G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
mitogen-activated protein kinase kinase 2
G00000181 (Mus musculus)

Databases (9)

Curated Gene
OTTHUMG00000071401 (Vega human gene)
ENSG00000126934 (Ensembl human gene)
5605 (Entrez Gene)
79 (G2Cdb plasticity & disease)
MAP2K2 (GeneCards)
601263 (OMIM)
Marker Symbol
HGNC:6842 (HGNC)
Protein Expression
3835 (human protein atlas)
Protein Sequence
P36507 (UniProt)

Synonyms (1)

  • MEK2

Literature (109)

Pubmed - other

  • 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

  • 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

  • Stromal cell-derived factor-1/CXCR4 enhanced motility of human osteosarcoma cells involves MEK1/2, ERK and NF-kappaB-dependent pathways.

    Huang CY, Lee CY, Chen MY, Yang WH, Chen YH, Chang CH, Hsu HC, Fong YC and Tang CH

    Department of Orthopaedic Surgery, China Medical University Beigang Hospital, Yun-Lin County, Taiwan.

    Osteosarcoma is characterized by a high malignant and metastatic potential. The chemokine stromal-derived factor-1alpha (SDF-1alpha) and its receptor, CXCR4, play a crucial role in adhesion and migration of human cancer cells. Integrins are the major adhesive molecules in mammalian cells, and has been associated with metastasis of cancer cells. Here, we found that human osteosarcoma cell lines had significant expression of SDF-1 and CXCR4 (SDF-1 receptor). Treatment of osteosarcoma cells with SDF-1alpha increased the migration and cell surface expression of alphavbeta3 integrin. CXCR4-neutralizing antibody, CXCR4 specific inhibitor (AMD3100) or small interfering RNA against CXCR4 inhibited the SDF-1alpha-induced increase the migration and integrin expression of osteosarcoma cells. Pretreated of osteosarcoma cells with MAPK kinase (MEK) inhibitor PD98059 inhibited the SDF-1alpha-mediated migration and integrin expression. Stimulation of cells with SDF-1alpha increased the phosphorylation of MEK and extracellular signal-regulating kinase (ERK). In addition, NF-kappaB inhibitor (PDTC) or IkappaB protease inhibitor (TPCK) also inhibited SDF-1alpha-mediated cell migration and integrin up-regulation. Stimulation of cells with SDF-1alpha induced IkappaB kinase (IKKalpha/beta) phosphorylation, IkappaB phosphorylation, p65 Ser(536) phosphorylation, and kappaB-luciferase activity. Furthermore, the SDF-1alpha-mediated increasing kappaB-luciferase activity was inhibited by AMD3100, PD98059, PDTC and TPCK or MEK1, ERK2, IKKalpha and IKKbeta mutants. Taken together, these results suggest that the SDF-1alpha acts through CXCR4 to activate MEK and ERK, which in turn activates IKKalpha/beta and NF-kappaB, resulting in the activations of alphavbeta3 integrins and contributing the migration of human osteosarcoma cells.

    Journal of cellular physiology 2009;221;1;204-12

  • 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 anaplastic 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 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

  • 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

  • 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

  • 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

  • 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

  • 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

  • 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, Cohen RB, 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 part 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

  • 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 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

  • 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

  • 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

  • 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

  • Systematic analysis of the protein interaction network for the human transcription machinery reveals the identity of the 7SK capping enzyme.

    Jeronimo C, Forget D, Bouchard A, Li Q, Chua G, Poitras C, Thérien C, Bergeron D, Bourassa S, Greenblatt J, Chabot B, Poirier GG, Hughes TR, Blanchette M, Price DH and Coulombe B

    Laboratory of Gene Transcription and Proteomics Discovery Platform, Institut de Recherches Cliniques de Montréal, Montréal, QC, Canada.

    We have performed a survey of soluble human protein complexes containing components of the transcription and RNA processing machineries using protein affinity purification coupled to mass spectrometry. Thirty-two tagged polypeptides yielded a network of 805 high-confidence interactions. Remarkably, the network is significantly enriched in proteins that regulate the formation of protein complexes, including a number of previously uncharacterized proteins for which we have inferred functions. The RNA polymerase II (RNAP II)-associated proteins (RPAPs) are physically and functionally associated with RNAP II, forming an interface between the enzyme and chaperone/scaffolding proteins. BCDIN3 is the 7SK snRNA methylphosphate capping enzyme (MePCE) present in an snRNP complex containing both RNA processing and transcription factors, including the elongation factor P-TEFb. Our results define a high-density protein interaction network for the mammalian transcription machinery and uncover multiple regulatory factors that target the transcription machinery.

    Funded by: Canadian Institutes of Health Research: 14309-3, 82851-1

    Molecular cell 2007;27;2;262-74

  • Selective role for RGS12 as a Ras/Raf/MEK scaffold in nerve growth factor-mediated differentiation.

    Willard MD, Willard FS, Li X, Cappell SD, Snider WD and Siderovski DP

    Department of Pharmacology, University of North Carolina, Chapel Hill, NC 27599-7365, USA.

    Regulator of G-protein signaling (RGS) proteins accelerate GTP hydrolysis by heterotrimeric G-protein alpha subunits and thus inhibit signaling by many G protein-coupled receptors. Several RGS proteins have a multidomain architecture that adds further complexity to their roles in cell signaling in addition to their GTPase-accelerating activity. RGS12 contains a tandem repeat of Ras-binding domains but, to date, the role of this protein in Ras-mediated signal transduction has not been reported. Here, we show that RGS12 associates with the nerve growth factor (NGF) receptor tyrosine kinase TrkA, activated H-Ras, B-Raf, and MEK2 and facilitates their coordinated signaling to prolonged ERK activation. RGS12 is required for NGF-mediated neurite outgrowth of PC12 cells, but not outgrowth stimulated by basic fibroblast growth factor. siRNA-mediated knockdown of RGS12 expression also inhibits NGF-induced axonal growth in dissociated cultures of primary dorsal root ganglia neurons. These data suggest that RGS12 may play a critical, and receptor-selective, role in coordinating Ras-dependent signals that are required for promoting and/or maintaining neuronal differentiation.

