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:2.7.12.2 (MEK)
18,161 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

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

Overexpression of the transmembrane protein-tyrosine phosphatase (PTPase) CD45 in nonhematopoietic cells results in decreased signaling through growth factor receptor tyrosine kinases. Consistent with these data, insulin receptor signaling is increased when the CD45-related PTPase LAR is reduced by antisense suppression in a rat hepatoma cell line. To test whether the hematopoietic cell-specific PTPase CD45 functions in a manner similar to LAR by negatively modulating insulin receptor signaling in hematopoietic cells, the insulin-responsive human multiple myeloma cell line U266 was isolated into two subpopulations that differed in CD45 expression. In CD45 nonexpressing (CD45-) cells, insulin receptor autophosphorylation was increased by 3-fold after insulin treatment when compared to CD45 expressing (CD45+) cells. This increase in receptor autophosphorylation was associated with similar increases in insulin-dependent tyrosine kinase activation. These receptor level effects were paralleled by postreceptor responses. Insulin-dependent tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) and Shc was 3-fold greater in CD45- cells. In addition, insulin-dependent IRS-1/phosphatidylinositol 3-kinase association and MAP kinase activation in CD45- cells were also 3-fold larger. While expression of CD45 was associated with a decrease in the responsiveness of early insulin receptor signaling, interleukin 6-dependent activation of mitogen-activated protein kinase kinase and mitogen-activated protein kinase was equivalent between CD45- and CD45+ cells. These observations indicate that CD45 can function as a negative modulator of growth factor receptor tyrosine kinases in addition to its well-established role as an activator of src family tyrosine kinases.
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PMID:The transmembrane protein-tyrosine phosphatase CD45 is associated with decreased insulin receptor signaling. 855 83

Elevated glucose concentrations have been reported to inhibit insulin receptor kinase activity. We studied the effects of high glucose on insulin action in Rat1 fibroblasts transfected with wild-type human insulin receptor (HIRcB) and a truncated receptor lacking the COOH-terminal 43 amino acids (delta CT). In both cell lines, 25 mM glucose impaired receptor and insulin receptor substrate-1 phosphorylation by 34%, but IGF-1 receptor phosphorylation was unaffected. Phosphatidylinositol 3-kinase activity and bromodeoxyuridine uptake were decreased by 85 and 35%, respectively. This was reversed by coincubation with a protein kinase C (PKC) inhibitor or microinjection of a PKC inhibitor peptide. Phosphopeptide mapping revealed that high glucose or PMA led to serine/threonine phosphorylation of similar peptides. Inhibition of the microtubule-associated protein (MAP) kinase cascade by the MAP kinase kinase inhibitor PD98059 did not reverse the impaired phosphorylation. We conclude that high glucose inhibits insulin action by inducing serine phosphorylation through a PKC-mediated mechanism at the level of the receptor at sites proximal to the COOH-terminal 43 amino acids. This effect is independent of activation of the MAP kinase cascade. Proportionately, the impairment of insulin receptor substrate-1 tyrosine phosphorylation is greater than that of the insulin receptor resulting in attenuated phosphatidylinositol 3-kinase activation and mitogenic signaling.
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PMID:Glucose-induced phosphorylation of the insulin receptor. Functional effects and characterization of phosphorylation sites. 860 15

The insulin receptor substrate-1 (IRS-1) is the major intracellular substrate of insulin and insulin-like growth factor-I (IGF-I) receptor tyrosine kinase activity, and this protein has been found to be overexpressed in human hepatocellular carcinomas. IRS-1 contains several src homology 2 (SH2) binding motifs that interact following tyrosyl phosphorylation with SH2-containing proteins, and this interaction may be essential for transmitting the growth signal from the cell surface to the nucleus. We have previously reported that overexpression of IRS-1 may induce neoplastic transformation of NIH 3T3 cells. This study examines the role of two SH2-containing molecules, namely the Grb2 adapter and Syp tyrosine phosphatase proteins as important components of the cellular transforming activity of IRS-1. Mutations of tyrosine 897 in the YVNI motif (Y897F) and of tyrosine 1180 in the YIDL motif (Y1180F) reduced the intracellular interaction of IRS-1 with Grb2 and Syp proteins, respectively. Furthermore, a single mutation at either Phe-897 or Phe-1180 substantially but not completely reduced IGF-I-dependent transforming activity of IRS-1, whereas creation of a double mutation of both tyrosine residues (Y897F/Y1180F) strikingly attenuated the transforming activity of IRS-1. Stable expression of the IRS-1 mutant constructs in NIH 3T3 cells was associated with a lower level of activation of the mitogen-activated protein kinase kinase (MAPKK)/MAPK cascade following IGF-I stimulation compared with cells stably transfected with the "wild-type" IRS-1 gene. These results suggest that IRS-1-induced cellular transformation requires an interaction with both Grb2 and Syp signal transduction molecules since neither interaction alone appears to be required, and this event subsequently leads to activation of the MAPKK/MAPK cascade.
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PMID:Neoplastic transformation induced by insulin receptor substrate-1 overexpression requires an interaction with both Grb2 and Syp signaling molecules. 866 27

