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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)

Exposure of 3T3-L1 cells to insulin stimulates a soluble, serine(threonine)-specific protein kinase that phosphorylates microtubule-associated protein 2 (MAP-2) in vitro. The enzyme, termed MAP kinase, was isolated from insulin-treated or control cells radiolabeled with 32Pi. A 40-kDa phosphoprotein was found to elute in exact correspondence with enzymatic activity during hydrophobic interaction and gel filtration chromatography of extracts from cells stimulated with insulin. Both MAP kinase activity and the phosphoprotein were absent in fractions prepared from untreated cells. The 32P incorporated into the 40-kDa protein was stable during treatment with alkali. Phospho amino acid analysis confirmed that the radiolabel was primarily incorporated into phosphotyrosine and to a lesser extent phosphothreonine. In addition, MAP kinase was incompletely but specifically adsorbed by antibodies to phosphotyrosine. We conclude, based on these data and additional studies from this laboratory, that MAP kinase is phosphorylated on tyrosine in vivo. The data are consistent with the possibility that MAP kinase may be a substrate for the insulin receptor or another insulin-regulated tyrosine kinase.
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PMID:Insulin-stimulated microtubule-associated protein kinase is phosphorylated on tyrosine and threonine in vivo. 328 75

AG-18, an inhibitor of protein-tyrosine kinases, was employed to study the role of tyrosine-phosphorylated proteins in insulin- and phorbol ester-induced signaling cascades. When incubated with Chinese hamster ovary cells overexpressing the insulin receptor, AG-18 reversibly inhibited insulin-induced tyrosine phosphorylation of insulin receptor substate-1, with minimal effects either on receptor autophosphorylation or on phosphorylation of Shc64. Under these conditions, AG-18 inhibited insulin-stimulated phosphorylation of the ribosomal protein S6, while no inhibition of insulin-induced activation of mitogen-activated protein kinase (MAPK) kinase or MAPK was detected. In contrast, 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced activation of MAPK kinase and MAPK and phosphorylation of S6 were inhibited by AG-18. This correlated with inhibition of TPA-stimulated tyrosine phosphorylation of several proteins, the most prominent ones being pp114 and pp120. We conclude that Tyr-phosphorylated insulin receptor substrate-1 is the main upstream regulator of insulin-induced S6 phosphorylation by p70s6k, whereas MAPK signaling seems to be activated in these cells primarily through the adaptor molecule Shc. In contrast, TPA-induced S6 phosphorylation is mediated by the MAPK/p90rsk cascade. A key element of this TPA-stimulated signaling pathway is an AG-18-sensitive protein-tyrosine kinase.
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PMID:Differential activation of mitogen-activated protein kinase and S6 kinase signaling pathways by 12-O-tetradecanoylphorbol-13-acetate (TPA) and insulin. Evidence for involvement of a TPA-stimulated protein-tyrosine kinase. 749 32

We studied a patient with severe insulin resistance and a remarkable decrease in the in vivo autophosphorylation of the insulin receptor. Using a polymerase chain reaction-single strand conformation polymorphism method and direct sequencing, we identified a heterozygous mutation substituting Gln for Arg1131 in the putative "catalytic loop" of the tyrosine kinase domain of the insulin receptor gene. The Gln1131 mutant receptor was expressed by transfection in Chinese hamster ovary cells and compared with cells expressing the wild-type insulin receptor. Both mutant and wild-type receptors were expressed on the cell surface and displayed similar insulin-binding affinity. The Gln1131 mutation impaired the activity of the receptor tyrosine kinase and inhibited the ability of insulin to phosphorylate the endogenous substrate insulin receptor substrate-I. In addition, the Gln1131 mutant receptor exhibited diminished tyrosine-phosphorylated phosphatidylinositol 3-kinase and myelin basic protein kinase activities compared with the wild-type cells. It also demonstrated a defective mediation of the insulin signal stimulating 2-deoxy-D-glucose transport and thymidine incorporation, resistance to endocytosis, and insulin-induced down-regulation. Unlike a previously described mutation in the putative catalytic loop of the receptor that substituted Glu for Ala1135, the Gln1131 mutation retained proteolytic cleavage of the proreceptor into separate subunits. Our results demonstrate that a naturally occurring mutation (R1131Q) in the putative catalytic loop of the insulin receptor results in severe impairment of the tyrosine kinase function in our patient. In addition, our results indicate that Arg1131 is important for receptor-mediated insulin action in vivo and suggest that the amino acids constituting the catalytic loop of protein kinases may possess different modes in order to retain kinase function.
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PMID:Substitution of glutamine for arginine 1131. A newly identified mutation in the catalytic loop of the tyrosine kinase domain of the human insulin receptor. 751 63

