Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.24 (mitogen-activated protein kinase)
 95,810 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

WC1 is a 215-kDa type 1 transmembrane glycoprotein, the expression of which is restricted to gammadelta T lymphocytes. The binding of an anti-WC1 monoclonal antibody (mAb) (SC-29) induces reversible growth arrest in proliferating interleukin (IL)-2-dependent gammadalta T lymphocytes and this study has examined the relevant biochemical mechanisms. WC1 binding activates multiple protein tyrosine phosphatases causing specific tyrosine dephosphorylation in the absence of calcium mobilization. One of the dephosphorylated proteins was identified as the MAP kinase erk2. Another phosphotyrosine protein of 70 kDa, found to coprecipitate with p85 phosphoinositol (PI)3-kinase was either dephosphorylated or uncoupled from the p85 PI 3-kinase immunoprecipitate after WC1 receptor binding by mAb SC-29. The anti-WC1-induced tyrosine dephosphorylation was reversed by stimulation of gammadelta T cells with concanavalin A or anti-CD3 mAb, demonstrating that at the biochemical level, mitogen stimulation is dominant to the growth-arresting effects of anti-WC1. It is therefore proposed that the activation of tyrosine phosphatases by WC1 binding and the resultant dephosphorylation of certain key signaling protein such as erk2 correlates with and may cause the induction of growth arrest in IL-2-dependent gammadelta T cells, without affecting the cells ability to respond to antigen. Possible mechanisms, which include the inhibition of IL-2 signal transduction pathways, are discussed.
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PMID:Growth arrest of gammadelta T cells induced by monoclonal antibody against WC1 correlates with activation of multiple tyrosine phosphatases and dephosphorylation of MAP kinase erk2. 907 14

In the present study we have investigated a possible role for the proline-rich SH2 domain protein Shb as a regulator of expression or activity of certain SH3 domain proteins and MAP kinase. The expression of the Shb binding proteins Eps8, Src, and p85 PI3-kinase, PI3-kinase activity, and MAP kinase activation were assessed in wild-type NIH3T3 cells and in NIH3T3 cells overexpressing the Shb cDNA. In addition, the expression of the SH3 domain STAT1 proteins was assessed in wild-type and Shb overexpressing cells. The Eps8 protein content and Eps8 mRNA steady-state levels were downregulated, whereas the protein contents of Src and p85 PI3-kinase were unaffected by Shb overexpression. There was, however, an increased basal PI3-kinase activity in Shb transfected cells after a 3-h serum starvation. Increased steady-state levels of STAT1 mRNA were accompanied by an increased STAT1 protein content in Shb overexpressing cells. Shb overexpression was not associated with an altered activation of p44 or p42 MAP kinases in response to PDGF stimulation. The data presented in this study suggest novel functions for the adaptor protein Shb regulating the expression of certain signal-transducing SH3 domain proteins and modulating PI3-kinase activity.
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PMID:Modulation of Src homology 3 proteins by the proline-rich adaptor protein Shb. 908 67

In the present study we examined the mechanism by which PAF activates MAPK in native cells such as guinea-pig neutrophils and P388D1 macrophage-like cells. We found that PAF activates MAPK through two distinct pathways. One calcium-dependent pathway that likely involves cPKC, and another calcium-independent but wortmannin-sensitive pathway. Using molecular biological methods we are presently examining whether hetrodimeric (p85/p110) type PI 3-kinase is the actual target of wortmannin involved in PAF mediated activation of MAPK.
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PMID:PAF-induced MAPK activation is inhibited by wortmannin in neutrophils and macrophages. 913 Nov 67

