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:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hepatocyte growth factor (HGF), also known as scatter factor (SF), is a polypeptide which induces motility and/or mitogenesis in epithelial cells. The receptor for HGF/SF, p190MET, is a two-chain transmembrane tyrosine kinase encoded by the MET proto-oncogene. To identify the cytoplasmic effectors involved in signal transduction we have produced the human HGF/SF receptor in insect cells (Sf9) by means of a recombinant baculovirus. Two 170-kDa forms of the receptor were synthesized in Sf9 cells: the uncleaved single-chain precursor (which is by far the more abundant) and the proteolytically processed two-chain molecule. Both receptor species are phosphorylated on tyrosine in vivo and are active kinases in vitro. The recombinant receptor binds and phosphorylates in vitro four known cytoplasmic transducers containing src homology region 2 (SH2) domains: the 85-kDa subunit of phosphatidylinositol 3-kinase (Pl 3-kinase), rasGAP, phospholipase-C gamma (PLC-gamma), and p59Fyn, a tyrosine kinase of the src family. In all cases the association is strictly dependent on tyrosine phosphorylation of the receptor, indicating that it occurs via specific interaction with the SH2 domains. These results show that the HGF/SF receptor has the sequence requirements for binding a spectrum of cytoplasmic transducers whose different combinations in target cells may result in the observed pleiotropic biological response.
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PMID:Autophosphorylation promotes complex formation of recombinant hepatocyte growth factor receptor with cytoplasmic effectors containing SH2 domains. 132 86

The receptor for hepatocyte growth factor, also known as scatter factor (HGF/SF), has recently been identified as the 190-kDa heterodimeric tyrosine kinase encoded by the MET proto-oncogene (p190MET). The signaling pathway(s) triggered by HGF/SF are unknown. In A549 cells, a lung epithelial cell line, nanomolar concentrations of HGF/SF induced tyrosine phosphorylation of the p190MET receptor. The autophosphorylated receptor coprecipitated with phosphatidylinositol 3-kinase (PI 3-kinase) activity. In GTL16 cells, a cell line derived from a gastric carcinoma, the p190MET receptor, overexpressed and constitutively phosphorylated on tyrosine, coprecipitated with PI 3-kinase activity and with the 85-kDa PI 3-kinase subunit. In these cells activation of protein kinase C or the increase of intracellular [Ca2+] inhibits tyrosine phosphorylation of the p190MET receptor as well as the association with both PI 3-kinase activity and the 85-kDa subunit of the enzyme. In an in vitro assay, tyrosine phosphorylation of the immobilized p190MET receptor was required for binding of PI 3-kinase from cell lysates. These data strongly suggest that the signaling pathway activated by the HGF/SF receptor includes generation of D-3-phosphorylated inositol phospholipids.
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PMID:The tyrosine-phosphorylated hepatocyte growth factor/scatter factor receptor associates with phosphatidylinositol 3-kinase. 171 89

Hepatocyte growth factor receptor is identified as a heterodimeric tyrosine kinase encoded by the c-met gene. This study was designed to determine how the c-met/hepatocyte growth factor receptor participates in the intracellular events involved in rat liver regeneration induced by administration of carbon tetrachloride. Expression of the rat c-met mRNA increased, peaking 24 h after carbon tetrachloride administration almost in parallel with MET protein expression. Histochemical studies demonstrated that expression of the rat c-met was enhanced in cells surrounding the damaged areas, and also that the distribution of cells expressing MET was almost in accordance with that of cells expressing proliferating cells nuclear antigen. The MET protein underwent intense tyrosine phosphorylation peaking at 12 h after carbon tetrachloride administration, and prior to DNA synthesis. Phospholipase C gamma and phosphatidylinositol 3-kinase, intracellular signal transducing molecules containing Src homology 2 domain, were associated with the MET protein following tyrosine phosphorylation in vivo. These observations suggest that expression and tyrosine phosphorylation of MET protein associated with signal transducing molecules may provide a mechanism whereby hepatocyte growth factor exerts its action on hepatocyte growth during rat liver regeneration induced by carbon tetrachloride administration.
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PMID:Expression and phosphorylation of rat c-met/hepatocyte growth factor receptor during rat liver regeneration. 749 89