    Funded by: NIGMS NIH HHS: GM062338, R01 GM062338, T32 GM007040; NINDS NIH HHS: R01 NS031768

    The EMBO journal 2007;26;8;2029-40

  • 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, Wilson 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

  • 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

  • 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

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

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

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

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

    Cell 2006;127;3;635-48

  • A probability-based approach for high-throughput protein phosphorylation analysis and site localization.

    Beausoleil SA, Villén J, Gerber SA, Rush J and Gygi SP

    Department of Cell Biology, Harvard Medical School, 240 Longwood Ave., Boston, Massachusetts 02115, USA.

    Data analysis and interpretation remain major logistical challenges when attempting to identify large numbers of protein phosphorylation sites by nanoscale reverse-phase liquid chromatography/tandem mass spectrometry (LC-MS/MS) (Supplementary Figure 1 online). In this report we address challenges that are often only addressable by laborious manual validation, including data set error, data set sensitivity and phosphorylation site localization. We provide a large-scale phosphorylation data set with a measured error rate as determined by the target-decoy approach, we demonstrate an approach to maximize data set sensitivity by efficiently distracting incorrect peptide spectral matches (PSMs), and we present a probability-based score, the Ascore, that measures the probability of correct phosphorylation site localization based on the presence and intensity of site-determining ions in MS/MS spectra. We applied our methods in a fully automated fashion to nocodazole-arrested HeLa cell lysate where we identified 1,761 nonredundant phosphorylation sites from 491 proteins with a peptide false-positive rate of 1.3%.

    Funded by: NHGRI NIH HHS: HG03456; NIGMS NIH HHS: GM67945

    Nature biotechnology 2006;24;10;1285-92

  • 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

  • Activation of mitogen-activated protein kinases by lysophosphatidylcholine-induced mitochondrial reactive oxygen species generation in endothelial cells.

    Watanabe N, Zmijewski JW, Takabe W, Umezu-Goto M, Le Goffe C, Sekine A, Landar A, Watanabe A, Aoki J, Arai H, Kodama T, Murphy MP, Kalyanaraman R, Darley-Usmar VM and Noguchi N

    Research Center for Advanced Science and Technology, University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904.

    Lysophosphatidylcholine (lysoPC) evokes diverse biological responses in vascular cells including Ca(2+) mobilization, production of reactive oxygen species, and activation of the mitogen-activated protein kinases, but the mechanisms linking these events remain unclear. Here, we provide evidence that the response of mitochondria to the lysoPC-dependent increase in cytosolic Ca(2+) leads to activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase through a redox signaling mechanism in human umbilical vein endothelial cells. ERK activation was attenuated by inhibitors of the electron transport chain proton pumps (rotenone and antimycin A) and an uncoupler (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), suggesting that mitochondrial inner membrane potential plays a key role in the signaling pathway. ERK activation was also selectively attenuated by chain-breaking antioxidants and by vitamin E targeted to mitochondria, suggesting that transduction of the mitochondrial hydrogen peroxide signal is mediated by a lipid peroxidation product. Inhibition of ERK activation with MEK inhibitors (PD98059 or U0126) diminished induction of the antioxidant enzyme heme oxygenase-1. Taken together, these data suggest a role for mitochondrially generated reactive oxygen species and Ca(2+) in the redox cell signaling path-ways, leading to ERK activation and adaptation of the pathological stress mediated by oxidized lipids such as lysoPC.

    Funded by: Medical Research Council: MC_U105663142; NHLBI NIH HHS: HL58031, R01 HL058031; NIEHS NIH HHS: ES10167, R01 ES010167

    The American journal of pathology 2006;168;5;1737-48

  • 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

  • Reduced stability of mitogen-activated protein kinase kinase-2 mRNA and phosphorylation of poly(A)-binding protein (PABP) in cells overexpressing PABP.

    Ma S, Musa T and Bag J

    Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.

    The poly(A)-binding protein (PABP) is an important regulator of mRNA translation and stability. The cellular level of PABP is controlled by regulating its mRNA translation by a feedback mechanism. The important aspect of this mechanism is that PABP binds to an adenosine-rich cis-element at the 5'-untranslated region of its own mRNA and inhibits its translation. To assess the importance of controlling the PABP level, we studied the effect of PABP overexpression on the transcription profile using the microarray technique. In PABP-overexpressing cells, 19 mRNAs showed a reduction in cellular levels due to reduced mRNA stability, and one showed an increase due to increased mRNA stability. Among these mRNAs, the MKK-2 mRNA encodes the protein kinase activator of ERK1/2 kinase, which is involved in the phosphorylation of eukaryotic initiation factor (eIF) 4E. As a result, mRNA translation may be regulated by the cellular level of MKK-2. In this study, we show that the abundance of the MKK-2 polypeptide is reduced in PABP-overexpressing cells. In these cells, the levels of phosphorylated PABP, eIF4E, and ERK2 are also reduced. Treatment of HeLa cells with the MKK-2 inhibitor U0126 reduced PABP phosphorylation, suggesting that the phosphorylation of PABP is mediated by the MKK-2/ERK signaling pathway. Thus, a novel signaling pathway involving MKK-2 and ERK1/2 may down-regulate the activity of PABP and eIF4E by controlling their phosphorylation and compensates for the effect of excess cellular PABP.

    The Journal of biological chemistry 2006;281;6;3145-56

  • 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

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

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

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

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

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

    Nature 2005;437;7062;1173-8

  • Human immunodeficiency virus type 1 Vpr-dependent cell cycle arrest through a mitogen-activated protein kinase signal transduction pathway.

    Yoshizuka N, Yoshizuka-Chadani Y, Krishnan V and Zeichner SL

    HIV and AIDS Malignancy Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20817, USA.

    The human immunodeficiency virus type 1 (HIV-1) Vpr protein has important functions in advancing HIV pathogenesis via several effects on the host cell. Vpr mediates nuclear import of the preintegration complex, induces host cell apoptosis, and inhibits cell cycle progression at G(2), which increases HIV gene expression. Some of Vpr's activities have been well described, but some functions, such as cell cycle arrest, are not yet completely characterized, although components of the ATR DNA damage repair pathway and the Cdc25C and Cdc2 cell cycle control mechanisms clearly play important roles. We investigated the mechanisms underlying Vpr-mediated cell cycle arrest by examining global cellular gene expression profiles in cell lines that inducibly express wild-type and mutant Vpr proteins. We found that Vpr expression is associated with the down-regulation of genes in the MEK2-ERK pathway and with decreased phosphorylation of the MEK2 effector protein ERK. Exogenous provision of excess MEK2 reverses the cell cycle arrest associated with Vpr, confirming the involvement of the MEK2-ERK pathway in Vpr-mediated cell cycle arrest. Vpr therefore appears to arrest the cell cycle at G(2)/M through two different mechanisms, the ATR mechanism and a newly described MEK2 mechanism. This redundancy suggests that Vpr-mediated cell cycle arrest is important for HIV replication and pathogenesis. Our findings additionally reinforce the idea that HIV can optimize the host cell environment for viral replication.

