Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.24 (mitogen-activated protein kinase)
95,810 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the germline of Caenorhabditis elegans hermaphrodites, meiotic cell cycle progression occurs in spatially restricted regions. Immediately after leaving the distal mitotic region, germ cells enter meiosis and thereafter remain in the pachytene stage of first meiotic prophase for an extended period. At the dorsoventral gonadal flexure, germ cells exit pachytene and subsequently become arrested in diakinesis. We have found that exit from pachytene is dependent on the function of three members of the MAP kinase signaling cascade. One of these genes, mek-2, is a newly identified C. elegans MEK (MAP kinase kinase). The other two genes, mpk-1/sur-1 (MAP kinase) and let-60 ras, were previously identified based on their roles in vulval induction and are shown here to act in combination with mek-2 to permit exit from pachytene. Through genetic mosaic analysis, we demonstrate that the expression of mpk-1/sur-1 is required within the germline to permit exit from pachytene.
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PMID:Three genes of the MAP kinase cascade, mek-2, mpk-1/sur-1 and let-60 ras, are required for meiotic cell cycle progression in Caenorhabditis elegans. 767 16

A PC-12 pheochromocytoma cell line is described with roughly equivalent levels of functional receptors for nerve growth factor (NGF), epidermal growth factor (EGF), and insulin. Each of these receptors undergoes autophosphorylation upon binding of their respective ligands, and causes the activation of phosphatidylinositol-3 kinase via a mechanism involving tyrosine phosphorylation. In the case of insulin, this activation is due to the tyrosine phosphorylation of its major cellular substrate, IRS-1. Despite the presence of functional receptors in these cells, insulin does not stimulate the activity of the mitogen-activated protein (MAP) kinase, despite a 5- to 8-fold activation observed with both NGF and EGF under the same conditions. This failure to activate MAP kinase was not due to the insulin-dependent dephosphorylation of the enzyme, but correlated with the lack of activation of the MAP kinase kinase, although this enzyme was also activated by NGF and EGF. Similarly, the activation of the raf and ras protooncogenes in these cells was not observed with insulin, whereas NGF and EGF produced marked activation. In addition, insulin-dependent induction of the c-fos protein was impaired, in comparison to NGF. In contrast to a lack of effect on the MAP kinase pathway, these PC-12 cells were metabolically responsive to insulin, exhibiting increases in glucose, lipid, and protein synthesis in response to the hormone. The differential responses of phosphorylation events to insulin, NGF, and EGF in these cells indicates that divergence of signaling pathways may occur at or near the insulin receptor.
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PMID:Divergence of signaling pathways for insulin in PC-12 pheochromocytoma cells. 768 84

To study the mechanism by which v-mos induces cell transformation, we generated a transformed rat cell line (DTM) containing two functional copies of mos, one encoding the p37v-mos of the m1 wild-type strain of Moloney murine sarcoma virus (Mo-MuSV) and the other the p85gag-mos fusion protein of the ts110 mutant of Moloney murine sarcoma virus. Subsequently, we isolated a revertant cell line (F-1) following transfection of DTM with a mutant retroviral construct (pIC4Neo) carrying a selectable marker. Like DTM, the F-1 revertant contained two integrated copies of v-mos, expressed mos containing viral RNA, and contained rescuable transforming viruses. The revertant did not grow in soft agar, showed a greatly reduced ability to form tumors in nude mice, and exhibited organized tubulin and actin structures similar to those found in normal cells. Revertant cells were resistant to retransformation by v-mos and v-raf but could be retransformed by v-ras. MAP kinase (ERK-2) and MAP kinase kinase (MKK-1) activity, which are constitutively elevated in v-mos- and v-raf-transformed cells, exhibits levels in the F-1 revertant similar to those seen in nontransformed cells. F-1 and normal REF-1 cells express elevated levels of protein phosphatases in comparison to DTM cells. In vivo treatment with okadaic acid, a potent protein phosphatase inhibitor, leads to an increase in MKK-1 and MAP kinase activity in F-1 cells but not in REF-1. The results support the hypothesis that mos acts through the MAP kinase cascade (MKK-1 and ERK-2) to induce cell transformation and that blocking v-mos activation of that cascade (possibly because of increased levels of phosphatase) prevents transformation.
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PMID:Transformation-resistant mos revertant is unable to activate MAP kinase kinase in response to v-mos or v-raf. 771 84

