin is a multifunctional signal scaffold protein. Using SPOT immobilized peptide arrays, coupled with scanning alanine substitution and mutagenesis, we show that the MAPK kinase, MEK1, interacts directly with betaarrestin1. Asp(26) and Asp(29) in the N-terminal domain of betaarrestin1 are critical for its binding to MEK1, whereas Arg(47) and Arg(49) in the N-terminal domain of MEK1 are critical for its binding to betaarrestin1. Wild-type FLAG-tagged betaarrestin1 co-immunopurifies with MEK1 in HEKB2 cells, whereas the D26A/D29A mutant does not. ERK-dependent phosphorylation at Ser(412) was compromised in the D26A/D29A-betaarrestin1 mutant. A cell-permeable, 25-mer N-stearoylated betaarrestin1 peptide that encompassed the N-domain MEK1 binding site blocked betaarrestin1/MEK1 association in HEK cells and recapitulated the altered phenotype seen with the D26A/D29A-betaarrestin1 in compromising the ERK-dependent phosphorylation of betaarrestin1. In addition, the MEK disruptor peptide promoted the ability of betaarrestin1 to co-immunoprecipitate with endogenous c-Src and clathrin, facilitating the isoprenaline-stimulated internalization of the beta(2)-adrenergic receptor.

    Funded by: Medical Research Council: G0400053, G0600765, G8604010

    The Journal of biological chemistry 2009;284;17;11425-35

  • Selective transcriptional down-regulation of human rhinovirus-induced production of CXCL10 from airway epithelial cells via the MEK1 pathway.

    Zaheer RS, Koetzler R, Holden NS, Wiehler S and Proud D

    Airway Inflammation Group, Institute of Infection, Immunity and Inflammation, University of Calgary, Calgary, Alberta, Canada.

    Human rhinovirus (HRV) infections can trigger exacerbations of lower airway diseases. Infection of airway epithelial cells induces production of a number of proinflammatory chemokines that may exacerbate airway inflammation, including CXCL10, a chemoattractant for type 1 lymphocytes and NK cells. Primary human bronchial epithelial cells and the BEAS-2B human bronchial epithelial cell line were used to examine the role of MAPK pathways in HRV-16-induced production of CXCL10. Surprisingly, PD98059 and U0126, two inhibitors of the MEK1/2-ERK MAPK pathway, significantly enhanced HRV-16-induced CXCL10 mRNA and protein. This enhancement was not seen with IFN-beta-induced production of 1f40 CXCL10. Studies using small interfering RNA revealed that knockdown of MEK1, but not MEK2, was associated with enhanced HRV-induced CXCL10 production. Promoter construct studies revealed that PD98059 and U0126 enhanced HRV-16-induced transcriptional activation of CXCL10. HRV-16-induced promoter activation was regulated by two NF-kappaB binding sites, kappaB1 and kappaB2, and by an IFN-stimulated response element. Inhibitors of the MEK1/2-ERK pathway did not alter HRV-16-induced activation of tandem repeat kappaB1 or kappaB2 constructs, nor did they alter HRV-16-induced nuclear translocation/binding of NF-kappaB to either kappaB1 or kappaB2 recognition sequences. Furthermore, PD98059 and U0126 did not alter phosphorylation or degradation of IkappaBalpha. In contrast, inhibitors of the MEK1/2-ERK pathway, and small interfering RNA knockdown of MEK1, enhanced nuclear translocation/binding of IFN regulatory factor (IRF)-1 to the IFN-stimulated response element recognition sequence in HRV-16 infected cells. We conclude that activation of MEK1 selectively down-regulates HRV-16-induced expression of CXCL10 via modulation of IRF-1 interactions with the gene promoter in human airway epithelial cells.

    Journal of immunology (Baltimore, Md. : 1950) 2009;182;8;4854-64

  • 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

  • Transforming growth factor beta1-mediated activation of the smooth muscle alpha-actin gene in human pulmonary myofibroblasts is inhibited by tumor necrosis factor-alpha via mitogen-activated protein kinase kinase 1-dependent induction of the Egr-1 transcriptional repressor.

    Liu X, Kelm RJ and Strauch AR

    Department of Physiology and Cell Biology and the Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University, Columbus, 43210, USA.

    Transforming growth factor (TGF) beta1 is a mediator of myofibroblast differentiation in healing wounds in which it activates transcription of the smooth muscle alpha-actin (SMalphaA) gene via dynamic interplay of nuclear activators and repressors. Targeting components of TGFbeta1 signaling may be an effective strategy for controlling myofibroblasts in chronic fibrotic diseases. We examined the ability of proinflammatory tumor necrosis factor (TNF)-alpha to antagonize TGFbeta1-mediated human pulmonary myofibroblast differentiation. TNF-alpha abrogated TGFbeta1-induced SMalphaA gene expression at the level of transcription without disrupting phosphorylation of regulatory Smads. Intact mitogen-activated protein kinase kinase (Mek)-extracellular signal-regulated kinase (Erk) kinase signaling was required for myofibroblast repression by TNF-alpha via induction of the early growth response factor-1 (Egr-1) DNA-binding protein. Egr-1 bound to the GC-rich SPUR activation element in the SMalphaA promoter and potently suppressed Smad3- and TGFbeta1-mediated transcription. Reduction in Smad binding to the SMalphaA promoter in TNF-alpha-treated myofibroblasts was accompanied by an increase in Egr-1 and YB-1 repressor binding, suggesting that the molecular mechanism underlying repression may involve competitive interplay between Egr-1, YB-1, and Smads. The ability of TNF-alpha to attenuate myofibroblast differentiation via modulation of a Mek1/Erk/Egr-1 regulatory axis may be useful in designing new therapeutic targets to offset destructive tissue remodeling in chronic fibrotic disease.

    Funded by: NHLBI NIH HHS: HL-054281, HL-085109, R01 HL054281, R01 HL085109

    Molecular biology of the cell 2009;20;8;2174-85

  • Sp1 and AP-1 regulate expression of the human gene VIL2 in esophageal carcinoma cells.

    Gao SY, Li EM, Cui L, Lu XF, Meng LY, Yuan HM, Xie JJ, Du ZP, Pang JX and Xu LY

    Department of Biochemistry and Molecular Biology, Shantou University, Shantou, China.

    Ezrin, encoded by VIL2, is a membrane-cytoskeletal linker protein that has been suggested to be involved in tumorigenesis. Ezrin expression in esophageal squamous cell carcinoma (ESCC) was described recently, but its clinical significance and the molecular mechanism underlying its regulated expression remain unclear. Thus, we retrospectively evaluated ezrin expression by immunohistochemistry in a tissue microarray representing 193 ESCCs. Ezrin overexpression in 90 of 193 tumors (46.6%) was associated with poor survival (p = 0.048). We then explored the mechanism by which ezrin expression is controlled in ESCC by assessing the transcriptional regulatory regions of human VIL2 by fusing deletions or site-directed mutants of the 5'-flanking region of the gene to a luciferase reporter. We found that the region -87/-32 containing consensus Sp1 (-75/-69) and AP-1 (-64/-58) binding sites is crucial for VIL2 promoter activity in esophageal carcinoma cells (EC109) derived from ESCC. AP-1 is comprised of c-Jun and c-Fos. Electrophoretic mobility shift and chromatin immunoprecipitation experiments demonstrated that Sp1 and c-Jun bound specifically to their respective binding sites within the VIL2 promoter. In addition, transient expression of Sp1, c-Jun, or c-Fos increased ezrin expression and VIL2 promoter activity. Use of selective inhibitors revealed that VIL2 transactivation required the MEK1/2 signal transduction pathway but not JNK or p38 MAPK. Taken together, we propose a possible signal transduction pathway whereby MEK1/2 phosphorylates ERK1/2, which phosphorylates Sp1 and AP-1 that in turn bind to their respective binding sites to regulate the expression of human VIL2 in ESCC cells.

    The Journal of biological chemistry 2009;284;12;7995-8004

  • 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

  • Ca(2+)/calmodulin-dependent protein kinase II promotes cell cycle progression by directly activating MEK1 and subsequently modulating p27 phosphorylation.

    Li N, Wang C, Wu Y, Liu X and Cao X

    Institute of Immunology and National Key Laboratory of Medical Immunology, Second Military Medical University, Shanghai, China. linan@immunol.org

    Cell proliferation is regulated by integration of multiple pathways, such as MAPK, phosphatidylinositol 3'-kinase, protein kinase C, and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) signaling, determining whether the cell proceeds into cell cycle progression. Recently, we have demonstrated that a novel endogenous CaMKII-inhibitory protein, hCaMKIINalpha, suppresses tumor growth by inducing cell cycle arrest via p27 stabilization, accompanied by MEK/ERK deactivation. The data indicate a potential link between Ca(2+)/CaMKII and other signaling pathways, such as MAPK signaling. However, the detailed mechanisms of cross-talks between these important pathways on cell cycle regulation have not been specified. Here we report that CaMKII, in colon adenocarcinoma cells, activates MEK/ERK, which is responsible for the phosphorylation and subsequent proteasomal degradation of p27, thus causing the promotion of the S-G(2)/M transition of cell cycle progression. Importantly, we found that CaMKII can bind to MEK1 and that active CaMKII directly phosphorylates MEK1 in vitro, which could be abrogated by CaMKII inhibitor. Besides, ERK2 can directly interact with and phosphorylate p27. This is the first demonstration that CaMKII interplays with MEK1 and regulates p27 phosphorylation in the cell cycle progression. These findings provide mechanistic evidence for the cross-talk between CaMKII and MAPK signaling, which converges in MEK/ERK activation in the regulation of cell cycle progression.

    The Journal of biological chemistry 2009;284;5;3021-7

  • 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

  • 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

  • Activation of MEK1 or MEK2 isoform is sufficient to fully transform intestinal epithelial cells and induce the formation of metastatic tumors.

    Voisin L, Julien C, Duhamel S, Gopalbhai K, Claveau I, Saba-El-Leil MK, Rodrigue-Gervais IG, Gaboury L, Lamarre D, Basik M and Meloche S

    Institut de Recherche en Immunologie et Cancérologie, Montreal, Quebec, Canada.

    Background: The Ras-dependent ERK1/2 MAP kinase signaling pathway plays a central role in cell proliferation control and is frequently activated in human colorectal cancer. Small-molecule inhibitors of MEK1/MEK2 are therefore viewed as attractive drug candidates for the targeted therapy of this malignancy. However, the exact contribution of MEK1 and MEK2 to the pathogenesis of colorectal cancer remains to be established.

    Methods: Wild type and constitutively active forms of MEK1 and MEK2 were ectopically expressed by retroviral gene transfer in the normal intestinal epithelial cell line IEC-6. We studied the impact of MEK1 and MEK2 activation on cellular morphology, cell proliferation, survival, migration, invasiveness, and tumorigenesis in mice. RNA interference was used to test the requirement for MEK1 and MEK2 function in maintaining the proliferation of human colorectal cancer cells.

    Results: We found that expression of activated MEK1 or MEK2 is sufficient to morphologically transform intestinal epithelial cells, dysregulate cell proliferation and induce the formation of high-grade adenocarcinomas after orthotopic transplantation in mice. A large proportion of these intestinal tumors metastasize to the liver and lung. Mechanistically, activation of MEK1 or MEK2 up-regulates the expression of matrix metalloproteinases, promotes invasiveness and protects cells from undergoing anoikis. Importantly, we show that silencing of MEK2 expression completely suppresses the proliferation of human colon carcinoma cell lines, whereas inactivation of MEK1 has a much weaker effect.

    Conclusion: MEK1 and MEK2 isoforms have similar transforming properties and are able to induce the formation of metastatic intestinal tumors in mice. Our results suggest that MEK2 plays a more important role than MEK1 in sustaining the proliferation of human colorectal cancer cells.

    BMC cancer 2008;8;337

  • WNK2 modulates MEK1 activity through the Rho GTPase pathway.

    Moniz S, Matos P and Jordan P

    Centre of Human Genetics, National Health Institute, Lisbon, Portugal.

    WNK protein kinases form a kinase subfamily expressed in multi-cellular organisms and the human genome encodes four distinct WNK genes. Human WNK2 has been recently identified as a cell growth regulator that modulates activation of the ERK1/2 protein kinase and is epigenetically silenced in gliomas. Here we provide mechanistic insight into how WNK2 affects ERK activation. W 1f40 e found that WNK2 depletion decreased RhoA activation and promoted GTP-loading of Rac1, leading to stimulation of the Rac1-effector PAK1, which is the kinase responsible for subsequent phosphorylation of MEK1 at serine 298, thereby increasing MEK affinity towards ERK1/2. We propose that WNK2 controls a RhoA-mediated cross-talk mechanism that regulates the efficiency with which MEK1 can activate ERK1/2 upon growth factor stimulation.

    Cellular signalling 2008;20;10;1762-8

  • Differences in activity and phosphorylation of MAPK enzymes in esophageal squamous cells of GERD patients with and without Barrett's esophagus.

    Zhang HY, Zhang X, Chen X, Thomas D, Hormi-Carver K, Elder F, Spechler SJ and Souza RF

    Department of Medcine, Veterans Affairs North tExas Health Care System and the University of Texas Southwestern Medical School, MC# 111B1, Dallas VA Medical Ctr., 4500 South Lancaster Rd., Dallas, TX 75216, USA.

    We hypothesized that, in esophageal squamous epithelial cells, there are differences among individuals in the signal transduction pathways activated by acid reflux that might underlie the development of Barrett's esophagus. To explore that hypothesis, we immortalized nonneoplastic, esophageal squamous cells from patients with gastroesophageal reflux disease (GERD) with (NES-B3T) and without (NES-G2T) Barrett's esophagus and used those cells to study acid effects on MAPK proteins. During endoscopy in patients with GERD with and without Barrett's esophagus, we took biopsy specimens from the distal squamous esophagus to study MAPK proteins before and after esophageal perfusion with 0.1 N HCl. We used immunoblotting and Western blotting to study MEK1/2 phosphorylation at two activating sites (serines 217/221), MEK1 phosphorylation at an inhibitory site (threonine 286), and MEK1/2 activity. After acid exposure, both cell lines exhibited increased MEK1/2 phosphorylation at the activating sites; the NES-B3T cells had higher levels of MEK1 phosphorylation at the inhibitory site, however, and only the NES-G2T cells showed an acid-induced increase in MEK1/2 activity. Similarly, in the squamous epithelium of patients with GERD with and without Barrett's esophagus, acid perfusion increased MEK1/2 phosphorylation at the activating sites in both patient groups; the Barrett's patients had higher levels of MEK1 phosphorylation at the inhibitory site, however, and only the patients without Barrett's demonstrated an acid-induced increase in ERK1/2 phosphorylation. In esophageal squamous cell lines and biopsies from patients with GERD with and without Barrett's esophagus, we have found differences in MAPK pathways activated by acid exposure. We speculate that these differences might underlie the development of Barrett's metaplasia.

    Funded by: NIDDK NIH HHS: DK63621

    American journal of physiology. Gastrointestinal and liver physiology 2008;295;3;G470-8

  • Major contribution of MEK1 to the activation of ERK1/ERK2 and to the growth of LS174T colon carcinoma cells.

    Shama J, Garcia-Medina R, Pouysségur J and Vial E

    Institute of Signaling Developmental Biology and Cancer, University of Nice, CNRS UMR 6543, Centre A. Lacassagne, 33 avenue de Valombrose, Nice 06189, France.

    Mammalian cells express two closely related MEK isoforms, MEK1 and MEK2, upstream of the ERK1/ERK2 MAPK module. Although genetic studies have suggested that MEK1 and MEK2 do not have overlapping functions in vivo, little is known about their specific contribution to the activation of ERKs and to tumor cell proliferation. We used Tet-inducible shRNA to investigate the independent role of MEK1 and MEK2 for the oncogenic and the serum-induced activation of ERK1 and ERK2 in LS174T colon carcinoma cells. We show that MEK1 is the main activator of both ERK1 and ERK2. MEK2 removal has no impact by itself but it can cooperate with MEK1 ablation for the inhibition of ERK1/2 activity. In addition, we show that MEK1 is the critical isoform regulating tumor cell proliferation in vitro and in vivo.

    Biochemical and biophysical research communications 2008;372;4;845-9

  • Novel MEK1 mutation identified by mutational analysis of epidermal growth factor receptor signaling pathway genes in lung adenocarcinoma.

    Marks JL, Gong Y, Chitale D, Golas B, McLellan MD, Kasai Y, Ding L, Mardis ER, Wilson RK, Solit D, Levine R, Michel K, Thomas RK, Rusch VW, Ladanyi M and Pao W

    Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.

    Genetic lesions affecting a number of kinases and other elements within the epidermal growth factor receptor (EGFR) signaling pathway have been implicated in the pathogenesis of human non-small-cell lung cancer (NSCLC). We performed mutational profiling of a large cohort of lung adenocarcinomas to uncover other potential somatic mutations in genes of this pathway that could contribute to lung tumorigenesis. We have identified in 2 of 207 primary lung tumors a somatic activating mutation in exon 2 of MEK1 (i.e., mitogen-activated protein kinase kinase 1 or MAP2K1) that substitutes asparagine for lysine at amino acid 57 (K57N) in the nonkinase portion of the kinase. Neither of these two tumors harbored known mutations in other genes encoding components of the EGFR signaling pathway (i.e., EGFR, HER2, KRAS, PIK3CA, and BRAF). Expression of mutant, but not wild-type, MEK1 leads to constitutive activity of extracellular signal-regulated kinase (ERK)-1/2 in human 293T cells and to growth factor-independent proliferation of murine Ba/F3 cells. A selective MEK inhibitor, AZD6244, inhibits mutant-induced ERK activity in 293T cells and growth of mutant-bearing Ba/F3 cells. We also screened 85 NSCLC cell lines for MEK1 exon 2 mutations; one line (NCI-H1437) harbors a Q56P substitution, a known transformation-competent allele of MEK1 originally identified in rat fibroblasts, and is sensitive to treatment with AZD6244. MEK1 mutants have not previously been reported in lung cancer and may provide a target for effective therapy in a small subset of patients with lung adenocarcinoma.

    Funded by: NCI NIH HHS: K08 CA097980, K08 CA097980-03, K08-CA097980, P01 CA129243, P01 CA129243-010003, R01 CA121210, R01 CA121210-02, R01-CA121210

    Cancer research 2008;68;14;5524-8

  • GRASP55 regulates Golgi ribbon formation.

    Feinstein TN and Linstedt AD

    Department of Biological Sciences, Carnegie Me e26 llon University, Pittsburgh, PA 15213, USA.

    Recent work indicates that mitogen-activated protein kinase kinase (MEK)1 signaling at the G2/M cell cycle transition unlinks the contiguous mammalian Golgi apparatus and that this regulates cell cycle progression. Here, we sought to determine the role in this pathway of Golgi reassembly protein (GRASP)55, a Golgi-localized target of MEK/extracellular signal-regulated kinase (ERK) phosphorylation at mitosis. In support of the hypothesis that GRASP55 is inhibited in late G2 phase, causing unlinking of the Golgi ribbon, we found that HeLa cells depleted of GRASP55 show a fragmented Golgi similar to control cells arrested in G2 phase. In the absence of GRASP55, Golgi stack length is shortened but Golgi stacking, compartmentalization, and transport seem normal. Absence of GRASP55 was also sufficient to suppress the requirement for MEK1 in the G2/M transition, a requirement that we previously found depends on an intact Golgi ribbon. Furthermore, mimicking mitotic phosphorylation of GRASP55 by using aspartic acid substitutions is sufficient to unlink the Golgi apparatus in a gene replacement assay. Our results implicate MEK1/ERK regulation of GRASP55-mediated Golgi linking as a control point in cell cycle progression.

    Funded by: NIGMS NIH HHS: GM-56779, R01 GM056779

    Molecular biology of the cell 2008;19;7;2696-707

  • NF-kappaB-dependent transcriptional activation in lung carcinoma cells by farnesol involves p65/RelA(Ser276) phosphorylation via the MEK-MSK1 signaling pathway.

    Joo JH and Jetten AM

    Cell Biology Section, LRB, Division of Intramural Research, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.

    In this study, we demonstrate that treatment of human lung adenocarcinoma H460 cells with farnesol induces the expression of a number of immune response and inflammatory genes, including IL-6, CXCL3, IL-1alpha, and COX-2. This response was dependent on the activation of the NF-kappaB signaling pathway. Farnesol treatment reduces the level of IkappaBalpha and induces translocation of p65/RelA to the nucleus, its phosphorylation at Ser(276), and transactivation of NF-kappaB-dependent transcription. Moreover, overexpression of IkappaBalpha or treatment with the NF-kappaB inhibitor caffeic acid phenethyl ester greatly diminishes the induction of inflammatory gene expression by farnesol. We provide evidence indicating that the farnesol-induced phosphorylation of p65/RelA at Ser(276) is important for optimal transcriptional activity of NF-kappaB. The MEK1/2 inhibitor U0126 and knockdown of MEK1/2 expression with small interfering RNAs effectively blocked the phosphorylation of p65/RelA(Ser(276)) but not that of Ser(536), suggesting that this phosphorylation is dependent on the activation of the MEK1/2-ERK1/2 pathway. We further show that inhibition of MSK1, a kinase acting downstream of MEK1/2-ERK1/2, by H89 or knockdown of MSK1 expression also inhibited phosphorylation of p65/RelA(Ser(276)), suggesting that this phosphorylation is dependent on MSK1. Knockdown of MEK1/2 or MSK1 expression inhibits farnesol-induced expression of CXCL3, IL-1alpha, and COX-2 mRNA. Our results indicate that the induction of inflammatory genes by farnesol is mediated by the activation of the NF-kappaB pathway and involves MEK1/2-ERK1/2-MSK1-dependent phosphorylation of p65/RelA(Ser(276)). The activation of the NF-kappaB pathway by farnesol might be part of a prosurvival response during farnesol-induced ER stress.

    Funded by: Intramural NIH HHS

    The Journal of biological chemistry 2008;283;24;16391-9

  • Phase I pharmacokinetic and pharmacodynamic study of the oral, small-molecule mitogen-activated protein kinase kinase 1/2 inhibitor AZD6244 (ARRY-142886) in patients with advanced cancers.

    Adjei AA, Franklin W, Morris C, Wilson D, Molina JR, Hanson LJ, Gore L, Chow L, Leong S, Maloney L, Gordon G, Simmons H, Marlow A, Litwiler K, Brown S, Poch G, Kane K, Haney J and Eckhardt SG

    Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263, USA. alex.adjei@roswellpark.org

    Purpose: To assess the tolerability, pharmacokinetics (PKs), and pharmacodynamics (PDs) of the mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor AZD6244 (ARRY-142886) in patients with advanced cancer.

    In part A, patients received escalating doses to determine the maximum-tolerated dose (MTD). In both parts, blood samples were collected to assess PK and PD parameters. In pa 7eb rt B, patients were stratified by cancer type (melanoma v other) and randomly assigned to receive the MTD or 50% MTD. Biopsies were collected to determine inhibition of ERK phosphorylation, Ki-67 expression, and BRAF, KRAS, and NRAS mutations.

    Results: Fifty-seven patients were enrolled. MTD in part A was 200 mg bid, but this dose was discontinued in part B because of toxicity. The 50% MTD (100 mg bid) was well tolerated. Rash was the most frequent and dose-limiting toxicity. Most other adverse events were grade 1 or 2. The PKs were less than dose proportional, with a median half-life of approximately 8 hours and inhibition of ERK phosphorylation in peripheral-blood mononuclear cells at all dose levels. Paired tumor biopsies demonstrated reduced ERK phosphorylation (geometric mean, 79%). Five of 20 patients demonstrated >or= 50% inhibition of Ki-67 expression, and RAF or RAS mutations were detected in 10 of 26 assessable tumor samples. Nine patients had stable disease (SD) for >or= 5 months, including two patients with SD for 19 (thyroid cancer) and 22 (uveal melanoma plus renal cancer) 28-day cycles.

    Conclusion: AZD6244 was well tolerated with target inhibition demonstrated at the recommended phase II dose. PK analyses supported twice-daily dosing. Prolonged SD was seen in a variety of advanced cancers. Phase II studies are ongoing.

    Funded by: NCI NIH HHS: 5P30CA006927, K12 CA090628, K12 CA090628-08, K24 CA106349, P30 CA006927, P30 CA046934, P30 CA46934

    Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2008;26;13;2139-46

  • Targeting the MEK1 cascade in lung epithelium inhibits proliferation and fibrogenesis by asbestos.

    Manning CB, Sabo-Attwood T, Robledo RF, Macpherson MB, Rincón M, Vacek P, Hemenway D, Taatjes DJ, Lee PJ and Mossman B 275 T

    Environmental Pathology Program, University of Vermont College of Medicine, Department of Pathology, 89 Beaumont Avenue, Burlington, VT 05405, USA.

    The extracellular signal-regulated kinases 1 and 2 (ERK1/2) are phosphorylated after inhalation of asbestos. The effect of blocking this signaling pathway in lung epithelium is unclear. Asbestos-exposed transgenic mice expressing a dominant-negative mitogen-activated protein kinase kinase-1 (dnMEK1) (i.e., the upstream kinase necessary for phosphorylation of ERK1/2) targeted to lung epithelium exhibited morphologic and molecular changes in lung. Transgene-positive (Tg+) (i.e., dnMEK1) and transgene-negative (Tg-) littermates were exposed to crocidolite asbestos for 2, 4, 9, and 32 days or maintained in clean air (sham controls). Distal bronchiolar epithelium was isolated using laser capture microdissection and mRNA analyzed for molecular markers of proliferation and Clara cell secretory protein (CCSP). Lungs and bronchoalveolar lavage fluids were analyzed for inflammatory and proliferative changes and molecular markers of fibrogenesis. Distal bronchiolar epithelium of asbestos-exposed wild-type mice showed increased expression of c-fos at 2 days. Elevated mRNA levels of histone H3 and numbers of Ki-67-labeled proliferating bronchiolar epithelial cells were decreased at 4 days in asbestos-exposed Tg+ mice. At 32 days, distal bronchioles normally composed of Clara cells in asbestos-exposed Tg+ mouse lungs exhibited nonreplicating ciliated and mucin-secreting cells as well as decreased mRNA levels of CCSP. Gene expression (procollagen 3-a-1, procollagen 1-a-1, and IL-6) linked to fibrogenesis was also increased in lung homogenates of asbestos-exposed Tg- mice, but reduced in asbestos-exposed Tg+ mice. These results suggest a critical role of MEK1 signaling in epithelial cell proliferation and lung remodeling after toxic injury.

    Funded by: NHLBI NIH HHS: P01 HL067004, P01 HL067004-05, P01 HL67004; NIEHS NIH HHS: T32 ES007122-25; PHS HHS: T32 07122

    American journal of respiratory cell and molecular biology 2008;38;5;618-26

  • Final stages of cytokinesis and midbody ring formation are controlled by BRUCE.

    Pohl C and Jentsch S

    Department of Molecular Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.

    Cytokinesis involves the formation of a cleavage furrow, followed by abscission, the cutting of the midbody channel, the final bridge between dividing cells. Recently, the midbody ring became known as central for abscission, but its regulation remains enigmatic. Here, we identify BRUCE, a 528 kDa multifunctional protein, which processes ubiquitin-conjugating activity, as a major regulator of abscission. During cytokinesis, BRUCE moves from the vesicular system to the midbody ring and serves as a platform for the membrane delivery machinery and mitotic regulators. Depletion of BRUCE in cell cultures causes defective abscission and cytokinesis-associated apoptosis, accompanied by a block of vesicular targeting and defective formation of the midbody and the midbody ring. Notably, ubiquitin relocalizes from midbody microtubules to the midbody ring during cytokinesis, and depletion of BRUCE disrupts this process. We propose that BRUCE coordinates multiple steps required for abscission and that ubiquitylation may be a crucial trigger.

    Cell 2008;132;5;832-45

  • Germline mutations of MEK in cardio-facio-cutaneous syndrome are sensitive to MEK and RAF inhibition: implications for therapeutic options.

    Senawong T, Phuchareon J, Ohara O, McCormick F, Rauen KA and Tetsu O

    Department of Pathology, School of Medicine, University of California, San Francisco, CA 94143-0128, USA.

    Cardio-facio-cutaneous (CFC) syndrome is a sporadic developmental disorder characterized by distinctive craniofacial features, heart defects, mental retardation and ectodermal abnormalities. We recently reported missense germline mutations in the genes MEK1 and MEK2 in patients with CFC. These mutations, including F53S and Y130C MEK1, and F57C MEK2, are the first naturally occurring mutations to be identified in these genes. This study reports data concerning the biochemical functions of the novel mutants, as well as the roles of these MEK genes in the MAPK signaling cascade. Our CFC MEK variants cannot induce ERK unless they are phosphorylated by RAF at two key serine residues in the regulatory loop. When we replaced the serine residues with alanines, ERK phosphorylation was significantly reduced in the presence of RAF. We did find that F57C MEK2 activation was less dependent on RAF signaling than the other mutants. This difference results in F57C MEK2 being resistant to the selective RAF inhibitor SB-590885. All three mutants are sensitive to the MEK inhibitor U0126. The majority of CFC cases result from mutations in B-RAF. A recent report indicates the possibility that cancer cells with activated B-RAF have enhanced, selective sensitivity to MEK inhibitors. Thus, regardless of mutations identified in an individual with CFC, MEK inhibition is a potential therapeutic approach for this population.

    Funded by: NICHD NIH HHS: HD048502

    Human molecular genetics 2008;17;3;419-30

  • Mutation and phenotypic spectrum in patients with cardio-facio-cutaneous and Costello syndrome.

