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)

We have studied the function of a mutant human insulin receptor in which two COOH-terminal autophosphorylation sites (Tyr-1316 and -1322) were replaced by phenylalanine (F/Y COOH-terminal 2 tyrosines (CT2)). In addition, we have also constructed a mutant receptor in which Lys-1018 in the ATP-binding site was changed to arginine (R/K 1018). Both the wild type insulin receptor (HIR) and the mutant receptors were expressed in Chinese hamster ovary (CHO) cells by stable transfection. Autophosphorylation of solubilized and partially purified F/Y CT2 was decreased by approximately 30% compared with the HIR. Tyrosine kinase activities of F/Y CT2 and HIR toward exogenous substrates were almost equal. When CHO cells transfected with F/Y CT2 (CHO-F/Y CT2) were stimulated with insulin, autophosphorylation of the beta-subunit of the insulin receptor and the phosphorylation of an endogenous substrate (pp185) in the intact cell were normal compared with cells expressing HIR (CHO-HIR). CHO-F/Y CT2 exhibited the same insulin sensitivity as CHO-HIR with respect to 2-deoxyglucose uptake. However, the dose-response curve of insulin-stimulated thymidine incorporation in CHO-F/Y CT2 was shifted to the left (approximately 5-7-fold) compared with that in CHO-HIR. There was no significant difference in insulin-like growth factor 1-stimulated thymidine incorporation between CHO-F/Y CT2 and CHO-HIR. Furthermore, the dose-response curve of insulin-stimulated kinase activity toward myelin basic protein in CHO-F/Y CT2 was also shifted to the left (approximately 5-fold) compared with that in CHO-HIR. Kinase assays in myelin basic protein-containing gels revealed that both species of MAP kinases (M(r) 44,000, 42,000) were more sensitive to activation by insulin in CHO-F/Y CT2 than in CHO-HIR. This observation was confirmed in immune complex kinase assays toward microtubule-associated protein 2 (MAP2) using specific antibodies against mitogen-activated protein (MAP) kinase. R/K 1018 mutant insulin receptors showed an absence of insulin-stimulated kinase activity and CHO cells transfected with R/K 1018 (CHO-R/K 1018) failed to enhance 2-deoxyglucose uptake or thymidine incorporation in response to insulin. In addition, R/K 1018 kinase-defective insulin receptors were unable to mediate insulin-stimulated MAP kinase activation. These data suggest that: 1) tyrosine kinase activity of the insulin receptor is required for activation of insulin-stimulated MAP kinases and 2) phosphorylation of COOH-terminal tyrosine residues may play an inhibitory role in mitogenic signaling through regulation of MAP kinases.
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PMID:Enhanced insulin-induced mitogenesis and mitogen-activated protein kinase activities in mutant insulin receptors with substitution of two COOH-terminal tyrosine autophosphorylation sites by phenylalanine. 161 80

Transgenic mice which overexpress kinase-deficient human insulin receptors in muscle were used to study the relationship between insulin receptor tyrosine kinase and the in vivo activation of several downstream signaling pathways. Intravenous insulin stimulated insulin receptor tyrosine kinase activity by 7-fold in control muscle versus < or = 1.5-fold in muscle from transgenic mice. Similarly, insulin failed to stimulate tyrosyl phosphorylation of receptor beta-subunits or insulin receptor substrate 1 (IRS-1) in transgenic muscle. Insulin substantially stimulated IRS-1-associated phosphatidylinositol (PI) 3-kinase in control versus absent stimulation in transgenic muscles. In contrast, insulin-like growth factor 1 modestly stimulated PI 3-kinase in both control and transgenic muscle. The effects of insulin to stimulate p42 mitogen-activated protein kinase and c-fos mRNA expression were also markedly impaired in transgenic muscle. Specific immunoprecipitation of human receptors followed by measurement of residual insulin receptors suggested the presence of hybrid mouse-human heterodimers. In contrast, negligible hybrid formation involving insulin-like growth factor 1 receptors was evident. We conclude that (i) transgenic expression of kinase-defective insulin receptors exerts dominant-negative effects at the level of receptor auto-phosphorylation and kinase activation; (ii) insulin receptor tyrosine kinase activity is required for in vivo insulin-stimulated IRS-1 phosphorylation, IRS-1-associated PI 3-kinase activation, phosphorylation of mitogen-activated protein kinase, and c-fos gene induction in skeletal muscle; (iii) hybrid receptor formation is likely to contribute to the in vivo dominant-negative effects of kinase-defective receptor expression.
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PMID:Impaired insulin signaling in skeletal muscles from transgenic mice expressing kinase-deficient insulin receptors. 775 7