Signal transduction pathways stimulated by insulin or insulin-like growth factor-I (IGF-I) were compared in transfected NIH3T3 fibroblast cell lines expressing the human insulin receptor, IGF-I receptor, or a chimeric IGF-I receptor with its carboxy-terminal tail replaced with that of the insulin receptor (approximately 1 x 10(6) receptors/cell). Although receptor autophosphorylation was very similar in the three cell lines overexpressing receptors (EC50 = 1-3 nM), there were differences detected in the protein tyrosine phosphorylation stimulated by insulin and IGF-I in these cells. Although no substrates specific for the insulin receptor were detected, phosphorylation of a 170-kilodalton (kDa; IRS-1) and a 70-kDa protein was 10 times more sensitive to insulin than to IGF-I (EC50 = 1.5-2.5 vs. 14-23 nM). The chimeric receptor stimulated significantly lower levels of phosphorylation of several proteins relative to the wild-type IGF-I receptor. Activation of phosphatidylinositol 3'-kinase paralleled phosphorylation of the 170- and 70-kDa proteins. Despite these differences in protein tyrosine phosphorylation, stimulation of mitogen-activated protein (MAP) kinase and DNA synthesis were very similar in the three cell lines overexpressing receptors. Little difference was detected in Shc phosphorylation or MAP kinase activation through the three receptors, although activation of MAP kinase was more efficiently coupled to the platelet-derived growth factor receptor than to any of the overexpressed receptors. All three receptors stimulated DNA synthesis to levels comparable to 10% serum, with similar sensitivities (EC50 = 1.5-3.5 nM).
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PMID:Insulin and insulin-like growth factor-I receptors similarly stimulate deoxyribonucleic acid synthesis despite differences in cellular protein tyrosine phosphorylation. 751 64

The exposure of mammalian cells to ultraviolet radiation (UV) may lead to DNA damage resulting in mutation and thus possibly cancer, while irradiation can further act as a potent tumor promoter. In addition UV induces p21ras-mediated signalling leading to activation of transcription factors such as AP-1 and NF-kappa B, as well as activation of the Src tyrosine kinase. This 'UV-response' has been well studied in mammalian cells and furthermore is conserved in yeast, however the most upstream components of this signal transduction pathway have remained elusive. Here we show that UV rapidly activates both the EGF receptor and insulin receptor, as shown by tyrosine phosphorylation of these receptors. We demonstrate that this activation is due to autophosphorylation as it only occurs in cells containing receptors with a functional kinase domain. We have further analysed the propagation of the UV-induced signal to downstream events such as, IRS-1 and Shc tyrosine phosphorylation, phosphatidylinositol 3-kinase activation, leukotriene synthesis, MAP kinase activation and gene induction all of which are activated by UV irradiation. Importantly, we demonstrate that in cells expressing a 'kinase-dead' receptor mutant the UV-response is inhibited, blocking leukotriene synthesis, MAP kinase activation and transcriptional induction. Furthermore, prior-stimulation of cells with UV appears to reduce further responsiveness to addition of growth factor suggesting a common signaling pathway. These data demonstrate a critical role for receptor-mediated events in regulating the response mammalian cells to UV exposure.
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PMID:UV activation of receptor tyrosine kinase activity. 754 96