Because the protein-tyrosine phosphatase (PTP) Syp associates with the tyrosine-phosphorylated platelet-derived growth factor beta receptor (beta PDGFR), the beta PDGFR is a likely Syp substrate. We tested this hypothesis by determining whether recombinant Syp (rSyp) and a control PTP, recombinant PTP1B (rPTP1B), were able to dephosphorylate the beta PDGFR. The beta PDGFR was phosphorylated at multiple tyrosine residues in an in vitro kinase assay and then incubated with increasing concentrations of rSyp or rPTP1B. While the receptor was nearly completely dephosphorylated by high concentrations of rPTP1B, receptor dephosphorylation by rSyp plateaued at approximately 50%. Two-dimensional phosphopeptide maps of the beta PDGFR demonstrated that rSyp displayed a clear preference for certain receptor phosphorylation sites; the most efficiently dephosphorylated sites were phosphotyrosines (Tyr(P)-771 and -751, followed by Tyr(P)740, while Tyr(P)-1021 and Tyr(P)-1009 were very poor substrates. In contrast, rPTP1B displayed no selectivity for the various rPTP1B displayed no selectivity for the various beta PDGFR tyrosine phosphorylation sites and dephosphorylated all of them with comparable efficiency. A Syp construct that lacked the SH2 domains was still able to discriminate between the various receptor phosphorylation sites, although less effectively than full-length Syp. These in vitro studies predicted that Syp can dephosphorylate the receptor in vivo. Indeed, we found that a beta PDGFR mutant (F1009) that associates poorly with Syp, had a much slower in vivo rate of receptor dephosphorylation than the wild type receptor. In addition, the GTPase-activating protein of Ras (GAP) and phosphatidylinositol 3-kinase were less stably associated with the wild type beta PDGFR than with the F1009 receptor. These findings are consistent with the in vitro experiments showign that Syp prefers to dephosphorylate sites on the beta PDGFR, that are important for binding phosphatidylinositol 3-kinase (Tyr(P)-740 and Tyr(P)-751) and GAP (Tyr(P)-771). These studies reveal that Syp is a substrate-selective PTP and that both the catalytic domain and the SH2 domains contribute to Syp's ability to choose substrates. Furthermore, it appears that Syp plays a role in PDGF-dependent intracellular signal relay by selectively dephosphorylating the beta PDGFR and thereby regulating the binding of a distinct group of receptor-associated signal relay enzymes.
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PMID:Identification of a putative Syp substrate, the PDGF beta receptor. 754 75

We have reported that overexpression of Neu leads to heregulin-stimulated neurite outgrowth and the tyrosine-phosphorylation of Neu and other cellular proteins in PC12 cells. Considering that Neu/ErbB2 alone is not able to functionally couple to heregulin, we looked for the possible involvement of ErbB3 in these neurite outgrowth and tyrosine phosphorylation responses. We found that heregulin stimulates the tyrosine phosphorylation of endogenous ErbB3 protein in PC12 cells and that this phosphorylation, like that of Neu, is greatly enhanced in cells that overexpress Neu. Furthermore, overexpression of ErbB3 in PC12 cells led to heregulin-stimulated neurite extension. In addition to becoming tyrosine-phosphorylated, Neu/ErbB2 and ErbB3 associate with each other, and each associates with the 85-kDa regulatory subunit (p85) of phosphatidylinositol 3-kinase in a heregulin-dependent manner. Thus, Neu/ErbB2 and ErbB3 appear to cooperate to mediate the heregulin signal in PC12 cells. Like heregulin, epidermal growth factor (EGF) also stimulate the tyrosine phosphorylation of both Neu and ErbB3. However, there are clear differences between the EGF- and heregulin-stimulated phosphorylations of ErbB3. In the heregulin response, two tyrosine-phosphorylated forms of ErbB3 are detected. Of these, only the more quickly migrating form (on SDS-polyacrylamide gel electrophoresis) is found to be associated with Neu, whereas the other, more slowly migrating form is uniquely capable of forming stable complexes with p85. In the EGF response, at least two tyrosine-phosphorylated forms of ErbB3 are detected, but these phosphoproteins have distinctly lower apparent molecular weights compared with the heregulin-stimulated ErbB3 phosphoproteins and do not complex with p85. Thus the formation of a stable ErbB3-p85 complex in PC12 cells is a unique outcome of heregulin signaling that correlates with the differences in cell morphology induced by the activated EGF receptor and the Neu tyrosine kinase.
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PMID:Heregulin-stimulated signaling in rat pheochromocytoma cells. Evidence for ErbB3 interactions with Neu/ErbB2 and p85. 764 63

Wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), inhibited adipocyte differentiation of 3T3-L1 fibroblasts induced by insulin/dexamethasone/IBMX (hormones/IBMX). Insulin as a key factor in the process of the adipocyte differentiation activated PI 3-kinase, Ras protein, and mitogen-activated protein kinase (MAP kinase, ERK) in 3T3-L1 fibroblasts. Pretreatment with wortmannin almost completely suppressed all these activations. These findings suggest that the sequential activation of PI 3-kinase, Ras protein, and MAP kinase is involved in the insulin signaling pathway(s) during differentiation by hormones/IBMX and in consequence of the inhibition of PI 3-kinase by wortmannin, the activation of Ras protein and MAP kinase which acts downstream of PI 3-kinase is suppressed and results in the inhibition of adipocyte differentiation.
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PMID:Wortmannin inhibits insulin-induced Ras and mitogen-activated protein kinase activation related to adipocyte differentiation in 3T3-L1 fibroblasts. 775 36

Hepatocyte growth factor/scatter factor (HGF/SF) is a heparin-binding polypeptide which shares structural domains with enzymes of the blood clotting cascade. HGF/SF is secreted by cells of mesodermal origin and has powerful mitogenic, motogenic and morphogenic activity on epithelial and endothelial cells. HGF/SF is produced as a biologically inactive single-chain precursor (pro-HGF/SF) most of which is sequestered on the cell surface or bound to the extracellular matrix. Maturation into the active alpha beta heterodimer results from proteolytic cleavage by a urokinase-type protease, which acts as a pro-HGF/SF convertase. The primary determinant for receptor binding appears to be located within the alpha-chain. The interaction of the alpha-chain with the receptor is sufficient for the activation of the signal cascade involved in the motility response. However, the complete HGF/SF protein seems to be required to elicit a mitogenic response. HGF/SF binds with high affinity to a transmembrane receptor, p190MET, encoded by the MET proto-oncogene. p190MET is the prototype of a distinct subfamily of heterodimeric tyrosine kinases, including the putative receptors Ron and Sea. The mature form of p190MET is a heterodimer of two disulfide-linked subunits (alpha and beta). The alpha-subunit is extracellular and heavily glycosylated. The beta-subunit consists of an extracellular portion involved in ligand binding, a membrane spanning segment, and a cytoplasmic tyrosine kinase domain. Both subunits derive from glycosylation and proteolytic cleavage of a common precursor of 170 kDa. In polarized epithelial cells the HGF/SF receptor is selectively exposed in the basolateral plasmalemma, where it is associated with detergent-insoluble components. Two Met isoforms, carrying an intact ligand binding domain but lacking the kinase domain due to truncation of the beta-subunit, arise from alternative post-transcriptional processing of the mature form. One truncated form is soluble and released from the cells. HGF/SF binding triggers tyrosine autophosphorylation of the receptor beta-subunit. Autophosphorylation on the major phosphorylation site Y1235 upregulates the kinase activity of the receptor, increasing the Vmax of the phosphotransfer reaction. Negative regulation of the kinase activity occurs through phosphorylation of a unique serine residue (S985) located in the juxtamembrane domain of the receptor. This phosphorylation is triggered by two distinct pathways involving either protein kinase C activation or increase in intracellular Ca2+ concentration. Upon ligand binding, the HGF/SF receptor recruits and activates several cytoplasmic effectors, including phosphatidylinositol 3-kinase (PI 3-K), phospholipase C-gamma (PLC-gamma), pp60c-Src, a tyrosine phosphatase, and a Ras-guanine nucleotide exchanger.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Identification of functional domains in the hepatocyte growth factor and its receptor by molecular engineering. 776 52