Insulin receptor substrate-1 (IRS-1) is one of the major substrates of insulin receptor tyrosine kinase and mediates various insulin signals downstream. In this study, we have examined the impact of three natural IRS-1 mutations identified in NIDDM patients (G971R, P170R, and M209T) on insulin signaling. G971R is located near src homology 2 protein binding sites, and P170R and M209T are located in the phosphotyrosine binding domain of IRS-1. 32D-IR cells, stably overexpressing human insulin receptor, were transfected with wild-type human IRS-1 cDNA (WT) or three mutant IRS-1 cDNAs and analyzed. All the cell lines expressing mutant IRS-1 showed a significant reduction in [3H]thymidine incorporation compared with WT. Upon insulin stimulation, cells expressing G971R showed a 39% decrease (P < 0.005) in phosphatidylinositol 3-kinase (PI 3-kinase) activity, a 43% decrease (P < 0.01) in binding of the 85-kDa regulatory subunit of PI 3-kinase, and a 22% decrease (P < 0.05) in mitogen-activated protein kinase activity compared with those expressing WT. Cells expressing P170R and M209T showed slight but significant decreases in PI 3-kinase activity (17 and 14%, respectively; both P < 0.05) and in binding of p85 (22 and 16%, respectively; both P < 0.05) and a greater decrease in mitogen-activated protein kinase activity (41 and 43%, respectively; both P < 0.005) compared with WT. After insulin stimulation, cells expressing P170R and M209T showed significant decreases in IRS-1 phosphorylation (37 and 42%, respectively; both P < 0.05) and in IRS-1 binding to the insulin receptor (48 and 53%, respectively; P < 0.01) compared with WT. G971R showed no changes in IRS-1 phosphorylation and in IRS-1 binding to the insulin receptor compared with WT. These data suggest that the impaired mitogenic response of P170R and M209T was mainly due to reduced binding to the insulin receptor, whereas the impaired response of G971R was mainly due to reduced association with PI 3-kinase p85.
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PMID:Impact of natural IRS-1 mutations on insulin signals: mutations of IRS-1 in the PTB domain and near SH2 protein binding sites result in impaired function at different steps of IRS-1 signaling. 916 61

The Met/Hepatocyte Growth Factor (HGF) receptor tyrosine kinase is oncogenically activated through a rearrangement that creates a hybrid gene Tpr-Met. The resultant chimeric p65(Tpr-Met) protein is constitutively phosphorylated on tyrosine residues in vivo and associates with a number of SH2-containing signaling molecules including the p85 subunit of PI-3 kinase and the Grb2 adaptor protein, which couples receptor tyrosine kinases to the Ras signaling pathway. Mutation of the binding site for Grb2 impairs the ability of Tpr-Met oncoprotein to transform fibroblasts, suggesting that the activation of the Ras/MAP kinase signaling pathway through Grb2 may be essential for cellular transformation. To test this hypothesis dominant-negative mutants of Grb2 with deletions of the SH3 domains were introduced into Tpr-Met transformed fibroblasts. Cells overexpressing the mutants were found to be morphologically reverted and exhibited reduced growth in soft agar. Surprisingly, the Grb2 mutants blocked activation of the JNK/SAPK but not MAP kinase activity induced by the Tpr-Met oncoprotein. Additionally, cells expressing dominant-negative Grb2 mutants had reduced PI-3-kinase activity and dominant-negative mutants of Rac1 blocked both Tpr-Met-induced transformation and activation of JNK. These experiments reveal a novel link between Met and the JNK pathway, which is essential for transformation by this oncogene.
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PMID:Activation of the JNK pathway is essential for transformation by the Met oncogene. 918 10

The isoform identity of activated protein kinase C (PKC) and its regulation were investigated in bacterial lipopolysaccharide (LPS)-treated human monocytes. Resolution of detergent-soluble lysates prepared from LPS-treated, peripheral blood monocytes using Mono Q anion-exchange chromatography revealed two principal peaks of myelin basic protein kinase activity. Immunoblotting and immunoprecipitation with isoform-specific anti-PKC antibodies showed that the major and latest eluting peak is accounted for by PKC-zeta. In addition to primary monocytes, activation of PKC-zeta in response to LPS was also observed in the human promonocytic cell lines, U937 and THP-1. Consistent with its identity as PKC-zeta, the kinase did not depend upon the presence of lipids, Ca2+, or diacylglycerol for activity. In addition, the kinase phosphorylates peptide epsilon and myelin basic protein with equal efficiency but phosphorylates Kemptide and protamine sulfate poorly. Translocation of PKC-zeta from the cytosolic to the particulate membrane fraction upon exposure of monocytes to LPS provided further evidence for activation of the kinase. Preincubation of monocytes with the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors, wortmannin or LY294002, abrogated LPS-induced activation of PKC-zeta. Furthermore, activation of PKC-zeta failed to occur in U937 cells transfected with a dominant negative mutant of the p85 subunit of PI 3-kinase. PKC-zeta activity was also observed to be enhanced in vitro by the addition of phosphatidylinositol 3,4,5P3. These findings are consistent with a model in which PKC-zeta is activated downstream of PI 3-kinase in monocytes in response to LPS.
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PMID:Phosphatidylinositol 3-kinase-dependent activation of protein kinase C-zeta in bacterial lipopolysaccharide-treated human monocytes. 919 53

