Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P51812 (mitogen-activated protein)
 10,636 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The immunosuppressant rapamycin inhibited proliferation of the H4IIEC hepatoma cell line. Rapamycin, but not its structural analog FK506, also inhibited the basal and insulin-stimulated activity of the p70 ribosomal protein S6 kinase. By contrast, insulin stimulation of the p85 Rsk S6 kinase and mitogen-activated protein (MAP) kinase activity were unaffected by drug. Rapamycin treatment of COS cells transfected with recombinant p70 S6 kinase completely inhibited the appearance of the hyperphosphorylated form of p70 S6 kinase concomitant with the inhibition of enzyme activity toward 40S subunits. Thus, rapamycin inhibits a signal transduction element that is necessary for the activation of p70 S6 kinase and mitogenesis but unnecessary for activation of p85 Rsk S6 kinase or MAP kinase.
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PMID:Rapamycin-induced inhibition of the 70-kilodalton S6 protein kinase. 138 Jan 82

The molecular structure of a rat hepatoma 70-kDa insulin/mitogen-stimulated S6 protein kinase, obtained by molecular cloning, is compared to that of a rat homolog of the 85-kDa Xenopus S6 protein kinase alpha; both kinases were cloned from H4 hepatoma cDNA libraries. The 70-kDa S6 kinase (calculated molecular mass of 59,186 Da) exhibits a single catalytic domain that is most closely related in amino acid sequence (56% identity) to the amino-terminal, kinase C-like domain of the rat p85 S6 kinase (calculated molecular mass of 82,695 Da); strong similarity extends through a further 67 residues carboxyl-terminal to the catalytic domain (40% identity), corresponding to a region also conserved among the kinase C family. Outside of this segment of approximately 330 amino acids, the structures of the p70 and p85 S6 kinases diverge substantially. The p70 S6 kinase is known to be activated through serine/threonine phosphorylation by unidentified insulin/mitogen-activated protein kinases. A model for the regulation of p70 S6 protein kinase activity is proposed wherein the low activity of the unphosphorylated enzyme results from the binding of a basic, inhibitory pseudosubstrate site (located carboxyl-terminal to the extended catalytic domain) to an acidic substrate binding region (located amino-terminal to the catalytic domain); substrate binding is thereby prevented. S6 kinase activation requires displacement of this inhibitory segment, which is proposed to occur consequent to its multiple phosphorylation. The putative autoinhibitory segment contains several serine and threonine residues, each followed directly by a proline residue. This motif may prevent autophosphorylation but permit transphosphorylation; two of these serine residues reside in a maturation promoting factor (MPF)/cdc-2 consensus motif. Thus, hormonal regulation of S6 kinase may involve the action of MPF/cdc-2 or protein kinases with related substrate specificity.
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PMID:Molecular structure of a major insulin/mitogen-activated 70-kDa S6 protein kinase. 223 64

Interleukin-13 (IL-13) and interleukin-4 (IL-4) are related in structure and function and are thought to share a common receptor component. We have investigated the signal transduction pathways activated by these two growth factors, as well as insulin, in cell-lines and primary cells of lymphohemopoietic origin. All three factors induced the tyrosine phosphorylation of a protein of 170 kDa (p170), which coimmunoprecipitated with the p85 subunit of P13'-kinase, via high affinity interactions mediated by the SH2 domains of p85. Antibodies raised against the entire insulin-receptor substrate-1 (IRS-1) protein immunoprecipitated p170 much less efficiently than they did IRS-1 from 3T3 cells. However, antibodies directed against the conserved pleckstrin homology domain of IRS-1 immunoprecipitated both p170 and IRS-1 with similar efficiency, suggesting they share structural similarities in this region. In lymphohemopoietic cells, IL-13, IL-4, and insulin failed to induce increased tyrosine phosphorylation of Shc, or its association with grb2, modification of Sos1, or activation of erk-1 and erk-2 mitogen-activated protein kinases, suggesting that p170 mediates downstream pathways distinct from those mediated by IRS-1. Both IL-13 and IL-4 induced low levels of tyrosine phosphorylation of Tyk-2 and Jak-1. IL-4 also activated the Jak-3-kinase, but, despite other similarities, IL-13 did not. Insulin failed to activate any of the known members of the Janus family of kinases. In that Jak-3 is reported to associate with the IL-2 gamma c chain, these data suggest that the IL-13 receptor does not utilize this subunit. However, both IL-13 and IL-4 induced tyrosine phosphorylation of the IL-4-140 kDa receptor chain, suggesting that this is a component of both receptors in these cells and accounts for the similarities in signaling pathways shared by IL-13 and IL-4.
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PMID:Interleukin-13 signal transduction in lymphohemopoietic cells. Similarities and differences in signal transduction with interleukin-4 and insulin. 774 81