Treatment of 3T3-L1 adipocytes with insulin resulted in activation of 2-deoxyglucose transport activity and translocation of glucose transporters (GLUT4 and GLUT1) from the cytoplasmic space to the plasma membrane. ML-9 (a myosin light chain kinase inhibitor) inhibited insulin stimulation of 2-deoxyglucose transport activity by 80% at 100 microM (IC50 = 27 microM) without affecting 2-deoxyglucose transport activity in the basal state. The inhibition was independent of extracellular Ca2+ concentration and almost fully reversible at 40 microM ML-9. ML-9 did not inhibit insulin-stimulated tyrosine phosphorylation of 95-kDa protein in the wheat germ agglutinin-purified preparation and of 95- and 160-kDa proteins in intact cells. However, ML-9 inhibited insulin-induced translocation of both GLUT4 and GLUT1 in a dose-dependent manner. The dose-response curves were similar to those observed for the inhibition of insulin stimulation of 2-deoxyglucose transport activity. Neither insulin nor ML-9 affected the phosphorylation state of both heavy and light chains of myosin. Therefore, it seems likely that ML-9 inhibits the insulin-induced translocation of glucose transporters at a step beyond the insulin receptor kinase activity by a mechanism different from that affecting phosphorylation of the myosin light chain. Phosphorylating activity of microtubule-associated protein 2 and myelin basic protein was stimulated by insulin, and this stimulation was not affected by ML-9. ML-9, however, inhibited the phosphorylating activity in vitro and insulin stimulation of the phosphorylating activity of ribosomal protein S6 in intact cells in a dose-dependent manner similar to that observed for the inhibition of insulin stimulation of glucose transport. These results suggest that mitogen-activated protein kinase may be one of the constituents in intracellular insulin signaling to the glucose transport system.
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PMID:Effects of ML-9 on insulin stimulation of glucose transport in 3T3-L1 adipocytes. 768 Mar 48

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 the mitogen-activated protein kinase (MAP kinase) isoforms ERK1 and ERK2 was investigated in rat adipocytes. Kinase activities were measured by using myelin basic protein as substrate after the isoforms were resolved by Mono Q chromatography or by immunoprecipitation with specific antibodies. Insulin increased the activity of both isoforms by 3- to 4-fold. The beta-adrenergic agonist isoproterenol was without effect in the absence of insulin but markedly reduced the increases in ERK1 and ERK2 activities produced by the hormone. MAP kinase activation was also attenuated by forskolin and glucagon, which increase intracellular cAMP, and by dibutyryl-cAMP, 8-bromo-cAMP, and 8-(4-chlorophenylthio)-cAMP. Thus, increasing cAMP is associated with decreased activation of MAP kinase by insulin. Forskolin also inhibited activation of MAP kinase by several agents (epidermal growth factor, phorbol 12-myristate 13-acetate, and okadaic acid) that act independently of insulin receptors. Moreover, forskolin did not inhibit insulin-stimulated tyrosine phosphorylation of the insulin receptor substrate IRS-1. Therefore, the inhibitory effect on MAP kinase did not result from compromised functioning of the insulin receptor. The inhibitory effect was not confined to adipocytes, as forskolin and dibutyryl-cAMP inhibited the increase in MAP kinase activity by phorbol 12-myristate 13-acetate in wild-type CHO cells. In contrast, these agents did not inhibit MAP kinase activity in mutant CHO cells (line 10248) that express a cAMP-dependent protein kinase resistant to activation by cAMP. Our results suggest that activation of cAMP-dependent protein kinase represents a general counter-regulatory mechanism for opposing MAP kinase activation.
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PMID:Increasing cAMP attenuates activation of mitogen-activated protein kinase. 769 90

Chimeric receptors encoding either the whole or a portion of the cytoplasmic domain of the drosophila insulin receptor (IR) with the extracellular domain of the human IR were expressed either transiently in COS cells or stably in Chinese hamster ovary cells and compared with the wild-type human IR. All three receptors bound insulin equally and exhibited an insulin-activated tyrosine kinase activity. The ability of the drosophila cytoplasmic domain to mediate the tyrosine phosphorylation of insulin receptor substrate 1, stimulate cell proliferation, and activate MAP kinase was found to be indistinguishable from that of the human IR. The chimeric drosophila receptors did not bind more phosphatidylinositol 3-kinase than the human IR, despite containing a C-terminal extension with potential tyrosine phosphorylation sites in the motif recognized by the SH2 domain of this enzyme. Thus, the essential signal-transducing abilities of the IR appear to have been conserved from invertebrates to mammals, despite the considerable differences in the sequences of these receptors.
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PMID:Comparison of the signaling abilities of the Drosophila and human insulin receptors in mammalian cells. 771 Oct 18

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


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