Tumor necrosis factor-alpha (TNF-alpha) is a proposed mediator of insulin resistance in obese/diabetic animals through its effects on tyrosine phosphorylation of the insulin receptor and its substrate, insulin receptor substrate-1. In this study, the acute effects of TNF-alpha on the mitogen-activated protein kinase (MAPK) signalling cascade were examined in cultured rat skeletal muscle cell line, L6. Insulin treatment of L6 cells resulted in a rapid increase in MAPK activity (> twofold in 5 min with 10 nM insulin). Prior treatment with TNF-alpha for 60 min blocked subsequent insulin-induced activation of MAPK in a dose- and time-dependent manner. Metabolic labelling studies with inorganic [32P]phosphate followed by immuno-precipitation of MAPK and its upstream activator, mitogen-activated protein kinase kinase, indicated decreased phosphorylation of MAPK and its kinase in response to insulin in cells exposed to TNF-alpha. This effect of TNF-alpha was not due to inhibition of insulin-stimulated p21ras-GTP loading or Raf-1 phosphorylation. Low concentrations (2 nM) of okadaic acid, a serine/threonine phosphatase inhibitor, prevented TNF-alpha-induced inhibition of MAPK and restored insulin's effect on MAPK activity, while orthovanadate (a tyrosine phosphatase inhibitor), inhibitor 2 (phosphatase-1 inhibitor) and FK506 (phosphatase-2B inhibitor) were ineffective. These results suggested an involvement of an okadaic-acid-sensitive serine/threonine phosphatase in TNF-alpha-induced blockade of insulin's effect on MAPK and/or its kinase. Therefore, we examined the effect of TNF-alpha on protein phosphatase-1 (PP-1) and protein phosphatase-2A (PP-2A) activities. As reported by us earlier, insulin rapidly stimulated PP-1 and concomitantly inhibited PP-2A activities in control cells. TNF-alpha treatment blocked insulin-induced activation of PP-1. In contrast to PP-1, TNF-alpha caused a 60% increase in PP-2A activity and insulin failed to prevent this TNF-alpha effect. The time course of PP-2A activation by TNF-alpha preceded the kinetics of inhibition of MAPK. Cell-permeable ceramide analogs mimicked the TNF-alpha effect on MAPK inhibition and PP-2A activation. We conclude that TNF-alpha abrogates the insulin effect on MAPK activation by increasing dephosphorylation of MAPK kinase via an activated phosphatase.
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PMID:Effect of tumor necrosis factor-alpha on insulin-stimulated mitogen-activated protein kinase cascade in cultured rat skeletal muscle cells. 866 40

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

Many studies suggest that insulin utilizes multiple signal transduction pathways. Insulin's effects are initiated by insulin binding to the insulin receptor, resulting in tyrosine phosphorylation of insulin receptor and intracellular substrates, such as insulin receptor substrate-1 (IRS-1), IRS-2, or Shc. We recently demonstrated that immediate-early gene egr-1 transcription was fully induced without phosphorylation of IRS-1 in Chinese hamster ovary cells (Harada, S., Smith, R. M., Smith, J. A., Shah, N. , Hu, D.-Q. & Jarett, L. (1995) J. Biol. Chem. 270, 26632-26638). In the present study, we examined the effects of insulin on immediate-early gene egr-1 and c-fos expression in 32D cells overexpressing the insulin receptor (32D/IR), IRS-1 (32D/IRS), or both (32D/IR+IRS) and compared these effects with insulin-induced tyrosine phosphorylation. Insulin (17 nM) increased egr-1 and c-fos expression in 32D/IR and 32D/IR+IRS cells, but not in parental cells or 32D/IRS cells, as determined by Northern blot analysis. Insulin treatment (5 min at 37 degrees C) markedly increased tyrosine phosphorylation of several proteins, including the insulin receptor, IRS-1, and Shc, in 32D/IR+IRS cells as determined by immunoprecipitation and Western blot analysis with anti-phosphotyrosine antibody. In contrast, only two tyrosine-phosphorylated proteins, i.e. insulin receptor and Shc, were detected in 32D/IR cells. These data suggest that insulin receptor and Shc phosphorylation is necessary for insulin-induced egr-1 and c-fos expression, but IRS-1 phosphorylation is not necessary or sufficient for the expression of these genes. Furthermore, the effect of specific inhibitors on insulin-induced egr-1 expression was examined. Wortmannin (25 nM), a phosphatidylinositol 3-kinase inhibitor, had no effect on insulin-induced egr-1 expression. In contrast, PD 98059 (30 microM), a mitogen-activated protein kinase kinase inhibitor, totally blocked egr-1 expression induced by insulin. These data indicate that mitogen-activated protein kinase activation, but not phosphatidylinositol 3-kinase activation, is involved in insulin-induced egr-1 expression. Taken together, insulin receptor tyrosine phosphorylation, Shc tyrosine phosphorylation, and mitogen-activated protein kinase activation appear to be the signal transduction pathway responsible for insulin-induced egr-1 expression in 32D cells. These data demonstrate that insulin has multiple signal transduction pathways that vary from cell to cell.
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PMID:Insulin-induced egr-1 and c-fos expression in 32D cells requires insulin receptor, Shc, and mitogen-activated protein kinase, but not insulin receptor substrate-1 and phosphatidylinositol 3-kinase activation. 893 74