Early signalling events between protein kinase C (PKC) activation and lymphokine transcription were compared between phorbol ester-sensitive and -resistant EL4 cell lines which do or do not respond with interleukin 2 (IL2) production, respectively. The earliest event detected in the sensitive cell line was a dramatic increase in the tyrosine phosphorylation of an 85,000 M(r) protein (p85; 30 s), followed by mobility shifts of raf-1, mitogen-activated protein kinase kinase (MEK), mitogen-activated protein (MAP) kinase, lck and ZAP-70 (within 5 min). In contrast, p85 was not detected in the resistant cell line and lck and raf-1 mobility shifts exhibited delayed kinetics. Both vanadate and okadaic acid blocked the phorbol ester-stimulated p85 tyrosine phosphorylation in the sensitive cell line, suggesting that a phosphatase activity downstream of PKC activation may be required for p85 tyrosine phosphorylation. Characterization of p85 and its regulation should help elucidate some of the earliest events in this PKC pathway.
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PMID:Rapid tyrosine phosphorylation of an 85,000 M(r) protein after phorbol ester stimulation of EL4 thymoma cells. 775 7

Insulin receptor substrate-1 (IRS-1) serves as the major immediate substrate of insulin/insulin-like growth factor (IGF)-1 receptors and following tyrosine phosphorylation binds to specific Src homology-2 (SH2) domain-containing proteins including the p85 subunit of phosphatidylinositol (PI) 3-kinase and GRB2, a molecule believed to link IRS-1 to the Ras pathway. To investigate how these SH2-containing signaling molecules interact to regulate insulin/IGF-1 action, IRS-1, glutathione S-transferase (GST)-SH2 domain fusion proteins and Ras proteins were microinjected into Xenopus oocytes. We found that pleiotropic insulin actions are mediated by IRS-1 through two independent, but convergent, pathways involving PI 3-kinase and GRB2. Thus, microinjection of GST-fusion proteins of either p85 or GRB2 inhibited IRS-1-dependent activation of mitogen-activated protein (MAP) and S6 kinases and oocyte maturation, although only the GST-SH2 of p85 reduced insulin-stimulated PI 3-kinase activation. Co-injection of a dominant negative Ras (S17N) with IRS-1 inhibited insulin-stimulated MAP and S6 kinase activation. Micro-injection of activated [Arg12,Thr59]Ras increased basal MAP and S6 kinase activities and sensitized the oocytes to insulin-stimulated maturation without altering insulin-stimulated PI 3-kinase. The Ras-enhanced oocyte maturation response, but not the elevated basal level of MAP and S6 kinase, was partially blocked by the SH2-p85, but not SH2-GRB2. These data strongly suggest that IRS-1 can mediate many of insulin's actions on cellular enzyme activation and cell cycle progression requires binding and activation of multiple different SH2-domain proteins.
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PMID:Interactive roles of Ras, insulin receptor substrate-1, and proteins with Src homology-2 domains in insulin signaling in Xenopus oocytes. 796 82