    Schulz AL, Albrecht B, Arici C, van der Burgt I, Buske A, Gillessen-Kaesbach G, Heller R, Horn D, Hübner CA, Korenke GC, König R, Kress W, Krüger G, Meinecke P, Mücke J, Plecko B, Rossier E, Schinzel A, Schulze A, Seemanova E, Seidel H, Spranger S, Tuysuz 1f3e B, Uhrig S, Wieczorek D, Kutsche K and Zenker M

    Institut für Humangenetik, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany.

    Cardio-facio-cutaneous (CFC) and Costello syndrome (CS) are congenital disorders with a significant clinical overlap. The recent discovery of heterozygous mutations in genes encoding components of the RAS-RAF-MAPK pathway in both CFC and CS suggested a similar underlying pathogenesis of these two disorders. While CFC is heterogeneous with mutations in BRAF, MAP2K1, MAP2K2 and KRAS, HRAS alterations are almost exclusively associated with CS. We carried out a comprehensive mutation analysis in 51 CFC-affected patients and 31 individuals with CS. Twelve different BRAF alterations were found in twenty-four patients with CFC (47.0%), two MAP2K1 mutations in five (9.8%) and two MAP2K2 sequence variations in three CFC-affected individuals (5.9%), whereas three patients had a KRAS alteration (5.9%). We identified four different missense mutations of HRAS in twenty-eight cases with CS (90.3%), while KRAS mutations were detected in two infants with a phenotype meeting criteria for CS (6.5%). In 14 informative families, we traced the parental origin of HRAS alterations and demonstrated inheritance of the mutated allele exclusively from the father, further confirming a paternal bias in the parental origin of HRAS mutations in CS. Careful clinical evaluation of patients with BRAF and MAP2K1/2 alterations revealed the presence of slight phenotypic differences regarding craniofacial features in MAP2K1- and MAP2K2-mutation positive individuals, suggesting possible genotype-phenotype correlations.

    Clinical genetics 2008;73;1;62-70

  • Biochemical characterization of novel germline BRAF and MEK mutations in cardio-facio-cutaneous syndrome.

    Rodriguez-Viciana P and Rauen KA

    UCSF Helen Diller Family, Comprehensive Cancer Center and Cancer Research Institute, University of California, San Francisco, California, USA.

    Cardio-facio-cutaneous syndrome (CFC) is a sporadic, complex developmental disorder involving characteristic craniofacial features, cardiac defects, ectodermal abnormalities, growth deficiency, hypotonia, and developmental delay. CFC is caused by alteration of activity through the mitogen-activated protein kinase (MAPK) pathway due to heterogeneous de novo germline mutations in B-Raf mutant proteins, MEK1 and MEK2. Approximately 75% of individuals with CFC have mutations in BRAF. In vitro functional studies demonstrate that many of these mutations confer increase activity upon the mutant protein as compared to the wildtype protein. However, as is seen cancer, some of the B-Raf mutant proteins are kinase impaired. Western blot analyses corroborate kinase assays as determined by mutant proteins phosphorylating downstream effectors MEK and ERK. Approximately 25% of individuals with CFC have mutations in either MEK1 or MEK2 that lead to increased MEK kinase activity as judged by increased phosphorylation of its downstream effector ERK. Unlike BRAF, no somatic mutations have ever been identified in MEK genes. The identification of novel germline BRAF and MEK mutations in CFC will help understand the pathophysiology of this syndrome. Furthermore, it will also provide insight to the normal function of B-Raf and MEK, and contribute to the knowledge of the role of the MAPK pathway in cancer. Since the MAPK pathway has been studied intensively in the context of cancer, numerous therapeutics that specifically target this pathway may merit investigation in this population of patients.

    Funded by: NICHD NIH HHS: HD048502

    Methods in enzymology 2008;438;277-89

  • Critical role of the phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase-3 signaling pathway in recovery from anthrax lethal toxin-induced cell cycle arrest and MEK cleavage in macrophages.

    Ha SD, Ng D, Pelech SL and Kim SO

    Department of Microbiology and Immunology, Siebens-Drake Research Institute, University of Western Ontario, London, Ontario, Canada N6G 2V4.

    Anthrax lethal toxin (LeTx) is a virulence factor causing immune suppression and toxic shock of Bacillus anthracis infected host. It inhibits cytokine production and cell proliferation/differentiation in various immune cells. This study showed that a brief exposure of LeTx caused a continual MEK1 cleavage and prevented tumor necrosis factor-alpha (TNF) production in response to lipopolysaccharide (LPS) in non-proliferating cells such as human peripheral blood mononuclear cells or mouse primary peritoneal macrophages. In human monocytic cell lines U-937 and THP-1, LeTx induced cell cycle arrest in G0-G1 phase by rapid down-regulation of cyclin D1/D2 and checkpoint kinase 1 through MEK1 inhibition. However, THP-1 cells adaptively adjusted to LeTx and overrode cell cycle arrest by activating the phosphatidylinositol 3-kinase/Akt signaling pathway. Inhibitory Ser-9 phosphorylation of glycogen synthase kinase 3beta (GSK3beta) by Akt prevented proteasome-mediated cyclin D1 degradation and induced cell cycle progress in LeTx-intoxicated THP-1 cells. Recovery from cell cycle arrest was required before recovering from on-going MEK1 cleavage and suppression of TNF production. Furthermore, pretreatment with LeTx or the GSK3-specific inhibitor SB-216763, or transfection with dominant active mutant Akt or degradation-defected mutant cyclin D1 protected cells from LeTx-induced cell cycle arrest, on-going MEK1 cleavage and suppression of TNF production. These results indicate that modulation of phosphatidylinositol 3-kinase/Akt/GSK3beta signaling cascades can be beneficial for protecting or facilitating recovery from cellular LeTx intoxication in cells that depend on basal MEK1 activity for proliferation.

    The Journal of biological chemistry 2007;282;50;36230-9

  • Mutation analysis of BRAF, MEK1 and MEK2 in 15 ovarian cancer cell lines: implications for therapy.

    Estep AL, Palmer C, McCormick F and Rauen KA

    Comprehensive Cancer Center and Cancer Research Institute, University of California at San Francisco, San Francisco, California, United States of America.

    Background: Among gynecologic cancers, ovarian cancer is the second most common and has the highest death rate. Cancer is a genetic disorder and arises due to the accumulation of somatic mutations in critical genes. An understanding of the genetic basis of ovarian cancer has implications both for early detection and for therapeutic intervention in this population of patients.

    Fifteen ovarian cancer cell lines, commonly used for in vitro experiments, were screened for mutations using bidirectional direct sequencing in all coding regions of BRAF, MEK1 and MEK2. BRAF mutations were identified in four of the fifteen ovarian cancer cell lines studied. Together, these four cell lines contained four different BRAF mutations, two of which were novel. ES-2 had the common B-Raf p.V600E mutation in exon 15 and Hey contained an exon 11 missense mutation, p.G464E. The two novel B-Raf mutants identified were a 5 amino acid heterozygous deletion p.N486-P490del in OV90, and an exon 4 missense substitution p.Q201H in OVCAR 10. One of the cell lines, ES-2, contained a mutation in MEK1, specifically, a novel heterozygous missense substitution, p.D67N which resulted from a nt 199 G-->A transition. None of the cell lines contained coding region mutations in MEK2. Functional characterization of the MEK1 mutant p.D67N by transient transfection with subsequent Western blot analysis demonstrated increased ERK phosphorylation as compared to controls.

    In this study, we report novel BRAF mutations in exon 4 and exon 12 and also report the first mutation in MEK1 associated with human cancer. Functional data indicate the MEK1 mutation may confer alteration of activation through the MAPK pathway. The significance of these findings is that BRAF and MEK1/2 mutations may be more common than anticipated in ovarian cancer which could have important implications for treatment of patients with this disease and suggests potential new therapeutic avenues.

    Funded by: NICHD NIH HHS: HD048502, K23 HD048502

    PloS one 2007;2;12;e1279

  • 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

  • Cardio-facio-cutaneous and Noonan syndromes due to mutations in the RAS/MAPK signalling pathway: genotype-phenotype relationships and overlap with Costello syndrome.

    Nava C, Hanna N, Michot C, Pereira S, Pouvreau N, Niihori T, Aoki Y, Matsubara Y, Arveiler B, Lacombe D, Pasmant E, Parfait B, Baumann C, Héron D, Sigaudy S, Toutain A, Rio M, Goldenberg A, Leheup B, Verloes A and Cavé H

    Department of Genetics, AP-HP, Hôpital Robert Debré, Paris, France.

    Cardio-facio-cutaneous (CFC) syndrome, Noonan syndrome (NS), and Costello syndrome (CS) are clinically related developmental disorders that have been recently linked to mutations in the RAS/MEK/ERK signalling pathway. This study was a mutation analysis of the KRAS, BRAF, MEK1 and MEK2 genes in a total of 130 patients (40 patients with a clinical diagnosis of CFC, 20 patients without HRAS mutations from the French Costello family support group, and 70 patients with NS without PTPN11 or SOS1 mutations). BRAF mutations were found in 14/40 (35%) patients with CFC and 8/20 (40%) HRAS-negative patients with CS. KRAS mutations were found in 1/40 (2.5%) patients with CFC, 2/20 (10%) HRAS-negative patients with CS and 4/70 patients with NS (5.7%). MEK1 mutations were found in 4/40 patients with CFC (10%), 4/20 (20%) HRAS-negative patients with CS and 3/70 (4.3%) patients with NS, and MEK2 mutations in 4/40 (10%) patients with CFC. Analysis of the major phenotypic features suggests significant clinical overlap between CS and CFC. The phenotype associated with MEK mutations seems less severe, and is compatible with normal mental development. Features considered distinctive for CS were also found to be associated with BRAF or MEK mutations. Because of its particular cancer risk, the term "Costello syndrome" should only be used for patients with proven HRAS mutation. These results confirm that KRAS is a minor contributor to NS and show that MEK is involved in some cases of NS, demonstrating a phenotypic continuum between the clinical entities. Although some associated features appear to be characteristic of a specific gene, no simple rule exists to distinguish NS from CFC easily.

    Journal of medical genetics 2007;44;12;763-71

  • Protein kinase WNK2 inhibits cell proliferation by negatively modulating the activation of MEK1/ERK1/2.

    Moniz S, Veríssimo F, Matos P, Brazão R, Silva E, Kotelevets L, Kotevelets L, Chastre E, Gespach C and Jordan P

    Centre of Human Genetics, Instituto Nacional de Saúde Dr Ricardo Jorge, Lisbon, Portugal.

    The recently identified subfamily of WNK protein kinases is characterized by a unique sequence variation in the catalytic domain and four related human WNK genes were identified. Here, we describe the cloning and functional analysis of the human family member WNK2. We show that the depletion of endogenous WNK2 expression by RNA interference in human cervical HeLa cancer cells led to the activation of the extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinases but, in contrast to the depletion of WNK1, had no effect on ERK5. Furthermore, expression of a kinase-dead WNK2-K207M mutant also activated ERK1/2 suggesting that WNK2 catalytic activity is required. Depletion of WNK2 expression increased G1/S progression and potentiated the cellular response to low epidermal growth factor concentrations. The molecular mechanism of ERK1/2 activation in WNK2-depleted cells lies downstream of the Raf kinases and involves MEK1 phosphorylation at serine 298 in both HeLa and HT29 colon cancer cells. This modification is linked to the upregulation of MEK1 activity toward ERK1/2. Together, these results provide evidence that WNK2 is involved in the modulation of growth factor-induced cancer cell proliferation through the MEK1/ERK1/2 pathway. The data identify WNK2 as a candidate tumor suppressor gene and suggest a coordinated activity of WNK kinases in the regulation of cell proliferation.

    Oncogene 2007;26;41;6071-81

  • An intrinsic ATPase activity of phospho-MEK-1 uncoupled from downstream ERK phosphorylation.

    Rominger CM, Schaber MD, Yang J, Gontarek RR, Weaver KL, Broderick T, Carter L, Copeland RA and May EW

    Department of Enzymology and Mechanistic Pharmacology, GlaxoSmithKline Pharmaceuticals, 1250 South Collegeville Road, Collegeville, PA 19426, USA.

    We have developed a highly sensitive assay of MEK-mediated ATP hydrolysis by coupling the formation of ADP to NADH oxidation through the enzymes pyruvate kinase and lactate dehydrogenase. Robust ATP hydrolysis is catalyzed by phosphorylated MEK in the absence of the protein substrate ERK. This ERK-uncoupled ATPase activity is dependent on the phosphorylation status of MEK and is abrogated by the selective MEK kinase inhibitor U0126. ADP production is concomitant with Raf-mediated phosphorylation of MEK. Based on this finding, a coupled Raf/MEK assay is developed for measuring the Raf activity. A kinetic treatment derived under steady-state assumptions is presented for the analysis of the reaction progress curve generated by this coupled assay. We have shown that inhibitory potency of selective Raf inhibitors can be determined accurately by this assay.

    Archives of biochemistry and biophysics 2007;464;1;130-7

  • 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

  • SMAD3 expression is regulated by mitogen-activated protein kinase kinase-1 in epithelial and smooth muscle cells.

    Ross KR, Corey DA, Dunn JM and Kelley TJ

    Department of Pediatrics, Case Western Reserve University, and Rainbow Babies and Children's Hospital, Cleveland, OH 44106-4948, USA.

    SMAD3 is a transcription factor that mediates TGF-beta1 signaling and is known to be important in many of the cellular processes that regulate fibrosis and inflammation. Although several studies have examined SMAD3 activation, little is known about the control of SMAD3 expression. It is well established that the mitogen-activated protein kinase (MAPK) pathway is responsive to TGF-beta1 stimulation and coordinates with SMAD signaling in many cases; therefore, the hypothesis of this study is that the MAPK pathway will be involved in the regulation of SMAD3 expression. Using a SMAD3 promoter construct, we demonstrate that inhibition of either c-Jun-N-terminal kinase (JNK) or p38 activity has little effect on SMAD3 promoter function. Inhibition of mitogen-activated protein kinase kinase-1 (MEK1) with either PD98059 or UO126, however, results in a substantial dose-dependent inhibition of SMAD3 promoter activity. Further studies confirm that promoter activity correlates with protein expression by demonstrating reduced SMAD3 protein expression in A549 cells and airway smooth muscle cells after treatment with MEK1 inhibitors. Positive regulation of SMAD3 expression is also demonstrated by expression of a constitutively active (ca)-MEK1 construct, where the presence of ca-MEK1 resulted in increased SMAD3 protein expression. These data lead to the conclusion that MEK1 is an important regulator of SMAD3 expression.

    Funded by: NCRR NIH HHS: KL2 RR024990

    Cellular signalling 2007;19;5;923-31

  • Molecular and clinical characterization of cardio-facio-cutaneous (CFC) syndrome: overlapping clinical manifestations with Costello syndrome.

    Narumi Y, Aoki Y, Niihori T, Neri G, Cavé H, Verloes A, Nava C, Kavamura MI, Okamoto N, Kurosawa K, Hennekam RC, LC, Gillessen-Kaesbach G, Wieczorek D, Lapunzina P, Ohashi H, Makita Y, Kondo I, Tsuchiya S, Ito E, Sameshima K, Kato K, Kure S and Matsubara Y

    Department of Medical Genetics, Tohoku University School of Medicine, Sendai, Japan.

    Cardio-facio-cutaneous (CFC) syndrome is a multiple congenital anomaly/mental retardation syndrome characterized by heart defects, a distinctive facial appearance, ectodermal abnormalities and mental retardation. Clinically, it overlaps with both Noonan syndrome and Costello syndrome, which are caused by mutations in two genes, PTPN11 and HRAS, respectively. Recently, we identified mutations in KRAS and BRAF in 19 of 43 individuals with CFC syndrome, suggesting that dysregulation of the RAS/RAF/MEK/ERK pathway is a molecular basis for CFC syndrome. The purpose of this study was to perform comprehensive mutation analysis in 56 patients with CFC syndrome and to investigate genotype-phenotype correlation. We analyzed KRAS, BRAF, and MAP2K1/2 (MEK1/2) in 13 new CFC patients and identified five BRAF and one MAP2K1 mutations in nine patients. We detected one MAP2K1 mutation in three patients and four new MAP2K2 mutations in four patients out of 24 patients without KRAS or BRAF mutations in the previous study [Niihori et al., 2006]. No mutations were identified in MAPK3/1 (ERK1/2) in 21 patients without any mutations. In total, 35 of 56 (62.5%) patients with CFC syndrome had mutations (3 in KRAS, 24 in BRAF, and 8 in MAP2K1/2). No significant differences in clinical manifestations were found among 3 KRAS-positive patients, 16 BRAF-positive patients, and 6 MAP2K1/2-positive patients. Wrinkled palms and soles, hyperpigmentation and joint hyperextension, which have been commonly reported in Costello syndrome but not in CFC syndrome, were observed in 30-40% of the mutation-positive CFC patients, suggesting a significant clinical overlap between these two syndromes.

    American journal of medical genetics. Part A 2007;143A;8;799-807

  • Mek1/2 MAPK kinases are essential for Mammalian development, homeostasis, and Raf-induced hyperplasia.

    Scholl FA, Dumesic PA, Barragan DI, Harada K, Bissonauth V, Charron J and Khavari PA

    Veterans Affairs Palo Alto Healthcare System, Palo Alto, CA 94304, USA.

    The p42/p44 mitogen-activated protein kinase (MAPK) cascade includes Ras, Raf, Mek, and Erk MAPK. To determine the effect of a full knockout at a single level of this signaling pathway in mammals, and to investigate functional redundancy between Mek1 and Mek2, we disrupted these genes in murine and human epidermis. Loss of either protein alone produced no phenotype, whereas combined Mek1/2 deletion in development or adulthood abolished Erk1/2 phosphorylation and led to hypoproliferation, apoptosis, skin barrier defects, and death. Conversely, a single copy of either allele was sufficient for normal development. Combined Mek1/2 loss also abolished Raf-induced hyperproliferation. Human tissue deficient in either Mek isoform was normal, whereas loss of both proteins led to hypoplasia, which was rescued by active Erk2 expression. These data indicate that Mek1/2 are functionally redundant in the epidermis, where they act as a linear relay in the MAPK pathway to mediate development and homeostasis.

    Funded by: NIAMS NIH HHS: AR43799, AR49737

    Developmental cell 2007;12;4;615-29

  • Roles of the MEK1/2 and AKT pathways in CXCL12/CXCR4 induced cholangiocarcinoma cell invasion.

    Leelawat K, Leelawat S, Narong S and Hongeng S

    Department of Surgery, Rajavithi Hospital, Bangkok 10400, Thailand. sckll@mahidol.ac.th

    Aim: To evaluate the expression of C-X-C motif chemokine receptor 4 (CXCR4) and its signaling cascades, which were previously identified as a key factor for cancer cell progression and metastasis, in cholangiocarcinoma cell lines.

    Methods: The expression of CXCR4 and its signaling cascades were determined in the cholangiocarcinoma cell lines (RMCCA1 and KKU100) by Western blotting. The invasion assays and the detection of actin polymerization were tested in these cholangiocarcinoma cells treated with CXC chemokine ligand -12 (CXCL12).

    Results: Expression of CXCR4 was detected in both cholangiocarcinoma cell lines and activation of CXCR4 with CXCL12 triggered the signaling via the extracellular signal-regulated kinase-1/2 (ERK1/2) and phosphoinositide 3-kinase (PI3K) and induction of cholangiocarcinoma cell invasion, and displayed high levels of actin polymerization. Addition of CXCR4 inhibitor (AMD3100) abrogated CXCL12-induced phosphorylation of MEK1/2 and Akt in these cells. Moreover, treatment with MEK1/2 inhibitor (U0126) or PI3K inhibitor (LY294002) also attenuated the effect of CXCL12-induced cholangiocarcinoma cell invasion.

    Conclusion: These results indicated that the activation of CXCR4 and its signaling pathways (MEK1/2 and Akt) are essential for CXCL12-induced cholangiocarcinoma cell invasion. This rises Implications on a potential role for the inhibition of CXCR4 or its signal cascades in the treatment of cholangiocarcinoma.

    World journal of gastroenterology 2007;13;10;1561-8

  • Proteomics analysis of protein kinases by target class-selective prefractionation and tandem mass spectrometry.

    Wissing J, Jänsch L, Nimtz M, Dieterich G, Hornberger R, Kéri G, Wehland J and Daub H

    Department of Cell Biology, Helmholtz Centre for Infection Research (HZI), Inhoffenstrasse 7, 38124 Braunschweig, Germany.

    Protein kinases constitute a large superfamily of enzymes with key regulatory functions in nearly all signal transmission processes of eukaryotic cells. However, due to their relatively low abundance compared with the vast majority of cellular proteins, currently available proteomics techniques do not permit the comprehensive biochemical characterization of protein kinases. To address these limitations, we have developed a prefractionation strategy that uses a combination of immobilized low molecular weight inhibitors for the selective affinity capture of protein kinases. This approach resulted in the direct purification of cell type-specific sets of expressed protein kinases, and more than 140 different members of this enzyme family could be detected by LC-MS/MS. Furthermore the enrichment technique combined with phosphopeptide fractionation led to the identification of more than 200 different phosphorylation sites on protein kinases, which often remain occluded in global phosphoproteome analysis. As the phosphorylation states of protein kinases can provide a readout for the signaling activities within a cellular system, kinase-selective phosphoproteomics based on the procedures described here has the potential to become an important tool in signal transduction analysis.

    Molecular & cellular proteomics : MCP 2007;6;3;537-47

  • Thermodynamics of nucleotide and non-ATP-competitive inhibitor binding to MEK1 by circular dichroism and isothermal titration calorimetry.

    Smith CK and Windsor WT

    Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033, USA. catherine.smith15@spcorp.com

    MEK1 is a member of the MAPK signal transduction pathway that responds to growth factors and cytokines. A wealth of information about the enzymatic activity of MEK1, its domain functions, and inhibitor action is available; however, the thermodynamic properties of the interaction between MEK1 and ligands, such as nucleotides and non-ATP-competitive inhibitors, have not been reported. This study describes the thermodynamic parameters for the binding interactions of MEK1, nucleotides, and non-ATP-competitive inhibitor complexes using temperature-dependent circular dichroism (TdCD) and isothermal titration calorimetry (ITC). Non-phosphorylated MEK1 (npMEK1) has a high affinity for both AMP-PNP and ADP (Kd approximately 2microM). The binding is enthalpically favored and Mg-dependent. The active, phosphorylated form of MEK1 (pMEK1) bound nucleotides with a similar high affinity (Kd approximately 2muM) and had a thermodynamic profile and Mg-dependence similar to that of the non-phosphorylated form. The non-ATP-competitive MEK1 inhibitors, U0126 and PD0325901, showed no preference for npMEK1 and pMEK1 by TdCD. TdCD results also showed that these inhibitors are more potent in the presence of the nucleotide than in its absence. The ternary complex, MEK1.PD0325901.nucleotide, showed synergistic binding as evidenced by a large, non-additive shift in the midpoint of the protein unfolding transition (Tm). This was apparent for both npMEK1 and pMEK1 using either ADP or AMP-PNP. ITC binding studies confirmed the synergistic binding effect. The ITC-determined affinity of nucleotide (AMP-PNP, ADP) binding to the npMEK1.PD0325901 complex was enhanced nearly 5-fold compared to nucleotide binding to npMEK1 alone. In addition, the affinity of PD0325901 binding to npMEK1.nucleotide complexes was increased nearly 10-fold relative to the affinity of PD0325901 for npMEK1 alone. These are the first thermodynamic binding studies that characterize the affinity of the allosteric non-ATP-competitive inhibitors U0126 and PD0325901 with and without the nucleotide. The results indicate these allosteric inhibitors have a dynamic range in the type of MEK1 activation states and nucleotide complexes that they can bind.

    Biochemistry 2007;46;5;1358-67

  • Interaction with MEK causes nuclear export and downregulation of peroxisome proliferator-activated receptor gamma.

    Burgermeister E, Chuderland D, Hanoch T, Meyer M, Liscovitch M and Seger R

    Department of Biological Regulation, The Weizmann Institute of Science, Rehovot 76100, Israel.

    The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) cascade plays a central role in intracellular signaling by many extracellular stimuli. One target of the ERK cascade is peroxisome proliferator-activated receptor gamma (PPARgamma), a nuclear receptor that promotes differentiation and apoptosis. It was previously demonstrated that PPARgamma activity is attenuated upon mitogenic stimulation due to phosphorylation of its Ser84 by ERKs. Here we show that stimulation by tetradecanoyl phorbol acetate (TPA) attenuates PPARgamma's activity in a MEK-dependent manner, even when Ser84 is mutated to Ala. To elucidate the mechanism of attenuation, we found that PPARgamma directly interacts with MEKs, which are the activators of ERKs, but not with ERKs themselves, both in vivo and in vitro. This interaction is facilitated by MEKs' phosphorylation and is mediated by the basic D domain of MEK1 and the AF2 domain of PPARgamma. Immunofluorescence microscopy and subcellular fractionation revealed that MEK1 exports PPARgamma from the nucleus, and this finding was supported by small interfering RNA knockdown of MEK1 and use of a cell-permeable interaction-blocking peptide, which prevented TPA-induced export of PPARgamma from the nucleus. Thus, we show here a novel mode of downregulation of PPARgamma by its MEK-dependent redistribution from the nucleus to the cytosol. This unanticipated role for the stimulation-induced nuclear shuttling of MEKs shows that MEKs can regulate additional signaling components besides the ERK cascade.

    Molecular and cellular biology 2007;27;3;803-17

  • Mitogen-activated protein kinase kinase 1-dependent Golgi unlinking occurs in G2 phase and promotes the G2/M cell cycle transition.

    Feinstein TN and Linstedt AD

    Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.

    Two controversies have emerged regarding the signaling pathways that regulate Golgi disassembly at the G(2)/M cell cycle transition. The first controversy concerns the role of mitogen-activated protein kinase activator mitogen-activated protein kinase kinase (MEK)1, and the second controversy concerns the participation of Golgi structure in a novel cell cycle "checkpoint." A potential simultaneous resolution is suggested by the hypothesis that MEK1 triggers Golgi unlinking in late G(2) to control G(2)/M kinetics. Here, we show that inhibition of MEK1 by RNA interference or by using the MEK1/2-specific inhibitor U0126 delayed the passage of synchronized HeLa cells into M phase. The MEK1 requirement for normal mitotic entry was abrogated if Golgi proteins were dispersed before M phase by treatment of cells with brefeldin A or if GRASP65, which links Golgi stacks into a ribbon network, was depleted. Imaging revealed that unlinking of the Golgi apparatus begins before M phase, is independent of cyclin-dependent kinase 1 activation, and requires MEK signaling. Furthermore, expression of the GRASP family member GRASP55 after alanine substitution of its MEK1-dependent mitotic phosphorylation sites inhibited both late G(2) Golgi unlinking and the G(2)/M transition. Thus, MEK1 plays an in vivo role in Golgi reorganization, which regulates cell cycle progression.

    Funded by: NIGMS NIH HHS: GM-56779, R01 GM056779

    Molecular biology of the cell 2007;18;2;594-604

  • MEK-ERK inhibition corrects the defect in VLDL assembly in HepG2 cells: potential role of ERK in VLDL-ApoB100 particle assembly.

    Tsai J, Qiu W, Kohen-Avramoglu R and Adeli K

    Division of Clinical Biochemistry, Hospital for Sick Children, University of Toronto, Ontario, Canada M5G 1X8.

    Objective: Hepatic VLDL assembly is defective in HepG2 cells, resulting in the secretion of immature triglyceride-poor LDL-sized apoB particles. We investigated the mechanisms underlying defective VLDL assembly in HepG2 and have obtained evidence implicating the MEK-ERK pathway.

    HepG2 cells exhibited considerably higher levels of the ERK1/2 mass and activity compared with primary hepatocytes. Inhibition of ERK1/2 using the MEK1/MEK2 inhibitor, U0126 (but not the inactive analogue) led to a significant increase in apoB secretion. In the presence of oleic acid, ERK1/2 inhibition caused a major shift in the lipoprotein distribution with a majority of particles secreted as VLDL, an effect independent of insulin. In contrast, overexpression of constitutively active MEK1 decreased apoB and large VLDL secretion. MEK1/2 inhibition significantly increased both cellular and microsomal TG mass, and mRNA levels for DGAT-1 and DGAT-2. In contrast to ERK, modulation of the PI3-K pathway or inhibition of the p38 MAP kinase, had no effect on lipoprotein density profile. Modulation of the MEK-ERK pathway in primary hamster hepatocytes led to changes in apoB secretion and altered the density profile of apoB-containing lipoproteins.

    Conclusions: Inhibition of the overactive ras-MEK-ERK pathway in HepG2 cells can correct the defect in VLDL assembly leading to the secretion of large, VLDL-sized particles, similar to primary hepatocytes, implicating the MEK-ERK cascade in VLDL assembly in the HepG2 model. Modulation of this pathway in primary hepatocytes also regulates apoB secretion and appears to alter the formation of VLDL-1 sized particles.

    Arteriosclerosis, thrombosis, and vascular biology 2007;27;1;211-8

  • Mitogen-activated protein kinase kinase 1/extracellular signal-regulated kinase (MEK-1/ERK) inhibitors sensitize reduced glucocorticoid response mediated by TNFalpha in human epidermal keratinocytes (HaCaT).