Insulin receptor substrate 1 (IRS-1) mediates the activation of a variety of signaling pathways by the insulin and insulin-like growth factor 1 receptors by serving as a docking protein for signaling molecules with SH2 domains. We and others have shown that in response to insulin stimulation IRS-1 binds GRB2/Sos and have proposed that this interaction is important in mediating Ras activation by the insulin receptor. Recently, it has been shown that the interleukin (IL)-4 receptor also phosphorylates IRS-1 and an IRS-1-related molecule, 4PS. Unlike insulin, however, IL-4 fails to activate Ras, extracellular signal-regulated kinases (ERKs), or mitogen-activated protein kinases. We have reconstituted the IL-4 receptor into an insulin-responsive L6 myoblast cell line and have shown that IRS-1 is tyrosine phosphorylated to similar degrees in response to insulin and IL-4 stimulation in this cell line. In agreement with previous findings, IL-4 failed to activate the ERKs in this cell line or to stimulate DNA synthesis, whereas the same responses were activated by insulin. Surprisingly, IL-4's failure to activate ERKs was not due to a failure to stimulate the association of tyrosine-phosphorylated IRS-1 with GRB2/Sos; the amounts of GRB2/Sos associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. Moreover, the amounts of phosphatidylinositol 3-kinase activity associated with IRS-1 were similar in insulin- and IL-4-stimulated cells. In contrast to insulin, however, IL-4 failed to induce tyrosine phosphorylation of Shc or association of Shc with GRB2. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Thus, ERK activation correlates with Shc tyrosine phosphorylation and formation of an Shc/GRB2 complex. Previous studies have indicated that activation of ERks in this cell line is dependent upon Ras since a dominant-negative Ras (Asn-17) blocks ERK activation by insulin. Our findings, taken in the context of previous work, suggest that binding of GRB2/Sos to Shc may be the predominant mechanism whereby insulin as well as cytokine receptors activate Ras.
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PMID:Association between GRB2/Sos and insulin receptor substrate 1 is not sufficient for activation of extracellular signal-regulated kinases by interleukin-4: implications for Ras activation by insulin. 786 67

As previous studies showed, PDGF-AA exerts a poor mitogenic effect on vascular smooth muscle cells. Simultaneous addition of insulin-like growth factor 1 (IGF-1), itself also poorly mitogenic, led to a significant increase in [3H]thymidine incorporation into the cell DNA as well as a strong increase in cell number. To explain the synergistic effect of PDGF-AA and IGF-1 on VSMC proliferation, we describe the effects of the two growth factors on distinct intracellular signals: on the activation of the signal proteins mitogen-activated protein kinase (MAPK) isoforms p42 and p44 and on the protein kinase C (PKC) isoforms alpha, delta, and epsilon, and on the induction of the transcription factor c-fos. PDGF-AA strongly activated the MAPK isoforms and PKC delta as well as the induction of c-fos. In contrast, IGF-1 exerted no effect on the signals induced by PDGF-AA, but strongly activated PKC epsilon isoform. Comparing this signal pattern to the one of the mitogenically potent PDGF isoform PDGF-BB, we found that PDGF-BB activated all of the signal proteins investigated.
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PMID:The synergistic effect of PDGF-AA and IGF-1 on VSMC proliferation might be explained by the differential activation of their intracellular signaling pathways. 866 Sep 39