Expression of the insulin receptor substrate-1 (IRS1) or Shc cDNA resulted in both increased protein and insulin-stimulated tyrosine phosphorylation of IRS1 and Shc proteins, respectively. Although expression of Shc had no significant effect on insulin-stimulated mitogen-activated protein (MAP) kinase gel shift or c-fos transcriptional activation, expression of IRS1 inhibited these responses. The effect of IRS1 expression on the formation of multisubunit signaling complexes was determined by a series of indirect co-immunoprecipitations. Grb2 immunoprecipitation from IRS1-transfected and insulin-treated cells demonstrated an increased coimmunoprecipitation of Syp and the p85 regulatory subunit of the phosphatidylinositol 3-kinase. Similarly, cell extracts immunoprecipitated with a p85 antibody displayed an increased co-immunoprecipitation of Syp and Grb2. However, expression of IRS1 increased the extent of Grb2 associated with IRS1 with a concomitant reduction in the amount of Grb2 associated with Shc. Furthermore, increased expression of Shc reduced the amount of Grb2 bound to IRS1 with a concomitant increase in Grb2 associated with Shc. Together, these data demonstrate that IRS1 and Shc compete for a limited cellular pool of Grb2, and insulin activation of MAP kinase and c-fos transcription predominantly occur through the Shc-Grb2 signaling pathway.
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PMID:Insulin receptor substrate-1 (IRS1) and Shc compete for a limited pool of Grb2 in mediating insulin downstream signaling. 798 51

Activation of mitogen-activated protein (MAP) kinase represents an important mechanism in hormonal regulation. To clarify the role of MAP kinase activation in insulin action, we compared the activation of the enzyme in Rat-1 cells transfected with wild-type (Hirc) and mutant insulin receptors in which the 2 carboxyl-terminal tyrosines were substituted with phenylalanine (Y/F2). Expression of the Y/F2 mutant receptor enhanced the responsiveness of MAP kinase to insulin. Moreover, the insulin responsiveness of the activator of this enzyme, MAP kinase kinase, was also increased in these cells. To explore the early signaling events that might account for this increase in responsiveness, we evaluated the tyrosine phosphorylation of the insulin receptor substrate, IRS-1, and its subsequent association with phosphatidylinositol (PI)-3 kinase. In both cell types, insulin led to a dose-dependent increase in the association of tyrosine phosphorylated IRS-1 with the SH2 domain of the p85 regulatory subunit of PI-3 kinase, and also increased the amount of PI kinase activity detected in anti-IRS-1 immunoprecipitates. The effect of insulin was significantly greater in Y/F2 cells, as determined in both assays. In previous studies, cells bearing this receptor mutant exhibited an identical metabolic response but enhanced mitogenic response to insulin when compared with wild-type receptor. These data provide further evidence for divergence of the mitogenic and metabolic signaling pathways at or near the insulin receptor.
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PMID:Mutation of the two carboxyl-terminal tyrosines in the insulin receptor results in enhanced activation of mitogen-activated protein kinase. 814 49

The mitogen response of p70/p85 S6 kinase (S6K) parallels that of mitogen-activated protein kinases (MAPK). However, S6K lies on a discrete signaling pathway from MAPK, since the immunosuppressant drug rapamycin inactivates S6K without affecting the MAPK cascade. Phosphatidylinositol 3-kinase operates upstream of S6K, but the intermediate effectors in this signaling pathway are unknown. We have identified an autoinhibitory domain in S6K that overrides the requirement of the amino terminus for the activation of S6K. The region between codons 58 and 77 is highly inhibitory, and its deletion results in constitutive kinase activation. Additionally, deletion of the first 77 codons confers mitogen independence and insensitivity to rapamycin. Rat1 cells expressing delta N77 S6K exhibit a distinctly abnormal morphology. This constitutively active mutant will provide a useful means of studying the effects of expressing unregulated S6K in cells. Subdeletion analysis of the amino terminus has defined two discrete domains in the N terminus of S6K--a domain between codons 1 and 58 is essential for the mitogen activation of S6K and confers rapamycin sensitivity; a second domain between codons 58 and 77 confers autoinhibition. We propose a model for the activation of S6 kinase in which mitogen-stimulated cellular factors interact with the amino terminus to negate the effects of the autoinhibitory domain.
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PMID:Constitutive activation of S6 kinase by deletion of amino-terminal autoinhibitory and rapamycin sensitivity domains. 852 22