    Onda K, Nagashima M, Kawakubo Y, Inoue S, Hirano T and Oka K

    Department of Clinical Pharmacology, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. knjond@ps.toyaku.ac.jp

    Glucocorticoids (GCs) are 1f40 essential drugs administered topically or systematically for the treatment of autoimmune skin diseases such as pemphigus. However, a certain proportion of patients does not respond well to GCs. Although studies on the relationship between cytokines and GC insensitivity in local tissues have attracted attention recently, little is known about the underlying mechanism(s) for GC insensitivity in epidermal keratinocytes. Here, we report that tumor necrosis factor (TNF) alpha reduces GC-induced transactivation of endogenous genes as well as a reporter plasmid which contains GC responsive element (GRE) in human epidermal keratinocyte cells (HaCaT). The GC insensitivity by TNFalpha was not accompanied by changes in mRNA expressions of GR isoforms (alpha or beta). However, we observed that mitogen-activated protein kinase kinase-1/extracellular signal-regulated kinase (MEK-1/ERK) inhibitors (PD98059 and U0126) significantly sensitized the GC-induced transactivation of anti-inflammatory genes (glucocorticoid-induced leucine zipper (GILZ) and mitogen-activated protein kinase phosphatase (MKP)-1) and FK506 binding protein (FKBP) 51 gene in the presence of TNFalpha. Additionally, we observed that TNFalpha reduced prednisolone (PSL)-dependent nuclear translocation of GR, which was restored by pre-treatment of MEK-1 inhibitors. This is the first study demonstrating a role of the MEK-1/ERK cascade in TNFalpha-mediated GC insensitivity. Our data suggest that overexpression of TNFalpha leads to topical GC insensitivity by reducing GR nuclear translocation in keratinocytes, and our findings also suggest that inhibiting the MEK-1/ERK cascade may offer a therapeutic potential for increasing GC efficacy in epidermis where sufficient inflammatory suppression is required.

    Biochemical and biophysical research communications 2006;351;1;266-72

  • MEK1/2 inhibition promotes Taxotere lethality in mammary tumors in vivo.

    Yacoub A, Gilfor D, Hawkins W, Park MA, Hanna D, Hagan MP, Curiel DT, Fisher PB, Grant S and Dent P

    Department of Biochemistry, Virginia Commonwealth University, Richmond, Virginia 23298, USA.

    Taxol (paclitaxel) and Taxotere (docetaxel) are considered as two of the most important anti-cancer chemotherapy drugs. The cytotoxic action of these drugs has been linked to their ability to inhibit microtubule depolymerization, causing growth arrest and subsequent cell death. Studies by a number of laboratories have also linked suppression of MEK1/2 signaling to enhanced Taxol toxicity in vitro and in vivo. The present study examined the interactions of the semi-synthetic taxane Taxotere with MEK1/2 inhibitors in epithelial tumor cells. In vitro colony formation studies demonstrated that Taxotere and the MEK1/2 inhibitor PD184352 interacted in a sequence dependent fashion to synergistically kill human mammary carcinoma cells (MDA-MB-231, MCF7) as well as in other tumor cell types; e.g. prostate and renal cell carcinoma. Athymic mice were implanted in the rear flank with either MDA-MB-231 or MCF7 cells and tumors permitted to form to a volume of approximately 100 mm3 prior to a two day exposure of either Vehicle, PD184352 (25 mg/kg), Taxotere (15 mg/kg) or the drug combination. Tumor volume was measured every other day and tumor growth determined over the following approximately 30 days. Transient exposure of MDA-MB-231 tumors or MCF7 tumors to PD184352 did not significantly alter tumor growth rate or the mean tumor volume in vivo approximately 15-30 days after drug administration. Transient Taxotere exposure of MDA-MB-231 or to a lesser extent MCF7, tumors modestly reduced the mean tumor volume in vivo approximately 15-30 days after drug administration. In contrast, combined treatment with PD184352 and Taxotere significantly reduced MDA-MB-231 and MCF7 tumor growth. The tumor control values for MDA-MB-231 cells and MCF7 cells were 0.43 and 0.71, respectively. Fractionated irradiation of MDA-MB-231 tumors during drug exposure or single dose irradiation prior to drug administration did not significantly further suppress tumor growth beyond that of cells exposed to Taxotere and MEK1/2 inhibitor. Single dose irradiation of tumors after drug exposure, however, caused a significant further suppression of tumor growth below that caused by drug exposure. These findings were also reflected in ex vivo colony formation analyses of isolated tumor cells. Collectively, these findings argue that Taxotere and MEK1/2 inhibitors have the potential to suppress mammary tumor growth in vivo which is enhanced by sequence-dependent exposure to ionizing radiation. Based on the cell lines used in these studies, our findings argue that the interaction of Taxotere and PD184352 is independent of p53 status, estrogen dependency, caspase 3 levels or oncogenic K-RAS expression.

    Funded by: NCI NIH HHS: P01 CA104177

    Cancer biology & therapy 2006;5;10;1332-9

  • The expression of the homologue of the Caenorhabditis elegans lin-45 raf is regulated in the motile stages of the plant parasitic nematode Meloidogyne artiellia.

    Cortese MR, Di Vito M and De Giorgi C

    Dipartimento di Biochimica e Biologia Molecolare, Via Orabona 4, 70126 Bari, Italy.

    The Ras-MAPK signal transduction pathway controls multiple developmental events and is involved in the processing of olfactory information in the free living nematode Caenorhabditis elegans 1efe . We have studied the Ras-MAPK pathway in the plant parasitic nematode Meloidogyne artiellia. The genes Mt-let-60, Mt-lin-45, Mt-mek-2 and Mt-mpk-1 have been isolated and sequenced. Each of them shows a high level of sequence similarity to its presumed ortholog in C. elegans and key functional domains are structurally conserved. Furthermore, we show that the M. artiellia recombinant MEK-2 protein can phosphorylate and activate the M. artiellia recombinant MPK-1 and the recombinant MEK-2 itself can be phosphorylated and activated by immunoprecipitated mammalian Raf. Surprisingly, the Mt-lin-45 message is not detectable in freshly emerged juveniles or in male specimens, suggesting that it may be quickly degraded in these life stages.

    Molecular and biochemical parasitology 2006;149;1;38-47

  • Activation of the NaCl- and drought-induced RD29A and RD29B promoters by constitutively active Arabidopsis MAPKK or MAPK proteins.

    Hua ZM, Yang X and Fromm ME

    Plant Science Initiative, University of Nebraska, Lincoln, NE 68508, USA. zhimingh@hotmail.com

    Mitogen-activated protein (MAP) kinases mediate cellular responses to a wide variety of stimuli. Activation of a MAP kinase (MAPK) occurs after phosphorylation by an upstream MAP kinase kinase (MAPKK). The Arabidopsis thaliana genome encodes 10 MKKs, but few of these have been shown directly to activate any of the 20 Arabidopsis MAPKs (AtMPKs) and NaCl-, drought- or abscisic acid (ABA)-induced genes RD29A or RD29B. We have constructed the constitutively activated form for nine of the 10 AtMKK proteins, and tested their ability to activate the RD29A and RD29B promoters and also checked the ability of the nine activ 1f40 ated AtMKK proteins to phosphorylate 11 of the AtMPK proteins in transient assays. The results show that three proteins, AtMKK1, AtMKK2 and AtMKK3, could activate the RD29A promoter, while these three and two additional AtMKK6/8 proteins could activate the RD29B promoter. Four other proteins, AtMKK7/AtMKK9 and AtMKK4/AtMKK5, can cause hypersensitive response (HR) in tobacco leaves using transient analysis. The activation of the RD29A promoter correlated with four uniquely activated AtMPK proteins. A novel method of activating AtMPK proteins by fusion to a cis-acting mutant of a human MAPK kinase MEK1 was used to confirm that specific members of the AtMPK gene family can activate the RD29A stress pathway.

    Plant, cell & environment 2006;29;9;1761-70

  • Effects of GM-CSF and M-CSF on tumor progression of lung cancer: roles of MEK1/ERK and AKT/PKB pathways.

    Uemura Y, Kobayashi M, Nakata H, Kubota T, Bandobashi K, Saito T and Taguchi H

    Department of Internal Medicine, Kochi Medical School, Kochi 783-8505, Japan. uemuray@med.kochi-u.ac.jp

    Several studies have demonstrated that colony-stimulating factors (CSFs) are closely associated with tumor progression, metastasis and invasion through autocrine or paracrine mechanism in lung cancer. However, biologic roles of CSFs are still unknown. Elucidating the biologic roles of CSFs and the regulatory mechanisms of tumor-specific behavior by CSFs raises the possibility of having a new therapeutic approach for lung cancer. We previously established two adenocarcinoma cell lines, A924 and A964 and a large cell carcinoma cell line MI-4. MI-4 and A924 constitutively produced an abundant dose of granulocyte macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF). We examined the effects of GM-CSF and M-CSF on tumor growth, death, and invasion in CSF-producing (A924 and MI-4) and non-producing lung cancer cells (A549 and A964). These cell lines demonstrated both GM-CSF and M-CSF receptor mRNA expression. In our study, GM-CSF seemed to have advantage for tumor proliferation and invasion in lung cancer cells. M-CSF seemed to have advantage for tumor invasion, but not proliferation. The tumor-specific phenotypes (proliferation, invasion and survival) up-regulated by GM-CSF and M-CSF were mediated through MEK/ERK and PI3k/Akt pathways. However, when MEK/ERK was activated by transfection of active form of MEK1 cDNA, the tumor-specific behavior was promoted in CSF-non-producing cells, whereas inhibited in CSF-producing cells though MEK/ERK activation increased constitutive GM-CSF production. MEK/ERK signaling regulated differently tumor-specific behavior between CSF-producing cells and CSF-non-producing cells.

    International journal of molecular medicine 2006;18;2;365-73

  • FGF-2 protects small cell lung cancer cells from apoptosis through a complex involving PKCepsilon, B-Raf and S6K2.

    Pardo OE, Wellbrock C, Khanzada UK, Aubert M, Arozarena I, Davidson S, Bowen F, Parker PJ, Filonenko VV, Gout IT, Sebire N, Marais R, Downward J and Seckl MJ

    Lung Cancer Biology Group, Cancer Research UK, Imperial College London, Hammersmith Hospitals Campus, Du Cane Road, London, UK.

    Patients with small cell lung cancer (SCLC) die because of chemoresistance. Fibroblast growth factor-2 (FGF-2) increases the expression of antiapoptotic proteins, XIAP and Bcl-X(L), and triggers chemoresistance in SCLC cells. Here we show that these effects are mediated through the formation of a specific multiprotein complex comprising B-Raf, PKCepsilon and S6K2. S6K1, Raf-1 and other PKC isoforms do not form similar complexes. RNAi-mediated downregulation of B-Raf, PKCepsilon or S6K2 abolishes FGF-2-mediated survival. In contrast, overexpression of PKCepsilon increases XIAP and Bcl-X(L) levels and chemoresistance in SCLC cells. In a tetracycline-inducible system, increased S6K2 kinase activity triggers upregulation of XIAP, Bcl-X(L) and prosurvival effects. However, increased S6K1 kinase activity has no such effect. Thus, S6K2 but not S6K1 mediates prosurvival/chemoresistance signalling.

    The EMBO journal 2006;25;13;3078-88

  • ERK and beyond: insights from B-Raf and Raf-1 conditional knockouts.

    Galabova-Kovacs G, Kolbus A, Matzen D, Meissl K, Piazzolla D, Rubiolo C, Steinitz K and Baccarini M

    Max F. Perutz Laboratories, University of Vienna, Vienna, Austria.

    The Raf/MEK/ERK cascade is a highly conserved signal transduction module whose activation reportedly results in a plethora of physiological outcomes. Depending on the cell type or the stimulus used, the pathway has been implicated in proliferation, differentiation, survival, and migration. Their wide range of activities renders the component of the Raf/MEK/ERK pathway prime candidates for molecule-targeted therapies, in particular, but not exclusively, in the context of cancer. Ras, Raf and MEK inhibitors have been developed, and some of them are in advanced clinical trials. Somewhat surprising in view of all this interest, our understanding of the fundamental biology of the ERK pathway in vivo is still scanty. Its investigation has been hampered by the fact that conventional targeting of many of these genes results in embryonic lethality. Recently, we and others have generated mouse strains that allow the conditional ablation of the genes coding for Raf-1, B-Raf and MEK-1. We are using these tools to identify the essential biological functions of these kinases, and to understand how the ERK pathway is wired in vivo. Here, we discuss some of the surprises yielded by the analysis of the role of B-Raf and Raf-1 and of their downstream effectors.

    Cell cycle (Georgetown, Tex.) 2006;5;14;1514-8

  • Yersinia YopJ acetylates and inhibits kinase activation by blocking phosphorylation.

    Mukherjee S, Li Y, Wang Y, Ball HL, Goldsmith EJ and Orth K

    Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

    Yersinia species use a variety of type III effector proteins to target eukaryotic signaling systems. The effector YopJ inhibits mitogen-activated protein kinase (MAPK) and the nuclear factor kappaB (NFkappaB) signaling pathways used in innate immune response by preventing activation of the family of MAPK kinases (MAPKK). We show that YopJ acted as an acetyltransferase, using acetyl-coenzyme A (CoA) to modify the critical serine and threonine residues in the activation loop of MAPKK6 and thereby blocking phosphorylation. The acetylation on MAPKK6 directly competed with phosphorylation, preventing activation of the modified protein. This covalent modification may be used as a general regulatory mechanism in biological signaling.

    Funded by: NIAID 1f40 NIH HHS: R01-AI056404; NIDDK NIH HHS: R21-DK072134

    Science (New York, N.Y.) 2006;312;5777;1211-4

  • Germline mutations in genes within the MAPK pathway cause cardio-facio-cutaneous syndrome.

    Rodriguez-Viciana P, Tetsu O, Tidyman WE, Estep AL, Conger BA, Cruz MS, McCormick F and Rauen KA

    Comprehensive Cancer Center and Cancer Research Institute, University of California, San Francisco, CA 94115, USA.

    Cardio-facio-cutaneous (CFC) syndrome is a sporadic developmental disorder involving characteristic craniofacial features, cardiac defects, ectodermal abnormalities, and developmental delay. We demonstrate that heterogeneous de novo missense mutations in three genes within the mitogen-activated protein kinase (MAPK) pathway cause CFC syndrome. The majority of cases (18 out of 23) are caused by mutations in BRAF, a gene frequently mutated in cancer. Of the 11 mutations identified, two result in amino acid substitutions that occur in tumors, but most are unique and suggest previously unknown mechanisms of B-Raf activation. Furthermore, three of five individuals without BRAF mutations had missense mutations in either MEK1 or MEK2, downstream effectors of B-Raf. Our findings highlight the involvement of the MAPK pathway in human development and will provide a molecular diagnosis of CFC syndrome.

    Funded by: NICHD NIH HHS: HD048502

    Science (New York, N.Y.) 2006;311;5765;1287-90

  • Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes.

    Kimura K, Wakamatsu A, Ota T, Nishikawa T, Yamashita R, Yamamoto J, Sekine M, Tsuritani K, Wakaguri H, Ishii S, Sugiyama T, Saito K, Isono Y, Irie R, Kushida N, Yoneyama T 5a8 , Otsuka R, Kanda K, Yokoi T, Kondo H, Wagatsuma M, Murakawa K, Ishida S, Ishibashi T, Takahashi-Fujii A, Tanase T, Nagai K, Kikuchi H, Nakai K, Isogai T and Sugano S

    Life Science Research Laboratory, Central Research Laboratory, Hitachi, Ltd., Kokubunji, Tokyo, 185-8601, Japan.

    By analyzing 1,780,295 5'-end sequences of human full-length cDNAs derived from 164 kinds of oligo-cap cDNA libraries, we identified 269,774 independent positions of transcriptional start sites (TSSs) for 14,628 human RefSeq genes. These TSSs were clustered into 30,964 clusters that were separated from each other by more than 500 bp and thus are very likely to constitute mutually distinct alternative promoters. To our surprise, at least 7674 (52%) human RefSeq genes were subject to regulation by putative alternative promoters (PAPs). On average, there were 3.1 PAPs per gene, with the composition of one CpG-island-containing promoter per 2.6 CpG-less promoters. In 17% of the PAP-containing loci, tissue-specific use of the PAPs was observed. The richest tissue sources of the tissue-specific PAPs were testis and brain. It was also intriguing that the PAP-containing promoters were enriched in the genes encoding signal transduction-related proteins and were rarer in the genes encoding extracellular proteins, possibly reflecting the varied functional requirement for and the restricted expression of those categories of genes, respectively. The patterns of the first exons were highly diverse as well. On average, there were 7.7 different splicing types of first exons per locus partly produced by the PAPs, suggesting that a wide variety of transcripts can be achieved by this mechanism. Our findings suggest that use of alternate promoters and consequent alternative use of first exons should play a pivotal role in generating the complexity required for the highly elaborated molecular systems in humans.

    Genome research 2006;16;1;55-65

  • Mitogen-activated protein kinase (MAPK)-docking sites in MAPK kinases function as tethers that are crucial for MAPK regulation in vivo.

    Grewal S, Molina DM and Bardwell L

    Department of Developmental and Cell Biology, 5205 McGaugh Hall, University of California Irvine, CA 92697-2300, USA.

    Docking sites on targets of mitogen-activated protein kinases (MAPKs) facilitate accurate and efficient substrate phosphorylation. MAPK/ERK kinases (MEKs, or MKKs), the upstream regulators of MAPKs, also contain N-terminal MAPK-docking sites, or 'D-sites'; however, the in vivo functions of MEK D-sites are incompletely understood. Here we found that the ability of constitutively-active human MEK1 and MEK2 to stimulate ERK phosphorylation and to induce the neoplastic transformation of NIH 3T3 cells required the integrity of the D-site. In addition, D-site mutants of otherwise wild-type MEK1/2 were unable to anchor unphosphorylated ERK2 in the cytoplasm. ERK activation, cytoplasmic anchoring and release were completely retained in 'swap' mutants in which MEK2's D-site was replaced with the D-site of MEK1 or yeast Ste7. Furthermore, these abilities were significantly retained when MEK2's D-site was moved to its C-terminus, or replaced by an unrelated MAPK-binding domain taken from the Ets-1 transcription factor. We conclude that the D-sites in MEKs are crucial for the activation of their cognate MAPKs in vivo, and that their primary function is to tether their cognate MAPKs near the MEK's kinase domain. This proximity effect is sufficient to explain the contribution that the D-site interaction makes to several biologically important signaling events.

    Funded by: NIGMS NIH HHS: GM60366, R01 GM060366, R01 GM060366-06

    Cellular signalling 2006;18;1;123-34

  • Modelling thymic HIV-1 Nef effects.

    Stove V and Verhasselt B

    Department of Clinical Chemistry, Microbiology and Immunology, Ghent University, Ghent University Hospital, 9000 Ghent, Belgium.

    The nef gene is conserved among primate lentiviruses and is one of the first viral genes that is transcribed following infection. This suggests a critical role for Nef in the virus life cycle and in the pathogenesis of lentiviral infections. In vitro, several functions have been described, including down regulation of CD4 and MHC class I surface expression, altered T-cell signaling and activation, and enhanced viral infectivity. However, the impact of these individual functions on viral pathogenicity in general, and thymic T cell production in particular, remains elusive. Here, we review the observations from experimental models that have been used to study the pathogenic effect of HIV-1 Nef on the thymus. These in vitro and in vivo studies have led to a better understanding of Nef's mechanism of action, although there still exists discord as to the contribution of Nef-mediated disturbance of thymopoiesis in the pathogenesis of AIDS.

    Current HIV research 2006;4;1;57-64

  • 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

  • Role of RANKL in physiological and pathological bone resorption and therapeutics targeting the RANKL-RANK signaling system.

    Tanaka S, Nakamura K, Takahasi N and Suda T

    Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, Tokyo, Japan. tanakas-ort@h.u-tokyo.ac.jp

    Osteoclasts are primary cells for physiological and pathological bone resorption, and receptor activator of nuclear factor-kappaB ligand (RANKL) is critically involved in the differentiation, activation, and survival of these cells. Recently, therapeutics for pathological bone destruction targeting RANKL pathways has attracted a great deal of attention. Herein, we review the recent advances in the research on osteoclast biology and discuss the advantages and disadvantages of anti-RANKL therapies.

    Immunological reviews 2005;208;30-49

  • 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, 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

  • Cdc2-mediated inhibition of epidermal growth factor activation of the extracellular signal-regulated kinase pathway during mitosis.

    Dangi S and Shapiro P

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

    Inhibition of general transcription and translation occurs during mitosis to preserve the high energy requirements needed for the dynamic structural changes that are occurring at this time of the cell cycle. Although the mitotic kinase Cdc2 appears to directly phosphorylate and inhibit key proteins directly involved in transcription and translation, the role of Cdc2 in regulating up-stream growth factor receptor-mediated signal transduction pathways is limited. In the present study, we examined mechanisms involved in uncoupling receptor-mediated activation of the extracellular signal-regulated (ERK) signaling pathway in mitotic cells. Treatment with epidermal growth factor (EGF) failed to activate the ERK pathway in mitotic cells, although partial activation of ERK could be achieved in mitotic cells treated with phorbol 12-myristate 13-acetate (PMA). The discrepancy between EGF and PMA-mediated ERK activation suggested that multiple events in the ERK pathway were regulated during mitosis. We show that Cdc2 inhibits EGF-mediated ERK activation through direct interaction and phosphorylation of several ERK pathway proteins, including the guanine nucleotide exchange factor, Sos-1, and Raf-1 kinase. Inhibition of Cdc2 activity with roscovitine in mitotic cells restored ERK activation by EGF and PMA. Similarly, mitotic inhibition of ERK activity in cells expressing active mutants of H-Ras and Raf-1 kinase could also be reversed following Cdc2 inhibition. In contrast, ERK activation in cells expressing active MEK1 was not inhibited during mitosis or affected by roscovitine. These data suggest that Cdc2 inhibits growth factor receptor-mediated ERK activation during mitosis by primarily targeting signaling proteins that are upstream of MEK1.

    The Journal of biological chemistry 2005;280;26;24524-31

  • Transformation by oncogenic RAS sensitizes human colon cells to TRAIL-induced apoptosis by up-regulating death receptor 4 and death receptor 5 through a MEK-dependent pathway.

    Drosopoulos KG, Roberts ML, Cermak L, Sasazuki T, Shirasawa S, Andera L and Pintzas A

    Laboratory of Signal Mediated Gene Expression, Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, Greece.

    RAS oncogenes play a major role in cancer development by activating an array of signaling pathways, most notably mitogen-activated protein kinases, resulting in aberrant proliferation and inhibition of apoptotic signaling cascades, rendering transformed cells resistant to extrinsic death stimuli. However, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is able to kill specific tumor cells through the engagement of its receptors, death receptor 4 (DR4) and death receptor 5 (DR5), and the activation of apoptotic pathways, providing promising targets for anticancer therapies. In this study, we show that TRAIL induces cell death in human colon adenocarcinoma cells in a MEK-dependent manner. We also report a prolonged MEK-dependent activation of ERK1/2 and increased c-FOS expression induced by TRAIL in this system. Our study reveals that transformation of the colon cell line Caco-2 by Ki- and mainly by Ha-ras oncogenes sensitizes these cells to TRAIL-induced apoptosis by causing specific MEK-dependent up-regulation of DR4 and DR5. These observations taken together reveal that RAS-MEK-ERK1/2 signaling pathway can sensitize cells to TRAIL-induced apoptosis by up-regulating DR4 and DR5 and overall imply that TRAIL-based therapeutic strategies using TRAIL agonists could be used in cases of human colon cancers bearing RAS mutations.

    The Journal of biological chemistry 2005;280;24;22856-67

  • 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

  • MEK1-dependent delayed expression of Fos-related antigen-1 counteracts c-Fos and p65 NF-kappaB-mediated interleukin-8 transcription in response to cytokines or growth factors.

    Hoffmann E, Thiefes A, Buhrow D, Dittrich-Breiholz O, Schneider H, Resch K and Kracht M

    Institute of Pharmacology, Medical School Hannover, Carl-Neuberg Strasse 1, D-30625 Hannover, Germany.

    Binding sites for the dimeric transcription factor activator protein (AP)-1 are found in numerous immunoregulatory and inflammatory genes. The precise mechanisms by which AP-1 activates or represses immune response genes and in particular the roles of individual AP-1 subunits in inflammatory responses are largely unknown. We report here that c-Fos and Fos-related ant 1f40 igen-1 (Fra-1), two inducible components of AP-1, are recruited to the endogenous interleukin (IL)-8 promoter in an IL-1-dependent manner. c-Fos activates IL-8 transcription and synergizes in this effect with p65 NF-kappaB. In contrast, Fra-1 strongly inhibits inducible IL-8 transcription. Fra-1 activation involves its stabilization, ubiquitination, and interaction with histone deacetylase-1. Blockade of MEK1 by PD98059 suppresses c-Fos and Fra-1 expression and, thus, affects two counteractive signals for IL-8 mRNA synthesis simultaneously. This disturbs the inducible recruitment of TATA box-binding protein and RNA polymerase II to the IL-8 promoter. Additional experiments reveal that, in conjunction with p65 NF-kappaB, the MEK1-ERK-dependent synthesis of c-Fos and Fra-1 serves to adjust the overall expression level of IL-8 in response to two of its physiological inducers, IL-1 and epidermal growth factor. Relative to c-Fos, the delayed recruitment of Fra-1 to the IL-8 promoter provides an example how AP-1 subunits may dampen excessive chemokine synthesis.

    The Journal of biological chemistry 2005;280;10;9706-18

  • ERK kinase inhibition stabilizes the aryl hydrocarbon receptor: implications for transcriptional activation and protein degradation.

    Chen S, Operaña T, Bonzo J, Nguyen N and Tukey RH

    The ultimate carcinogen and metabolite of benzo-[a]pyrene-7,8-dihydrodiol, benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide (+/-), stimulates apoptosis, and this process can be blocked by extracellular signal-regulated kinase (Erk) kinase inhibitors. However, we show here that Erk kinase inhibitors were unable to prevent B[a]P-7,8-dihydrodiol-induced apoptosis, leading us to speculate that Erk kinases are linked to regulation of the aryl hydrocarbon (Ah) receptor. Cotreatment of hepa1c1c7 cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and Erk kinase inhibitor PD98059, U0126, or SL327 led to enhanced nuclear accumulation of Ah receptor but with a reduced capacity to complement TCDD induction of Cyp1a1. This is explained in part by the ability of Erk kinase inhibitors to alter the steady-state levels of cellular Ah receptor, a result that leads to a dramatic induction in detectable receptor levels. These changes in cellular Ah receptor levels are associated with delayed degradation of the Ah receptor because TCDD-initiated degradation is reversed when cells are co-treated with TCDD and Erk kinase inhibitors. Erk kinase is linked to Ah receptor expression, as demonstrated by reductions in total Ah receptor levels after overexpression of constitutively active MEK1. In addition, Erk kinase activity modulates the transcriptional response because MEK1 overexpression enhances TCDD-initiated transactivation potential of the receptor. Thus, Erk kinase activity facilitates ligand-initiated transcriptional activation while targeting the Ah receptor for degradation. Immunoprecipitation experiments of the Ah receptor indicate that Erk kinase activity is associated with the receptor. It is interesting that the carboxyl region of the Ah receptor is associated with the transactivation region as well as the site for ubiquitination, indicating that Erk kinase-dependent phosphorylation targets the carboxyl region of the receptor.

    Funded by: NIEHS NIH HHS: ES10337

    The Journal of biological chemistry 2005;280;6;4350-9

  • Arsenic trioxide (ATO) and MEK1 inhibition synergize to induce apoptosis in acute promyelocytic leukemia cells.

    Lunghi P, Tabilio A, Lo-Coco F, Pelicci PG, Pelicci P and Bonati A

    Department of Clinical Sciences, Section of Hemato-Oncology, University of Parma, Parma, Italy. p.lunghi@libero.it

    Recent studies suggest that components of the prosurvival signal transduction pathways involving the Ras-mitogen-activated protein kinase (MAPK) can confer an aggressive, apoptosis-resistant phenotype to leukemia cells. In this study, we report that acute promyelocytic leukemia (APL) cells exploit the Ras-MAPK activation pathway to phosphorylate at Ser112 and to inactivate the proapoptotic protein Bad, delaying arsenic trioxide (ATO)-induced apoptosis. Both in APL cell line NB4 and in APL primary blasts, the inhibition of extracellular signal-regulated kinases 1/2 (ERK1/2) and Bad phosphorylation by MEK1 inhibitors enhanced apoptosis in ATO-treated cells. We isolated an arsenic-resistant NB4 subline (NB4-As(R)), which showed stronger ERK1/2 activity (2.7-fold increase) and Bad phosphorylation (2.4-fold increase) compared to parental NB4 cells in response to ATO treatment. Upon ATO exposure, both NB4 and NB4-As(R) cell lines doubled protein levels of the death antagonist Bcl-xL, but the amount of free Bcl-xL that did not heterodimerize with Bad was 1.8-fold greater in NB4-As(R) than in the parental line. MEK1 inhibitors dephosphorylated Bad and inhibited the ATO-induced increase of Bcl-xL, overcoming ATO resistance in NB4-As(R). These results may provide a rationale to develop combined or sequential MEK1 inhibitors plus ATO therapy in this clinical setting.

    Leukemia 2005;19;2;234-44

  • Nef: "necessary and enforcing factor" in HIV infection.

    Joseph AM, Kumar M and Mitra D

    National Centre for Cell Science, Ganeshkhind, Pune-411007, India.