    Journal of virology 2005;79;17;11366-81

  • 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

  • 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. 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

  • 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

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

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

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

    Funded by: PHS HHS: N01-C0-12400

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

  • Large-scale characterization of HeLa cell nuclear phosphoproteins.

    Beausoleil SA, Jedrychowski M, Schwartz D, Elias JE, Villén J, Li J, Cohn MA, Cantley LC and Gygi SP

    Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

    Determining the site of a regulatory phosphorylation event is often essential for elucidating specific kinase-substrate relationships, providing a handle for understanding essential signaling pathways and ultimately allowing insights into numerous disease pathologies. Despite intense research efforts to elucidate mechanisms of protein phosphorylation regulation, efficient, large-scale identification and characterization of phosphorylation sites remains an unsolved problem. In this report we describe an application of existing technology for the isolation and identification of phosphorylation sites. By using a strategy based on strong cation exchange chromatography, phosphopeptides were enriched from the nuclear fraction of HeLa cell lysate. From 967 proteins, 2,002 phosphorylation sites were determined by tandem MS. This unprecedented large collection of sites permitted a detailed accounting of known and unknown kinase motifs and substrates.

    Funded by: NHGRI NIH HHS: HG00041, K22 HG000041, T32 HG000041; NIGMS NIH HHS: GM67945, GMS6203, R01 GM056203, R01 GM067945

    Proceedings of the National Academy of Sciences of the United States of America 2004;101;33;12130-5

  • Dystroglycan, a scaffold for the ERK-MAP kinase cascade.

    Spence HJ, Dhillon AS, James M and Winder SJ

    The Beatson Institute for Cancer Research, CRUK Beatson Laboratories, Switchback Road, Glasgow G61 1BD, UK.

    Dystroglycan is an important cell adhesion receptor linking the actin cytoskeleton, via utrophin and dystrophin, to laminin in the extracellular matrix. To identify adhesion-related signalling molecules associated with dystroglycan, we conducted a yeast two-hybrid screen and identified mitogen-activated protein (MAP) kinase kinase 2 (MEK2) as a beta-dystroglycan interactor. Pull-down experiments and localization studies substantiated a physiological link between beta-dystroglycan and MEK and localized MEK with dystroglycan in membrane ruffles. Moreover, we also identified active extracellular signal-regulated kinase (ERK), the downstream kinase from MEK, as another interacting partner for beta-dystroglycan and localized both active ERK and dystroglycan to focal adhesions in fibroblast cells. These studies suggest a role for dystroglycan as a multifunctional adaptor or scaffold capable of interacting with components of the ERK-MAP kinase cascade including MEK and ERK. These findings have important implications for our understanding of the role of dystroglycan in normal cellular processes and in disease states such as muscular dystrophy.

    EMBO reports 2004;5;5;484-9

  • 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

    Blood 2004;103;7;2636-44

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

    Powell CS, Wright MM and Jackson RM

    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

    American journal of physiology. Lung cellular and molecular physiology 2004;286;4;L826-33

  • 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

  • Complete sequencing and characterization of 21,243 full-length human cDNAs.

    Ota T, Suzuki Y, Nishikawa T, Otsuki T, Sugiyama T, Irie R, Wakamatsu A, Hayashi K, Sato H, Nagai K, Kimura K, Makita H, Sekine M, Obayashi M, Nishi T, Shibahara T, Tanaka T, Ishii S, Yamamoto J, Saito K, Kawai Y, Isono Y, Nakamura Y, Nagahari K, Murakami K, Yasuda T, Iwayanagi T, Wagatsuma M, Shiratori A, Sudo H, Hosoiri T, Kaku Y, Kodaira H, Kondo H, Sugawara M, Takahashi M, Kanda K, Yokoi T, Furuya T, Kikkawa E, Omura Y, Abe K, Kamihara K, Katsuta N, Sato K, Tanikawa M, Yamazaki M, Ninomiya K, Ishibashi T, Yamashita H, Murakawa K, Fujimori K, Tanai H, Kimata M, Watanabe M, Hiraoka S, Chiba Y, Ishida S, Ono Y, Takiguchi S, Watanabe S, Yosida M, Hotuta T, Kusano J, Kanehori K, Takahashi-Fujii A, Hara H, Tanase TO, Nomura Y, Togiya S, Komai F, Hara R, Takeuchi K, Arita M, Imose N, Musashino K, Yuuki H, Oshima A, Sasaki N, Aotsuka S, Yoshikawa Y, Matsunawa H, Ichihara T, Shiohata N, Sano S, Moriya S, Momiyama H, Satoh N, Takami S, Terashima Y, Suzuki O, Nakagawa S, Senoh A, Mizoguchi H, Goto Y, Shimizu F, Wakebe H, Hishigaki H, Watanabe T, Sugiyama A, Takemoto M, Kawakami B, Yamazaki M, Watanabe K, Kumagai A, Itakura S, Fukuzumi Y, Fujimori Y, Komiyama M, Tashiro H, Tanigami A, Fujiwara T, Ono T, Yamada K, Fujii Y, Ozaki K, Hirao M, Ohmori Y, Kawabata A, Hikiji T, Kobatake N, Inagaki H, Ikema Y, Okamoto S, Okitani R, Kawakami T, Noguchi S, Itoh T, Shigeta K, Senba T, Matsumura K, Nakajima Y, Mizuno T, Morinaga M, Sasaki M, Togashi T, Oyama M, Hata H, Watanabe M, Komatsu T, Mizushima-Sugano J, Satoh T, Shirai Y, Takahashi Y, Nakagawa K, Okumura K, Nagase T, Nomura N, Kikuchi H, Masuho Y, Yamashita R, Nakai K, Yada T, Nakamura Y, Ohara O, Isogai T and Sugano S

    Helix Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan.