It is now well-recognized that the mitogen-activated protein (MAP) kinase cascade facilitates signaling from an activated tyrosine kinase receptor to the nucleus. In fact, an increasing number of extracellular effectors have been reported to activate the MAP kinase cascade, with a significant number of cellular responses attributed to this activation. We set out to explore how two extracellular effectors, basic fibroblast growth factor (bFGF) and insulin-like growth factor 1 (IGF-1), which have both been reported to activate MAP kinase, generate quite distinct cellular responses in C2C12 myoblasts. We demonstrate here that bFGF, which is both a potent mitogen and inhibitor of myogenic differentiation, is a strong MAP kinase agonist. By contrast, IGF-1, which is equally mitogenic for C2C12 cells but ultimately enhances the differentiated phenotype, is a weak activator of the MAP kinase cascade. We further demonstrate that IGF-1 is a potent activator of both insulin receptor substrate IRS-1 tyrosyl phosphorylation and association of IRS-1 with activated phosphatidylinositol 3-kinase (PI 3-kinase). Finally, use of the specific MAP kinase kinase inhibitor, PD098059, and wortmannin, a PI 3-kinase inhibitor, suggests the existence of an IGF-1-induced, MAP kinase-independent signaling event which contributes to the mitogenic response of this factor, whereas bFGF-induced mitogenesis appears to strongly correlate with activation of the MAP kinase cascade.
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PMID:Stimulation of C2C12 myoblast growth by basic fibroblast growth factor and insulin-like growth factor 1 can occur via mitogen-activated protein kinase-dependent and -independent pathways. 888 26

The role of insulin-like growth factor 1 (IGF-1) in preventing apoptosis was examined in differentiated PC12 cells. Induction of differentiation was achieved using nerve growth factor, and apoptosis was provoked by serum withdrawal. After 4-6 h of serum deprivation, apoptosis was initiated, concomitant with a 30% decrease in cell number and a 75% decrease in MTT activity. IGF-1 was capable of preventing apoptosis at concentrations as low as 10(-9) M and as early as 4 h. The phosphatidylinositol 3' (PI3')-kinase inhibitors wortmannin (at concentrations of 10(-8) M) and LY294002 (10(-6) M) blocked the effect of IGF-1. The pp70 S6 kinase (pp70S6K) inhibitor rapamycin (10(-8) M) was, however, less effective in blocking IGF-1 action. Moreover, stable transfection of a dominant-negative p85 (subunit of PI3'-kinase) construct in PC12 cells enhanced apoptosis provoked by serum deprivation. Interestingly, in the cells overexpressing the dominant-negative p85 protein, IGF-1 was still capable of inhibiting apoptosis, suggesting the existence of a second pathway involved in the IGF-1 effect. Blocking the mitogen-activated protein kinase pathway with the specific mitogen-activated protein kinase/extracellular-response kinase kinase inhibitor PD098059 (10(-5) M) inhibited the IGF-1 effect. When wortmannin and PD098059 were given together, the effect was synergistic. The results presented here suggest that IGF-1 is capable of preventing apoptosis by activation of multiple signal transduction pathways.
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PMID:Insulin-like growth factor 1 inhibits apoptosis using the phosphatidylinositol 3'-kinase and mitogen-activated protein kinase pathways. 899 41