Activated Ras initiates a cascade of sequential phosphorylation events, including the protein kinases Raf, MEK, and MAP kinase. The Let-60 Ras-mediated signal transduction pathway controls vulval induction in Caenorhabditis elegans. Both Lin-45 Raf and Sur-1 MAP kinase have been determined to be essential factors during vulval induction; however, the C. elegans mek gene has not been identified. In this paper, we have cloned a C. elegans mek gene, mek-2, and demonstrated that the MEK-2 protein possesses the biochemical properties of MAP kinase kinases: The C. elegans MEK-2 protein can phosphorylate and activate a human MAP kinase (ERK1), and MEK-2 itself can be phosphorylated and activated by immunoprecipitated mammalian Raf. The mek-2 gene plays a key role in the let-60 ras-mediated vulval induction pathway, as loss-of-function mutations in the gene (ku114 and h294) significantly reduce the signal transmitted through Ras. mek-2(ku114) completely suppressed the Multivulva (Muv) phenotype of a hyperactive let-60 ras mutation, and animals homozygous for mek-2(ku114) also displayed a partial larval lethal phenotype. Animals homozygous for mek-2(h294) exhibited a highly penetrant sterile and Vulvaless phenotype. Microinjection of a gain-of-function mek-2 mutation resulted in Muv and other mutant phenotypes, whereas microinjection of a dominant-negative mutation not only suppressed the Muv phenotype of an activated let-60 ras mutation but also caused an egg-laying defective phenotype in otherwise wild type animals. Our results demonstrate that mek-2 acts between lin-45 raf and sur-1/mpk-1 in a signal transduction pathway used in the control of vulval differentiation and other developmental events.
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PMID:MEK-2, a Caenorhabditis elegans MAP kinase kinase, functions in Ras-mediated vulval induction and other developmental events. 772 90

An evolutionarily conserved signal transduction pathway that utilizes a receptor tyrosine kinase and a Ras protein mediates the induction of vulval cell fates in the nematode Caenorhabditis elegans. We sought new genes that function in this pathway by screening for suppressors of the Multivulva phenotype caused by a mutation that activates the let-60 ras gene. Seven such suppressor mutations defined a new gene involved in vulval induction. We named this gene mek-2, because its predicted protein product is most similar to MEK, a protein-serine/threonine and tyrosine kinase. mek-2 mutations can be arranged in an allelic series. A probable null mutation eliminated vulval induction, and the strongest mutations alter codons conserved in most or all protein kinases. Our genetic analysis showed that mek-2 functions downstream of let-60 ras and is required for ras-mediated signal transduction in vivo. The MEK-2 protein may interact with the products of the lin-45 raf and mpk-1 MAP kinase genes, which also mediate vulval induction.
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PMID:The Caenorhabditis elegans gene mek-2 is required for vulval induction and encodes a protein similar to the protein kinase MEK. 772 91

Activation of mitogen-activated protein kinase (MAP kinase) plays an important role in the cellular effects of nerve growth factor (NGF). Although the precise pathway by which NGF activates MAP kinase is not clear, several enzymes have been identified that may form a linear phosphorylation cascade, in which MAP kinase is activated by MAP kinase kinase (MEK). A key enzyme that links the ras-GTP complex to MEK is widely believed to be the raf kinase. However, immunoprecipitation experiments in PC-12 cells revealed that raf is not the major NGF-dependent MEK kinase [Zheng, Ohmichi, Saltiel and Guan (1994) Biochemistry 33, 5595-5599]. We have identified a protein kinase from PC-12 cells that catalyses both the phosphorylation and activation of MEK. This activity is stimulated 3-fold in cells treated with NGF. The partial purification on FPLC and characterization of this MEK kinase indicate that it is distinct from raf, MEK, MAP kinase and other previously described NGF-stimulated protein kinases. The activity of this enzyme is unaffected by direct addition to the assay of heparin, staurosporine, K252A and the heat-stable cyclic AMP-dependent kinase peptide inhibitor, but is slightly inhibited by NaF and calcium ions. Comparison of its behaviour on gel permeation and sucrose-density gradients indicates a molecular mass in the region of 50,000 Da. Moreover, isoelectric focusing of the enzyme revealed a pI of approx. 7.3. The kinase activity is specific for ATP as substrate with a Km of 11 microM, and requires Mg2+ as a cofactor. Analysis of the activation of this enzyme in PC-12 cells transfected with a dominant inhibitory mutant of p21ras suggests that this MEK kinase resides downstream of ras in the MAP kinase activation pathway. Moreover, site-directed mutation of the residues on MEK that are phosphorylated by raf does not completely abrogate phosphorylation by the MEK kinase, suggesting that this enzyme may share some phosphorylation sites with raf, but also phosphorylates MEK on other sites.
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PMID:Nerve growth factor stimulates a novel protein kinase in PC-12 cells that phosphorylates and activates mitogen-activated protein kinase kinase (MEK). 773 91