The mitogen-activated protein kinase (MAP kinase) is a key participant in growth factor-stimulated intracellular events such as proliferation and differentiation. We and others have previously described a cross-talk between the MAP kinase pathway and the cAMP pathway. Indeed, in several cell lines and, in particular in fibroblasts, an increase in the level of cAMP produced an inhibition of MAP kinase together with decreased cell proliferation. In contrast, in PC12 cells, cAMP induced an increase in the NGF-induced activation of MAP kinase concomitantly with augmented NGF-induced differentiation. Therefore, it has been proposed that the cellular context is important for the nature of the cAMP effects on growth factor-stimulated MAP kinase activity. Here we show that the type of tyrosine kinase receptor stimulated also participates in the nature of the cAMP effect. Thus, in NIH3T3 fibroblasts expressing NGF receptors (NIH3T3/trk cells) we found that cAMP potentiates NGF-stimulated ERK1 and MEK1 activities, whereas in NIH3T3 fibroblasts expressing insulin receptors (NIH3T3/IR cells) we saw no effect of cAMP on the activation of insulin-stimulated ERK1 and MEK1. In PC12 cells and in Rat1 fibroblasts expressing insulin receptors (PC12/IR and Rat1/IR cells) we observed, respectively, a potentiation and an inhibition of insulin-stimulated ERK1 activity. In addition, cAMP does not seem to modify the basal nor growth factor-stimulated She or IRS-1 tyrosine phosphorylation in the different cell lines studied. Finally, we observed that cAMP inhibited serum- and insulin-induced, but not NGF-induced, cell proliferation in NIH3T3 cells. However, cAMP potentiated insulin-stimulated cell differentiation in PC12/IR cells. These results led us to conclude that the cAMP effect on cell proliferation in NIH3T3 fibroblasts and PC12/IR cells appears to be correlated, in part, with the effect of cAMP on the MAP kinase pathway, but by itself this pathway cannot fully account for these observations.
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PMID:The effect of cyclic adenosine monophosphate on the mitogen-activated protein kinase pathway depends on both the cell type and the type of tyrosine kinase-receptor. 904 17

Insulin acts on its target tissues by specific interaction with the cell surface insulin receptor (IR). The IR possesses an intrinsic tyrosine kinase (TK) activity which is stimulated by insulin binding. This TK activity is required for many aspects of insulin signalling. We had earlier reported that human plasma alpha 2-HS glycoprotein (alpha 2-HSG) inhibits insulin-stimulated mitogenesis at the level of IR-TK (Mol Endo 7: 1445-1455, 1993). In the present study, using recombinant alpha 2-HSG, which possesses 50-100 times the specific activity of plasma alpha 2-HSG, we have further investigated the molecular basis of this effect. We examined the insulin-stimulated Ras signalling pathway in Chinese Hamster Ovary cells overexpressing the human IR. alpha 2-HSG inhibits insulin-induced tyrosine phosphorylation of IRS-1 and the subsequent association of GRB2, as well as Sos, with IRS-1. This inhibition results in reduced guanine nucleotide exchange in p21ras. alpha 2-HSG also inhibits the stimulation of Raf phosphorylation, in response to insulin, leading to inhibition of MEK activity. In a parallel pathway, alpha 2-HSG also inhibits insulin-induced tyrosine phosphorylation of Shc. However, alpha 2-HSG does not affect any of the metabolic actions of insulin rested in these cells. These results suggest that, while insulin's mitogenic effects can be abolished by inhibition of insulin-induced IR-TK, propagation of signals for metabolic activities might utilize alternate of rescue mechanisms.
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PMID:Recombinant human alpha 2-HS glycoprotein inhibits insulin-stimulated mitogenic pathway without affecting metabolic signalling in Chinese hamster ovary cells overexpressing the human insulin receptor. 911 49


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