    The Human Immunodeficiency Virus -1 (HIV-1) Nef protein that was originally identified as a viral negative factor is a 27kDa myristoylated protein. However, this so called dispensable viral protein has emerged as one of the most important proteins for viral life cycle. Nef not only establishes the host cell environment suitable for viral replication and pathogenesis but also facilitates the progression of the infection into disease. Previous efforts have been focussed to explain how Nef down modulates host cell receptors like CD4 and MHC-1 molecules, thereby helping the virus to evade host defense and to increase viral infectivity. Nef also ably modulates specific processes like apoptosis in favour of viral life cycle other than being the stimulus for cell activation and signal transduction pathways. After much maligning over its reported positive or negative functions on the HIV-1 Long Terminal Repeat (LTR) promoter, the Nef protein is now perceived to enhance viral replication and infection through a combination of different effector functions. Recent reports emphasize a role for Nef in viral gene expression and place it in a prime position to oversee and optimize viral replication. Nef may do so by enhancing Tat mediated gene expression from the LTR by activating signalling pathways that result in a concomitant increase in the activation of general transcription factors, and also by mediating translocation of repression factors from the nucleus. Thus, Nef not only enhances infection but also plays an important role in viral replication and pathogenesis.

    Current HIV research 2005;3;1;87-94

  • Selective inhibitors of MEK1/ERK44/42 and p38 mitogen-activated protein kinases potentiate apoptosis induction by sulindac sulfide in human colon carcinoma cells.

    Sun Y and Sinicrope FA

    Division of Gastroenterology/Hepatology, Mayo Clinic, 200 First Street Southwest, Rochester, MN 55905, USA.

    The nonsteroidal anti-inflammatory drug (NSAID) sulindac prevents experimental colon cancer and can regress precancerous polyps in humans. Sulindac sulfide inhibits cyclooxygenase (COX)-mediated prostaglandin synthesis and retards the growth of cultured colon cell lines primarily by inducing apoptosis. Given the known role of mitogen-activated protein kinase (MAPK) in signal transduction and the regulation of cell survival and death, we determined the effect of sulindac sulfide on MAPK activation, COX-2 expression, and apoptosis induction in HCA-7 human colon cancer cells. Sulindac sulfide treatment was associated with activation of ERKp44/42 and p38 MAPK in a dosage- and time-dependent manner, and also activated upstream MEK. Similar results were seen in HCT-15 cells and also with the selective COX-2 inhibitor NS398. ERKp44/42 and p38 activation were accompanied by an induction of COX-2 protein expression. Selective inhibitors of sulindac sulfide-induced ERKp44/42 (PD98059) and p38 MAPK (SB203580) activation also suppressed the induction of COX-2 by this NSAID. Furthermore, both MAPK inhibitors significantly augmented sulindac sulfide-induced apoptosis, as did suppression of constitutive COX-2 using antisense oligonucleotides. In conclusion, MEK/ERK and p38 MAPK activation mediate COX-2 induction by sulindac sulfide. Selective inhibitors of these MAPKs potentiate apoptosis induction by this NSAID, suggesting a novel strategy for the prevention or treatment of colorectal cancer.

    Funded by: NIDDK NIH HHS: R01 DK56378

    Molecular cancer therapeutics 2005;4;1;51-9

  • VEGF production by primary human renal proximal tubular cells: requirement of HIF-1, PI3-kinase and MAPKK-1 signaling.

    Hellwig-Bürgel T, Stiehl DP, Katschinski DM, Marxsen J, Kreft B and Jelkmann W

    Institute of Physiology, University of Lübeck, Germany. hellwig@physio.uni-luebe 12fe ck.de

    Renal proximal tubular epithelial cells (PTEC) respond to hypoxia exposure or interleukin-1beta (IL-1beta) treatment with increased vascular endothelial growth factor (VEGF) production. With respect to O2 deprivation, the hypoxia-inducible factor 1alpha/ beta (HIF-1) is the most important transcription factor driving VEGF mRNA expression. HIF-1 is also activated by IL-1beta and may thus be involved in the stimulation of VEGF production by this cytokine. However, the molecular mechanisms of HIF-1 dependent VEGF synthesis are poorly understood. Herein, human PTEC in primary culture were challenged by hypoxic incubation and/or IL-1beta treatment in absence or presence of specific phosphatidylinositol 3-kinase (PI3K) or mitogen activated protein kinase kinase-1 (MAPKK-1) inhibitors for assay of VEGF protein, VEGF mRNA and detection of HIF-1alpha by Western Blotting, EMSA and fluorescence microscopy. In addition, the activities of PI3K and MAPKK-1 were studied following hypoxia and IL-1beta treatment of the cultures. The study shows that PI3K but not MAPKK-1 inhibition resulted in the loss of hypoxic and IL-1beta induced HIF-1alpha accumulation, whereas VEGF synthesis was reduced by either intervention. Thus, PI3K signaling is required for HIF-1alpha accumulation and VEGF synthesis, whereas MAPKK-1 signaling is required for VEGF synthesis only. Furthermore, hypoxia alone was sufficient to activate PI3K in PTEC in contrast to MAPKK-1, whose activity was lowered in hypoxia.

    Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology 2005;15;1-4;99-108

  • Combined disruption of both the MEK/ERK and the IL-6R/STAT3 pathways is required to induce apoptosis of multiple myeloma cells in the presence of bone marrow stromal cells.

    Chatterjee M, Stühmer T, Herrmann P, Bommert K, Dörken B and Bargou RC

    Department of Hematology, Oncology, and Tumorimmunology, Robert Rössle Cancer Clinic and HELIOS-Clinics at the Max Delbrück Center for Molecular Medicine, Charité, Humboldt University, Campus Berlin-Buch, Lindenberger Weg 80, 13122 Berlin, Germany.

    The interleukin-6 receptor (IL-6R)/signal transducer and activator of transcription 3 (STAT3) pathway contributes to the pathogenesis of multiple myeloma (MM) and protects MM cells from apoptosis. However, MM cells survive the IL-6R blockade if they are cocultured with bone marrow stromal cells (BMSCs), suggesting that the BM microenvironment stimulates IL-6-independent pathways that exert a pro-survival effect. The goal of this study was to investigate the underlying mechanism. Detailed pathway analysis revealed that BMSCs stimulate STAT3 via the IL-6R, and mitogen-activated protein (MAP) kinases via IL-6R-independent mechanisms. Abolition of MEK1,2 activity with PD98059, or ERK1,2 small interfering RNA knockdown, was insufficient to induce apoptosis. However, the combined disruption of the IL-6R/STAT3 and MEK1,2/ERK1,2 pathways led to strong induction of apoptosis even in the presence of BMSCs. This effect was observed with MM cell lines and with primary MM cells, suggesting that the BMSC-induced activation of MEK1,2/ERK1,2 renders MM cells IL-6R/STAT3 independent. Therefore, in the presence of cells from the BM micro-environment, combined targeting of different (and independently activated) pathways is required to efficiently induce apoptosis of MM cells. This might have direct implications for the development of future therapeutic strategies for MM.

    Blood 2004;104;12;3712-21

  • 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

  • Structures of human MAP kinase kinase 1 (MEK1) and MEK2 describe novel noncompetitive kinase inhibition.

    Ohren JF, Chen H, Pavlovsky A, Whitehead C, Zhang E, Kuffa P, Yan C, McConnell P, Spessard C, Banotai C, Mueller WT, Delaney A, Omer C, Sebolt-Leopold J, Dudley DT, Leung IK, Flamme C, Warmus J, Kaufman M, Barrett S, Tecle H and Hasemann CA

    Department of Discovery Technologies, Pfizer Global Research & Development, 2800 Plymouth Road, Ann Arbor, Michigan 48105, USA.

    MEK1 and MEK2 are closely related, dual-specificity tyrosine/threonine protein kinases found in the Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) signaling pathway. Approximately 30% of all human cancers have a constitutively activated MAPK pathway, and constitutive activation of MEK1 results in cellular transformation. Here we present the X-ray structures of human MEK1 and MEK2, each determined as a ternary complex with MgATP and an inhibitor to a resolution of 2.4 A and 3.2 A, respectively 194d . The structures reveal that MEK1 and MEK2 each have a unique inhibitor-binding pocket adjacent to the MgATP-binding site. The presence of the potent inhibitor induces several conformational changes in the unphosphorylated MEK1 and MEK2 enzymes that lock them into a closed but catalytically inactive species. Thus, the structures reported here reveal a novel, noncompetitive mechanism for protein kinase inhibition.

    Nature structural & molecular biology 2004;11;12;1192-7

  • Integrin alphav-mediated inactivation of p53 controls a MEK1-dependent melanoma cell survival pathway in three-dimensional collagen.

    Bao W and Strömb 1c62 lad S

    Department of Laboratory Medicine, Karolinska Institutet, Stockholm, 141 86, Sweden.

    Integrin alphav is required for melanoma cell survival and tumor growth in various models. To elucidate integrin alphav-mediated melanoma cell survival mechanisms, we used a three-dimensional (3D) collagen gel model mimicking the pathophysiological microenvironment of malignant melanoma in the dermis. We found that integrin alphav inactivated p53 and that suppression of p53 activity by dominant negative p53 or p53-small interfering RNA obviated the need for integrin alphav for melanoma cell survival in 3D-collagen and for tumor growth in vivo. This indicates that integrin alphav-mediated inactivation of p53 functionally controls melanoma cell survival. Furthermore, we found that melanoma cell integrin alphav was required for MAPK kinase (MEK) 1 and extracellular signal-regulated kinase (ERK)1/2 activity in 3D-collagen, whereas inhibition of MEK1 activity induced apoptosis. Surprisingly, MEK1 and ERK1/2 activities were restored in integrin alphav-negative melanoma cells by suppression of p53, whereas concomitant block of MEK1 induced apoptosis. This suggests that integrin alphav controls melanoma cell survival in 3D-collagen through a pathway involving p53 regulation of MEK1 signaling.

    The Journal of cell biology 2004;167;4;745-56

  • 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

  • Molecular cross-talk between MEK1/2 and mTOR signaling during recovery of 293 cells from hypertonic stress.

    Naegele S and Morley SJ

    Biochemistry Laboratory, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, United Kingdom.

    To investigate the role for initiation factor phosphorylation in de novo translation, we have studied the recovery of human kidney cells from hypertonic stress. Previously, we have demonstrated that hypertonic shock causes a rapid inhibition of protein synthesis, the disaggregation of polysomes, the dephosphorylation of eukaryotic translation initiation factor (eIF)4E, 4E-BP1, and ribosomal protein S6, and increased association of 4E-BP1 with eIF4E. The return of cells to isotonic medium promotes a transient activation of Erk1/2 and the phosphorylation of initiation factors, promoting an increase in protein synthesis that is independent of a requirement for eIF4E phosphorylation. As de novo translation is associated with the phosphorylation of 4E-BP1, we have investigated the role of the signaling pathways required for this event by the use of cell-permeable inhibitors. Surprisingly, although rapamycin, RAD001, wortmannin, and LY294002 inhibited the phosphorylation of 4E-BP1 and its release from eIF4E, they did not prevent the recovery of translation rates. These data suggest that only a small proportion of the available eIF4F complex is required for maximal translation rates under these conditions. Similarly, prevention of Erk1/2 activity alone with low concentrations of PD184352 did not impinge upon de novo translation until later times of recovery from salt shock. However, U0126, which prevented the phosphorylation of Erk1/2, ribosomal protein S6, TSC2, and 4E-BP1, attenuated de novo protein synthesis in recovering cells. These results indicate that the phosphorylation of 4E-BP1 is mediated by both phosphatidylinositol 3-kinase-dependent rapamycin-sensitive and Erk1/2-dependent signaling pathways and that activation of either pathway in isolation is sufficient to promote de novo translation.

    The Journal of biological chemistry 2004;279;44;46023-34

  • MEK1 and MEK2, different regulators of the G1/S transition.

    Ussar S and Voss T

    Boehringer Ingelheim Austria GmbH, Dr. Boehringer Gasse 5-11, A-1121 Vienna.

    The ERK cascade is activated by hormones, cytokines, and growth factors that result in either proliferation or growth arrest depending on the duration and intensity of the ERK activation. Here we provide evidence that the MEK1/ERK module preferentially provides proliferative signals, whereas the MEK2/ERK module induces growth arrest at the G1/S boundary. Depletion of either MEK subtype by RNA interference generated a unique phenotype. The MEK1 knock down led to p21cip1 induction and to the appearance of cells with a senescence-like phenotype. Permanent ablation of MEK1 resulted in reduced colony formation potential, indicating the importance of MEK1 for long term proliferation and survival. MEK2 deficiency, in contrast, was accompanied by a massive induction of cyclin D expression and, thus, CDK4/6 activation followed by nucleophosmin hyperphosphorylation and centrosome over-amplification. Our results suggest that the two MEK subtypes have distinct ways to contribute to a regulated ERK activity and cell cycle progression.

    The Journal of biological chemistry 2004;279;42;43861-9

  • Human tribbles, a protein family controlling mitogen-activated protein kinase cascades.

    Kiss-Toth E, Bagstaff SM, Sung HY, Jozsa V, Dempsey C, Caunt JC, Oxley KM, Wyllie DH, Polgar T, Harte M, O'neill LA, Qwarnstrom EE and Dower SK

    Cardiovascular Research Unit, University of Sheffield, Northern General Hospital, Sheffield S5 7AU, United Kingdom. E.Kiss-Toth@sheffield.ac.uk

    Control of mitogen-activated protein kinase (MAPK) cascades is central to regulation of many cellular responses. We describe here human tribbles homologues (Htrbs) that control MAPK activity. MAPK kinases interact with Trbs and regulate their steady state levels. Further, Trbs selectively regulate the activation of extracellular signal-regulated kinases, c-Jun NH2-terminal kinases, and p38 MAPK with different relative levels of activity for the three classes of MAPK observed depending on the level of Trb expression. These results suggest that Trbs control both the extent and the specificity of MAPK kinase activation of MAPK.

    The Journal of biological chemistry 2004;279;41;42703-8

  • Phosphorylation of actopaxin regulates cell spreading and migration.

    Clarke DM, Brown MC, LaLonde DP and Turner CE

    Department of Cell Biology and Developmental Biology, State University of New York Upstate Medical University, Syracuse, NY 13210, USA.

    Actopaxin is an actin and paxillin binding protein that localizes to focal adhesions. It regulates cell spreading and is phosphorylated during mitosis. Herein, we identify a role for actopaxin phosphorylation in cell spreading and migration. Stable clones of U2OS cells expressing actopaxin wild-type (WT), nonphosphorylatable, and phosphomimetic mutants were developed to evaluate actopaxin function. All proteins targeted to focal adhesions, however the nonphosphorylatable mutant inhibited spreading whereas the phosphomimetic mutant cells spread more efficiently than WT cells. Endogenous and WT actopaxin, but not the nonphosphorylatable mutant, were phosphorylated in vivo during cell adhesion/spreading. Expression of the nonphosphorylatable actopaxin mutant significantly reduced cell migration, whereas expression of the phosphomimetic increased cell migration in scrape wound and Boyden chamber migration assays. In vitro kinase assays demonstrate that extracellular signal-regulated protein kinase phosphorylates actopaxin, and treatment of U2OS cells with the MEK1 inhibitor UO126 inhibited adhesion-induced phosphorylation of actopaxin and also inhibited cell migration.

    Funded by: NHLBI NIH HHS: R01 HL070244

    The Journal of cell biology 2004;166;6;901-12

  • MLK3 is required for mitogen activation of B-Raf, ERK and cell proliferation.

    Chadee DN and Kyriakis

    The Molecular Cardiology Research Institute, Department of Medicine, Tufts-New England Medical Center and The Department of Medicine, Tufts University School of Medicine, 750 Washington Street, Box 8486, Boston, MA 02111, USA.

    The ERK group of mitogen-activated protein kinases (MAPKs) is essential for cell proliferation stimulated by mitogens, oncogenic ras and raf (ref. 1). All MAPKs are activated by MAP3K/MEK/MAPK core pathways and the Raf proto-oncoproteins, especially B-Raf, are ERK-specific MAP3Ks (refs 1-3). Mixed lineage kinase-3 (MLK3) is a MAP3K that was thought to be a cytokine-activated, and comparatively selective, regulator of the JNK group of MAPKs (refs 1, 4-6). Here we report that silencing of mlk3 by RNAi suppressed mitogen and cytokine activation not only of JNK but of ERK and p38 as well. Silencing mlk3 also blocked mitogen-stimulated phosphorylation of B-Raf at Thr 598 and Ser 601, a step required for B-Raf activation. Furthermore, silencing mlk3 prevented serum-stimulated cell proliferation and the proliferation of tumour cells bearing either oncogenic Ki-Ras or loss-of-function neurofibromatosis-1 (NF1) or NF2 mutations. The proliferation of tumour cells containing activating B-raf or raf-1 mutations was unaffected by silencing mlk3. Our results define an unexpected role for MLK3 in mitogen regulation of B-Raf, ERK and cell proliferation.

    Nature cell biology 2004;6;8;770-6

  • MEK is a key modulator for TLR5-induced interleukin-8 and MIP3alpha gene expression in non-transformed human colonic epithelial cells.

    Rhee SH, Keates AC, Moyer MP and Pothoulakis C

    The Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.

    Flagellin, a specific ligand for Toll-like receptor 5 (TLR5), is a molecular pattern associated with several bacterial species. Recently, TLR signaling has been intensively studied. However, TLR5-associated signaling in non-transformed colonocytes has not been investigated. Here we studied the expression of cytokines induced by flagellin in non-transformed human colonic NCM460 cells and the signaling mechanisms mediating these responses. Cytokine expression array experiments showed that exposure of the cells to flagellin (100 ng/ml) for 12 h increased the expression of interleukin (IL)-8 and macrophage-inflammatory protein 3alpha (MIP3alpha) in a TLR5-specific manner. Flagellin also activated MAP kinases (ERK1/2, JNK, and p38) and degraded IkappaBalpha. Dominant negative MEK1 (a kinase that activates ERK1/2) blocked flagellin-stimulated IL-8 and MIP3alpha transcriptional activity, while the MEK-specific inhibitors PD98059 and U0126 reduced protein production of these cytokines. Conversely, transfection with a constitutively active MEK1 increased IL-8 and MIP3alpha transcriptional activity in a NFkappaB-independent manner. Furthermore, overexpression of the constitutively active MEK1 induced IL-8 and MIP3alpha protein production. We also demonstrated that C-terminal coiled-coil and TRAF-C domains of TRAF6, unable to mediate NFkappaB activation, are involved in MEK-mediated IL-8 and MIP3alpha expression. Thus, in non-transformed human colonocytes, MEK activation following flagellin/TLR5 engagement is a key modulator for NFkappaB-independent, IL-8 and MIP3alpha expression.

    Funded by: NIDDK NIH HHS: DK 33506, DK 47343, DK 60729

    The Journal of biological chemistry 2004;279;24;25179-88

  • MEK1-induced Golgi dynamics during cell cycle progression is partly mediated by Polo-like kinase-3.

    Xie S, Wang Q, Ruan Q, Liu T, Jhanwar-Uniyal M, Guan K and Dai W

    Department of Medicine, New York Medical College, Valhalla, NY 10595, USA.

    MEK1, a gene product that regulates cell growth and differentiation, also plays an important role in Golgi breakdown during the cell cycle. We have recently shown that polo-like kinase (Plk3) is Golgi localized and involved in Golgi dynamics during the cell cycle. To study the mode of action of Plk3 in the Golgi fragmentation cascade, we examined functional as well as physical interactions between Plk3 and MEK1/ERKs. In HeLa cells, although a significant amount of Plk3 signals dispersed in a manner similar to those of Golgi during mitosis concentrated Plk3 was detected at spindle poles, which colocalized with phospho-MEKs and phospho-ERKs. Pull-down assays showed that Plk3 physically interacted with MEK1 and ERK2. Nocodazole activated Plk3 and its activation was blocked by MEK-specific inhibitors (PD98059 or U0126). Moreover, transfection of activated MEK1 resulted in an enhanced kinase activity of Plk3; Plk3-induced fragmentation of Golgi stacks was significantly reduced after treatment with MEK inhibitors. Consistently, ectopic expression of activated MEK1, but not kinase-dead MEK1(K97R), stimulated Plk3 to induce Golgi breakdown and the stimulation was not observed in cells expressing Plk3(K52R). Furthermore, PLK3(-/-) murine embryonic fibroblast cells exhibited a significantly less fragmentation of the Golgi complex than that in wild-type cells after exposed to nocodazole. Thus, our studies strongly suggest that Plk3 may be a key protein kinase mediating MEK1 function in the Golgi fragmentation pathway during cell division.

    Funded by: NCI NIH HHS: R01-CA74229

    Oncogene 2004;23;21;3822-9

  • Glycogen synthase kinase-3beta is tyrosine-phosphorylated by MEK1 in human skin fibroblasts.

    Takahashi-Yanaga F, Shiraishi F, Hirata M, Miwa Y, Morimoto S and Sasaguri T

    Department of Clinical Pharmacology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan. yanaga@clipharm.med.kyushu-u.ac.jp

    Glycogen synthase kinase-3beta (GSK-3beta) can be associated with several proteins in cell. We analyzed the immunoprecipitates by an anti-GSK-3beta antibody from cell lysate of human fibroblasts and found that this protein was co-precipitated with mitogen-activated protein kinase kinase (MEK1/2). U0126, a MEK1/2 inhibitor, inhibited tyrosine phosphorylation of GSK-3beta, suggesting that MEK1/2 was involved in the phosphorylation of Tyr(216) in GSK-3beta. In vitro kinase assay was carried out using a recombinant human active MEK1 and we found that GSK-3beta was phosphorylated on Tyr(216) by this kinase in a dose- and time-dependent manner. Further, the pretreatment of fibroblasts with U0126 inhibited serum-induced nuclear translocation of GSK-3beta. These results suggested that MEK1/2 induces tyrosine phosphorylation of GSK-3beta and this cellular event might induce nuclear translocation of GSK-3beta. This is the first report to suggest that MEK1/2 phosphorylates not only ERK1/2 but also GSK-3beta.

    Biochemical and biophysical research communications 2004;316;2;411-5

  • MEK1,2 response element mediates angiotensin II-stimulated plasminogen activator inhibitor-1 promoter activation.

    Chen HC and Feener EP

    Research Division, Joslin Diabetes Center, Harvard Medical School, Boston, MA 02215, USA.

    The MEK1,2 (MAPK/ERK kinase 1 and 2) pathway mediates the up-regulation of plasminogen activator inhibitor-1 (PAI-1) expression in vascular smooth muscle cells by a variety of hormones, including angiotensin II. Transfection of constitutively active MEKK-1, an upstream activator of the mitogen-activated protein (MAP) kinase pathways, was used to isolate an enhancer element located between -89 and -50 bp in PAI-1 promoter that was activated by MEKK-1 and selectively blocked by the MEK1,2 inhibitor PD98059. Mutational analysis revealed that the MEKK-1 response element (MRE) contained 2 cis-acting Sp1- and AP-1-like sequences, located between -75 to -70 and -63 to -52 bp, respectively. Overexpression of Sp1 enhanced MEKK-1-induced MRE promoter activity and a dominant-negative c-Fos blocked this Sp1 response. The combination of Sp1 and c-Jun or c-Fos was required to activate this MRE. Angiotensin II (Ang II) stimulation increased c-Fos, c-Jun, and Sp1 binding to the MRE by 100-, 4.9-, and 1.9-fold, respectively, and these responses were inhibited by PD98059 and AT1 receptor antagonist candesartan. Intravenous Ang II infusion in rats increased aortic c-Fos binding to the MRE. This MRE sequence mediated a 4-fold increase of MEK1,2-dependent PAI-1/luciferase mRNA expression by angiotensin II stimulation. This report identifies the MEK1,2 response element that mediates angiotensin II-stimulated PAI-1 promoter activation and shows that activation of this element requires Sp1 and AP-1 co-activation.

    Funded by: NIDDK NIH HHS: DK 36836, DK 48358, P30 DK036836

    Blood 2004;103;7;2636-44

  • RhoA binds to the amino terminus of MEKK1 and regulates its kinase activity.

    Gallagher ED, Gutowski S, Sternweis PC and Cobb MH

    Department of Pharmacology, University of Texas Southwestern Medical Center, 53 180a 23 Harry Hines Boulevard, Dallas, TX 75390-9041, USA.

    MEKK1 is a mitogen-activated protein kinase kinase kinase (MAP3K) that can regulate the c-Jun amino-terminal kinase (JNK) MAP kinase cascade. MEKK1 is comprised of a kinase domain and a long amino-terminal regulatory domain. This amino-terminal domain has a scaffold function in that it can assemble modules of the JNK and ERK MAP kinase cascades. Recently, we have demonstrated that MEKK1 binds to p115 Rho GTPase-activating protein, which has GTPase-activating protein activity toward RhoA. Thus, we tested whether Rho GTPases interact with the regulatory domain of MEKK1. RhoA, but not Rac or Cdc42, binds to a site in the aminoterminal one-third of MEKK1, which includes its PHD domain. The interaction is prevented by mutation of the essential cysteine in the MEKK1 PHD domain. Rho-GTP stimulates the kinase activity of full-length MEKK1 as much as 10-fold toward MEK4 but does not appear to be ubiquitinated by MEKK1 under conditions that result in modification of ERK2. In summary, we have characterized a novel point at which Rho GTPases impinge upon the regulation and function of MEKK1.

    Funded by: NIGMS NIH HHS: GM 31954, GM 56498

    The Journal of biological chemistry 2004;279;3;1872-7

  • 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

  • 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

  • Mutation of B-Raf in human choroidal melanoma cells mediates cell proliferation and transformation through the MEK/ERK pathway.

    Calipel A, Lefevre G, Pouponnot C, Mouriaux F, Eychène A and Mascarelli F

    Institut Biomédical des Cordeliers, INSERM U450, 15 rue de l'Ecole de Médecine, 75006 Paris, France.

    The BRAF gene, encoding a mitogen-activated protein kinase kinase kinase, is mutated in several human cancers, with the highest incidence occurring in cutaneous melanoma. The activating V599E mutation accounted for 80% of all mutations detected in cutaneous melanoma cell lines. Reconstitution experiments have shown that this mutation increases ectopically expressed B-Raf kinase activity and induces NIH3T3 cell transformation. Here we used tumor-derived cell lines to characterize the activity of endogenous mutated B-Raf protein and assess its specific role in transformation. We show that three cell lines (OCM-1, MKT-BR, and SP-6.5) derived from human choroidal melanoma, the most frequent primary ocular neoplasm in humans, express B-Raf containing the V599E mutation. These melanoma cells showed a 10-fold increase in endogenous B-RafV599E kinase activity and a constitutive activation of the MEK/ERK pathway that is independent of Ras. This, as well as melanoma cell proliferation, was strongly diminished by siRNA-mediated depletion of the mutant B-Raf protein. Moreover, blocking B-RafV599E-induced ERK activation by different experimental approaches significantly reduced cell proliferation and anchorage-independent growth of melanoma cells. Finally, quantitative immunoblot analysis allowed us to identify signaling and cell cycle proteins that are differentially expressed between normal melanocytes and melanoma cells. Although the expression of signaling molecules was not sensitive to U0126 in melanoma cells, the expression of a cluster of cell cycle proteins remained regulated by the B-RafV599E/MEK/ERK pathway. Our results pinpoint this pathway as an important component in choroidal melanoma cell lines.

    The Journal of biological chemistry 2003;278;43;42409-18

  • MEK/ERK pathway is aberrantly active in Hodgkin disease: a signaling pathway shared by CD30, CD40, and RANK that regulates cell proliferation and survival.

    Zheng B, Fiumara P, Li YV, Georgakis G, Snell V, Younes, Vauthey JN, Carbone A and Younes A

    Department of Lymphoma/Myeloma, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Unit 429, Houston, TX 77030, USA.

    The mitogen-activated protein kinase (MAPK) (also called extracellular signal-regulated kinase [ERK]) pathway has been implicated in malignant transformation and in the regulation of cellular growth and proliferation of several tumor types, but its expression and function in Hodgkin disease (HD) are unknown. We report here that the active phosphorylated form of MAPK/ERK is aberrantly expressed in cultured and primary HD cells. Inhibition of the upstream MAPK kinase (also called MEK) by the small molecule UO126 inhibited the phosphorylation of ERK and demonstrated a dose- and time-dependent antiproliferative activity in HD cell lines. UO126 modulated the levels of several intracellular proteins including B-cell lymphoma protein 2 (Bcl-2), myeloid cell leukemia-1 (Mcl-1) and caspase 8 homolog FLICE-inhibitory protein (cFLIP), and induced G2M cell-cycle arrest or apoptosis. Furthermore, UO126 potentiat 836 ed the activity of apoliprotein 2/tumor necrosis factor-related apoptosis-inducing ligand (APO2L/TRAIL) and chemotherapy-induced cell death. Activation of CD30, CD40, and receptor activator of nuclear kappabeta (RANK) receptors in HD cells by their respective ligands increased ERK phosphorylation above the basal level and promoted HD cell survival. UO126 inhibited basal and ligand-induced ERK phosphorylation, and inhibited ligand-induced cell survival of HD cell lines. These findings provide a proof-of-principle that inhibition of the MEK/ERK pathway may have therapeutic value in HD.

    Blood 2003;102;3;1019-27

  • p38 Mitogen-activated protein kinase pathway suppresses cell survival by inducing dephosphorylation of mitogen-activated protein/extracellular signal-regulated kinase kinase1,2.

    Li SP, Junttila MR, Han J, Kähäri VM and Westermarck J

    Turku Centre for Biotechnology, University of Turku, Finland.