    As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

    Nature genetics 2004;36;1;40-5

  • MAPK-activated protein kinase-2 participates in p38 MAPK-dependent and ERK-dependent functions in human neutrophils.

    Coxon PY, Rane MJ, Uriarte S, Powell DW, Singh S, Butt W, Chen Q and McLeish KR

    Department of Medicine, University of Louisville Health Sciences Center, Louisville, KY 40202, USA.

    Many neutrophil responses, including chemotaxis, exocytosis, respiratory burst activity and chemokine synthesis, are mediated by p38 MAPK. MAPK-activated protein kinase-2 (MK2) is activated by p38 MAPK in human neutrophils. The present study tested the hypothesis that MK2 mediates multiple p38 MAPK-dependent responses in human neutrophils by comparing the effect of the p38 MAPK inhibitor, SB203580, with an MK2 inhibitory peptide. Both SB203580 and MK2 inhibitory peptide attenuated respiratory burst activity, exocytosis, and chemotaxis. Lipopolysaccharide (LPS)-induced IL-8 production was inhibited by SB203580, but not by the MK2 inhibitory peptide. Inhibition of chemotaxis and respiratory burst activity by SB203580 was less than that of MK2 inhibitory peptide. Inhibition of extracellular signal-regulated kinase (ERK) activity by PD98059 attenuated superoxide release and chemotaxis, and simultaneous treatment with SB203580 and PD98059 demonstrated additive inhibition. ERK phosphorylated MK2 in vitro and activated MK2 in f-methionyl-leucyl-phenylalanine (FMLP)-stimulated neutrophils. These data suggest that MK2 mediates both ERK- and p38 MAPK-dependent neutrophil responses.

    Cellular signalling 2003;15;11;993-1001

  • Stabilization of urokinase and urokinase receptor mRNAs by HuR is linked to its cytoplasmic accumulation induced by activated mitogen-activated protein kinase-activated protein kinase 2.

    Tran H, Maurer F and Nagamine Y

    Friedrich Miescher Institute for Biomedical Research, Novartis Research Foundation, CH-4058 Basel, Switzerland.

    The mRNAs of urokinase plasminogen activator (uPA) and its receptor, uPAR, contain instability-determining AU-rich elements (AREs) in their 3' untranslated regions. The cellular proteins binding to these RNA sequences (ARE(uPA/uPAR)) are not known. We show here that the mRNA-stabilizing factor HuR functionally interacts with these sequences. HuR stabilized an ARE(uPA)-containing RNA substrate in vitro and stabilized in HeLa Tet-off cells both endogenous uPA and uPAR mRNAs and a beta-globin reporter mRNA containing the ARE(uPA). RNAi-mediated depletion of HuR in BT-549 and MDA-MB-231 cells significantly reduced the steady-state levels of endogenous uPA and uPAR mRNAs. Furthermore, we show that a constitutively active form of mitogen-activated protein kinase-activated protein kinase 2 (MK2), MK2-EE, has an ARE-mRNA-stabilizing effect that correlates with its ability to enhance the cytoplasmic accumulation of endogenous HuR, but not in cells cotransfected with a dominant negative version of MK2, MK2-K76R. These effects were mimicked by hydrogen peroxide treatment (oxidative stress), which resulted in the phosphorylation of endogenous MK2. In addition, hydrogen peroxide treatment enhanced the cytoplasmic binding of HuR to the ARE(uPA), which was abrogated in cells transfected with MK2-K76R. These results indicate a role for HuR and MK2 in regulating the expression of uPA and uPAR genes at the posttranscriptional level.

    Molecular and cellular biology 2003;23;20;7177-88

  • 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

  • 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

  • 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

  • Interferon-gamma induces C/EBP beta expression and activity through MEK/ERK and p38 in T84 colon epithelial cells.

    Salmenperä P, Hämäläinen S, Hukkanen M and Kankuri E

    Department of Pharmacology, Institute of Biomedicine, FIN-00014 University of Helsinki, Finland.

    We investigated the role of IFN-gamma and MAPKs on the expression and activity of the transcription factor CCAAT/enhancer-binding protein-beta (C/EBP beta) in the T84 colon epithelial cell line. IFN-gamma induced the expression and activity of C/EBP beta and subsequently increased the secretion of IL-6 from these cells. Treatment with the p38 inhibitor SB-203580, the MEK1 and MEK2 inhibitor U-0126, or the translational inhibitor cycloheximide inhibited the induction of C/EBP beta and IL-6 by IFN-gamma, whereas the MEK1 inhibitor PD-98059 or the tyrosine kinase inhibitor genistein had no effect. These results suggest a role for MEK2 and p38 in IFN-gamma-mediated signal transduction and induction of C/EBP beta expression and activity associated with interleukin-6 (IL-6) secretion in colon epithelial cells.

    American journal of physiology. Cell physiology 2003;284;5;C1133-9

  • The p38 and MK2 kinase cascade phosphorylates tuberin, the tuberous sclerosis 2 gene product, and enhances its interaction with 14-3-3.

    Li Y, Inoki K, Vacratsis P and Guan KL

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

    Tuberous sclerosis complex (TSC) is a genetic disease caused by mutations in either TSC1 or TSC2 tumor suppressor genes. TSC1 and TSC2 (also known as hamartin and tuberin, respectively) form a functional complex and negatively regulate cell growth by inhibiting protein synthesis. 14-3-3 binds to TSC2 and may inhibit TSC2 function. We have reported previously that phosphorylation of serine 1210 (Ser(1210)) in TSC2 is essential for 14-3-3 binding. Here we show that serum and anisomycin enhance the interaction between TSC2 and 14-3-3 by stimulating phosphorylation of Ser(1210). Activation of p38 MAP kinase (p38) is essential for the stimulating effect of serum and anisomycin although p38 is not directly responsible for the phosphorylation of Ser(1210) in TSC2. Both in vitro and in vivo experiments demonstrate that the p38-activated kinase MK2 (also known as MAPKAPK2) is directly responsible for the phosphorylation of Ser(1210). Our data show that anisomycin stimulates phosphorylation of Ser(1210) of TSC2 via the p38-MK2 kinase cascade. Phosphorylation of TSC2 by MK2 creates a 14-3-3 binding site and thus regulates the cellular function of the TSC2 tumor suppressor protein.