In view of the potent mitogenic effect exerted by insulin in human colonic cells, we used Caco-2 cells transfected with an activated (Val12) human Ha-ras gene or the polyoma middle T (PyMT) oncogene, a constitutive activator of pp60c-src tyrosine kinase activity, to investigate the effect of oncogenic p21ras and PyMT/pp60c-src on insulin mitogenic signaling. As compared to vector control Caco-2 cells, both oncogene-transfected cells exhibited: 1) a lost of response to insulin's stimulatory effect on mitogen-activated protein (MAP) kinase activity and cell proliferation, both of which were constitutively increased; 2) a decrease in insulin receptor (IR) affinity and insulin-stimulated exogenous tyrosine kinase activity, which resulted, at least in part, from increased protein kinase C (PKC) activity (4), since both IR alterations were partially corrected by PKC down-regulation; and 3) a decrease in both insulin receptor mRNA level and insulin receptor number, which was independent of PKC since it persisted after PKC down-regulation. In conclusion, oncogenic p21ras and PyMT/pp60c-src abolished insulin mitogenic signaling in Caco-2 cells through mechanisms involving (i) constitutive activation of MAP kinase, and (ii) marked decreases in both insulin receptor function and expression which were mediated by PKC-dependent and PKC-independent pathways respectively. This is the first evidence that, when oncogenically activated, p21ras and pp60c-src not only exert a negative control on insulin receptor function but also repress insulin receptor gene expression in human colonic cells.
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PMID:[Oncogenic activation of p21(ras) and pp60(c-src) in human colonic Caco-2 cells decreases insulin receptor function and expression through protein kinase C-dependent and independent pathways]. 773 71

Insulin stimulation of differentiated 3T3-L1 adipocytes or Chinese hamster ovary cells expressing high levels of the insulin receptor resulted in a time-dependent decrease in the electrophoretic mobility of SOS on sodium dodecyl sulfate-polyacrylamide gels. The reduction in SOS mobility was completely reversed by alkaline phosphatase treatment, and the in vitro phosphorylation of SOS by mitogen-activated protein kinase resulted in a decrease of electrophoretic mobility identical to that following in vivo insulin stimulation. Immunoprecipitation of Grb2 followed by SOS immunoblotting demonstrated a disassociation of the SOS-Grb2 complex that paralleled the decrease in SOS electrophoretic mobility. Similarly, SOS immunoprecipitation followed by Grb2 immunoblotting also indicated an uncoupling of the SOS-Grb2 complex. Further, incubation of whole-cell extracts with glutathione-S-transferase-Grb2 fusion proteins demonstrated that insulin stimulation resulted in a decreased affinity of SOS for Grb2. In contrast, the dissociation of SOS from Grb2 did not affect the interactions between Grb2 and tyrosine-phosphorylated Shc. In addition to insulin, several other agents which activate the mitogen-activated protein kinase pathway (platelet-derived growth factor, serum, and phorbol ester) also resulted in the uncoupling of the SOS-Grb2 complex. Consistent with these results, expression of v-ras and v-raf resulted in a constitutive decrease in the association between SOS and Grb2. Together, these data suggest a molecular mechanism accounting for the transient activation of ras due to the uncoupling of the SOS-Grb2 complex following SOS phosphorylation.
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PMID:Insulin-stimulated disassociation of the SOS-Grb2 complex. 773 60

We previously purified a protein factor, named REKS (Ras-dependent Extracellular Signal-regulated Kinase (ERK)/mitogen-activated protein kinase Kinase (MEK) Stimulator), from Xenopus eggs by use of a cell-free assay system in which recombinant GTP gamma S (guanosine 5'-(3-O-thio)triphosphate)-Ki-Ras activates recombinant MEK. By use of this assay system, we purified here bovine REKS to near homogeneity from the cytosol fraction of bovine brain by successive chromatographies of Mono S, Mono Q, GTP gamma S-glutathione S-transferase-Ha-Ras-coupled glutathione-agarose, and Mono Q columns. It was composed of three proteins with masses of about 95, 32, and 30 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 95-, 32-, and 30-kDa proteins were identified by immunoblot analysis to be B-Raf protein kinase, 14-3-3 protein, and 14-3-3 protein, respectively. Moreover, the REKS activity was specifically immunoprecipitated by an anti-B-Raf antibody. Bovine REKS was activated by lipid-modified GTP gamma S-Ki-Ras far more effectively than by a lipid-unmodified one. Lipid-modified GDP-Ki-Ras was inactive. Exogenous addition of 14-3-3 proteins stimulated further the REKS activity both in the presence and absence of GTP gamma S-Ki-Ras. These results indicate that at least one of the direct targets of Ras is B-Raf complexed with 14-3-3 proteins in bovine brain.
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PMID:Purification of a Ras-dependent mitogen-activated protein kinase kinase kinase from bovine brain cytosol and its identification as a complex of B-Raf and 14-3-3 proteins. 774 15

The let-60 ras gene of Caenorhabditis elegans is required for multiple aspects of development. The vulvar differentiation pathway is the most intensively studied of these, but the ras pathway has now been shown to also be essential for male spicule development. Using vulval differentiation, molecular genetic techniques are now being used to study structure/function relationships of particular signaling components and to identify new positively and negatively acting proteins of Ras-mediated signaling pathways. Mutations affecting LET-23, a receptor tyrosine kinase homolog, which cause tissue-specific defects have been localized to the carboxyl terminus. SH2 domain specificity has been analyzed through Src/SEM-5 chimeric proteins in transgenic nematodes. A mitogen-activated protein kinase that acts downstream of LET-60 Ras in vulval differentiation has been identified. Negative regulatory genes have been cloned and found to encode novel proteins and a clathrin adaptor protein.
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PMID:Ras pathways in Caenorhabditis elegans. 774 23


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