    Raf/mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)1,2/extracellular signal-regulated kinase1,2 and MKK3,6/p38 mitogen-activated protein kinase pathways play an important role in cellular survival and apoptosis. The results of this study identify novel mechanisms to explain the opposing effects of these pathways in the regulation of apoptosis induction. Our results show that activation of p38 by adenoviral expression of MKK3b or arsenite treatment was followed by rapid dephosphorylation of MEK1,2 and subsequent apoptosis in human skin fibroblasts. Inhibition of p38 activity by SB203580 and adenoviral expression of dominant-negative forms of p38 potently inhibited MEK1,2 dephosphorylation and apoptosis. Strikingly, p38-mediated dephosphorylation of MEK1,2, was not detected in a series of transformed human cell lines. Taken together, we provide evidence for mechanisms unidentified previously that negatively regulates survival signaling during apoptosis induction. In addition, we show that in all transformed cell lines we have studied thus far, the function of this pathway is impaired.

    Cancer research 2003;63;13;3473-7

  • Distribution, levels, and activation of MEK1 in Alzheimer's disease.

    Zhu X, Sun Z, Lee HG, Siedlak SL, Perry G and Smith MA

    Institute of Pathology, Case Western Reserve University, Cleveland, Ohio 44106, USA.

    Extracellular-signal-regulated kinase (ERK) has been implicated in the pathogenesis of Alzheimer's disease (AD), but the upstream cascade leading to ERK activation has not been elucidated. In this study, we focused on one of the physiological activators of ERK, mitogen-activated protein kinase (MAPK)/ERK kinase 1 (MEK1). Although there was no significant difference in the level and distribution of total MEK1 between AD and age-matched control cases, increased levels of activated phospho-MEK1 were specifically localized to neuronal intracytoplasmic granular structures in severe AD (Braak stage V-VI). The considerable overlap between MEK1 and its downstream effector, phospho-ERK, suggests both a functional and mechanistic link. Nuclear localization of phospho-MEK1 was a prominent feature in both mild AD cases (Braak stage III-IV) and control cases with limited pathology (Braak stage I-II). Since MEK1 is normally cytoplasmic due to the active export from nucleus because of the presence of nuclear export signal in its amino-terminus, we suspect that the apparent nuclear accumulation of phospho-MEK1 in AD patients at early stages suggests that abnormal nuclear trafficking may contribute to the pathogenesis of AD. By immunoblot analyses, phospho-MEK1 was significantly increased in AD over control cases. Together, these findings lend further credence to the notion that the ERK pathway is dysregulated in AD and also indicate an active role for this pathway in disease pathogenesis.

    Funded by: NINDS NIH HHS: NS38648

    Journal of neurochemistry 2003;86;1;136-42

  • Inhibition of caspase-9 through phosphorylation at Thr 125 by ERK MAPK.

    Allan LA, Morrice N, Brady S, Magee G, Pathak S and Clarke PR

    Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, Scotland, UK.

    Many pro-apoptotic signals activate caspase-9, an initiator protease that activates caspase-3 and downstream caspases to initiate cellular destruction. However, survival signals can impinge on this pathway and suppress apoptosis. Activation of the Ras-Raf-MEK-ERK mitogen-activated protein kinase (MAPK) pathway is associated with protection of cells from apoptosis and inhibition of caspase-3 activation, although the targets are unknown. Here, we show that the ERK MAPK pathway inhibits caspase-9 activity by direct phosphorylation. In mammalian cell extracts, cytochrome c-induced activation of caspases-9 and -3 requires okadaic-acid-sensitive protein phosphatase activity. The opposing protein kinase activity is overcome by treatment with the broad-specificity kinase inhibitor staurosporine or with inhibitors of MEK1/2. Caspase-9 is phosphorylated at Thr 125, a conserved MAPK consensus site targeted by ERK2 in vitro, in a MEK-dependent manner in cells stimulated with epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA). Phosphorylation at Thr 125 is sufficient to block caspase-9 processing and subsequent caspase-3 activation. We suggest that phosphorylation and inhibition of caspase-9 by ERK promotes cell survival during development and tissue homeostasis. This mechanism may also contribute to tumorigenesis when the ERK MAPK pathway is constitutively activated.

    Nature cell biology 2003;5;7;647-54

  • 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

  • Differential transcriptional regulation by human immunodeficiency virus type 1 and gp120 in human astrocytes.

    Galey D, Becker K, Haughey N, Kalehua A, Taub D, Woodward J, Mattson MP and Nath A

    Department of Neurology, Johns Hopkins School of Medicine, Baltimore, Maryland 21287, USA.

    Astrocytes may be infected with the human immunodeficiency virus type 1 (HIV-1) or exposed to the HIV protein gp120, yet their role in the pathogenesis of HIV dementia is largely unknown. To characterize the effects of HIV on astrocytic transcription, microarray analysis and ribonuclease protection assays (RPA) were performed. Infection of astrocytes by HIV or treatment with gp120 had differential and profound effects on gene transcription. Of the 1153 oligonucleotides on the immune-based array, the expression of 108 genes (53 up; 55 down) and 82 genes (32 up; 50 down) were significantly modulated by gp120 and HIV infection respectively. Of the 1153 oligonucleotides on the neuro-based array, 58 genes (25 up; 33 down) and 47 genes (17 up; 30 down) were significantly modulated by gp120 and HIV infection respectively. Chemokine and cytokine induction occurred predominantly by HIV infection, whereas gp120 had no significant effect. These results were confirmed by RPA. The authors conclude that profound alterations of astrocytic function occur in response to HIV infection or interaction with viral proteins, suggesting that astrocytes may play an important role in the pathogenesis of HIV dementia.

    Funded by: NCRR NIH HHS: RR15592; NINDS NIH HHS: NS38428, NS39253

    Journal of neurovirology 2003;9;3;358-71

  • Mechanism of mitosis-specific activation of MEK1.

    Harding A, Giles N, Burgess A, Hancock JF and Gabrielli BG

    Cancer Biology Program, Centre for Immunology and Cancer Research, University of Queensland, Queensland 4102, Australia.

    Activation of cyclin B-Cdc2 is an absolute requirement for entry into mitosis, but other protein kinase pathways that also have mitotic functions are activated during G(2)/M progression. The MAPK cascade has well established roles in entry and exit from mitosis in Xenopus, but relatively little is known about the regulation and function of this pathway in mammalian mitosis. Here we report a detailed analysis of the activity of all components of the Ras/Raf/MEK/ERK pathway in HeLa cells during normal G(2)/M. The focus of this pathway is the dramatic activation of an endomembrane-associated MEK1 without the corresponding activation of the MEK substrate ERK. This is because of the uncoupling of MEK1 activation from ERK activation. The mechanism of this uncoupling involves the cyclin B-Cdc2-dependent proteolytic cleavage of the N-terminal ERK-binding domain of MEK1 and the phosphorylation of Thr(286). These results demonstrate that cyclin B-Cdc2 activity regulates signaling through the MAPK pathway in mitosis.

    The Journal of biological chemistry 2003;278;19;16747-54

  • p38alpha isoform Mxi2 binds to extracellular signal-regulated kinase 1 and 2 mitogen-activated protein kinase and regulates its nuclear activity by sustaining its phosphorylation levels.

    Sanz-Moreno V, Casar B and Crespo P

    Departamento de Biología Molecular, Universidad de Cantabria, Santander 39011, Spain.

    Mxi2 is a p38alpha splice isoform that is distinctively activated by mitogenic stimuli. Here we show that Mxi2 immunoprecipitates carry a kinase activity that is persistently activated by epidermal growth factor in a fashion regulated by Ras, Raf, and MEK. We demonstrate that this kinase activity can be attributed not to Mxi2 but rather to extracellular signal-regulated kinases 1 and 2 (ERK1/2), which coimmunoprecipitated with Mxi2 both by ectopic expression and in a physiological environment like the kidney. Furthermore, we provide evidence that Mxi2-ERK interaction has profound effects on ERK function, demonstrating that Mxi2 prolongs the duration of the ERK signal by sustaining its phosphorylation levels. Interestingly, we show that the effects of Mxi2 on ERK are restricted to nuclear events. Mxi2 potently up-regulates ERK-mediated activation of the transcription factors Elk1 and HIF1alpha but has no effect on the activity of ERK cytoplasmic substrates RSK2 and cPLA(2), induced by epidermal growth factor or by MEK. Overall, our findings point to Mxi2 as a unique member of the p38 family that may have an unprecedented role in the regulation of the functions of ERK mitogen-activated protein kinases.

    Molecular and cellular biology 2003;23;9;3079-90

  • Identification of novel ERK2 substrates through use of an engineered kinase and ATP analogs.

    Eblen ST, Kumar NV, Shah K, Henderson MJ, Watts CK, Shokat KM and Weber MJ

    Department of Microbiology and Cancer Center, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA. mjw@virginia.edu

    The mitogen-activated protein kinases are key regulators of cellular organization and function. To understand the mechanisms(s) by which these ubiquitous kinases affect specific cellular changes, it is necessary to identify their diverse and numerous substrates in different cell contexts and compartments. As a first step in achieving this goal, we engineered a mutant ERK2 in which a bulky amino acid residue in the ATP binding site (glutamine 103) is changed to glycine, allowing this mutant to utilize an analog of ATP (cyclopentyl ATP) that cannot be used by wild-type ERK2 or other cellular kinases. The mutation did not inhibit ERK2 kinase activity or substrate specificity in vitro or in vivo. This method allowed us to detect only ERK2-specific phosphorylations within a mixture of proteins. Using this ERK2 mutant/analog pair to phosphorylate ERK2-associated proteins in COS-1 cells, we identified the ubiquitin ligase EDD (E3 identified by differential display) and the nucleoporin Tpr (translocated promoter region) as two novel substrates of ERK2, in addition to the known ERK2 substrate Rsk1. To further validate the method, we present data that confirm that ERK2 phosphorylates EDD in vitro and in vivo. These results not only identify two novel ERK2 substrates but also provide a framework for the future identification of numerous cellular targets of this important signaling cascade.

    Funded by: NCI NIH HHS: CA39076, CA40042

    The Journal of biological chemistry 2003;278;17;14926-35

  • Identification of targets for calcium signaling through the copine family of proteins. Characterization of a coiled-coil copine-binding motif.

    Tomsig JL, Snyder SL and Creutz CE

    Department of Pharmacology, University of Virginia, Charlottesville 22908, USA.

    We provide evidence that copines, members of a ubiquitous family of calcium-dependent, membrane-binding proteins, may represent a universal transduction pathway for calcium signaling because we find copines are capable of interacting with a wide variety of "target" proteins including MEK1, protein phosphatase 5, and the CDC42-regulated kinase, that are themselves components of intracellular signaling pathways. The copine target proteins were identified by yeast two-hybrid screening and the interactions were verified in vitro using purified proteins. In the majority of cases the copine binds to a domain of the target protein that is predicted to form a characteristic coiled-coil. A consensus sequence for the coiled-coil copine-binding site was derived and found to have predictive value for identifying new copine targets. We also show that interaction with copines may result in recruitment of target proteins to membrane surfaces and regulation of the enzymatic activities of target proteins.

    Funded by: NIGMS NIH HHS: GM53266

    The Journal of biological chemistry 2003;278;12;10048-54

  • Anthrax lethal factor proteolysis and inactivation of MAPK kinase.

    Chopra AP, Boone SA, Liang X and Duesbery NS

    Laboratory of Developmental Cell Biology, Van Andel Research Institute, Grand Rapids, Michigan 49503, USA.

    Anthrax lethal toxin produced by the bacterium Bacillus anthracis is the major cause of death in animals infected with anthrax. One component of this toxin, lethal factor (LF), inactivates members of the mitogen-activated protein kinase kinase or MEK family through proteolysis of their NH(2) termini. However, neither the substrate requirements for LF cleavage nor the mechanism by which proteolysis inactivates MEK have been demonstrated. By means of deletion mutant analysis and site-directed mutagenesis, we have identified an LFIR (LF interacting region) in the COOH-terminal kinase domain of MEK1 adjacent to the proline-rich region, which is essential for LF-mediated proteolysis of MEK. Point mutations in this region block proteolysis but do not alter the kinase activity of MEK. Similar mutations in MEK6 also prevent proteolysis, indicating that this region is functionally conserved among MEKs. In addition, NH(2)-terminal proteolysis of MEK1 by LF was found to reduce not only the affinity of MEK1 for its substrate mitogen-activated protein kinase but also its intrinsic kinase activity, indicating that the NH(2)-terminal end of MEK is important not only for substrate interaction but also for catalytic activity.

    Funded by: PHS HHS: N01-C0-74101

    The Journal of biological chemistry 2003;278;11;9402-6

  • Phosphatidylinositol 3-kinase and mek1/2 are necessary for insulin-like growth factor-I-induced vascular endothelial growth factor synthesis in prostate epithelial cells: a role for hypoxia-inducible factor-1?

    Burroughs KD, Oh J, Barrett JC and DiAugustine RP

    Hormones and Cancer Group, Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.

    Due to the importance of vascular endothelial growth factor (VEGF) in the neovascularization of solid tumors, a clear understanding of how VEGF is regulated in normal and tumor cells is warranted. We investigated insulin-like growth factor (IGF)-I-stimulated signaling pathways that increase the rate of VEGF synthesis in primary cultures of normal prostate epithelial cells (PrEC). IGF-I increased the secretion of VEGF(165) into PrEC growth medium and stimulated transcription of a reporter gene driven by a 1.5-kb region of the VEGF promoter. Inhibition of either phosphatidylinositol 3-kinase (PI3-K) or Mek1/2 signaling pathways completely abrogated the IGF-I-induced increase in VEGF secretion and promoter activity, indicating a dependence on coordinate signaling from both pathways to produce this effect. Levels of the transcription factors hypoxia-inducible factor (HIF)-1 and Fos were elevated in response to IGF-I in a PI3-K-dependent and Mek1/2-dependent manner, respectively. The expression of an activator protein (AP)-1 dominant negative in an immortalized prostate epithelial cell line PZ-HPV-7 suppressed the IGF-I-induced increase in VEGF promoter activity. Mutation of the hypoxia response element (HRE), which mediates hypoxic stimulation of VEGF transcription, did not inhibit the effect of IGF-I on the VEGF promoter, despite the fact that this mutation inhibited PI3-K-stimulated VEGF promoter activity in prostate cancer cells. These data indicate that PI3-K signaling does not increase VEGF transcription through transactivation by HIF-1 at the HRE in normal PrEC. This work also suggests that an additional signal, not stimulated by IGF-I in PrEC, is needed for HIF-1 to stimulate transcription from the VEGF HRE.

    Molecular cancer research : MCR 2003;1;4;312-22

  • Witowsky JA and Johnson GL

    Department of Pharmacology, University of Colorado Health Sciences Center and University of Colorado Cancer Center, Denver, Colorado 80262, USA.

    MEKK1 is a MAPK kinase kinase that is activated in response to stimuli that alter the cytoskeleton and cell shape. MEKK1 phosphorylates and activates MKK1 and MKK4, leading to ERK1/2 and JNK activation. MEKK1 has a plant homeobox domain (PHD) that has been shown to have E3 ligase activity. (Lu, Z., Xu, S., Joazeiro, C., Cobb, M. H., and Hunter, T. (2002) Mol. Cell 9, 945-956). MEKK1 kinase activity is required for ubiquitylation of MEKK1. MEKK1 ubiquitylation is inhibited by mutation of cysteine 441 to alanine (C441A) within the PHD. The functional consequence of MEKK1 ubiquitylation is the inhibition of MEKK1 catalyzed phosphorylation of MKK1 and MKK4 resulting in inhibition of ERK1/2 and JNK activation. The C441A mutation within the PHD of MEKK1 prevents ubiquitylation and preserves the ability of MEKK1 to catalyze MKK1 and MKK4 phosphorylation. MEKK1 ubiquitylation represents a mechanism for inhibiting the ability of a protein kinase to phosphorylate substrates and regulate downstream signaling pathways.

    Funded by: NIDDK NIH HHS: DK37871

    The Journal of biological chemistry 2003;278;3;1403-6

  • Increased expression of epidermal growth factor receptor induces sequestration of extracellular signal-related kinases and selective attenuation of specific epidermal growth factor-mediated signal transduction pathways.

    Habib AA, Chun SJ, Neel BG and Vartanian T

    Department of Neurology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA. ahabib@caregroup.harvard.edu

    Increased expression of the epidermal growth factor receptor (EGFR) is common in cancer and correlates with neoplastic progression. Although the biology of this receptor has been the subject of intense investigation, surprisingly little is known about how increased expression of the wild-type EGFR affects downstream signal transduction in cells. We show that increasing the expression of the receptor results in dramatic shifts in signaling with attenuation of EGF-induced Ras, extracellular signal-related kinases (ERKs), and Akt activation, as well as amplification of STAT1 and STAT3 signaling. In this study, we focus on the mechanism of attenuated ERK signaling and present evidence suggesting that the mechanism of attenuated ERK signaling in EGFR-overexpressing cells is a sequestration of ERKs at the cell membrane in EGFR-containing complexes. Increased expression of the EGFR results in an aberrant localization of ERKs to the cell membrane. Furthermore, ERKs become associated with the EGFR in a physical complex in EGFR-overexpressing cells but not in control cells. The EGFR-ERK association is detected in unstimulated cells or on exposure to a low concentration of EGF; under these conditions, ERK activation is minimal. Exposure of these cells to saturating concentrations of EGF results in a decreased membrane localization of ERKs, a concomitant dissociation of ERKs from the EGFR, and restores ERK activation. A similar association can be detected between the EGFR and MEK1 in receptor-overexpressing cells, suggesting that multiple components of the ERK signaling pathway may become trapped in complexes with the EGFR. These findings can be demonstrated in cells transfected to express high levels of the EGFR as well as in cancer cells which naturally overexpress the EGFR and, thus, may be representative of altered EGFR signaling in human cancer.

    Funded by: NCI NIH HHS: K08 CA78741; NINDS NIH HHS: NS02028

    Molecular cancer research : MCR 2003;1;3;219-33

  • Up-regulation of mitogen-activated protein kinases ERK1/2 and MEK1/2 is associated with the progression of neurofibrillary degeneration in Alzheimer's disease.

    Pei JJ, Braak H, An WL, Winblad B, Cowburn RF, Iqbal K and Grundke-Iqbal I

    Karolinska Institutet, NEUROTEC, Division of Experimental Geriatrics, Novum, KFC Plan 4, Novum, S-141 86, Huddinge, Sweden. jin-jing.pei@neurotec.ki.se

    The abnormal hyperphosphorylation of tau in Alzheimer's disease (AD) has been proposed to involve the extracellular-signal-regulated protein kinase (ERK) of the mitogen-activated protein (MAP) kinase family. ERK is phosphorylated and thereby activated by MAP kinase kinase (MEK). In the present study, we determined the intracellular and regional distribution of the active forms of both MEK1/2 and ERK1/2, i.e. p-MEK1/2 and p-ERK1/2 in the entorhinal, hippocampal, and temporal cortices of 49 brains staged for neurofibrillary changes according to Braak and Braak's protocol. We found that p-MEK1/2 and p-ERK1/2 were present in the initial stages of neurofibrillary degeneration in the projecting neurons in the transentorhinal region, and extended into other brain regions co-incident with the progressive sequence of neurofibrillary changes up to and including Braak stage VI. It appeared that the accumulation of p-MEK1/2 and p-ERK1/2 was initiated in the cytoplasm of pretangle neurons in varying size granules, which grew into large aggregates co-existing with the progressive development of neurofibrillary tangles. Accumulation of p-MEK1/2 and p-ERK1/2 was found in cases with stages I-III neurofibrillary degeneration, which were devoid of amyloid deposition. These data provide direct in situ evidence consistent with the possible involvement of MAP kinase pathway in the hyperphosphorylation o 1f40 f tau and the presence of this lesion before deposition of beta-amyloid in AD.

    Funded by: NIA NIH HHS: AG08076, AG19158; NINDS NIH HHS: NS18105

    Brain research. Molecular brain research 2002;109;1-2;45-55

  • Signaling pathways triggered by HIV-1 Tat in human monocytes to induce TNF-alpha.

    Bennasser Y, Badou A, Tkaczuk J and Bahraoui E

    Laboratoire d'Immuno-Virologie, EA 3038, Université Paul Sabatier 118, route de Narbonne, 31062, Toulouse Cedex, France.

    In this study we investigated the signaling pathways triggered by Tat in human monocyte to induce TNF-alpha. In monocytes, the calcium, the PKA, and the PKC pathways are highly implicated in the expression of cytokine genes. Thus, these three major signaling pathways were investigated. Our data show that (i) PKC and calcium pathways are required for TNF-alpha production, whereas the PKA pathway seems to be not involved; (ii) downstream from PKC, activation of NFkappaB is essential while ERK1/2 MAP kinases, even though activated by Tat, are not directly involved in the pathway signaling leading to TNF-alpha production.

    Virology 2002;303;1;174-80

  • Identification of a novel mitogen-activated protein kinase kinase activation domain recognized by the inhibitor PD 184352.

    Delaney AM, Printen JA, Chen H, Fauman EB and Dudley DT

    Cell Biology, Discovery Technologies, Pfizer Global Research and Development, Ann Arbor Laboratories, Ann Arbor, Michigan 48105, USA.

    Utilizing a genetic screen in the yeast Saccharomyces cerevisiae, we identified a novel autoactivation region in mammalian MEK1 that is involved in binding the specific MEK inhibitor, PD 184352. The genetic screen is possible due to the homology between components of the yeast pheromone response pathway and the eukaryotic Raf-MEK-ERK signaling cascade. Using the FUS1::HIS3 reporter as a functional readout for activation of a reconstituted Raf-MEK-ERK signaling cascade, randomly mutagenized MEK variants that were insensitive to PD 184352 were obtained. Seven single-base-change mutations were identified, five of which mapped to kinase subdomains III and IV of MEK. Of the seven variants, only one, a leucine-to-proline substitution at amino acid 115 (Leu115Pro), was completely insensitive to PD 184352 in vitro (50% inhibitory concentration >10 micro M). However, all seven mutants displayed strikingly high basal activity compared to wild-type MEK. Overexpression of the MEK variants in HEK293T cells resulted in an increase in mitogen-activated protein (MAP) kinase phosphorylation, a finding consistent with the elevated basal activity of these constructs. Further, treatment with PD 184352 failed to inhibit Leu115Pro-stimulated MAP kinase activation in HEK293T cells, whereas all other variants had some reduction in phospho-MAP kinase levels. By using cyclic AMP-dependent protein kinase (1CDK) as a template, an MEK homology model was generated, with five of the seven identified residues clustered together, forming a potential hydrophobic binding pocket for PD 184352. Additionally, the model allowed identification of other potential residues that would interact with the inhibitor. Directed mutation of these residues supported this region's involvement with inhibitor binding.

    Molecular and cellular biology 2002;22;21;7593-602

  • Human immunodeficiency virus type 1 (HIV-1) tat induces nitric-oxide synthase in human astroglia.

    Liu X, Jana M, Dasgupta S, Koka S, He J, Wood C and Pahan K

    Department of Oral Biology, University of Nebraska Medical Center, Lincoln, Nebraska 68583, USA.

    Human immunodeficiency virus type 1 (HIV-1) infection is known to cause neuronal injury and dementia in a significant proportion of patients. However, the mechanism by which HIV-1 mediates its deleterious effects in the brain is poorly defined. The present study was undertaken to investigate the effect of the HIV-1 tat gene on the expression of inducible nitric-oxide synthase (iNOS) in human U373MG astroglial cells and primary astroglia. Expression of the tat gene as RSV-tat but not that of the CAT gene as RSV-CAT in U373MG astroglial cells led to the induction of NO production and the expression of iNOS protein and mRNA. Induction of NO production by recombinant HIV-1 Tat protein and inhibition of RSV-tat-induced NO production by anti-Tat antibodies suggest that RSV-tat-induced production of NO is dependent on Tat and that Tat is secreted from RSV-tat-transfected astroglia. Similar to U373MG astroglial cells, RSV-tat also induced the production of NO in human primary astroglia. The induction of human iNOS promoter-derived luciferase activity by the expression of RSV-tat suggests that RSV-tat induces the transcription of iNOS. To understand the mechanism of induction of iNOS, we investigated the role of NF-kappaB and C/EBPbeta, transcription factors responsible for the induction of iNOS. Activation of NF-kappaB as well as C/EBPbeta by RSV-tat, stimulation of RSV-tat-induced production of NO by the wild type of p65 and C/EBPbeta, and inhibition of RSV-tat-induced production of NO by deltap65, a dominant-negative mutant of p65, and deltaC/EBPbeta, a dominant-negative mutant of C/EBPbeta, suggest that RSV-tat induces iNOS through the activation of NF-kappaB and C/EBPbeta. In addition, we show that extracellular signal-regulated kinase (ERK) but not that p38 mitogen-activated protein kinase (MAPK) is involved in RSV-tat induced production of NO. Interestingly, PD98059, an inhibitor of the ERK pathway, and deltaERK2, a dominant-negative mutant of ERK2, inhibited RSV-tat-induced production of NO through the inhibition of C/EBPbeta but not that of NF-kappaB. This study illustrates a novel role for HIV-1 tat in inducing the expression of iNOS in human astrocytes that may participate in the pathogenesis of HIV-associated dementia.

    Funded by: NCI NIH HHS: CA76958; NCRR NIH HHS: P20 RR015635, P20-RR15635; NINDS NIH HHS: NS39940, R01 NS039940, R01 NS039940-01A1

    The Journal of biological chemistry 2002;277;42;39312-9

  • HIV envelope induces a cascade of cell signals in non-proliferating target cells that favor virus replication.

    Cicala C, Arthos J, Selig SM, Dennis G, Hosack DA, Van Ryk D, Spangler ML, Steenbeke TD, Khazanie P, Gupta N, Yang J, Daucher M, Lempicki RA and Fauci AS

    Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA. ccicala@nih.gov

    Certain HIV-encoded proteins modify host-cell gene expression in a manner that facilitates viral replication. These activities may contribute to low-level viral replication in nonproliferating cells. Through the use of oligonucleotide microarrays and high-throughput Western blotting we demonstrate that one of these proteins, gp120, induces the expression of cytokines, chemokines, kinases, and transcription factors associated with antigen-specific T cell activation in the absence of cellular proliferation. Examination of transcriptional changes induced by gp120 in freshly isolated peripheral blood mononuclear cells and monocyte-derived-macrophages reveals a broad and complex transcriptional program conducive to productive infection with HIV. Observations include the induction of nuclear factor of activated T cells, components of the RNA polymerase II complex including TFII D, proteins localized to the plasma membrane, including several syntaxins, and members of the Rho protein family, including Cdc 42. These observations provide evidence that envelope-mediated signaling contributes to the productive infection of HIV in suboptimally activated T cells.

    Proceedings of the National Academy of Sciences of the United States of America 2002;99;14;9380-5

  • Activation of a Src-dependent Raf-MEK1/2-ERK signaling pathway is required for IL-1alpha-induced upregulation of beta-defensin 2 in human middle ear epithelial cells.

    Moon SK, Lee HY, Li JD, Nagura M, Kang SH, Chun YM, Linthicum FH, Ganz T, Andalibi A and Lim DJ

    Gonda Department of Cell and Molecular Biology, House Ear Institute, 2100 West 3rd Street, Los Angeles, CA 90057, USA.

    beta-defensin 2 is produced by a variety of epithelial cell types in the body and exhibits potent antimicrobial activity against a variety of pathogens, including the bacteria that are most commonly associated with otitis media (OM). The human beta-defensin 2 (hBD-2) gene is an NF-kappa B regulated gene and a variety of proinflammatory stimuli can induce its expression. Although the presence of molecules of innate immunity such as lysozyme and lactoferrin has been demonstrated in the middle ear, to date there have been no reports on the expression of beta-defensin 2. In the present study, we demonstrate that beta-defensin 2 is expressed in the middle ear mucosa of humans and rats. We also show that it is expressed in a human middle ear epithelial cell line and that its expression is induced by proinflammatory stimuli such as interleukin 1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF-alpha), and lipopolysaccharide (LPS). Moreover, we demonstrate that the transcriptional activation of hBD-2 gene by IL-1 alpha is mediated through an Src-dependent Raf-MEK1/2-ERK signaling pathway.

    Funded by: NIDCD NIH HHS: R01-DC5025

    Biochimica et biophysica acta 2002;1590;1-3;41-51

  • Laminin-10/11 and fibronectin differentially prevent apoptosis induced by serum removal via phosphatidylinositol 3-kinase/Akt- and MEK1/ERK-dependent pathways.

    Gu J, Fujibayashi A, Yamada KM and Sekiguchi K

    Division of Protein Chemistry, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.

    Cell adhesion to the extracellular matrix inhibits apoptosis, but the molecular mechanisms underlying the signals transduced by different matrix components are not well understood. Here, we examined integrin-mediated antiapoptotic signals from laminin-10/11 in comparison with those from fibronectin, the best characterized extracellular adhesive ligand. We found that the activation of protein kinase B/Akt in cells adhering to laminin-10/11 can rescue cell apoptosis induced by serum removal. Consistent with this, wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, or ectopic expression of a dominant-negative mutant of Akt selectively accelerated cell death upon serum removal. In contrast to laminin-10/11, fibronectin rescued cells from serum depletion-induced apoptosis mainly through the extracellular signal-regulated kinase pathway. Cell survival on fibronectin but not laminin was significantly reduced by treatment with PD98059, a specific inhibitor of mitogen- or extracellular signal-regulated kinase kinase-1 (MEK1) and by expression of a dominant-negative mutant of MEK1. Laminin-10/11 was more potent than fibronectin in preventing apoptosis induced by serum depletion. These results, taken together, demonstrate laminin-10/11 potency as a survival factor and demonstrate that different extracellular matrix components can transduce distinct survival signals through preferential activation of subsets of multiple integrin-mediated signaling pathways.