    The Journal of biological chemistry 2003;278;16;13663-71

  • 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

  • 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 of 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

  • 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

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

    Robinson FL, Whitehurst 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

  • Identification of interaction between MEK2 and A-Raf-1.

    Yin XL, Chen S, Yan J, Hu Y and Gu JX

    Box 103, Gene Research Center, Medical Center of Fudan University (Former Shanghai Medical University), Shanghai 200032, PR China.

    Mitogen-activated protein (MAP) kinases are activated by dual-specificity kinases, termed MEKs. Using MEK2 as bait in yeast two-hybrid screening, besides c-Raf and KSR, A-Raf was identified as a novel partner that interacts with MEK2. This interaction was confirmed by in vitro binding assay. Further investigation indicates that regions critical for this interaction were located between residues 255 and 606 that represent the kinase domain of A-Raf.

    Biochimica et biophysica acta 2002;1589;1;71-6

  • 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

  • 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

  • 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 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

  • 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

  • Proteomic analysis of NMDA receptor-adhesion protein signaling complexes.

    Husi H, Ward MA, Choudhary JS, Blackstock WP and Grant SG

    Centre for Genome Research, Centre for Neuroscience, University of Edinburgh, West Mains Road, Edinburgh EH9 3JQ, UK.

    N-methyl-d-aspartate receptors (NMDAR) mediate long-lasting changes in synapse strength via downstream signaling pathways. We report proteomic characterization with mass spectrometry and immunoblotting of NMDAR multiprotein complexes (NRC) isolated from mouse brain. The NRC comprised 77 proteins organized into receptor, adaptor, signaling, cytoskeletal and novel proteins, of which 30 are implicated from binding studies and another 19 participate in NMDAR signaling. NMDAR and metabotropic glutamate receptor subtypes were linked to cadherins and L1 cell-adhesion molecules in complexes lacking AMPA receptors. These neurotransmitter-adhesion receptor complexes were bound to kinases, phosphatases, GTPase-activating proteins and Ras with effectors including MAPK pathway components. Several proteins were encoded by activity-dependent genes. Genetic or pharmacological interference with 15 NRC proteins impairs learning and with 22 proteins alters synaptic plasticity in rodents. Mutations in three human genes (NF1, Rsk-2, L1) are associated with learning impairments, indicating the NRC also participates in human cognition.

    Nature neuroscience 2000;3;7;661-9

  • 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

  • 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

  • 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

  • Inhibition of the mitogen-activated protein kinase kinase superfamily by a Yersinia 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

  • Extracellular regulated kinases (ERK) 1 and ERK2 are authentic substrates for the dual-specificity protein-tyrosine phosphatase VHR. A novel role in down-regulating the ERK pathway.

    Todd JL, Tanner KG and Denu JM

    Oregon Health Sciences University, Department of Biochemistry and Molecular Biology L224, Portland, Oregon 97201-3098, USA.

    The mammalian dual-specificity protein-tyrosine phosphatase VHR (for VH1-related) has been identified as a novel regulator of extracellular regulated kinases (ERKs). To identify potential cellular substrates of VHR, covalently immobilized mutant VHR protein was employed as an affinity trap. A tyrosine-phosphorylated protein(s) of approximately 42 kDa was specifically adsorbed by the affinity column and identified as ERK1 and ERK2. Subsequent kinetic analyses and transfection studies demonstrated that VHR specifically dephosphorylates and inactivates ERK1 and ERK2 in vitro and in vivo. Only the native structure of phosphorylated ERK was recognized by VHR and was inactivated with a second-order rate constant of 40,000 M-1 s-1. VHR was found to dephosphorylate endogenous ERK, but not p38 and JNK. Immunodepletion of endogenous VHR eliminated the dephosphorylation of cellular ERK. Transfection studies in COS-1 cells demonstrated that in vivo phosphorylation of epidermal growth factor-stimulated ERK depended on VHR protein levels. Overexpression above endogenous levels of VHR led to accelerated ERK inactivation, but did not alter the normal activation of ERK. Unique among reported mitogen activated protein kinase phosphatases, VHR is constitutively expressed, localized to the nucleus, and tyrosine-specific. This study is the first to report the identification of authentic substrates of dual-specificity phosphatases utilizing affinity absorbents and is the first to identify a nuclear, constitutively expressed, and tyrosine-specific ERK phosphatase. The data strongly suggest that VHR is responsible for the rapid inactivation of ERK following stimulation and for its repression in quiescent cells.

    The Journal of biological chemistry 1999;274;19;13271-80

  • Mitogen-activated protein kinase kinase 2 activation is essential for progression through the G2/M checkpoint arrest in cells exposed to ionizing radiation.

    Abbott DW and Holt JT

    Vanderbilt University Departments of Cell Biology and Pathology and the Vanderbilt University Cancer Center, Nashville, Tennessee 37232, USA.

    An increasing body of evidence suggests that mitogen-induced activation of the RAF/ERK signaling pathway is functionally separate from the stress-induced activation of the SEK/JNK/p38 signaling pathway. In general, stress stimuli strongly activate the p38s and the JNKs while only weakly activating ERK1 and ERK2. However, a number of independent groups have now shown that the RAF/ERK signaling pathway is strongly activated by ionizing radiation. In this work, we examine this paradox. We show that both mitogen-activated protein (MAP) kinase kinase 1 (MEK1) and MAP kinase kinase 2 (MEK2) are activated by ionizing radiation. Blockage of this activation through the use of dominant negative MEK2 increases sensitivity of the cell to ionizing radiation and decreases the ability of a cell to recover from the G2/M cell cycle checkpoint arrest. Blocking MEK2 activation does not affect double-strand DNA break repair, however. Although MEK1 is activated to a lesser extent by ionizing radiation, expression of a dominant negative MEK1 does not affect radiation sensitivity of the cell, the G2/M checkpoint of the cell, or double-strand break repair. Because ionizing radiation leads to a different cell cycle arrest (G2/M arrest) than that typically seen with other stress stimuli, and because we have shown that MEK2 can affect G2/M checkpoint kinetics, these results provide an explanation for the observation that the MEKs can be strongly activated by ionizing radiation and only weakly activated by other stressful stimuli.