Xenopus postvitellogenic oocytes resume meiosis in vitro upon exposure to insulin or insulin-like growth factor 1 (IGF-1) via a ras-dependent pathway, whereas stage IV (600 micron < diameter < 1000 micron) oocytes cannot. The aim of the present study was to determine which event(s) of the transduction pathway from IGF-1 receptor to maturation-promoting factor (MPF) activation is deficient in the small, vitellogenic, oocytes to explain their inability to undergo germinal vesicle breakdown (GVB) after insulin treatment. We thus analyzed the effect of insulin on the Ras/Raf-dependent mitogen-activated protein kinase cascade because of its crucial role prior to MPF activation. The effect of insulin on pp39mos synthesis in stage IV oocytes was also studied since this protein kinase participates in the mitogen-activated protein kinase (MAPK) pathway as a MAPKK kinase like Raf. Contrary to what is observed in postvitellogenic oocytes, MAPK was not activated in insulin-treated stage IV oocytes even 20 hr after the stimulation. This was not caused by the absence of MAPK activators like MEK (MAPKK), Raf, or Ras, but rather by the inability of insulin to activate Ras. Interestingly, injection of constitutively active raf mRNA as well as oncogenic Ras protein, Ha-Ras lys12, in stage IV oocytes resulted in MAPK activation, whereas neither Mos accumulation nor GVB occurred, suggesting that the Ras --> Raf --> MAPKK --> MAPK cascade was functional but that MAPK activation alone was not sufficient for the mitogenic signal to proceed further down in the pathway leading to MPF activation. Treatment of stage IV oocytes with insulin did not stimulate Mos synthesis either, indicating a dysfunction in the "Mos synthesis machinery." The present results show that incompetence of Xenopus stage IV oocytes to activate MPF in response to insulin is primarily due to the inability of the peptide to activate Ras and to stimulate pp39mos synthesis and secondarily to a deficiency in the mitogenic pathway that connects MAPK to MPF activation.
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PMID:Comparative effects of insulin on the activation of the Raf/Mos-dependent MAP kinase cascade in vitellogenic versus postvitellogenic Xenopus oocytes. 924 17

In the present study, we showed that Chinese hamster ovary (CHO) cells transfected with human central cannabinoid receptor (CB1) exhibit high constitutive activity at both levels of mitogen-activated protein kinase (MAPK) and adenylyl cyclase. These activities could be blocked by the CB1-selective ligand, SR 141716A, that functions as an inverse agonist. Moreover, binding studies showed that guanine nucleotides decreased the binding of the agonist CP-55,940, an effect usually observed with agonists, whereas it enhanced the binding of SR 141716A, a property of inverse agonists. Unexpectedly, we found that CB1-mediated effects of SR 141716A included inhibition of MAPK activation by pertussis toxin-sensitive receptor-tyrosine kinase such as insulin or insulin-like growth factor 1 receptors but not by pertussis toxin-insensitive receptor-tyrosine kinase such as the fibroblast growth factor receptor. We also observed similar results when cells were stimulated with Mas-7, a mastoparan analog, that directly activates the Gi protein. Furthermore, SR 141716A inhibited guanosine 5'-0-(thiotriphosphate) uptake induced by CP-55,940 or Mas-7 in CHO-CB1 cell membranes. This indicates that, in addition to the inhibition of autoactivated CB1, SR 141716A can deliver a biological signal that blocks the Gi protein and consequently abrogates most of the Gi-mediated responses. By contrast, SR 141716A had no effect on MAPK activation by insulin or IGF1 in CHO cells lacking CB1 receptors, ruling out the possibility of a direct interaction of SR 141716A with the Gi protein. This supports the notion that the Gi protein may act as a negative intracellular signaling cross-talk molecule. From these original results, which considerably enlarge the biological properties of the inverse agonist, we propose a novel model for receptor/ligand interactions.
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PMID:A selective inverse agonist for central cannabinoid receptor inhibits mitogen-activated protein kinase activation stimulated by insulin or insulin-like growth factor 1. Evidence for a new model of receptor/ligand interactions. 926 84