    The Journal of biological chemistry 2002;277;22;19922-8

  • Kinase suppressor of Ras (KSR) is a scaffold which facilitates mitogen-activated protein kinase activation in vivo.

    Nguyen A, Burack WR, Stock JL, Kortum R, Chaika OV, Afkarian M, Muller WJ, Murphy KM, Morrison DK, Lewis RE, McNeish J and Shaw AS

    Department of Pathology and Immunology, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

    While scaffold proteins are thought to be key components of signaling pathways, their exact function is unknown. By preassembling multiple components of signaling cascades, scaffolds are predicted to influence the efficiency and/or specificity of signaling events. Here we analyze a potential scaffold of the Ras/mitogen-activated protein kinase (MAPK) pathway, kinase suppressor of Ras (KSR), by generating KSR-deficient mice. KSR-deficient mice were grossly normal even though ERK kinase activation was attenuated to a degree sufficient to block T-cell activation and inhibit tumor development. Consistent with its role as a scaffold, high-molecular-weight complexes containing KSR, MEK, and ERK were lost in the absence of KSR. This demonstrates that KSR is a bona fide scaffold that is not required for but enhances signaling via the Ras/MAPK signaling pathway.

    Funded by: NCI NIH HHS: CA90400, R01 CA090400; NIDDK NIH HHS: DK52809, R01 DK052809

    Molecular and cellular biology 2002;22;9;3035-45

  • Identification of novel point mutations in ERK2 that selectively disrupt binding to MEK1.

    Robinson FL, AW, Raman M and Cobb MH

    Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390-9041, USA.

    Extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) are essential components of pathways through which signals received at membrane receptors are converted into specific changes in protein function and gene expression. As with other members of the mitogen-activated protein (MAP) kinase family, ERK1 and ERK2 are activated by phosphorylations catalyzed by dual-specificity protein kinases known as MAP/ERK kinases (MEKs). MEKs exhibit stringent specificity for individual MAP kinases. Indeed, MEK1 and MEK2 are the only known activators of ERK1 and ERK2. ERK2 small middle dotMEK1/2 complexes can be detected in vitro and in vivo. The biochemical nature of such complexes and their role in MAP kinase signaling are under investigation. This report describes the use of a yeast two-hybrid screen to identify point mutations in ERK2 that impair its interaction with MEK1/2, yet do not alter its interactions with other proteins. ERK2 residues identified in this screen are on the surface of the C-terminal domain of the kinase, either within or immediately preceding alpha-helix G, or within the MAP kinase insert. Some mutations identified in this manner impaired the two-hybrid interaction of ERK2 with both MEK1 and MEK2, whereas others had a predominant effect on the interaction with either MEK1 or MEK2. Mutant ERK2 proteins displayed reduced activation in HEK293 cells following epidermal growth factor treatment, consistent with their impaired interaction with MEK1/2. However, ERK2 proteins containing MEK-specific mutations retained kinase activity, and were similar to wild type ERK2 in their activation following overexpression of constitutively active MEK1. Unlike wild type ERK2, proteins containing MEK-specific point mutations were constitutively localized in the nucleus, even in the presence of overexpressed MEK1. These data suggest an essential role for the MAP kinase insert and residues within or just preceding alpha-helix G in the interaction of ERK2 with MEK1/2.

    Funded by: NIDDK NIH HHS: DK34128

    The Journal of biological chemistry 2002;277;17;14844-52

  • HIV Nef increases T cell ERK MAP kinase activity.

    Schrager JA, Der Minassian V and Marsh JW

    Laboratory of Molecular Biology, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland 20892-4034, USA.

    The human immunodeficiency regulatory protein Nef enhances viral replication and is central to viral pathogenesis. Although Nef has displayed a capacity to associate with a diverse assortment of cellular molecules and to increase T cell activity, the biochemical activity of Nef in T cells remains poorly defined. In this report we examine the bioactivity of Nef in primary CD4 T cells and, in particular, focus on the biochemical pathways known to be central to T cell activity. The extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase pathway was dramatically affected by Nef expression with increases in ERK, MEK, and Elk induction. The capacity of Nef to increase the MAP kinase pathway activity was dependent on T cell receptor stimulation. By increasing ERK MAP kinase activity, Nef is functionally associated with a kinase known to affect T cell activity, viral replication, and viral infectivity.

    The Journal of biological chemistry 2002;277;8;6137-42

  • Different domains of the mitogen-activated protein kinases ERK3 and ERK2 direct subcellular localization and upstream specificity in vivo.

    Robinson MJ, Xu Be BE, Stippec S and Cobb MH

    Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9041, USA.

    Extracellular signal-regulated kinase 3 (ERK3) is a member of the mitogen-activated protein (MAP) kinase family. ERK3 is most similar in its kinase catalytic domain to ERK2, yet it displays many unique properties. Among these, unlike ERK2, which translocates to the nucleus following activation, ERK3 is constitutively localized to the nucleus, despite the lack of a defined nuclear localization sequence. We created two chimeras between ERK2 and the catalytic domain of ERK3 (ERK3DeltaC), and some mutants of these chimeras, to examine the basis for the different behaviors of these two MAP kinase family members. We find the following: 1) the N-terminal folding domain of ERK3 functions in phosphoryl transfer reactions with the C-terminal folding domain of ERK2; 2) the C-terminal halves of ERK2 and ERK3DeltaC are primarily responsible for their subcellular localization in resting cells; and 3) the N-terminal folding domain of ERK2 is required for its activation in cells, its interaction with MEK1, and its accumulation in the nucleus.

    Funded by: NIDDK NIH HHS: DK34128

    The Journal of biological chemistry 2002;277;7;5094-100

  • Phosphorylation of MEK1 by cdk5/p35 down-regulates the mitogen-activated protein kinase pathway.

    Sharma P, Veeranna, Sharma M, Amin ND, Sihag RK, Grant P, Ahn N, Kulkarni AB and Pant HC

    Laboratory of Neurochemistry, NINDS, National Institutes of Health, Bethesda, Maryland 20892, USA.

    Cyclin-dependent protein kinase 5 (cdk5), a member of the cdk family, is active mainly in postmitotic cells and plays important roles in neuronal development and migration, neurite outgrowth, and synaptic transmission. In this study we investigated the relationship between cdk5 activity and regulation of the mitogen-activated protein (MAP) kinase pathway. We report that cdk5 phosphorylates the MAP kinase kinase-1 (MEK1) in vivo as well as the Ras-activated MEK1 in vitro. The phosphorylation of MEK1 by cdk5 resulted in inhibition of MEK1 catalytic activity and the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. In p35 (cdk5 activator) -/- mice, which lack appreciable cdk5 activity, we observed an increase in the phosphorylation of NF-M subunit of neurofilament proteins that correlated with an up-regulation of MEK1 and ERK1/2 activity. The activity of a constitutively active MEK1 with threonine 286 mutated to alanine (within a TPXK cdk5 phosphorylation motif in the proline-rich domain) was not affected by cdk5 phosphorylation, suggesting that Thr286 might be the cdk5/p35 phosphorylation-dependent regulatory site. These findings support the hypothesis that cdk5 and the MAP kinase pathway cross-talk in the regulation of neuronal functions. Moreover, these data and the recent studies of Harada et al. (Harada, T., Morooka, T., Ogawa, S., and Nishida, E. (2001) Nat. Cell Biol. 3, 453-459) have prompted us to propose a model for feedback down-regulation of the MAP kinase signal cascade by cdk5 inactivation of MEK1.

    The Journal of biological chemistry 2002;277;1;528-34

  • MEK-ERK pathway is expressed but not activated in high proliferating, self-renewing cord blood hematopoietic progenitors.

    Bonati A, Lunghi P, Gammaitoni L, Pinelli S, Ridolo E, Dall'Aglio PP, Piacibello W and Aglietta M

    The Institute of Medical Pathology, University of Parma Medical School, Italy. antbonny@ipruniv.cce.unipr.it

    Introduction: The expression, activity and functions of mitogen-activated protein (MAP) kinases in primary human hematopoietic progenitors (HP) have not yet been fully clarified.

    To perform our experiments we used a stroma-free cell culture system in which the combination of FLT3 ligand (FL), stem cell factor (SCF) and thrombopoietin (TPO) induces massive expansion and proliferation of cord blood HP. The addition of IL-3 results in a rapid decrease of HP due to the prevalence of maturation and cell death. To detect extracellular regulated kinase (ERK) immunoenzymatic activity we recovered HP from FL, SCF and TPO stimulated long term cultures (LTC) after four weeks of culture. Some samples were recovered 16 h after addition of IL-3 to the LTC. We selectively immunoprecipitated p44/42 ERK kinase from 245 microg of cell lysates. We then analysed dual-phosphorylation of ERK-activating kinase-kinase (p45 MEK1/2) and of p44 ERK1 and p42 ERK2, and investigated MEK and ERK expression.

    Results: ERK activity, MEK1, and p42 and p44 ERK dual-phosphorylation were undetectable in the expanding, greatly proliferating and self-renewing HP. However, after addition of IL-3 sustained (still detectable 16 h after the stimulus) and high levels of ERK activity and dual-phosphorylation of the kinases were seen. The levels of MEK and ERK expression were stable in the different phases.

    Conclusions: These findings add new information on the intracellular mechanisms of HP and help explain the very low levels of hematopoietic toxicity recently seen when treating cancer with down-modulators of ERK activity.

    The hematology journal : the official journal of the European Haematology Association 2002;3;2;105-13

  • 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

  • The roles of phosphatidylinositol 3-kinase and protein kinase Czeta for thrombopoietin-induced mitogen-activated protein kinase activation in primary murine megakaryocytes.

    Rojnuckarin P, Miyakawa Y, Fox NE, Deou J, Daum G and Kaushansky K

    Division of Hematology, University of Washington School of Medicine, Seattle, Washington 98195, USA.

    Thrombopoietin (TPO) stimulates a network of intracellular signaling pathways that displays extensive cross-talk. We have demonstrated previously that the ERK/mitogen-activated protein kinase pathway is important for TPO-induced endomitosis in primary megakaryocytes (MKs). One known pathway by which TPO induces ERK activation is through the association of Shc with the penultimate phosphotyrosine within the TPO receptor, Mpl. However, several investigators found that the membrane-proximal half of the cytoplasmic domain of Mpl is sufficient to activate ERK in vitro and support base-line megakaryopoiesis in vivo. Using BaF3 cells expressing a truncated Mpl (T69Mpl) as a tool to identify non-Shc/Ras-dependent signaling pathways, we describe here novel mechanisms of TPO-induced ERK activation mediated, in part, by phosphoinositide 3-kinase (PI3K). Similar to cells expressing full-length receptor, PI3K was activated by its incorporation into a complex with IRS2 or Gab2. Furthermore, the MEK-phosphorylating activity of protein kinase Czeta (PKCzeta) was also enhanced after TPO stimulation of T69Mpl, contributing to ERK activity. PKCzeta and PI3K also contribute to TPO-induced ERK activation in MKs, confirming their physiological relevance. Like in BaF3 cells, a TPO-induced signaling complex containing p85PI3K is detectable in MKs expressing T61Mpl and is probably responsible for PI3K activation. These data demonstrate a novel role of PI3K and PKCzeta in steady-state megakaryopoiesis.

    The Journal of biological chemistry 2001;276;44;41014-22

  • C-TAK1 regulates Ras signaling by phosphorylating the MAPK scaffold, KSR1.

    1f40 Müller J, Ory S, Copeland T, Piwnica-Worms H and Morrison DK

    Regulation of Cell Growth Laboratory, Center for Cancer Research, NCI-Frederick, Frederick, MD 21702, USA.

    Kinase suppressor of Ras (KSR) is a conserved component of the Ras pathway that interacts directly with MEK and MAPK. Here we show that KSR1 translocates from the cytoplasm to the cell surface in response to growth factor treatment and that this process is regulated by Cdc25C-associated kinase 1 (C-TAK1). C-TAK1 constitutively associates with mammalian KSR1 and phosphorylates serine 392 to confer 14-3-3 binding and cytoplasmic sequestration of KSR1 in unstimulated cells. In response to signal activation, the phosphorylation state of S392 is reduced, allowing the KSR1 complex to colocalize with activated Ras and Raf-1 at the plasma membrane, thereby facilitating the phosphorylation reactions required for the activation of MEK and MAPK.

    Molecular cell 2001;8;5;983-93

  • Pro-inflammatory and pro-oxidant properties of the HIV protein Tat in a microglial cell line: attenuation by 17 beta-estradiol.

    Bruce-Keller AJ, Barger SW, Moss NI, Pham JT, Keller JN and Nath A

    Department of Anatomy, University of Kentucky, Lexington, Kentucky, USA. abruce@pop.uky.edu

    Microglia are activated in humans following infection with human immunodeficiency virus (HIV), and brain inflammation is thought to be involved in neuronal injury and dysfunction during HIV infection. Numerous studies indicate a role for the HIV regulatory protein Tat in HIV-related inflammatory and neurodegenerative processes, although the specific effects of Tat on microglial activation, and the signal transduction mechanisms thereof, have not been elucidated. In the present study, we document the effects of Tat on microglial activation and characterize the signal transduction pathways responsible for Tat's pro-inflammatory effects. Application of Tat to N9 microglial cells increased multiple parameters of microglial activation, including superoxide production, phagocytosis, nitric oxide release and TNF alpha release. Tat also caused activation of both p42/p44 mitogen activated protein kinase (MAPK) and NF kappa B pathways. Inhibitor studies revealed that Tat-induced NF kappa B activation was responsible for increased nitrite release, while MAPK activation mediated superoxide release, TNF alpha release, and phagocytosis. Lastly, pre-treatment of microglial cells with physiological concentrations of 17 beta-estradiol suppressed Tat-mediated microglial activation by interfering with Tat-induced MAPK activation. Together, these data elucidate specific components of the microglial response to Tat and suggest that Tat could contribute to the neuropathology associated with HIV infection through microglial promulgation of oxidative stress.

    Funded by: NCRR NIH HHS: P20RR15592; NINDS NIH HHS: NS39398

    Journal of neurochemistry 2001;78;6;1315-24

  • Hydrophobic as well as charged residues in both MEK1 and ERK2 are important for their proper docking.

    Xu Be, Stippec S, Robinson FL and Cobb MH

    Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390-9041, USA.

    Docking between MEK1 and ERK2 is required for their stable interaction and efficient signal transmission. The MEK1 N terminus contains the ERK docking or D domain that consists of conserved hydrophobic and basic residues. We mutated the hydrophobic and basic residues individually and found that loss of either type reduced MEK1 phosphorylation of ERK2 in vitro and its ability to bind to ERK2 in vivo. Moreover, ERK2 was localized in both the cytoplasm and the nucleus when co-expressed with MEK1 that had mutations in either the hydrophobic or the basic residues. We then identified two conserved hydrophobic residues on ERK2 that play roles in docking with MEK1. Mutating these residues to alanine reduced the interaction of ERK2 with MEK1 in cells. These mutations also reduced the phosphorylation of MEK1 by ERK2 but had little effect on phosphorylation of MBP by ERK2. Finally, we generated docking site mutants in ERK2-MEK1 fusion proteins. Although the mutation of the MEK1 D domain significantly reduced ERK2-MEK1 activity, mutations of the putatively complementary acidic residues and hydrophobic residues on ERK2 did not change its activity. However, both types of mutations decreased the phosphorylation of Elk-1 caused by ERK2-MEK1 fusion proteins. These findings suggest complex interactions of MEK1 D domains with ERK2 that influence its activation and its effects on substrates.

    Funded by: NIDDK NIH HHS: DK34128

    The Journal of biological chemistry 2001;276;28;26509-15

  • Regulation of stress-responsive mitogen-activated protein (MAP) kinase pathways by TAO2.

    Chen Z and Cobb MH

    Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9041, USA.

    Previous studies demonstrated that in vitro the protein kinase TAO2 activates MAP/ERK kinases (MEKs) 3, 4, and 6 toward their substrates p38 MAP kinase and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK). In this study, we examined the ability of TAO2 to activate stress-sensitive MAP kinase pathways in cells and the relationship between activation of TAO2 and potential downstream pathways. Over-expression of TAO2 activated endogenous JNK/SAPK and p38 but not ERK1/2. Cotransfection experiments suggested that TAO2 selectively activates MEK3 and MEK6 but not MEKs 1, 4, or 7. Coimmunoprecipitation demonstrated that endogenous TAO2 specifically associates with MEK3 and MEK6 providing one mechanism for preferential recognition of MEKs upstream of p38. Sorbitol, and to a lesser extent, sodium chloride, Taxol, and nocodazole increased TAO2 activity toward itself and kinase-dead MEKs 3 and 6. Activation of endogenous TAO2 during differentiation of C2C12 myoblasts paralleled activation of p38 but not JNK/SAPK, consistent with the idea that TAO2 is a physiological regulator of p38 under certain circumstances.

    Funded by: NIGMS NIH HHS: GM53032

    The Journal of biological chemistry 2001;276;19;16070-5

  • Stimulation of p300-mediated transcription by the kinase MEKK1.

    See RH, Calvo D, Shi Y, Kawa H, Luke MP, Yuan Z and Shi Y

    Department of Pathology, Harvard Medical School and Department of Radiation Biology, Harvard School of Public Health, Boston, Massachusetts 02115.

    p300 and CREB-binding protein (CBP) are related transcriptional coactivators that possess histone acetyltransferase activity. Inactivation of p300/CBP is part of the mechanism by which adenovirus E1A induces oncogenic transformation of cells. Recently, the importance of p300/CBP has been demonstrated directly in several organisms including mouse, Drosophila, and Caenorhabditis elegans where p300/CBP play an indispensable role in differentiation, in patterning, and in cell fate determination and proliferation during development. CBP/p300s are modified by phosphorylation during F9 cell differentiation as well as adenovirus infection, suggesting that phosphorylation may play a role in the regulation of p300/CBP activity. Here we show that the mitogen-activated/extracellular response kinase kinase 1 (MEKK1) enhances p300-mediated transcription. We identify several domains within p300 that can respond to MEKK1-induced transcriptional activation. Interestingly, activation of p300-mediated transcription by MEKK1 does not appear to require the downstream kinase JNK and may involve either a direct phosphorylation of p300 by MEKK1 or by other non-JNK MEKK1-directed downstream kinases. Finally, we present evidence that p300 is important for MEKK1 to induce apoptosis. Taken together, these results identify MEKK1 as a kinase that is likely to be involved in the regulation of the transactivation potential of p300 and support a role of p300 in MEKK1-induced apoptosis.

    Funded by: NIGMS NIH HHS: GM58012

    The Journal of biological chemistry 2001;276;19;16310-7

  • A conserved docking site in MEKs mediates high-affinity binding to MAP kinases and cooperates with a scaffold protein to enhance signal transmission.

    Bardwell AJ, Flatauer LJ, Matsukuma K, Thorner J and Bardwell L

    Department of Developmental and Cell Biology, University of California, Irvine, California 92697, USA. bardwell@uci.edu

    The recognition of mitogen-activated protein kinases (MAPKs) by their upstream activators, MAPK/ERK kinases (MEKs), is crucial for the effective and accurate transmission of many signals. We demonstrated previously that the yeast MAPKs Kss1 and Fus3 bind with high affinity to the N terminus of the MEK Ste7, and proposed that a conserved motif in Ste7, the MAPK-docking site, mediates this interaction. Here we show that the corresponding sequences in human MEK1 and MEK2 are necessary and sufficient for the direct binding of the MAPKs ERK1 and ERK2. Mutations in MEK1, MEK2, or Ste7 that altered conserved residues in the docking site diminished binding of the cognate MAPKs. Furthermore, short peptides corresponding to the docking sites in these MEKs inhibited MEK1-mediated phosphorylation of ERK2 in vitro. In yeast cells, docking-defective alleles of Ste7 were modestly compromised in their ability to transmit the mating pheromone signal. This deficiency was dramatically enhanced when the ability of the Ste5 scaffold protein to associate with components of the MAPK cascade was also compromised. Thus, both the MEK-MAPK docking interaction and binding to the Ste5 scaffold make mutually reinforcing contributions to the efficiency of signaling by this MAPK cascade in vivo.

    Funded by: NIGMS NIH HHS: GM21841, GM60366, R01 GM021841, R01 GM060366, R01 GM060366-01

    The Journal of biological chemistry 2001;276;13;10374-86

  • Extracellular Tat activates c-fos promoter in low serum-starved CD4+ T cells.

    Gibellini D, Re MC, Ponti C, Celeghini C, Melloni E, La Placa M and Zauli G

    Department of Clinical and Experimental Medicine, Microbiology Section, University of Bologna, Via Massarenti 9, 40138 Bologna, Italy. rabbiloew@hotmail.com

    The regulatory human immunodeficiency virus-1 (HIV-1) Tat protein shows pleiotropic effects on the survival and growth of both HIV-1-infected and uninfected CD4+ T lymphocytes. In this study, we have demonstrated that low concentrations (10 ng/ml) of extracellular Tat protein induce the expression of both c-fos mRNA and protein in serum-starved Jurkat CD4+ lymphoblastoid T cells. Using deletion mutants, we demonstrates that the SRE, CRE and, to a lesser extent, also the SIE domains (all placed in the first 356 bp of c-fos promoter) play a key role in mediating the response to extracellular Tat. Moreover, the ability of Tat to activate the transcriptional activity of c-fos promoter was consistently decreased by pretreatment with the ERK/MAPK kinase inhibitor PD98058. Activation of c-fos is functional as demonstrated by induction of the AP-1 transcription factor, which is involved in the regulation of critical genes for the activation of T lymphocytes, such as interleukin 2. The Tat-mediated induction of c-fos and AP-1 in uninfected lymphoid T cells may contribute to explain the immune hyperactivation that characterizes the progression to autoimmuno deficiency syndrome and constitutes the optimal environment for HIV-1 replication, occurring predominantly in activated/proliferating CD4+ T cells.

    British journal of haematology 2001;112;3;663-70

  • 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

  • A novel 14-kilodalton protein interacts with the mitogen-activated protein kinase scaffold mp1 on a late endosomal/lysosomal compartment.

    Wunderlich W, Fialka I, Teis D, Alpi A, Pfeifer A, Parton RG, Lottspeich F and Huber LA

    Research Institute of Molecular Pathology, A-1030 Vienna, Austria.

    We have identified a novel, highly conserved protein of 14 kD copurifying with late endosomes/lysosomes on density gradients. The protein, now termed p14, is peripherally associated with the cytoplasmic face of late endosomes/lysosomes in a variety of different cell types. In a two-hybrid screen with p14 as a bait, we identified the mitogen-activated protein kinase (MAPK) scaffolding protein MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) partner 1 (MP1) as an interacting protein. We confirmed the specificity of this interaction in vitro by glutathione S-transferase pull-down assays and by coimmunoprecipitation, cosedimentation on glycerol gradients, and colocalization. Moreover, expression of a plasma membrane-targeted p14 causes mislocalization of coexpressed MP1. In addition, we could reconstitute protein complexes containing the p14-MP1 complex associated with ERK and MEK in vitro.The interaction between p14 and MP1 suggests a MAPK scaffolding activity localized to the cytoplasmic surface of late endosomes/lysosomes, thereby combining catalytic scaffolding and subcellular compartmentalization as means to modulate MAPK signaling within a cell.

    The Journal of cell biology 2001;152;4;765-76

  • Activation of endothelial cell mitogen activated protein kinase ERK(1/2) by extracellular HIV-1 Tat protein.

    Rusnati M, Urbinati C, Musulin B, Ribatti D, Albini A, Noonan D, Marchisone C, Waltenberger J and Presta M

    University of Brescia, Italy.

    Extracellular Tat protein, the transactivating factor of the human immunodeficiency virus type 1 (HIV-1), modulates gene expression, growth, and angiogenic activity in endothelial cells by interacting with the vascular endothelial growth factor (VEGF) receptor-2 (Flk-1/KDR). Recombinant Tat protein, produced as glutathione-S-transferase chimera (GST-Tat), activates mitogen-activated protein kinase (MAPK) ERK(1/2) in human, murine, and bovine endothelial cells whereas GST is ineffective. In bovine aortic endothelial cells, GST-Tat and the 165 amino acid VEGF isoform (VEGF165) induce transient ERK(1/2) phosphorylation with similar potency and kinetics. The synthetic peptide Tat(41-60), but not peptides Tat(1-21) and Tat(71-86), causes ERK(1/2) phosphorylation, thus implicating Tat/KDR interaction in the activation of this signalling pathway. Accordingly, GST-Tat induces ERK(1/2) phosphorylation in KDR-transfected porcine aortic endothelial cells but not in parental cells. MAPK kinase inhibitors PD098059 and U0126 prevent ERK(1/2) phosphorylation by Tat. However, they do not affect the angiogenic activity exerted by Tat in the murine Matrigel plug and chick embryo chorioallantoic membrane assays. Blocking of MAPK kinase activity impairs instead the angiogenic response to VEGF165 and to fibroblast growth factor-2 (FGF-2). Our data demonstrate that ERK(1/2) activation following the interaction of HIV-1 Tat protein with endothelial cell Flk-1/KDR receptor does not represent an absolute requirement for a full angiogenic response to this growth factor that appears to utilize mechanism(s) at least in part distinct from those triggered by other prototypic angiogenic growth factors.

    Endothelium : journal of endothelial cell research 2001;8;1;65-74

  • 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

  • Susceptibility of mitogen-activated protein kinase kinase family members to proteolysis by anthrax lethal factor.

    Vitale G, Bernardi L, Napolitani G, Mock M and Montecucco C

    Centro CNR Biomembrane and Dipartimento di Scienze Biomediche, Università di Padova, Via Trieste 75, 35121 Padova, Italy. gvitale@makek.dstb.uniud.it

    The lethal factor (LF) produced by toxigenic strains of Bacillus anthracis is a Zn(2+)-endopeptidase that cleaves the mitogen-activated protein kinase kinases (MAPKKs) MEK1, MEK2 and MKK3. Using genetic and biochemical approaches, we have extended the study of LF proteolytic specificity to all known MAPKK family members and found that LF also cleaves MKK4, MKK6 and MKK7, but not MEK5. The peptide bonds hydrolysed by LF within all MAPKKs were identified. Cleavage invariably occurs within the N-terminal proline-rich region preceding the kinase domain, thus disrupting a sequence involved in directing specific protein-protein interactions necessary for the assembly of signalling complexes. Alignment of the sequences flanking the site of cleavage reveals the occurrence of some consensus motifs: position P2 and P1' are occupied by hydrophobic residues and at least one basic residue is present between P4 and P7. The implications of these findings for the biochemical activity and functional specificity of LF are discussed.

    The Biochemical journal 2000;352 Pt 3;739-45

  • Tat protein of human immunodeficiency virus type 1 induces interleukin-10 in human peripheral blood monocytes: implication of protein kinase C-dependent pathway.

    Badou A, Bennasser Y, Moreau M, Leclerc C, Benkirane M and Bahraoui E

    Laboratoire d'Immuno-Virologie EA3038, Université Paul Sabatier, 31062 Toulouse Cedex, France.

    The clinical manifestations observed in human immunodeficiency virus type 1 (HIV-1)-infected patients are primarily due to the capacity of the virus and its components to inactivate the immune system. HIV-1 Tat protein could participate in this immune system disorder. This protein is secreted by infected cells of HIV-infected patients and is free in the plasma, where it can interact and be taken up by both infected and noninfected cells. In asymptomatic patients infected by HIV-1, production of interleukin-10 (IL-10), a highly immunosuppressive cytokine, is associated with disease progression to AIDS. In the present work, we tested the capacity of Tat to induce IL-10 production by peripheral blood monocytes of healthy donors. The results show that Tat causes the production of IL-10 in a dose- and stimulation time-dependent manner. Investigations of the mechanisms involved in signal transduction show that (i) the calcium pathway is not or only slightly involved in Tat-induced IL-10 production, (ii) the protein kinase C pathway plays an essential role, and (iii) monocyte stimulation by Tat results in the intranuclear translocation of transcription factor NF-kappaB and in the induction of phosphorylation of the mitogen-activated protein kinases ERK1 and ERK2; activation of these two potential substrates of protein kinase C is required for the production of IL-10. Finally, our results suggest that the effect of Tat is exerted at the membrane level and that the active domain is located within N-terminal residues 1 to 45. This production of IL-10 induced by Tat could participate in the progression of HIV infection to AIDS.

    Journal of virology 2000;74;22;10551-62

  • Induction of the chemokines interleukin-8 and IP-10 by human immunodeficiency virus type 1 tat in astrocytes.

    Kutsch O, Oh J, Nath A and Benveniste EN

    Department of Cell Biology, The University of Alabama at Birmingham, Birmingham, Alabama 35294-0005, USA.

    A finding commonly observed in human immunodeficiency virus type 1 (HIV-1)-infected patients is invasion of the brain by activated T cells and infected macrophages, eventually leading to the development of neurological disorders and HIV-1-associated dementia. The recruitment of T cells and macrophages into the brain is likely the result of chemokine expression. Indeed, earlier studies revealed that levels of different chemokines were increased in the cerebrospinal fluid of HIV-1-infected patients whereas possible triggers and cellular sources for chemokine expression in the brain remain widely undefined. As previous studies indicated that HIV-1 Tat, the retroviral transactivator, is capable of inducing a variety of cellular genes, we investigated its capacity to induce production of chemokines in astrocytes. Herein, we demonstrate that HIV-1 Tat(72aa) is a potent inducer of MCP-1, interleukin-8 (IL-8), and IP-10 expression in astrocytes. Levels of induced IP-10 protein were sufficiently high to induce chemotaxis of peripheral blood lymphocytes. In addition, Tat(72aa) induced IL-8 expression in astrocytes. IL-8 mRNA induction was seen less then 1 h after Tat(72aa) stimulation, and levels remained elevated for up to 24 h, leading to IL-8 protein production. Tat(72aa)-mediated MCP-1 and IL-8 mRNA induction was susceptible to inhibition by the MEK1/2 inhibitor UO126 but was only modestly decreased by the inclusion of the p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190. In contrast, Tat-mediated IP-10 mRNA induction was suppressed by SB202190 but not by the MEK1/2 inhibitor UO126. These findings indicate that MAPKs play a major role in Tat(72aa)-mediated chemokine induction in astrocytes.