    Funded by: NCI NIH HHS: R01CA51735; NIGMS NIH HHS: 5T32GM07347

    The Journal of biological chemistry 1999;274;5;2732-42

  • JNKK1 organizes a MAP kinase module through specific and sequential interactions with upstream and downstream components mediated by its amino-terminal extension.

    Xia Y, Wu Z, Su B, Murray B and Karin M

    Laboratory of Gene Regulation and Signal Transduction, Department of Pharmacology, University of California San Diego, La Jolla, California 92093-0636 USA.

    MAP kinase (MAPK) cascades are composed of a MAPK, MAPK kinase (MAPKK), and a MAPKK kinase (MAPKKK). Despite the existence of numerous components and ample opportunities for crosstalk, most MAPKs are specifically and distinctly activated. We investigated the basis for specific activation of the JNK subgroup of MAPKs. The specificity of JNK activation is determined by the MAPKK JNKK1, which interacts with the MAPKKK MEKK1 and JNK through its amino-terminal extension. Inactive JNKK1 mutants can disrupt JNK activation by MEKK1 or tumor necrosis factor (TNF) in intact cells only if they contain an intact amino-terminal extension. Mutations in this region interfere with the ability of JNKK1 to respond to TNF but do not affect its activation by physical stressors. As JNK and MEKK1 compete for binding to JNKK1 and activation of JNKK1 prevents its binding to MEKK1, activation of this module is likely to occur through sequential MEKK1:JNKK1 and JNKK1:JNK interactions. These results underscore a role for the amino-terminal extension of MAPKKs in determination of response specificity.

    Funded by: NHLBI NIH HHS: HL 35018, P01 HL035018

    Genes & development 1998;12;21;3369-81

  • 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

  • Activation of the 41/43 kDa mitogen-activated protein kinase signaling pathway is required for hepatocyte growth factor-induced cell scattering.

    Tanimura S, Chatani Y, Hoshino R, Sato M, Watanabe S, Kataoka T, Nakamura T and Kohno M

    Laboratory of Cell Regulation, School of Pharmaceutical Sciences, Nagasaki University, Japan.

    Hepatocyte growth factor (HGF) markedly induced the spreading, dissociation and scattering of Madin-Darby canine kidney epithelial cells (MDCK) and human stomach adenocarcinoma cells (TMK1). Scattering of MDCK and TMK1 cells was induced by 12-O-tetradecanoyl-phorbol-13-acetate (PMA) and epidermal growth factor (EGF), respectively. In all these agent-stimulated cells, rapid activation of Raf-1, MAP kinase/ERK kinase (MEK), 41/43 kDa MAP kinases and p90rsk was commonly observed. In contrast, PMA neither induced the scattering nor activation of all these kinases in TMK1 cells. Pretreatment of MDCK and TMK1 cells with 2-(2-amino-3-methoxyphenyl) choromone (AMPC), a specific inhibitor of MEK, selectively inhibited the HGF-, PMA- and EGF-stimulated activities of MEK, 41/43 kDa MAP kinases and p90rsk in a dose dependent manner. AMPC-pretreatment, however, did not affect HGF-, PMA- or EGF-induced activation of Raf-1, nor HGF-induced activation of phosphatidylinositol 3-kinase in these cells. Importantly, HGF-, PMA- and EGF-induced scattering of MDCK and TMK1 cells was inhibited at doses of AMPC similar to those that gave comparable levels of inhibition of the activities of MEK, 41/43 kDa MAP kinases and p90rsk. These results suggest that activation of the 41/43 kDa MAP kinase signaling pathway is required for the motility response of MDCK and TMK1 cells induced by agents such as HGF, PMA and EGF.

    Oncogene 1998;17;1;57-65

  • 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

  • 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

  • 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

  • Murine Ksr interacts with MEK and inhibits Ras-induced transformation.

    Denouel-Galy A, Douville EM, Warne PH, Papin C, Laugier D, Calothy G, Downward J and Eychène A

    Unité Mixte de Recherche 146 du CNRS Institut Curie Centre Universitaire Laboratoire 110 91405, Orsay Cédex, France.

    Background: Ksr (kinase supressor of Ras) was identified as a regulator of the Ras-MAP kinase (mitogen-activated protein kinase) pathway by genetic screens in Drosophila and Caenorhabditis elegans. Ksr is a kinase with similarities to the three conserved regions of Raf kinases, especially within the kinase domain. To investigate whether these structural similarities correlated with common functional properties, we examined the ability of mKsr-1, the murine homolog of Ksr, to interact with components of the vertebrate MAP kinase pathway.

    Results: In the yeast two-hybrid interaction assay, mKsr-1 did not bind to either Ras, B-Raf or Raf-1, but interacted strongly with both MEK-1 and MEK-2, activators of MAP kinase. The Ksr-MEK interaction was confirmed by co-immunoprecipitation experiments. Ectopically expressed mKsr-1 co-precipitated with endogenous MEK-1 in COS-1 cells, and endogenous Ksr and MEK co-precipitated from PC12 cells. Phosphorylation of MEK by mKsr-1 was not detected, however. In contrast, the MEK subpopulation complexed with mKsr-1 in COS-1 cells or PC12 cells did not display kinase activity. This ability of Ksr to block MEK in an inactive form correlated with a biological response: mKsr-1 did not transform NIH3T3 cells, and, furthermore, mKsr-1 reduced Ras-induced transformation. Similarly, mKsr-1 inhibited the proliferation of embryonic neuroretina cells induced by Ras and B-Raf but not that induced by MEK.

    Conclusions: Our results suggest a novel mechanism for Ksr in regulating the MAP kinase pathway, at least in part through an ability to interact with MEK.

    Current biology : CB 1998;8;1;46-55

  • Fine mapping of Noonan/cardio-facio cutaneous syndrome in a large family.

    Legius E, Schollen E, Matthijs G and Fryns JP

    Center for Human Genetics, Leuven, Belgium.