Activation of alpha1 adrenergic receptors stimulates mitogenesis in human vascular smooth muscle cells (HVSMCs). To examine signaling pathways by which activation of alpha1 receptors may induce mitogenesis in HVSMCs, we have found that alpha1 receptor stimulated-DNA synthesis and activation of mitogen-activated protein (MAP) kinase are blocked by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase). To determine directly if activation of alpha1 receptors stimulated PI 3-kinase, in vitro assays of kinase activity were performed in immunocomplexes precipitated by an antibody against the p85 alpha subunit of PI 3-kinase. Noradrenaline stimulated a time- and concentration-dependent activation of PI 3-kinase in the presence of a beta adrenergic receptor antagonist. Noradrenaline-stimulated PI 3-kinase activation was blocked by antagonists of alpha1 receptors and by pertussis toxin, suggesting that alpha1 receptors activate PI 3-kinase via a pertussis toxin-sensitive G protein. Direct activation of protein kinase C by a phorbol ester did not stimulate PI 3-kinase; also, a Ca2+ L-channel blocker did not inhibit noradrenaline-stimulated PI 3-kinase activity. Increased PI 3-kinase activity was detected in both anti-Ras and anti-phosphotyrosine immunoprecipitates from noradrenaline-stimulated HVSMCs. Moreover, noradrenaline stimulated formation of active Ras-GTP complexes. Because blockade of PI 3-kinase by wortmannin inhibited formation of this complex, this result suggests that Ras might be a target of PI 3-kinase. Noradrenaline stimulated tyrosine phosphorylation of the p85 subunit of PI 3-kinase, and a phosphorylated tyrosine protein could be co-immunoprecipitated with anti-p85 of PI 3-kinase. These results demonstrate that stimulation of alpha1 receptors activates PI 3-kinase in HVSMCs and that alpha1 receptor-activated PI 3-kinase is associated with an increase in active Ras-GTP and activation of tyrosine protein phosphorylation. These pathways may contribute to alpha1 receptor-stimulated mitogenic responses including activation of MAP kinase and DNA synthesis in HVSMCs.
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PMID:Alpha1 adrenergic receptors activate phosphatidylinositol 3-kinase in human vascular smooth muscle cells. Role in mitogenesis. 862 43

Okadaic acid has been described previously as being a negative regulator of insulin signaling, as it inhibits insulin stimulation of glucose transport. In addition, this drug induces on insulin receptor substrate-1 (IRS-1) a decrease in tyrosine phosphorylation, concomitantly with an increase in serine/threonine phosphorylation. The present work was aimed at the identification of the serine/threonine residues that, upon phosphorylation, might be involved in modulating insulin signaling. To this end, we studied double-point mutants of IRS-1, in which serines 612/632 and 662/731 were replaced with alanine. These are four plausible sites of phosphorylation by mitogen-activated protein kinases and are in the immediate proximity of tyrosine residues, which are potential sites of interaction with phosphatidylinositol 3-kinase Src homology 2 domains. Using transient expression in 293 EBNA cells, we demonstrate that serines 612, 632, 662, and 731 and mitogen-activated protein kinases are not involved in the okadaic acid effect on IRS-1. Rather, these serines appear to play a role in modulating basal and insulin-stimulated IRS-1 tyrosine phosphorylation, association of IRS-1, with p85, and phosphatidylinositol 3-kinase activity in the IRS-1.p85 immune complex, since mutation of these sites enhances these events. Our findings suggest the existence of an IRS-1 desensitization mechanism resulting from serine/threonine phosphorylation, occurring at least on serines 612, 632, 662, and 731.
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PMID:Phosphorylation of insulin receptor substrate-1 on multiple serine residues, 612, 632, 662, and 731, modulates insulin action. 862 71


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