    Funded by: NIMH NIH HHS: MH55795; NINDS NIH HHS: NS29719, NS36765, R01 NS036765

    Journal of virology 2000;74;19;9214-21

  • Control sites of ribosomal S6 kinase B and persistent activation through tumor necrosis factor.

    Tomás-Zuber M, Mary JL and Lesslauer W

    Department PRPN and Department PRPV of F. Hoffmann-LaRoche, Ltd., CH-4070 Basel, Switzerland.

    RSKB, a 90-kDa ribosomal S6 protein kinase family (RSK) member with two complete catalytic domains connected by a linker, is activated through p38- and ERK-mitogen-activated protein kinases. The N-terminal kinases of RSKs phosphorylate substrates; activation requires phosphorylation of linker and C-terminal kinase sites. Unlike other RSKs, the activation loop phosphorylation sites of both catalytic domains of RSKB, Ser(196) and Thr(568), were required for activity. RSKB activation depended on phosphorylation of linker Ser(343) and Ser(360) and associated with phosphorylation of nonconserved Ser(347), but Ser(347)-deficient RSKB retained partial activity. The known protein kinase A and protein kinase C inhibitors, H89 and Ro31-8220, blocked RSKB activity. Treatment of HeLa cells with tumor necrosis factor, epidermal growth factor, phorbol 12-myristate 13-acetate, and ionomycin but not with insulin resulted in strong activation of endogenous RSKB. High RSKB activity and Ser(347)/Ser(360) phosphorylation persisted for 3 h in tumor necrosis factor-treated cells, in contrast to the short bursts of p38, ERK, and RSK1-3 activities. In conclusion, a variety of stimuli induced phosphorylation and activation of RSKB through both p38 and ERK pathways; the persistence of activation indicated that RSKB selectively escaped cell mechanisms causing rapid deactivation of upstream p38 and ERK and other RSKs.

    The Journal of biological chemistry 2000;275;31;23549-58

  • Activation of mitogen-activated protein kinases is required for alpha1-adrenergic agonist-induced cell scattering in transfected HepG2 cells.

    Spector M, Nguyen VA, Sheng X, He L, Woodward J, Fan S, Baumgarten CM, Kunos G, Dent P and Gao B

    Department of Pharmacology and Toxicology, Medical College of Virginia of Virginia Commonwealth University, Richmond 23298, USA.

    Activation of alpha1B-adrenergic receptors ((alpha1B)AR) by phenylephrine (PE) induces scattering of HepG2 cells stably transfected with the (alpha1B)AR (TFG2 cells). Scattering was also observed after stimulation of TFG2 cells with phorbol myristate acetate (PMA) but not with hepatocyte growth factor/scatter factor, epidermal growth factor, or insulin. PMA but not phenylephrine rapidly activated PKCalpha in TFG2 cells, and the highly selective PKC inhibitor bisindolylmaleimide (GFX) completely abolished PMA-induced but not PE-induced scattering. PE rapidly activated p44/42 mitogen-activated protein kinase (MAPK), p38 MAPK, c-Jun N-terminal kinase (JNK), and AP1 (c-fos/c-jun). Selective blockade of p42/44 MAPK activity by PD98059 or by transfection of a MEK1 dominant negative adenovirus significantly inhibited the PE-induced scattering of TFG2 cells. Selective inhibition of p38 MAPK by SB203850 or SB202190 also blocked PE-induced scattering, whereas treatment of TFG2 cells with the PI3 kinase inhibitors LY294002 or wortmannin did not inhibit PE-induced scattering. Blocking JNK activation with a dominant negative mutant of JNK or blocking AP1 activation with a dominant negative mutant of c-jun (TAM67) significantly inhibited PE-induced cell scattering. These data indicate that PE-induced scattering of TFG2 cells is mediated by complex mechanisms, including activation of p42/44 MAPK, p38 MAPK, and JNK. Cell spreading has been reported to play important roles in wound repair, tumor invasion, and metastasis. Therefore, catecholamines acting via the (alpha1)AR may modulate these physiological and pathological processes.

    Funded by: NCI NIH HHS: R29CA72681; NHLBI NIH HHS: R01 HL046764-08; NIAAA NIH HHS: R01AA12637, R03AA11823

    Experimental cell research 2000;258;1;109-20

  • Mapping of atypical protein kinase C within the nerve growth factor signaling cascade: relationship to differentiation and survival of PC12 cells.

    Wooten MW, Seibenhener ML, Neidigh KB and Vandenplas ML

    Department of Biological Sciences, Program in Cell and Molecular Biosciences, Auburn University, AL 36849, USA. mwwooten@ag.auburn.edu

    The pathway by which atypical protein kinase C (aPKC) contributes to nerve growth factor (NGF) signaling is poorly understood. We previously reported that in PC12 cells NGF-induced activation of mitogen-activated protein kinase (MAPK) occurs independently of classical and nonclassical PKC isoforms, whereas aPKC isoforms were shown to be required for NGF-induced differentiation. NGF-induced activation of PKC-iota was observed to be dependent on phosphatidylinositol 3-kinase (PI3K) and led to coassociation of PKC-iota with Ras and Src. Expression of dominant negative mutants of either Src (DN2) or Ras (Asn-17) impaired activation of PKC-iota by NGF. At the level of Raf-1, neither PKC-iota nor PI3 kinase was required for activation; however, PKC-iota could weakly activate MEK. Inhibitors of PKC-iota activity and PI3K had no effect on NGF-induced MAPK or p38 activation but reduced NGF-stimulated c-Jun N-terminal kinase activity. Src, PI3K, and PKC-iota were likewise required for NGF-induced NF-kappaB activation and cell survival, whereas Ras was not required for either survival or NF-kappaB activation but was required for differentiation. IKK existed as a complex with PKC-iota, Src and IkappaB. Consistent with a role for Src in regulating NF-kappaB activation, an absence of Src activity impaired recruitment of PKC-iota into an IKK complex and markedly impaired NGF-induced translocation of p65/NF-kappaB to the nucleus. These findings reveal that in PC12 cells, aPKCs comprise a molecular switch to regulate differentiation and survival responses coupled downstream to NF-kappaB. On the basis of these findings, Src emerges as a critical upstream regulator of both PKC-iota and the NF-kappaB pathway.

    Funded by: NINDS NIH HHS: R01 NS033661, R02-NS33661

    Molecular and cellular biology 2000;20;13;4494-504

  • ERK1b, a 46-kDa ERK isoform that is differentially regulated by MEK.

    Yung Y, Yao Z, Hanoch T and Seger R

    Department of Biological Regulation, The Weizmann Institute of Science, Rehovot 76100, Israel.

    We identified a 46-kDa ERK, whose kinetics of activation was similar to that of ERK1 and ERK2 in most cell lines and conditions, but showed higher fold activation in response to osmotic shock and epidermal growth factor treatments of Ras-transformed cells. We purified and cloned this novel ERK (ERK1b), which is an alternatively spliced form of ERK1 with a 26-amino acid insertion between residues 340 and 341 of ERK1. When expressed in COS7 cells, ERK1b exhibited kinetics of activation and kinase activity similar to those of ERK1. Unlike the uniform pattern of expression of ERK1 and ERK2, ERK1b was detected only in some of the tissues examined and seems to be abundant in the rat and human heart. Interestingly, in Ras-transformed Rat1 cells, there was a 7-fold higher expression of ERK1b, which was also more responsive than ERK1 and ERK2 to various extracellular treatments. Unlike ERK1 and ERK2, ERK1b failed to interact with MEK1 as judged from its nuclear localization in resting cells overexpressing ERK1b together with MEK1 or by lack of coimmunoprecipitation of the two proteins. Thus, ERK1b is a novel 46-kDa ERK isoform, which seems to be the major ERK isoform that responds to exogenous stimulation in Ras-transformed cells probably due to its differential regulation by MEK.

    The Journal of biological chemistry 2000;275;21;15799-808

  • Differential requirement for p56lck in HIV-tat versus TNF-induced cellular responses: effects on NF-kappa B, activator protein-1, c-Jun N-terminal kinase, and apoptosis.

    Manna SK and Aggarwal BB

    Cytokine Research Section, Department of Bioimmunotherapy, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA.

    HIV-tat protein, like TNF, activates a wide variety of cellular responses, including NF-kappa B, AP-1, c-Jun N-terminal kinase (JNK), and apoptosis. Whether HIV-tat transduces these signals through the same mechanism as TNF is not known. In the present study we investigated the role of the T cell-specific tyrosine kinase p56lck in HIV-tat and TNF-mediated cellular responses by comparing the responses of Jurkat T cells with JCaM1 cells, an isogeneic lck-deficient T cell line. Treatment with HIV-tat protein activated NF-kappa B, degraded I kappa B alpha, and induced NF-kappa B-dependent reporter gene expression in a time-dependent manner in Jurkat cells but not in JCaM1 cells, suggesting the critical role of p56lck kinase. These effects were specific to HIV-tat, as activation of NF-kappa B by PMA, LPS, H2O2, and TNF was minimally affected. p56lck was also found to be required for HIV-tat-induced but not TNF-induced AP-1 activation. Similarly, HIV-tat activated the protein kinases JNK and mitogen-activated protein kinase kinase in Jurkat cells but not in JCaM1 cells. HIV-tat also induced cytotoxicity, activated caspases, and reactive oxygen intermediates in Jurkat cells, but not in JCaM1 cells. HIV-tat activated p56lck activity in Jurkat cells. Moreover, the reconstitution of JCaM1 cells with p56lck tyrosine kinase reversed the HIV-tat-induced NF-kappa B activation and cytotoxicity. Overall, our results demonstrate that p56lck plays a critical role in the activation of NF-kappa B, AP-1, JNK, and apoptosis by HIV-tat protein but has minimal or no role in activation of these responses by TNF.

    Journal of immunology (Baltimore, Md. : 1950) 2000;164;10;5156-66

  • 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 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

  • HIV-1 Tat increases endothelial solute permeability through tyrosine kinase and mitogen-activated protein kinase-dependent pathways.

    Oshima T, Flores SC, Vaitaitis G, Coe LL, Joh T, Park JH, Zhu Y, Alexander B and Alexander JS

    Department of Molecular and Cellular Physiology, Louisiana State University Health Science Center, Shreveport 71130-3932, USA.

    Objective: HIV-1 infection is associated with alterations of several vascular endothelial functions including adhesion molecule expression, growth, and vascular permeability. The bases of these errors are not known, but might involve secretion of the HIV-1 derived transcription factor 'Tat-1'. This study investigated Tat-1 mediated endothelial barrier changes and second message regulation of this phenomenon.

    Methods: We exposed human umbilical vein endothelial cell monolayers to Tat-1 (0-150 ng/ml) for up to 48 h and measured resulting changes in monolayer permeability. We also investigated the role of tyrosine and mitogen activated protein (MAP) kinases, and protein kinase G using the pharmacological blockers genistein, PD98059 and KT5823 respectively.

    Results: Tat-1 significantly reduced monolayer barrier and increased albumin permeability within 24 h. Tat-1 also stimulated tyrosine phosphorylation of multiple endothelial proteins, disorganized junctional phosphotyrosine staining and increased the number of these immunostaining structures. The increased permeability produced by Tat-1 was blocked by genistein and PD98059, but not by KT5823. Genistein and PD98059 pretreatment also prevented the changes in phosphotyrosine immunostaining produced by Tat-1 and blocked phosphorylation of several proteins including MAP kinase.

    Conclusion: These results suggest that HIV may dysregulate endothelial barrier through the effects of Tat-1. These blocker experiments suggest that the effects of Tat are transcription/translation-dependent. These data demonstrate that Tat increases endothelial albumin permeability in vitro through tyrosine kinase and MAP kinase, but not protein kinase G pathways.

    Funded by: NHLBI NIH HHS: HL47615, HL59785; NIDDK NIH HHS: DK43785

    AIDS (London, England) 2000;14;5;475-82

  • Ribosomal subunit kinase-2 is required for growth factor-stimulated transcription of the c-Fos gene.

    Bruning JC, Gillette JA, Zhao Y, Bjorbaeck C, Kotzka J, Knebel B, Avci H, Hanstein B, Lingohr P, Moller DE, Krone W, Kahn CR and Muller-Wieland D

    Klinik II und Poliklinik für Innere Medizin and Center of Molecular Medicine der Universität zu Köln, Joseph Stelzmann Strasse 9, 50931 Cologne, Germany.

    Ribosomal subunit kinases (Rsk) have been implicated in the regulation of transcription by phosphorylating and thereby activating numerous transcription factors, such as c-Fos, cAMP responsive element binding protein (CREB), and nuclear receptors. Here we describe the generation and characterization of immortalized embryonic fibroblast cell lines from mice in which the Rsk-2 gene was disrupted by homologous recombinant gene targeting. Rsk-2-deficient (knockout or KO) cell lines have no detectable Rsk-2 protein, whereas Rsk-1 expression is unaltered as compared with cell lines derived from wild-type control mice. KO cells exhibit a major reduction in platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF)-1-stimulated expression of the immediate-early gene c-Fos. This results primarily from a reduced transcriptional activation of the ternary complex factor Elk-1 and reduced activation of the serum response factor. The reduced Elk-1 activation in KO cells occurs despite normal activation of the mitogen-activated protein kinase pathway and normal PDGF- and IGF-1-stimulated Elk-1 phosphorylation. By contrast, PDGF- and IGF-1-stimulated phosphorylation and transcriptional activation of CREB is unaltered in KO cells. Thus Rsk-2 is required for growth factor-stimulated expression of c-Fos and transcriptional activation of Elk-1 and the serum response factor, but not for activation of CREB or the mitogen-activated protein kinase pathway in response to PDGF and IGF-1 stimulation.

    Funded by: NIDDK NIH HHS: DK33201, R01 DK033201

    Proceedings of the National Academy of Sciences of the United States of America 2000;97;6;2462-7

  • Binding of stromal derived factor-1alpha (SDF-1alpha) to CXCR4 chemokine receptor in normal human megakaryoblasts but not in platelets induces phosphorylation of mitogen-activated protein kinase p42/44 (MAPK), ELK-1 transcription factor and serine/threonine kinase AKT.

    Majka M, Ratajczak J, Kowalska MA and Ratajczak MZ

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

    The aim of this study was to identify pathways which are involved in signal transduction from the CXCR4 receptor stimulated by stromal derived factor-1alpha (SDF-1alpha) in human malignant hematopoietic cells and normal megakaryoblasts. First, we found that activation of CXCR4 in human T cell lines (Jurkat and ATL-2) rapidly induced phosphorylation of mitogen-activated protein kinases (MAPK) (p44 ERK-1 and p42 ERK-2). Next, we became interested in CXCR4-mediated signaling in normal hematopoietic cells, and employed human megakaryoblasts, which highly express CXCR4 as a model. We found that stimulation of these cells with SDF-1alpha led to the phosphorylation of MAPK and serine/threonine kinase AKT as well. Activation of MAPK further led to the phosphorylation of the nuclear transcription factor ELK-1. Phosphorylation of ELK-1 in megakaryoblasts implies that phosphorylated MAPK translocate from cytoplasm into the nucleus where they may phosphorylate some nuclear proteins. Note that neither MAPK nor AKT was phosphorylated in normal human platelets after stimulation by SDF-1. We conclude that both MAPK and AKT are involved in signal transduction pathways from the CXCR4 receptor in malignant and normal human hematopoietic cells. The biological consequences of MAPK, ELK-1 and AKT phosphorylation in megakaryoblasts after stimulation with SDF-1alpha require further studies.

    Funded by: NHLBI NIH HHS: R01 HL61796-01

    European journal of haematology 2000;64;3;164-72

  • Chromosome mapping of the human genes encoding the MAP kinase kinase MEK1 (MAP2K1) to 15q21 and MEK2 (MAP2K2) to 7q32.

    Meloche S, Gopalbhai K, Beatty BG, Scherer SW and Pellerin J

    Research Centre, Centre hospitalier de l'Université de Montréal and Department of Pharmacology, University of Montreal, Quebec, Canada. meloches@ere.umontreal.ca

    Activation of the ERK mitogen-activated protein (MAP) kinase pathway has been implicated in the regulation of cell growth, differentiation and senescence. In this pathway, the MAP kinases ERK1/ERK2 are phosphorylated and activated by the dual-specificity kinases MEK1 and MEK2, which in turn are activated by serine phosphorylation by a number of MAP kinase kinase kinases. We report here the chromosomal localization of the human genes encoding the MAP kinase kinase isoforms MEK1 and MEK2. Using a combination of fluorescence in situ hybridization, somatic cell hybrid analysis, DNA sequencing and yeast artificial chromosome (YAC) clone analysis, we have mapped the MEK1 gene (MAP2K1) to chromosome 15q21. We also present evidence for the presence of a MEK1 pseudogene on chromosome 8p21. The MEK2 gene (MAP2K2) was mapped to chromosome 7q32 by fluorescence in situ hybridization and YAC clone analysis.

    Cytogenetics and cell genetics 2000;88;3-4;249-52

  • The CXC chemokine stromal cell-derived factor activates a Gi-coupled phosphoinositide 3-kinase in T lymphocytes.

    Sotsios Y, Whittaker GC, Westwick J and Ward SG

    Department of Pharmacy and Pharmacology, Bath University, Claverton Down, Bath, Avon, United Kingdom.

    The cellular effects of stromal cell-derived factor-1 (SDF-1) are mediated primarily by binding to the CXC chemokine receptor-4. We report in this study that SDF-1 and its peptide analogues induce a concentration- and time-dependent accumulation of phosphatidylinositol-(3,4,5)-trisphosphate (PtdIns(3,4,5)P3) in Jurkat cells. This SDF-1-stimulated generation of D-3 phosphoinositide lipids was inhibited by pretreatment of the cells with an SDF-1 peptide antagonist or an anti-CXCR4 Ab. In addition, the phosphoinositide 3 (PI 3)-kinase inhibitors wortmannin and LY294002, as well as the Gi protein inhibitor pertussis toxin, also inhibited the SDF-1-stimulated accumulation of PtdIns(3,4,5)P3. The effects of SDF-1 on D-3 phosphoinositide lipid accumulation correlated well with activation of the known PI 3-kinase effector protein kinase B, which was also inhibited by wortmannin and pertussis toxin. Concentrations of PI 3-kinase inhibitors, sufficient to inhibit PtdIns(3,4,5)P3 accumulation, also inhibited chemotaxis of Jurkat and peripheral blood-derived T lymphocytes in response to SDF-1. In contrast, SDF-1-stimulated actin polymerization was only partially inhibited by PI 3-kinase inhibitors, suggesting that while chemotaxis is fully dependent on PI 3-kinase activation, actin polymerization requires additional biochemical inputs. Finally, SDF-1-stimulated extracellular signal-related kinase (ERK)-1/2 mitogen-activated protein kinase activation was inhibited by PI 3-kinase inhibitors. In addition, the mitogen-activated protein/ERK kinase inhibitor PD098059 partially attenuated chemotaxis in response to SDF-1. Hence, it appears that ERK1/2 activation is dependent on PI 3-kinase activation, and both biochemical events are involved in the regulation of SDF-1-stimulated chemotaxis.

    Funded by: Wellcome Trust

    Journal of immunology (Baltimore, Md. : 1950) 1999;163;11;5954-63

  • 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

  • Inhibition of the mitogen-activated protein kinase kinase superfamily by a Yersini 1eba a effector.

    Orth K, Palmer LE, Bao ZQ, Stewart S, Rudolph AE, Bliska JB and Dixon JE

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

    The bacterial pathogen Yersinia uses a type III secretion system to inject several virulence factors into target cells. One of the Yersinia virulence factors, YopJ, was shown to bind directly to the superfamily of MAPK (mitogen-activated protein kinase) kinases (MKKs) blocking both phosphorylation and subsequent activation of the MKKs. These results explain the diverse activities of YopJ in inhibiting the extracellular signal-regulated kinase, c-Jun amino-terminal kinase, p38, and nuclear factor kappa B signaling pathways, preventing cytokine synthesis and promoting apoptosis. YopJ-related proteins that are found in a number of bacterial pathogens of animals and plants may function to block MKKs so that host signaling responses can be modulated upon infection.

    Funded by: NIAID NIH HHS: AI35175; PHS HHS: 18024

    Science (New York, N.Y.) 1999;285;5435;1920-3

  • Extracellular HIV-1 Tat protein differentially activates the JNK and ERK/MAPK pathways in CD4 T cells.

    Mischiati C, Pironi F, Milani D, Giacca M, Mirandola P, Capitani S and Zauli G

    Department of Morphology and Embryology, University of Ferrara, Italy.

    Objective: To investigate the intracellular signals elicited by extracellular HIV-1 Tat protein in lymphoid CD4 T cells.

    Methods: CD4 Jurkat T cells were treated with a series of glutathione S-transferase (GST)-Tat fusion proteins: full-length two-exon GST-Tat (GST-Tat2E); one-exon Tat, in which the second exon of Tat was deleted (GST-Tat1E); two-exon Tat, in which the seven arginine residues have been changed to alanine residues (GST-TatArg(mut)), GST-TatdeltaN, which shows a deletion of the N-terminal 21 amino acids. The cells were either treated with soluble GST-Tat proteins or seeded on plates coated with GST-Tat proteins immobilized on plastic. At various time points, Jurkat cells were lysed and examined for c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) activity.

    Results: Soluble and immobilized GST-Tat2E, but not GST-Tat1E, GST-TatArg(mut) and GST-TatdeltaN, activated JNK in a dose-dependent manner, induced a rapid phosphorylation of c-Jun on Ser63 and promoted the de novo synthesis of c-Jun protein. Moreover, both GST-Tat2E and GST-Tat1E also stimulated ERK/MAPK. However, the activation of JNK was maximal at concentrations of 100 nM of GST-Tat2E and was blocked by the S6-kinase inhibitor rapamycin, whereas the activation of ERK/MAPK was already maximal at 1 nM of GST-Tat2E and was enhanced by rapamycin.

    Conclusions: Tat-mediated activation of JNK requires the second exon of Tat, which is dispensable for the activation of ERK/MAPK. The ability to stimulate JNK and ERK/MAPK does not require Tat internalization.

    AIDS (London, England) 1999;13;13;1637-45

  • Myosin light chain kinase functions downstream of Ras/ERK to promote migration of urokinase-type plasminogen activator-stimulated cells in an integrin-selective manner.

    Nguyen DH, Catling AD, Webb DJ, Sankovic M, Walker LA, Somlyo AV, Weber MJ and Gonias SL

    Department of Biochemistry and Molecular Genetics, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.

    Urokinase-type plasminogen activator (uPA) activates the mitogen activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK) 1 and 2, in diverse cell types. In this study, we demonstrate that uPA stimulates migration of MCF-7 breast cancer cells, HT 1080 fibrosarcoma cells, and uPAR-overexpressing MCF-7 cells by a mechanism that depends on uPA receptor (uPAR)-ligation and ERK activation. Ras and MAP kinase kinase (MEK) were necessary and sufficient for uPA-induced ERK activation and stimulation of cellular migration, as demonstrated in experiments with dominant-negative and constitutively active mutants of these signaling proteins. Myosin light chain kinase (MLCK) was also required for uPA-stimulated cellular migration, as determined in experiments with three separate MLCK inhibitors. When MCF-7 cells were treated with uPA, MLCK was phosphorylated by a MEK-dependent pathway and apparently activated, since serine-phosphorylation of myosin II regulatory light chain (RLC) was also increased. Despite the transient nature of ERK phosphorylation, MLCK remained phosphorylated for at least 6 h. The uPA-induced increase in MCF-7 cell migration was observed selectively on vitronectin-coated surfaces and was mediated by a beta1-integrin (probably alphaVbeta1) and alphaVbeta5. When MCF-7 cells were transfected to express alphaVbeta3 and treated with uPA, ERK was still phosphorylated; however, the cells did not demonstrate increased migration. Neutralizing the function of alphaVbeta3, with blocking antibody, restored the ability of uPA to promote cellular migration. Thus, we have demonstrated that uPA promotes cellular migration, in an integrin-selective manner, by initiating a uPAR-dependent signaling cascade in which Ras, MEK, ERK, and MLCK serve as essential downstream effectors.

    Funded by: NCI NIH HHS: CA-53462, R01 CA053462

    The Journal of cell biology 1999;146;1;149-64

  • Blockade of the MAP kinase pathway suppresses growth of colon tumors in vivo.

    Sebolt-Leopold JS, Dudley DT, Herrera R, Van Becelaere K, Wiland A, Gowan RC, Tecle H, Barrett SD, Bridges A, Przybranowski S, Leopold WR and Saltiel AR

    Department of Cell Biology, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert, Ann Arbor, Michigan 48105, USA.

    The mitogen-activated protein kinase pathway is thought to be essential in cellular growth and differentiation. Here we report the discovery of a highly potent and selective inhibitor of the upstream kinase MEK that is orally active. Tumor growth was inhibited as much as 80% in mice with colon carcinomas of both mouse and human origin after treatment with this inhibitor. Efficacy was achieved with a wide range of doses with no signs of toxicity, and correlated with a reduction in the levels of activated mitogen-activated protein kinase in excised tumors. These data indicate that MEK inhibitors represent a promising, noncytotoxic approach to the clinical management of colon cancer.

    Nature medicine 1999;5;7;810-6

  • Human mitogen-activated protein kinase kinase kinase mediates the stress-induced activation of mitogen-activated protein kinase cascades.

    Chan-Hui PY and Weaver R

    Amgen, Department of Inflammation Research, 3200 Walnut Street, Boulder, CO 80301, USA. povying@stratabio.com

    The mitogen-activated protein kinase (MAPK) cascades represent one of the important signalling mechanisms in response to environmental stimuli. We report the identification of a human MAPK kinase kinase, MAPKKK4, via sequence similarity with other MAPKKKs. When truncated MAPKKK4 (DeltaMAPKKK4) was overexpressed in HEK293 cells, it was constitutively active and induced the activation of endogenous p38alpha, c-Jun N-terminal kinase (JNK)1/2 and extracellular signal-regulated kinase (ERK)2 in vivo. Kinase-inactive DeltaMAPKKK4 partly inhibited the activation of p38alpha, JNK1/2 and ERK2 induced by stress, tumour necrosis factor alpha or epidermal growth factor, suggesting that MAPKKK4 might be physiologically involved in all three MAPK cascades. Co-expressed MAP kinase kinase (MKK)-1, MKK-4, MKK-3 and MKK-6 were activated in vivo by DeltaMAPKKK4. All of the above MKKs purified from Escherichia coli were phosphorylated and activated by DeltaMAPKKK4 immunoprecipitates in vitro. When expressed by lower plasmid doses, DeltaMAPKKK4 preferentially activated MKK-3 and p38alpha in vivo. Overexpression of DeltaMAPKKK4 did not activate the NF-kappaB pathway. Immunoprecipitation of endogenous MAPKKK4 by specific antibodies showed that MAPKKK4 was activated after the treatment of K562 cells with various stress conditions. As a broadly distributed kinase, MAPKKK4 might serve as a stress responder. MAPKKK4 is 91% identical with the recently described murine MEKK-4beta and might be its human homologue. It is also identical with the recently cloned human MAP three kinase 1 except for the lack of an internal sequence homologous to the murine MEKK-4alpha isoform. Differences in the reported functional activities of the three kinases are discussed.

    The Biochemical journal 1998;336 ( Pt 3);599-609

  • 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

  • MP1: a MEK binding partner that enhances enzymatic activation of the MAP kinase cascade.

    Schaeffer HJ, Catling AD, Eblen ST, Collier LS, Krauss A and Weber MJ

    Department of Microbiology and Cancer Center, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA.

    Signal transduction is controlled both by regulation of enzyme activation and by organization of enzymatic complexes with nonenzymatic adapters, scaffolds, and anchor proteins. The extracellular signal-regulated kinase (ERK) cascade is one of several evolutionarily conserved mitogen-activated protein (MAP) kinase cascades important in the regulation of growth, apoptosis, and differentiation. A two-hybrid screen was conducted to identify nonenzymatic components of this signaling cascade that might be important in regulating its activity. A protein called MP1 (MEK Partner 1) was identified that bound specifically to MEK1 and ERK1 and facilitated their activation. When overexpressed in cultured cells, MP1 enhanced activation of ERK1 and activation of a reporter driven by the transcription factor Elk-1. Expression of MP1 in cells increased binding of ERK1 to MEK1. MP1 apparently functions as an adapter to enhance the efficiency of the MAP kinase cascade.

    Funded by: NCI NIH HHS: CA39076; NIGMS NIH HHS: GM47332

    Science (New York, N.Y.) 1998;281;5383;1668-71

  • Human immunodeficiency virus tat modulates the Flk-1/KDR receptor, mitogen-activated protein kinases, and components of focal adhesion in Kaposi's sarcoma cells.