    Noonan syndrome (NS) is an autosomal dominant condition with facial dysmorphy, congenital cardiac defects and short stature. A gene for NS has previously been linked to a 14 cM region in 12q24. We performed linkage analysis in a four generation Belgian family with NS in some individuals and cardio-facio-cutaneous (CFC) syndrome in others. Clinical data and linkage data in this family indicate that NS and CFC syndrome result from a variable expression of the same genetic defect. We report a maximum lod score of 4.43 at zero recombination for marker D12S84 in 12q24. A crossover in this pedigree narrows the candidate gene region for NS to a 5 cM interval between markers D12S84 and D12S1341.

    European journal of human genetics : EJHG 1998;6;1;32-7

  • 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

  • Activation of mitogen-activating protein kinase by glucose is not required for insulin secretion.

    Khoo S and Cobb MH

    University of Texas Southwestern Medical Center, Department of Pharmacology, 5323 Harry Hines Boulevard, Dallas, TX 75235-9041, USA.

    In the insulinoma cell line INS-1, a model system for glucose-regulated insulin secretion, the mitogen-activating protein (MAP) kinases/extracellular signal-regulated protein kinases, ERK1 and ERK2 are activated up to 15-fold by physiological concentrations of glucose, in the range of 3-12 mM. The related MAP kinase family members, the c-Jun-N-terminal kinases/stress-activated protein kinases are insensitive to glucose, while the p38 MAP kinase is slightly glucose responsive (1.5-fold). ERK activation is dependent on glucose metabolism and the subsequent increase in calcium influx. Inhibiting activation of ERK1 and ERK2 with the MEK1/2 inhibitor PD98059 has no effect on insulin secretion, indicating that ERK activity is not necessary for secretion under these conditions. Glucose activates ERK1 and ERK2 in cytosolic and purified nuclear fractions of INS-1 cells and more of each is found in nuclei from glucose-treated cells. These findings suggest that some of the glucose-dependent actions of ERKs will be exerted in the nucleus.

    Funded by: NIDDK NIH HHS: DK34128, R01 DK034128, R37 DK034128

    Proceedings of the National Academy of Sciences of the United States of America 1997;94;11;5599-604

  • 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

  • Chemotactic peptide-induced activation of MEK-2, the predominant isoform in human neutrophils. Inhibition by wortmannin.

    Downey GP, Butler JR, Brumell J, Borregaard N, Kjeldsen L, Sue-A-Quan AK and Grinstein S

    Department of Medicine, Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada.

    Exposure of neutrophils to a variety of agonists including chemoattractant peptides and cytokines induces degranulation and activation of the oxidative burst which are required for bacterial killing. The signaling pathways regulating these important functions are incompletely characterized. Mitogen-activated protein (MAP) kinases, which include the extracellular signal-regulated kinases (ERKs), are activated rapidly in neutrophils, suggesting that they may regulate cell activation. We found that neutrophils express two isoforms of MAP/ERK kinase (MEK), mixed-function kinases that are responsible for phosphorylation and activation of ERK. Like MEK-1, MEK-2 was found to reside in the cytosol both before and after stimulation. Studies were undertaken to define the relative abundance and functional contribution of MEK-1 and MEK-2 in neutrophils and to characterize the signaling pathways leading to their activation. Although the abundance of the two isoforms was similar, the activity of MEK-2 was at least 3-fold greater than that of MEK-1. A rise in cytosolic [Ca2+] was insufficient for MEK stimulation, and blunting the [Ca2+] change with intracellular chelators failed to prevent receptor-mediated activation of either isoform, implying that cytosolic Ca2+ transients are not necessary. In contrast, both MEK-1 and MEK-2 were activated by exposure of cells to protein kinase C (PKC) agonists. Conversely, PKC antagonists inhibited the chemotactic stimulation of both isoforms, suggesting that PKC was required for their activation. Despite these similarities, clear differences were also found in the pathways leading to activation of the MEK isoforms. In particular, MEK-2 was considerably more sensitive than MEK-1 to the phosphatidylinositol 3-kinase inhibitor wortmannin. Phosphorylation and activation of ERK-1 and ERK-2 were also reduced by this inhibitor. In summary, MEK-2 is stimulated in formyl-methionyl-leucyl-phenylalanine-treated neutrophils, where it appears to be functionally the predominant isoform. The time course and inhibitor sensitivity of MEK-2 activation parallel those of several components of the microbicidal response, suggesting a signaling role of the MEK-ERK pathway.

    The Journal of biological chemistry 1996;271;35;21005-1011

  • Identification of signalling proteins interacting with B-Raf in the yeast two-hybrid system.

    Papin C, Denouel A, Calothy G and Eychène A

    Unité Mixte de Recherche 146 du CNRS, Institut Curie, Orsay, France.

    Recent studies suggested the existence of Ras/B-Raf/ MEK-1 complexes and a critical role for B-Raf in regulating the MAP kinase/ERKs signalling pathway. We report, here, that both Ras and MEK-1 proteins interact physically with B-Raf proteins in the yeast two-hybrid system. In addition, by screening a mouse brain cDNA library, we isolated additional B-Raf interacting proteins. These include three members of the 14-3-3 proteins family (eta, theta and zeta) and the MEK-2 protein. We also show that c-Raf-1, previously reported to interact with beta and zeta 14-3-3 proteins, also interacts with eta and theta 14-3-3 proteins in the two-hybrid system. By using different portions of the B-Raf protein, we mapped the regions of the protein involved in these interactions. Specifically, we have characterized B-Raf specific sequences required for an efficient interaction with MEK proteins. We show that, consequently, B-Raf interacts with MEK-1 and MEK-2 with a better affinity than does c-Raf-1, thus strengthening the notion that B-Raf is a stronger MEK activator than c-Raf-l. Our results also suggest that a MEK specific sequence, not present in MAP kinase kinases which are not activated by members of the Raf family, is required for the interaction with Raf proteins.

    Oncogene 1996;12;10;2213-21

  • 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 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

  • Initial assessment of human gene diversity and expression patterns based upon 83 million nucleotides of cDNA sequence.

    Adams MD, Kerlavage AR, Fleischmann RD, Fuldner RA, Bult CJ, Lee NH, Kirkness EF, Weinstock KG, Gocayne JD, White O et al.

    Institute for Genomic Research, Rockville, Maryland 20850, USA.