Inorganic phosphate (Pi) is a major regulator of cell metabolism. The Pi transport activity in the plasma membrane is a main determinant of the intracellular level of this ion. In bone-forming cells, Pi transport is important for the calcification of the bone matrix. In this study, the effect of platelet-derived growth factor (PDGF) on Pi transport activity and the signaling mechanism involved in this cellular response were analyzed. The results indicate that PDGF is a potent and selective stimulator of sodium-dependent Pi transport in the mouse calvaria-derived MC3T3-E1 osteoblast-like cells. The change in Pi transport induced by PDGF-BB was dependent on translational processes and affected the Vmax of the Pi transport system. These observations suggested that enhanced Pi transport activity in response to PDGF resulted from insertion of newly synthesized Pi transporters in the plasma membrane. The role of activation of mitogen activated protein (MAP) kinase, phospholipase C (PLC)gamma or phosphatidylinositol 3-kinase (PI-3-kinase), in mediating this effect of PDGF, was investigated. A selective inhibitor of the PDGF receptor tyrosine kinase activity (CGP 53716) completely blocked PDGF-induced protein tyrosine phosphorylation of several proteins including the PDGF receptor, PLCgamma, MAP kinase, and association of the p85 subunit of PI-3'-kinase. Associated with this effect, the increase in Pi transport induced by PDGF was completely blunted by 5 microM CGP 53716. Inhibition of MAP kinase activity by cAMP agonists did not influence Pi transport stimulation induced by PDGF. However, inhibitors of protein kinase C completely blocked this response. A selective inhibitor of PI-3-kinase, LY294002, also significantly reduced this effect of PDGF. In summary, these results indicate that PDGF is a potent and selective stimulator of Pi transport in osteoblastic cells. The mechanism responsible for this effect is not mediated by MAP kinase but involves tyrosine phosphorylation-dependent activation of PLCgamma and PI-3-kinase.
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PMID:Platelet-derived growth factor stimulates sodium-dependent Pi transport in osteoblastic cells via phospholipase Cgamma and phosphatidylinositol 3' -kinase. 924 Jul 23

The(1) regulatory mechanism of glucose uptake in 3T3-L1 adipocytes was investigated with the use of recombinant adenovirus vectors encoding various dominant negative proteins. Infection with a virus encoding a mutant regulatory subunit of phosphoinositide (PI) 3-kinase that does not bind the 110-kDa catalytic subunit (delta p85) inhibited the insulin-induced increase in PI 3-kinase activity co-precipitated by antibodies to phosphotyrosine and glucose uptake in a virus dose-dependent manner. Overexpression of a dominant negative RAS mutant in which Asp57 is replaced with tyrosine (RAS57Y) or of a dominant negative SOS mutant that lacks guanine nucleotide exchange activity (delta SOS) abolished the insulin-induced increase in mitogen-activated protein kinase activity, but had no effect on PI 3-kinase activity or glucose uptake. Although GH and hyperosmolarity attributable to 300 mM sorbitol each promoted glucose uptake and translocation of glucose transporter (GLUT)4 to an extent comparable to that of insulin, these stimuli triggered little or no association of PI 3-kinase activity with tyrosine-phosphorylated proteins. Overexpression of delta p85 or treatment of cells with wortmannin, an inhibitor of PI 3-kinase activity, had no effect on glucose uptake or translocation of GLUT4 stimulated by GH or hyperosmolarity. Moreover, overexpression of delta SOS or RAC17N also did not affect the increase in glucose uptake induced by these stimuli. A serine/threonine kinase Akt, a constitutively active mutant of which was previously shown to stimulate glucose uptake, is activated by insulin, GH, and hyperosmolarity to approximately 4-fold, approximately 2.1-fold, and approximately 2.3-fold over basal level, respectively. These results suggest that insulin-induced but neither GH- or hyperosmolarity-induced glucose uptake is PI 3-kinase-dependent, and neither RAS nor RAC is required for glucose uptake induced by these stimuli in 3T3-L1 adipocytes.
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PMID:Phosphoinositide 3-kinase is required for insulin-induced but not for growth hormone- or hyperosmolarity-induced glucose uptake in 3T3-L1 adipocytes. 928 70