    Ganju RK, Munshi N, Nair BC, Liu ZY, Gill P and Groopman JE

    Divisions of Experimental Medicine and Hematology/Oncology, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, Boston, Massachusetts 02115, USA.

    Kaposi's sarcoma (KS) spindle cell growth and spread have been reported to be modulated by various cytokines as well as the human immunodeficiency virus (HIV) gene product Tat. Recently, HIV-1 Tat has been shown to act like a cytokine and bind to the Flk-1/KDR receptor for the vascular endothelial growth factor A (VEGF-A), which is expressed by KS cells. We have characterized signal transduction pathways stimulated by HIV-1 Tat upon its binding to surface receptors on KS cells. We observed that stimulation in KS 38 spindle cells resulted in tyrosine phosphorylation and activation of the Flk-1/KDR receptor. We also report that HIV-1 Tat treatment enhanced the phosphorylation and association of proteins found in focal adhesions, such as the related adhesion focal tyrosine kinase RAFTK, paxillin, and p130(cas). Further characterization revealed the activation of mitogen-activated protein kinase, c-Jun amino-terminal kinase (JNK), and Src kinase. HIV-1 Tat contains a basic domain which can interact with growth factor tyrosine kinase receptors and a classical RGD sequence which may bind to and activate the surface integrin receptors for fibronectin and vitronectin. We observed that stimulation of KS cells with basic as well as RGD sequence-containing Tat peptides resulted in enhanced phosphorylation of RAFTK and activation of MAP kinase. These studies reveal that Tat stimulation activates a number of signal transduction pathways that are associated with cell growth and migration.

    Funded by: NHLBI NIH HHS: HL 43510, HL 53745, HL 55187, R01 HL053745

    Journal of virology 1998;72;7;6131-7

  • Modulation of HIV-1 infectivity by MAPK, a virion-associated kinase.

    Jacqué JM, Mann A, Enslen H, Sharova N, Brichacek B, Davis RJ and Stevenson M

    University of Massachusetts Medical School, Program in Molecular Medicine, Worcester, MA 01605, USA.

    Infection of a cell by human immunodeficiency virus type 1 (HIV-1) results in the formation of a reverse transcription complex in which viral nucleic acids are synthesized. Efficient disengagement of the reverse transcription complex from the cell membrane and subsequent nuclear translocation require phosphorylation of reverse transcription complex components by a virion-associated kinase. In this study, we identify the virion-associated kinase as mitogen-activated protein kinase (ERK/MAPK). Upon density gradient fractionation, MAPK, but not its activating kinase MEK, co-sedimented with viral particles. Expression of a constitutively active, but not kinase-inactive, MEK1 in virus producer cells was able to activate virion-associated MAPK in trans. Stimulation of virion-associated MAPK activity in trans by the mitogen phorbol myristate acetate (PMA) increased viral infectivity. Conversely, suppression of virion-associated MAPK by specific inhibitors of the MAPK cascade markedly impaired viral infectivity. These studies demonstrate regulation of an early step in HIV-1 infection by the host cell MAPK signal transduction pathway.

    The EMBO journal 1998;17;9;2607-18

  • Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor.

    Duesbery NS, Webb CP, Leppla SH, Gordon VM, Klimpel KR, Copeland TD, Ahn NG, Oskarsson MK, Fukasawa K, Paull KD and Vande Woude GF

    Advanced BioScience Laboratories-Basic Research Program, National Cancer Institute-Frederick Cancer Research and Development Center, Post Office Box B, Frederick, MD 21702.

    Anthrax lethal toxin, produced by the bacterium Bacillus anthracis, is the major cause of death in animals infected with anthrax. One component of this toxin, lethal factor (LF), is suspected to be a metalloprotease, but no physiological substrates have been identified. Here it is shown that LF is a protease that cleaves the amino terminus of mitogen-activated protein kinase kinases 1 and 2 (MAPKK1 and MAPKK2) and that this cleavage inactivates MAPKK1 and inhibits the MAPK signal transduction pathway. The identification of a cleavage site for LF may facilitate the development of LF inhibitors.

    Science (New York, N.Y.) 1998;280;5364;734-7

  • 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

  • Extracellular HIV-1 Tat protein induces the rapid Ser133 phosphorylation and activation of CREB transcription factor in both Jurkat lymphoblastoid T cells and primary peripheral blood mononuclear cells.

    Gibellini D, Bassini A, Pierpaoli S, Bertolaso L, Milani D, Capitani S, La Placa M and Zauli G

    Institute of Microbiology, University of Bologna, Italy.

    Extracellular HIV-1 Tat protein (0.1-100 ng/ml) induced a rapid (peak at 30 min) increase in the Ser133 phosphorylation levels of the transcription factor CREB in serum-starved Jurkat cells, as revealed by Western blot and indirect immunofluorescence analyses. Nuclear cAMP-responsive element (CRE) binding activity in electrophoretic mobility shift assays was constitutive in unstimulated Jurkat cells, showing only a small increase upon Tat treatment. However, transient transfection experiments performed with various chloramphenicol acetyl-transferase (CAT) constructs showed that Tat produced a fourfold induction of CAT activity only in the presence of a CRE-dependent CAT construct. Moreover, the use of plasmids encoding for GAL4-CREB fusion proteins demonstrated that Tat induction of pG4-CAT reporter gene required the CREB moiety of the GAL4-CREB fusion protein and that Ser133 CREB was essential for Tat activity. Extracellular Tat also stimulated Ser133 CREB phosphorylation in freshly isolated PBMC; this effect was completely blocked by either staurosporin, a broad-spectrum inhibitor of various protein kinases, or PD 98059, a specific inhibitor of mitogen-activated protein kinases (MAPK). Furthermore, extracellular Tat induced a rapid (peak at 5-15 min) stimulation of the MAPK catalytic activity in primary PBMC. Altogether, these findings suggest that HIV-1 Tat protein activates CREB in lymphoid cells through a signal cascade involving the MAPK pathway.

    Journal of immunology (Baltimore, Md. : 1950) 1998;160;8;3891-8

  • Cross-cascade activation of ERKs and ternary complex factors by Rho family proteins.

    Frost JA, Steen H, Shapiro P, Lewis T, Ahn N, Shaw PE and Cobb MH

    U.T. Southwestern Medical Center, Department of Pharmacology, Dallas 75235-9041, USA.

    Mitogens promote cell growth through integrated signal transduction networks that alter cellular metabolism, gene expression and cytoskeletal organization. Many such signals are propagated through activation of MAP kinase cascades partly regulated by upstream small GTP-binding proteins. Interactions among cascades are suspected but not defined. Here we show that Rho family small G proteins such as Rac1 and Cdc42hs, which activate the JNK/SAPK pathway, cooperate with Raf-1 to activate the ERK pathway. This causes activation of ternary complex factors (TCFs), which regulate c-fos gene expression through the serum response element. Examination of ERK pathway kinases shows that neither MEK1 nor Ras will synergize with Rho-type proteins, and that only MEK1 is fully activated, indicating that MEKs are a focal point for cross-cascade regulation. Rho family proteins utilize PAKs for this effect, as expression of an active PAK1 mutant can substitute for Rho family small G proteins, and expression of an interfering PAK1 mutant blocks Rho-type protein stimulation of ERKs. PAK1 phosphorylates MEK1 on Ser298, a site important for binding of Raf-1 to MEK1 in vivo. Expression of interfering PAK1 also reduces stimulation of TCF function by serum growth factors, while expression of active PAK1 enhances EGF-stimulated MEK1 activity. This demonstrates interaction among MAP kinase pathway elements not previously recognized and suggests an explanation for the cooperative effect of Raf-1 and Rho family proteins on cellular transformation.

    Funded by: NIDDK NIH HHS: DK34128; NIGMS NIH HHS: GM16026, GM53032

    The EMBO journal 1997;16;21;6426-38

  • Tat protein from HIV-1 activates MAP kinase in granular neurons and glial cells from rat cerebellum.

    Menegon A, Leoni C, Benfenati F and Valtorta F

    DIBIT San Raffaele Scientific Institute, Department of Medical Pharmacology, University of Milan, Italy.

    We have investigated the effect of extracellularly applied Tat protein of the human immunodeficiency virus type 1 (HIV-1) on tyrosine phosphorylation processes, which represent a major signal transduction pathway of cells of the central nervous system. Primary cultures of rat cerebellar astrocytes or granule cells were incubated with synthetic Tat (10 ng/ml) for various periods of time and analyzed for their phosphotyrosine content by Western blotting. In both types of cultures Tat was able to induce the phosphorylation of mitogen-activated protein kinase (MAP kinase) on tyrosine residues, although with different kinetics and isoform specificity. In addition, in neuronal cells, but not in astrocytes, Tat increased the phosphotyrosine content of Shc, a protein involved in signal transduction downstream of receptor tyrosine kinase activation. This study shows that Tat applied extracellularly is able to induce the generation of intracellular signals in neuronal as well as glial cells.

    Biochemical and biophysical research communications 1997;238;3;800-5

  • Tat protein induces self-perpetuating permissivity for productive HIV-1 infection.

    Li CJ, Ueda Y, Shi B, Borodyansky L, Huang L, Li YZ and Pardee AB

    Division of Cell Growth and Regulation, Harvard Medical School, 44 Binney Street, Boston, MA 02115, USA. cjli-mbcrr.harvard.edu

    We report that human immunodeficiency virus type 1 (HIV-1) has evolved a self-perpetuating mechanism to actively generate cells permissive for productive and cytopathic infection. Only activated T cells can be productively infected, which leads to their rapid depletion (2 x 10(9)/day in an infected individual). Establishment of productive HIV-1 infection therefore requires continual activations from the large pool of quiescent T cells. Tat protein, which is secreted by infected cells, activated uninfected quiescent T cells in vitro and in vivo. These Tat-activated uninfected cells became highly permissive for productive HIV-1 infection. Activation of primary T cells by Tat protein involved integrin receptors and was associated with activation of mitogen-activated protein kinases, including ERK1 and JNK kinase. Accordingly, these primary T cells progressed from G0 to the late G1 phase of the cell cycle.

    Funded by: NIAID NIH HHS: AI-35576

    Proceedings of the National Academy of Sciences of the United States of America 1997;94;15;8116-20

  • 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

  • Actin-binding protein-280 binds the stress-activated protein kinase (SAPK) activator SEK-1 and is required for tumor necrosis factor-alpha activation of SAPK in melanoma cells.

    Marti A, Luo Z, Cunningham C, Ohta Y, Hartwig J, Stossel TP, Kyriakis JM and Avruch J

    Diabetes Unit and Medical Services, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 021291, USA.

    SEK-1, a dual specificity protein kinase that serves as one of the immediate upstream activators of the stress-activated protein kinases (SAPKs), associates specifically with the actin-binding protein, ABP-280, in vitro and in situ. SEK-1 binds to the carboxyl-terminal rod segment of ABP-280, upstream of the ABP carboxyl-terminal dimerization domain. Activation of SEK-1 in situ increases the SEK-1 activity bound to ABP-280 without changing the amount of SEK-1 polypeptide bound. The influence of ABP-280 on SAPK regulation was evaluated in human melanoma cells that lack ABP-280 expression, and in stable transformants of these cells expressing wild type ABP, or an actin-binding but dimerization-deficient mutant ABP (ABPDeltaCT109). ABP-280-deficient cells show an activation of SAPK in response to most stimuli that is comparable to that seen in ABP-280-replete cells; ABP-280-deficient cells, however, fail to show the brisk tumor necrosis factor-alpha (TNF-alpha) activation of SAPK seen in ABP-replete cells and have an 80% reduction in SAPK activation by lysophosphatidic acid. Expression of the dimerization-deficient mutant ABP-280 fails to correct the defective SAPK response to lysophosphatidic acid, but essentially normalizes the TNF-alpha activation of SAPK. Thus, a lack of ABP-280 in melanoma cells causes a defect in the regulation of SAPK that is selective for TNF-alpha and is attributable to the lack of ABP-280 polypeptide itself rather than to the disordered actin cytoskeleton that results therefrom. ABP-280 participates in TNF-alpha signal transduction to SAPKs, in part through the binding of SEK-1.

    The Journal of biological chemistry 1997;272;5;2620-8

  • Chromosomal localization of four MAPK signaling cascade genes: MEK1, MEK3, MEK4 and MEKK5.

    Rampoldi L, Zimbello R, Bortoluzzi S, Tiso N, Valle G, Lanfranchi G and Danieli GA

    Biology Department, University of Padova, Italy.

    The mitogen-activated protein kinase (MAPK) signaling cascade is one of the most important mechanisms for the cytoplasmic transduction of extracellular signals. We report the chromosomal localization of the human MEK1, MEK3, MEK4 and MEKK5 genes, involved in the MAPK cascade. Using radiation hybrid mapping, MEK1 was assigned to chromosome 15q22.1 --> q22.33, MEK3 to chromosome 17q11.2, MEK4 to chromosome 17p12, and MEKK5 to chromosome 6q22.33.

    Cytogenetics and cell genetics 1997;78;3-4;301-3

  • Regulation and interaction of pp90(rsk) isoforms with mitogen-activated protein kinases.

    Zhao Y, Bjorbaek C and Moller DE

    Department of Medicine, Beth Israel Hospital and Harvard Medical School, Boston, Massachusetts 02215, USA.

    Each of the three known mammalian 90-kDa S6 kinase (pp90(rsk)) isoforms (RSK1, RSK2, and RSK3) was expressed in transfected cells and further characterized. The kinase activity (immunocomplex toward S6 peptide) of each isoform was activated by in vivo growth factor (epidermal growth factor (EGF)) stimulation; RSK1 was more responsive (10-15-fold) versus RSK2 and RSK3 (2-4-fold). Pretreatment with PD98059 (MEK1 inhibitor) partially (80%) blocked EGF-mediated ERK1 activation and had similar effects on EGF stimulation of each ribosomal S6 kinase (RSK). Cotransfection with dominant-negative MEK1 inhibited activation of each RSK; furthermore, the kinase activity of RSK1, RSK2, and RSK3 was markedly increased by cotransfection with constitutively active MEK1. A specific association between mitogen-activated protein kinases (MAPKs) (ERK1 and ERK2) and RSK isoforms was tested by MAPK immunoblotting after immunoprecipitation of RSKs. ERK1 and ERK2 were present in RSK3 (and to a lesser extent, RSK2) immunoprecipitates, but were absent in RSK1 immunoprecipitates. Both dephosphorylated (from quiescent cells) and phosphorylated (from stimulated cells) MAPKs were associated with RSK2 and RSK3. Deletion mutants of RSK3 were characterized: the C terminus (33 residues) was shown to be required for association with MAPKs. The kinase activity of RSK1 or RSK2 was enhanced by in vitro incubation with ERK1. In contrast, RSK3 activity was not affected by exposure to ERK1. Furthermore, MAPKs in RSK3 immunoprecipitates were phosphorylated by purified MEK1; however, RSK3 kinase activity was unaffected. We conclude that 1) the MEK1-MAPK signaling pathway is both necessary and sufficient for in vivo growth factor-mediated activation of all three RSK isoforms; 2) RSK isoforms differ with respect to growth factor responsiveness and their physical association with MAPK; and 3) formation of the MAPK.RSK complex is mediated by the RSK C terminus.

    Funded by: PHS HHS: R01 45874-02

    The Journal of biological chemistry 1996;271;47;29773-9

  • 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

  • Characterization of ERK1 activation site mutants and the effect on recognition by MEK1 and MEK2.

    Butch ER and Guan KL

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

    To discern MEK1 and MEK2 specificity for their substrate, extracellular signal-regulated kinase (ERK), site-directed mutagenesis was performed on the amino acid residues flanking the regulatory phosphorylation sites of ERK1. These ERK1 mutants were analyzed for the ability to act as a substrate for MEK1 and MEK2. Based on both phosphorylation and activation analyses, the mutants could be divided into four classes: 1) dramatically decreased phosphorylation and activation, 2) enhanced basal kinase activity, 3) preferentially enhanced phosphorylation of tyrosine and decreased phosphorylation of threonine, and 4) increased threonine phosphorylation with an increase in activation. In general, the residues proximal to the regulatory phosphorylation sites of ERK1 had greater influence on both phosphorylation and activation. This is consistent with the highly specific recognition of the ERK1 regulatory sites by MEK. Mutation of Arg-208 or Thr-207 to an alanine residue significantly altered the relative phosphorylation on Thr-202 and Tyr-204. The Arg-208 to alanine mutant increased the phosphorylation of Tyr-204 approximately 4-fold yet almost completely eliminated the phosphorylation on Thr-202. In contrast, mutation of Gly-199 to alanine resulted in an increased phosphorylation of Thr-202 relative to Tyr-204. This suggests that both Gly-199 and Arg-208 play important roles in determining the relative phosphorylation of Thr-202 and Tyr-204. Our results demonstrate that residues in the phosphorylation lip of ERK play an important role in the recognition and phosphorylation by MEK.

    Funded by: NIA NIH HHS: 5T32AG00114; NIGMS NIH HHS: GM51586

    The Journal of biological chemistry 1996;271;8;4230-5

  • Activation of MEK-1 and SEK-1 by Tpl-2 proto-oncoprotein, a novel MAP kinase kinase kinase.

    Salmeron A, Ahmad TB, Carlile GW, Pappin D, Narsimhan RP and Ley SC

    Division of Cellular Immunology, National Institute of Medical Research, London, UK.

    The Tpl-2 protein serine/threonine kinase was originally identified, in a C-terminally deleted form, as the product of an oncogene associated with the progression of Moloney murine leukemia virus-induced T cell lymphomas in rats. The kinase domain of Tpl-2 is homologous to the Saccharomyces cerevisiae gene product, STE11, which encodes a MAP kinase kinase kinase. This suggested that Tpl-2 might have a similar activity. Consistent with this hypothesis, immunoprecipitated Tpl-2 and Tpl-2deltaC (a C-terminally truncated mutant) phosphorylated and activated recombinant fusion proteins of the mammalian MAP kinase kinases, MEK-1 and SEK-1, in vitro. Furthermore, transfection of Tpl-2 into COS-1 cells or Jurkat T cells. markedly activated the MAP kinases, ERK-1 and SAP kinase (JNK), which are substrates for MEK-1 and SEK-1, respectively. Tpl-2, therefore, is a MAP kinase kinase kinase which can activate two MAP kinase pathways. After Raf and Mos, Tpl-2 is the third serine/threonine oncoprotein kinase that has been shown to function as a direct activator of MEK-1.

    The EMBO journal 1996;15;4;817-26

  • 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 fact 1f40 or 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

  • Activation of two isoforms of mitogen-activated protein kinase kinase in response to epidermal growth factor and nerve growth factor.

    Moriguchi T, Gotoh Y and Nishida E

    Department of Genetics and Molecular Biology, Kyoto University, Japan.

    Mitogen-activated protein kinase kinase (MAPKK) is a dual-specificity protein kinase which phosphorylates and activates mitogen-activated protein kinase (MAPK). cDNAs encoding two isoforms of MAPKK, MAPKK1 and MAPKK2 (also known as MEK1 and MEK2), have been cloned in mammalian cells. To analyze the characteristics of MAPKK1 and MAPKK2 individually, we have produced specific anti-MAPKK serum against each isoform. MAPKK1 and MAPKK2 have apparent molecular masses of 45 kDa and 47 kDa, respectively, on SDS/polyacrylamide gel electrophoresis. In mouse tissues, MAPKK1 was highly enriched in brain, while MAPKK2 was present relatively evenly. In rat fibroblastic 3Y1 cells, epidermal growth factor (EGF) treatment induced activation of both MAPKK1 and MAPKK2. Immunoprecipitation experiments have shown that the time courses of activation and deactivation of both isoforms of MAPKK were superimposed. In PC12 cells, both MAPKK1 and MAPKK2 were activated in response to nerve growth factor (NGF) as well as EGF, and the time courses of activation and deactivation of both isoforms were indistinguishable from each other in the NGF-stimulated cells and also in the EGF-stimulated cells. Furthermore, localization of both MAPKK1 and MAPKK2 in the cytoplasm was unchanged in response to EGF and NGF. Thus, the same or quite similar mechanisms may operate in the regulation of the activation and deactivation of two isoforms of MAPKK, and both kinases might have redundant functions when expressed in the same cell.

    European journal of biochemistry 1995;234;1;32-8

  • MEKK1 phosphorylates MEK1 and MEK2 but does not cause activation of mitogen-activated protein kinase.

    Xu S, Robbins D, Frost J, Dang A, Lange-Carter C and Cobb MH

    Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235-9041, USA.

    A constitutively active fragment of rat MEK kinase 1 (MEKK1) consisting of only its catalytic domain (MEKK-C) expressed in bacteria quantitatively activates recombinant mitogen-activated protein (MAP) kinase/extracellular signal-regulated protein kinase (ERK) kinases 1 and 2 (MEK1 and MEK2) in vitro. Activation of MEK1 by MEKK-C is accompanied by phosphorylation of S218 and S222, which are also phosphorylated by the protein kinases c-Mos and Raf-1. MEKK1 has been implicated in regulation of a parallel but distinct cascade that leads to phosphorylation of N-terminal sites on c-Jun; thus, its role in the MAP kinase pathway has been questioned. However, in addition to its capacity to phosphorylate MEK1 in vitro, MEKK-C interacts with MEK1 in the two-hybrid system, and expression of mouse MEKK1 or MEKK-C in mammalian cells causes constitutive activation of both MEK1 and MEK2. Neither cotransfected nor endogenous ERK2 is highly activated by MEKK1 compared to its stimulation by epidermal growth factor in spite of significant activation of endogenous MEK. Thus, other as yet undefined mechanisms may be involved in determining information flow through the MAP kinase and related pathways.

    Funded by: NIDDK NIH HHS: DK01918, DK34128; NIGMS NIH HHS: GM16926

    Proceedings of the National Academy of Sciences of the United States of America 1995;92;15;6808-12

  • The MAPK signaling cascade.

    Seger R and Krebs EG

    Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel.

    The transmission of extracellular signals into their intracellular targets is mediated by a network of interacting proteins that regulate a large number of cellular processes. Cumulative efforts from many laboratories over the past decade have allowed the elucidation of one such signaling mechanism, which involves activations of several membranal signaling molecules followed by a sequential stimulation of several cytoplasmic protein kinases collectively known as mitogen-activated protein kinase (MAPK) signaling cascade. Up to six tiers in this cascade contribute to the amplification and specificity of the transmitted signals that eventually activate several regulatory molecules in the cytoplasm and in the nucleus to initiate cellular processes such as proliferation, differentiation, and development. Moreover, because many oncogenes have been shown to encode proteins that transmit mitogenic signals upstream of this cascade, the MAPK pathway provides a simple unifying explanation for the mechanism of action of most, if not all, nonnuclear oncogenes. The pattern of MAPK cascade is not restricted to growth factor signaling and it is now known that signaling pathways initiated by phorbol esters, ionophors, heat shock, and ligands for seven transmembrane receptors use distinct MAPK cascades with little or no cross-reactivity between them. In this review we emphasize primarily the first MAPK cascade to be discovered that uses the MEK and ERK isoforms and describe their involvement in different cellular processes.

    Funded by: NIDDK NIH HHS: DK-42528; NIGMS NIH HHS: GM-42508

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 1995;9;9;726-35

  • 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 inter 1f40 action 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

  • 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

  • MAP kinase binds to the NH2-terminal activation domain of c-Myc.

    Gupta S and Davis RJ

    Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester 01605.

    The transcription factor c-Myc is a substrate for phosphorylation by MAP kinases. Here we demonstrate that MAP kinase binds to c-Myc. The NH2-terminal region (residues 1-100) is necessary and sufficient for this interaction. Binding to c-Myc is not dependent on the state of MAP kinase activation. However, the c-Myc/MAP kinase complex is disrupted by ATP. Together, these observations indicate that substrate binding interactions contribute to the specificity of phosphorylation by MAP kinases.

    Funded by: NCI NIH HHS: CA58396

    FEBS letters 1994;353;3;281-5

  • 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

  • Activation of MEK family kinases requires phosphorylation of two conserved Ser/Thr residues.

    Zheng CF and Guan KL

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

    MEK is a family of dual specific protein kinases which activate the extracellular signal-regulated kinases by phosphorylation of threonine and tyrosine residues. MEK itself is activated via serine phosphorylation by upstream activator kinases, including c-raf, mos and MEK kinase. Here, we report the activation phosphorylation sites of human MEK1 and yeast STE7 kinase as determined by a combination of biochemical and genetic approaches. In human MEK1, substitution of either serine residue 218 or 222 with alanine completely abolished its activation by epidermal growth factor-stimulated Swiss 3T3 cell lysates or immunoprecipitated c-raf, suggesting that both serine residues are required for MEK1 activation. Phosphopeptide analysis demonstrated that serine residues 218 and 222 of human MEK1 are the primary sites for phosphorylation by c-raf. These two serine residues are highly conserved in all members of the MEK family, including the yeast STE7 gene product, a MEK homolog in the yeast mating pheromone response pathway. Mutation of the corresponding residues in STE7 completely abolished the biological functions of this gene. These data demonstrate that MEK is activated by phosphorylation of two adjacent serine/threonine residues and this activation mechanism is conserved in the MEK family kinases.

    Funded by: NCRR NIH HHS: M01-RR00042

    The EMBO journal 1994;13;5;1123-31

  • Properties of MEKs, the kinases that phosphorylate and activate the extracellular signal-regulated kinases.

    Zheng CF and Guan KL

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

    Activation of extracellular signal-regulated kinase (ERK) or mitogen-activated protein kinase by MEK (mitogen-activated protein kinase or extracellular signal-regulated kinase kinase) is an essential event in the mitogenic growth factor signal transduction. We now demonstrate that three recombinant MEKs (MEK1, MEK2, MEK3) show remarkably different activity toward recombinant ERK1 and ERK2. MEK2 is the most active ERK activator. The recombinant MEK1 has an activity approximately seven times lower than that of MEK2. MEK3, which is identical to MEK1 except for missing an internal 26-amino acid residue and probably represents an alternative splicing product of MEK1, shows neither autophosphorylation nor ERK-activating activity. Recombinant MEK1 and MEK2 can be activated by epidermal growth factor-stimulated SWISS3T3 cell lysate. MEK1 and MEK2 can also be activated by autophosphorylation. Autophosphorylation of MEKs correlates with their ability to phosphorylate and activate ERKs. Phosphorylation of MEK is also stimulated by ERK. Phosphoamino acid analysis showed that ERK1 preferentially phosphorylated threonine residue of MEKs. MEKs complex with ERKs in vitro. Interestingly, MEK3 also forms a complex with ERK1, although it is totally inactive as an ERK activator.

    Funded by: NCRR NIH HHS: M01RR00042

    The Journal of biological chemistry 1993;268;32;23933-9

  • 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

  • Cloning and characterization of two distinct human extracellular signal-regulated kinase activator kinases, MEK1 and MEK2.

    Zheng CF and Guan KL

    Department of Biological Chemistry, University of Michigan, Ann Arbor 48109.

    Mitogen-induced signal transduction is mediated by a cascade of protein phosphorylation and dephosphorylation. One of the immediate responses of mitogen stimulation is the activation of a family of protein kinases known as mitogen-activated protein kinase or extracellular signal-regulated kinase (ERK). MEK (MAP kinase or ERK kinase) is the immediate upstream activator kinase of ERK. Two cDNAs, MEK1 and MEK2, were cloned and sequenced. MEK1 and MEK2 encode 393 and 400 amino acid residues, respectively. The human MEK1 shares 99% amino acid sequence identity with the murine MEK1 and 80% with human MEK2. Both MEK1 and MEK2 were expressed in Escherichia coli and shown to be able to activate recombinant human ERK1 in vitro. The purified MEK2 protein stimulated threonine and tyrosine phosphorylation on ERK1 and concomitantly activated ERK1 kinase activity more than 100-fold. The recombinant MEK2 showed lower activity as an ERK activator as compared with MEK purified from tissue. However, the recombinant MEK2 can be activated by serum-stimulated cell extract in vitro. MEKs, in a manner similar to ERKs, are likely to consist of a family of related proteins playing critical roles in signal transduction.

    The Journal of biological chemistry 1993;268;15;11435-9

  • Human T-cell mitogen-activated protein kinase kinases are related to yeast signal transduction kinases.

    Seger R, Seger D, Lozeman FJ, Ahn NG, Graves LM, Campbell JS, Ericsson L, Harrylock M, Jensen AM and Krebs EG

    Department of Pharmacology, University of Washington, Seattle 98195.

    Mitogen-activated protein (MAP) kinase kinases, intermediates in a growth factor-stimulated protein kinase cascade, are dual specificity protein kinases that specifically phosphorylate and activate MAP kinases in response to extracellular signals. Here, we report the cloning of two forms of cDNA that encode this protein from human T-cells. MKK1a encodes a protein with predicted molecular size of 43,439 Da. Overexpression of this clone in COS cells led to elevated levels of protein and phorbol ester-stimulated MAP kinase kinase activity, confirming that MKK1a encodes the predicted protein. MKK1b, which appears to be an alternatively spliced form of the MKK1a gene, encodes a protein with predicted molecular size of 40,745 Da. Northern analysis revealed that the MKK1 cDNA hybridizes with a single 2.6-kilobase mRNA species in all human tissues examined. Sequence comparison shows homology to a group of yeast kinases that participate in signal transduction and to subdomain XI of other dual specificity kinase.

    Funded by: NIDDK NIH HHS: DK 42528; NIGMS NIH HHS: GM 42508

    The Journal of biological chemistry 1992;267;36;25628-31

Gene lists (8)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
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
L00000035 G2C Homo sapiens Pocklington H4 Human orthologues of cluster 4 (mouse) from Pocklington et al (2006) 8
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© 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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