The serine/threonine protein kinase encoded by the Akt proto-oncogene is catalytically inactive in serum-starved primary and immortalized fibroblasts. Here we show that Akt and the Akt-related kinase AKT2 are activated by PDGF. The activation was rapid and specific, and it was abrogated by mutations in the Akt Pleckstrin homology (PH) domain. The Akt activation was also shown to depend on PDGFR beta tyrosines Y740 and Y751, which bind phosphatidylinositol 3-kinase (PI 3-kinase) upon phosphorylation. Moreover, Akt activation was blocked by the PI 3-kinase-specific inhibitor wortmannin and the dominant inhibitory N17Ras. Conversely, Akt activity was induced following the addition of phosphatidylinositol-3-phosphate to Akt immunoprecipitates from serum-starved cells in vitro. These results identify Akt as a novel target of PI 3-kinase and suggest that the Akt PH domain may be a mediator of PI 3-kinase signaling.
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PMID:The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. 777 14

Vascular endothelial cell growth factor (VEGF), an endothelial cell-specific mitogen that plays an important role in angiogenesis, promotes the tyrosine phosphorylation of at least 11 proteins in bovine aortic endothelial cells (BAEC). Proteins immunoprecipitated from lysates of control- and VEGF-stimulated BAEC with antisera to phospholipase C-gamma (PLC-gamma) were fractionated by SDS-polyacrylamide gel electrophoresis and transferred to Immobilon-P. Evaluation of the Western blots with antisera to phosphotyrosine demonstrated that PLC-gamma and two proteins (100 and 85 kDa) that associate with PLC-gamma were phosphorylated in response to VEGF. By using antisera specific to other mediators of signal transduction that contain SH2 domains for immunoprecipitation, it was demonstrated that VEGF promotes phosphorylation of phosphatidylinositol 3-kinase, Ras GTPase activating protein (GAP), and the oncogenic adaptor protein NcK. Proteins of M(r) consistent with the VEGF receptors Flt-1 and Flk-1/KDR were also tyrosine phosphorylated in stimulated cells. Tyrosine-phosphorylated Nck, PLC-gamma, and two GAP-associated proteins, p190 and p62, were in GAP immunoprecipitates of VEGF-stimulated BAEC, and tyrosine-phosphorylated NcK was in phosphatidylinositol 3-kinase immunoprecipitates. These observations suggest that VEGF promotes formation of multimeric aggregates of VEGF receptors with proteins that contain SH2 domains and activate various signaling pathways. VEGF-promoted proliferation of endothelial cells and tyrosine phosphorylation of SH2 domain containing signaling molecules were inhibited by the tyrosine kinase inhibitor genistein.
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PMID:Vascular endothelial cell growth factor promotes tyrosine phosphorylation of mediators of signal transduction that contain SH2 domains. Association with endothelial cell proliferation. 789 17

Src-homology region 2 (SH2) domains are stretches of about 100 amino acids which are found to be structurally conserved in a number of signaling molecules. These regions have been shown to bind with high affinity to phosphotyrosine residues within activated receptor tyrosine kinases. Here we report the bacterial expression and purification of individual N-terminal SH2 (NSH2) domains of phosphatidylinositol 3-kinase (PI-3K) binding subunit (p85) and Ras GTPase activating protein (GAP) in amounts suitable for structure-function studies. The p85NSH2 domain stains dark purple and absorbs around 620-640 nm with Stains-all, a dye known to bind to calcium binding proteins. This effect was not observed for the GAPNSH2 domain. Circular dichroism analysis of the N-terminal SH2 domain of these proteins shows that p85NSH2, but not GAPNSH2, undergoes a significant dose-dependent change in conformation in the presence of increasing calcium concentrations. Moreover, the conformational change of p85NSH2 induced by calcium could be replicated by addition of a phosphorylated hexapeptide (DYpMDMK) representing the alpha-PDGFR binding site for p85. Limited proteolysis studies showed a significant calcium-dependent increase in protection of p85NSH2 but not GAPNSH2 from degradation by subtilisin. Our results further indicate that holmium, a trivalent lanthanide ion, which has been previously shown to substitute for calcium, could also protect the p85NSH2 domain from proteolysis even at 10-fold lower concentrations. In vitro binding studies using purified preparations of activated alpha-PDGFR show that calcium did not affect the binding of GAPNSH2 domains to activated alpha-PDGFR.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Comparison of calcium-dependent conformational changes in the N-terminal SH2 domains of p85 and GAP defines distinct properties for SH2 domains. 829 2


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