    In an effort to identify new genes and analyse their expression patterns, 174,472 partial complementary DNA sequences (expressed sequence tags (ESTs)), totalling more than 52 million nucleotides of human DNA sequence, have been generated from 300 cDNA libraries constructed from 37 distinct organs and tissues. These ESTs have been combined with an additional 118,406 ESTs from the database dbEST, for a total of 83 million nucleotides, and treated as a shotgun sequence assembly project. The assembly process yielded 29,599 distinct tentative human consensus (THC) sequences and 58,384 non-overlapping ESTs. Of these 87,983 distinct sequences, 10,214 further characterize previously known genes based on statistically significant similarity to sequences in the available databases; the remainder identify previously unknown genes. Thirty tissues were sampled by over 1,000 ESTs each; only eight genes were matched by ESTs from all 30 tissues, and 227 genes were represented in 20 or more of the tissues sampled with more than 1,000 ESTs. Approximately 40% of identified human genes appear to be associated with basic energy metabolism, cell structure, homeostasis and cell division, 22% with RNA and protein synthesis and processing, and 12% with cell signalling and communication.

    Nature 1995;377;6547 Suppl;3-174

  • Independent human MAP-kinase signal transduction pathways defined by MEK and MKK isoforms.

    Dérijard B, Raingeaud J, Barrett T, Wu IH, Han J, Ulevitch RJ and Davis RJ

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

    Mammalian mitogen-activated protein (MAP) kinases include extracellular signal-regulated protein kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38 subgroups. These MAP kinase isoforms are activated by dual phosphorylation on threonine and tyrosine. Two human MAP kinase kinases (MKK3 and MKK4) were cloned that phosphorylate and activate p38 MAP kinase. These MKK isoforms did not activate the ERK subgroup of MAP kinases, but MKK4 did activate JNK. These data demonstrate that the activators of p38 (MKK3 and MKK4), JNK (MKK4), and ERK (MEK1 and MEK2) define independent MAP kinase signal transduction pathways.

    Funded by: NCI NIH HHS: CA58396; NIAID NIH HHS: AI15136; NIGMS NIH HHS: GM37696

    Science (New York, N.Y.) 1995;267;5198;682-5

  • 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

  • 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

  • Identification and characterization of a new mammalian mitogen-activated protein kinase kinase, MKK2.

    Wu J, Harrison JK, Dent P, Lynch KR, Weber MJ and Sturgill TW

    Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville 22908.

    Mitogen-activated protein (MAP) kinases are serine/threonine protein kinases activated by dual phosphorylation on threonine and tyrosine residues. A MAP kinase kinase (MKK1 or MEK1) has been identified as a dual-specificity protein kinase that is sufficient to phosphorylate MAP kinases p42mapk and p44mapk on the regulatory threonine and tyrosine residues. Because of the multiplicity of MAP kinase isoforms and the diverse circumstances and agonists leading to their activation, we thought it unlikely that a single MKK could accommodate this complexity. Indeed, two protein bands with MKK activity have previously been identified after renaturation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We now report the molecular cloning and characterization of a second rat MAP kinase kinase cDNA, MKK2. MKK2 cDNA contains an open reading frame encoding a protein of 400 amino acids, 7 residues longer than MKK1 (MEK1). The amino acid sequence of MKK2 is 81% identical to that of MKK1, but nucleotide sequence differences occur throughout the aligned MKK2 and MKK1 cDNAs, indicating that MKK2 is the product of a distinct gene. MKK1 and MKK2 mRNAs are expressed differently in rat tissues. Both cDNAs when expressed in COS cells displayed the ability to phosphorylate and activate p42mapk and p44mapk, both MKK1 and MKK2 were activated in vivo in response to serum, and both could be phosphorylated and activated by the v-Raf protein in vitro. However, differences between MKK1 and MKK2 in sites of phosphorylation by proline-directed protein kinases predict differences in feedback regulation.

    Funded by: NIDDK NIH HHS: DK38942, DK41077; NIGMS NIH HHS: GM47332

    Molecular and cellular biology 1993;13;8;4539-48

  • Molecular cloning, expression, and characterization of the human mitogen-activated protein kinase p44erk1.

    Charest DL, Mordret G, Harder KW, Jirik F and Pelech SL

    Biomedical Research Centre, University of British Columbia, Vancouver, Canada.

    p44erk1 is a member of a family of tyrosyl-phosphorylated and mitogen-activated protein (MAP) kinases that participate in cell cycle control. A full-length erk1 cDNA was isolated from a human hepatoma cell line (Hep G2) library. The erk1 cDNA clone shared approximately 96% predicted amino acid identity with partial sequences of rodent erk1 cognates, and the erk1 gene was assigned to human chromosome 16 by hybrid panel analysis. Human erk1 expressed in Escherichia coli as a glutathione S-transferase fusion (GST-Erk1) protein was substantially phosphorylated on tyrosine in vivo. It underwent further autophosphorylation in vitro (up to 0.01 mol of P per mol) at the regulatory Tyr-204 site and at additional tyrosine and serine residues. Threonine autophosphorylation, presumably at the regulatory Thr-202 site, was also detected weakly when the recombinant kinase was incubated in the presence of manganese, but not in the presence of magnesium. Before and after cleavage of the GST-Erk1 protein with thrombin, it exhibited a relatively high level of myelin basic protein phosphotransferase activity, which could be reduced eightfold by treatment of the kinase with the protein-tyrosine phosphatase CD45, but not by treatment with the protein-serine/threonine phosphatase 2A. The protein-tyrosine kinase p56lck catalyzed phosphorylation of GST-Erk1 at two autophosphorylations sites, including Tyr-204, and at a novel site. A further fivefold stimulation of the myelin basic protein phosphotransferase activity of the GST-Erk1 was achieved in the presence of a partially purified MAP kinase kinase from sheep platelets. Under these circumstances, there was primarily an enhancement of the tyrosine phosphorylation of GST-Erk1. This MAP kinase kinase also similarly phosphorylated a catalytically compromised version of GST-Erk1 in which Lys-71 was converted to Ala by site-directed mutagenesis.

    Molecular and cellular biology 1993;13;8;4679-90

  • 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

Gene lists (4)

Gene List Source Species Name Description Gene count
L00000015 G2C Homo sapiens Human NRC Human orthologues of mouse NRC adapted from Collins et al (2006) 186
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000035 G2C Homo sapiens Pocklington H4 Human orthologues of cluster 4 (mouse) from Pocklington et al (2006) 8
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
© 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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