The human insulin receptor substrate-1 (hIRS-1) is a key intracellular protein involved in various cytokine signaling pathways associated with cell growth. We have previously demonstrated that stable transfection and overexpression of hIRS-1 in human hepatoblastoma cells in vitro leads to the constitutive activation of the mitogen-activated protein kinase (MAPK) cascade. In this setting, hIRS-1 acts as a dominant oncogene and will induce neoplastic transformation of NIH 3T3 cells. In the present study, the biologic effects of hIRS-1 overexpression in the liver was analyzed using both clinical tumor samples and a newly developed transgenic mouse model. We have found that approximately 40% of 22 human hepatocellular carcinoma (HCC) tumors had enhanced (>200%) hIRS-1 gene expression compared with adjacent non-involved liver tissue. There was a significant relationship between the level of hIRS-1 overexpression and the tumor size; this finding suggests a possible role for hIRS-1 in tumor progression. To determine if downstream signal transduction cascades were activated by overexpression of hIRS-1 in hepatocytes, we established a transgenic mouse model using an hIRS-1 construct driven by an albumin promoter/enhancer element to direct liver specific expression. The overexpressed hIRS-1 protein was found to be tyrosyl phosphorylated and interacted with downstream SH2-containing molecules such as the p85 subunit of phosphatidylinositol-3 kinase (PI3K), Grb2 adaptor, and SHP2 phosphatase proteins. The functional consequences of hIRS-1 overexpression were reflected by constitutive activation of both the MAPK and PI3K signal transduction cascades. More important, overexpression of hIRS-1 in the transgenic liver led to increased hepatocyte DNA synthesis. Our findings indicate that hIRS-1 overexpression induces downstream signaling molecules associated with hepatocyte growth and may potentially enhance tumor progression of HCC.
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PMID:Biological effects of human insulin receptor substrate-1 overexpression in hepatocytes. 930 88

Peroxovanadiums (pVs) are potent protein tyrosine phosphatase (PTP) inhibitors with insulin-mimetic properties in vivo and in vitro. We have established the existence of an insulin receptor kinase (IRK)-associated PTP whose inhibition by pVs correlates closely with IRK tyrosine phosphorylation, activation, and downstream signaling. pVs have also been shown to activate various tyrosine kinases (TKs) that could participate in activation of the insulin-signaling pathway. In the present study we have sought to determine whether pV-induced IRK tyrosine phosphorylation requires the intrinsic kinase activity of the IRK, and whether IRK activation is necessary to realize the early steps in the insulin-signaling cascade. To address this we evaluated the effect of a pure pV compound, bis peroxovanadium 1,10-phenanthroline [bpV(phen)], in HTC rat hepatoma cells overexpressing normal (HTC-IR) or kinase-deficient (HTC-M1030) mutant IRKs. We showed that at a dose of 0.1 mM, but not 1 mM, bpV(phen) induced IRK-dependent events. Thus, 0.1 mM bpV(phen) increased tyrosine phosphorylation and IRK activity in HTC-IR but not HTC-M1030 cells. Tyrosine phosphorylation of insulin signal-transducing molecules was promoted in HTC-IR but not HTC-M1030 cells by bpV(phen). The association of p185 and p60 with the src homology-2 (SH2) domains of Syp and the p85-regulatory subunit of phosphatidylinositol 3'-kinase was induced by bpV(phen) in HTC-IR, but not in HTC-M1030 cells, as was insulin receptor substrate-1-associated phosphatidylinositol 3'-kinase activity. Thus autophosphorylation and activation of the IRK by bpV(phen) is effected by the IRK itself, and the early events in the insulin- signaling cascade follow from this activation event. This establishes a critical role for PTP(s) in the regulation of IRK activity. bpV(phen) could be distinguished from insulin only in its ability to activate ERK1 in HTC-M1030 cells, thus indicating that this event is IRK independent, consistent with our previous hypothesis that bpV(phen) inhibits a PTP involved in the negative regulation of mitogen-activated protein kinases.
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PMID:Early signaling events triggered by peroxovanadium [bpV(phen)] are insulin receptor kinase (IRK)-dependent: specificity of inhibition of IRK-associated protein tyrosine phosphatase(s) by bpV(phen). 941 95


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