We have compared the influence of insulin-like growth factor 1 (IGF-1) on pituitary gene expression in the rat cell lines GH4C1 and GH3. Incubation with IGF-1 increased PRL messenger RNA (mRNA) levels in GH4C1 cells by 4- to 5-fold but decreased the levels of PRL transcripts in GH3 cells. In addition, the levels of GH-mRNA that were not affected by IGF-1 in GH4C1 cells were significantly inhibited by the growth factor in GH3 cells. IGF-1 also decreased PRL and GH-mRNA response to T3, retinoic acid, and Fk in GH3 cells. Stability of PRL or GH transcripts was not altered by IGF-1 in GH3 cells, suggesting that the inhibitory effect is exerted at a transcriptional level. The pituitary-specific transcription factor GHF-1/Pit-1 activates both the GH and PRL promoters. As analyzed by Western blot, IGF-1 did not alter GHF-1/Pit-1 protein levels in GH4C1 cells but reduced the levels of the transcription factor in GH3 cells. This decrease is secondary to a reduction of GHF-1/Pit-1 transcripts in IGF-1-treated GH3 cells. Thus, a different effect of IGF-1 on the expression of GHF-1/Pit-1 in GH3 and GH4C1 cells is likely involved in the different regulation of GH and PRL gene in both cell types. IGF-1 increases the activity of the PRL promoter in transient transfection assays in GH4C1 cells by a Ras-dependent mechanism. Expression of oncogenic Ras(Val12) mimics the effect of IGF-1, and the dominant negative Ras(Asn17) blocks IGF-1-mediated stimulation of the PRL promoter in GH4C1 cells. Although IGF-1 did not stimulate the PRL promoter in GH3 cells, Ras(Val12) strongly activated the promoter in these cells. Hence, the machinery to activate Ras-dependent signaling is intact in GH3 cells. Moreover, IGF-1 stimulates the mitogen-activated protein kinase in GH3 cells, showing that the components linking the IGF-1 receptor to Ras are also active. These results suggest that, in addition to the Ras/mitogen-activated protein kinase pathway, IGF-1 could activate a different pathway and that the combination of both is required to elicit PRL gene expression by the growth factor. This second pathway may be defective in GH3 cells that respond to Ras but not to IGF-1.
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PMID:Differential regulation of pituitary-specific gene expression by insulin-like growth factor 1 in rat pituitary GH4C1 and GH3 cells. 938 30

Both insulin-like growth factor 1 (IGF-1) and fibroblast growth factor 2 (FGF-2) are key modulators of skeletal myoblast differentiation. The critical signaling pathways used by either IGF-1 or FGF-2 to inhibit differentiation have not been determined. In this study, we show that both IGF-1 and FGF-2 inhibit the differentiation of 23A2 myoblasts and that both stimulate signaling through mitogen-activated protein kinase (MAPK) kinase (MEK) to MAPK roughly 8-fold in 23A2 myoblasts. We used the selective chemical inhibitor of MEK, PD 098059, to determine if signaling by MEK is required by IGF-1 or FGF-2 to inhibit differentiation. PD 098059 did not affect the ability of 23A2 myoblasts to differentiate. Addition of PD 098059 to the culture medium 10 min before the addition of IGF-1 or FGF-2 completely blocked the signal from MEK to MAPK and restored the ability of the 23A2 myoblasts to differentiate in the presence of either IGF-1 or FGF-2. The peak of signaling through MEK to MAPK in response to either IGF-1 or FGF-2 occurred within the first hour with maximal activation observed after 10 min. This signal remained elevated (at roughly 70% above basal) for at least 48 h. PD 098059 was added to the culture 60 min after IGF-1 or FGF-2 to test whether this initial peak of signaling was sufficient for the inhibition of differentiation. The restoration of myogenic potential seen when cells were preincubated with PD 098059 was essentially identical to that seen when PD 098059 was added to cultures after the initial peak of signaling from MEK to MAPK, suggesting that persistent signaling through MEK is required for the inhibition of differentiation by either IGF-1 or FGF-2.
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PMID:Mitogen-activated protein kinase kinase (MEK) activity is required for inhibition of skeletal muscle differentiation by insulin-like growth factor 1 or fibroblast growth factor 2. 952 64


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