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)

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

Photoaffinity labeling has been used to identify amino acids involved in recognition of protein substrates by the protein-tyrosine phosphatase PTP1. The photoactive amino acid p-benzoylphenylalanine (Bpa) was incorporated into a phosphotyrosine-containing peptide derived from epidermal growth factor autophosphorylation site Tyr992 (EGFR988 998). This peptide photoinactivated PTP1 in a time- and concentration-dependent manner. Three lines of evidence indicate that the interaction between PTP1 and the photoaffinity label was specific: 1) photoinactivation was inhibited in the presence of a non-Bpa-containing peptide from EGFR Tyr992 in molar excess. 2) The photoaffinity label-containing phosphopeptide was rapidly dephosphorylated by PTP1 with kinetic constants similar to those of the non-Bpa-containing peptide under identical conditions. 3) After complete photoinactivation, the level of incorporation of radioactive photoaffinity label into PTP1 was approximately 0.9 mol of label/mol of enzyme, consistent with a 1:1 stoichiometry of photolabeling. Radiolabeled peptide was used to identify sites of cross-linking to PTP1. Bpa peptide-PTP1 was digested with trypsin, and radioactive fragments were purified by high performance liquid chromatography (HPLC) and analyzed by Edman sequencing. In two parallel experiments which were analyzed using different HPLC columns, a site in the alpha2 region of PTP1, most likely Ile23, was labeled by the Tyr992-derived peptide. The results are discussed in light of the crystal structure of human PTP1B and suggest that an additional mode of substrate recognition must exist for PTP1 catalysis.
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PMID:Determinants of substrate recognition in the protein-tyrosine phosphatase, PTP1. 862 92

Using transient overexpression and microinjection approaches, we examined SHPTP2's function in growth factor signaling. Overexpression of catalytically inactive SHPTP2 (PTP2CS) but not catalytically inactive SHPTP1, inhibited mitogen-activated protein (MAP) kinase activation and Elk-1 transactivation following epidermal growth factor (EGF) stimulation of 293 cells. An SHPTP2 mutant with both C-terminal tyrosyl phosphorylation sites converted to phenylalanine (PTP2YF) was also without effect; moreover, PTP2YF rescued PTP2CS-induced inhibition of EGF-induced Elk-1 transactivation. PTP2CS did not inhibit transactivation by activated Ras, suggesting that SHPTP2 acts upstream of or parallel to Ras. Neither PTP2CS nor PTP2YF inhibited platelet-derived growth factor (PDGF)-induced Elk-1 transactivation. Thus, protein-tyrosine phosphatase activity, but not tyrosyl phosphorylation of SHPTP2, is required for the immediate-early responses to EGF but not to PDGF. To determine whether SHPTP2 is required later in the cell cycle, we assessed S-phase entry in NIH 3T3 cells microinjected with anti-SHPTP2 antibodies or with a glutathione S-transferase (GST) fusion protein encoding both SH2 domains (GST-SH2). Microinjection of anti-SHPTP2 antibodies prior to stimulation inhibited EGF- but no PDGF- or serum-induced S-phase entry. Anti-SHPTP2 antibodies or GST-SH2 fusion protein could inhibit EGF-induced S-phase entry for up to 8 h after EGF addition. Although MAP kinase activation was detected shortly after EGF stimulation, no MAP kinase activation was detected around the restriction point. Therefore, SHPTP2 is absolutely required for immediate-early and late events induced by some, but not all, growth factors, and the immediate-early and late signal transduction pathways regulated by SHPTP2 are distinguishable.
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PMID:Multiple requirements for SHPTP2 in epidermal growth factor-mediated cell cycle progression. 862 63

Rapid tyrosine phosphorylation of key cellular proteins is a crucial event in signal transduction. The regulatory role of protein-tyrosine phosphatases (PTPs) in this process was explored by studying the effects of a powerful PTP inhibitor, pervanadate, on the activation of the mitogen-activated protein (MAP) kinase cascade. Treatment of HeLa cells with pervanadate resulted in a marked inhibition of PTP activity, accompanied by a drastic increase in tyrosine phosphorylation of cellular proteins. The increased tyrosine phosphorylation coincided with the activation of the MAP kinase cascade as indicated by enzymatic activity assays of MEK (MAP kinase/ERK-kinase) and MAP kinase and gel mobility shift analyses of Raf-1 and MAP kinase. The activation was sustained but reversible. Upon removal of pervanadate, both tyrosine phosphorylation and MAP kinase activation declined to basal levels. Therefore, inhibition of PTP activity is sufficient per se to initiate a complete MAP kinase activation program.
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PMID:Activation of mitogen-activated protein (MAP) kinase pathway by pervanadate, a potent inhibitor of tyrosine phosphatases. 870 41

Vascular endothelial cell growth factor (VEGF) binds to and promotes the activation of one of its receptors, KDR. Once activated, KDR induces the tyrosine phosphorylation of cytoplasmic signaling proteins that are important to endothelial cell proliferation. In human umbilical vein endothelial cells (HUVECs), tumor necrosis factor (TNF) inhibits the phosphorylation and activation of KDR. The ability of TNF to diminish VEGF-stimulated KDR activity was impaired by sodium orthovanadate, suggesting that the inhibitory activity of TNF was mediated by a protein-tyrosine phosphatase. KDR-initiated responses specifically associated with endothelial cell proliferation, mitogen-activated protein kinase activation and DNA synthesis, were also inhibited by TNF, and this was reversed by sodium orthovanadate. Stimulation of HUVECs with TNF induced association of the SHP-1 protein-tyrosine phosphatase with KDR, identifying this phosphatase as a candidate negative regulator of VEGF signal transduction. Heterologous receptor inactivation mediated by a protein-tyrosine phosphatase provides insight into how TNF may inhibit endothelial cell proliferative responses and modulate angiogenesis in pathological settings.
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PMID:Tumor necrosis factor employs a protein-tyrosine phosphatase to inhibit activation of KDR and vascular endothelial cell growth factor-induced endothelial cell proliferation. 1075 29

c-Jun N-terminal kinase (JNK) regulates gene expression in response to various extracellular stimuli. JNK can be activated by the tumor promoting agent, 12-O-tetradecanoylphorbol-13-acetate (TPA) in normal human oral keratinocytes but not in human keratinocytes that have been immortalized (HOK-16B and HaCaT) or transformed (HOK-16B-Bap-T) nor in a cervical carcinoma cell line (HeLa). The refractory JNK activation response to TPA is not due a defect in the JNK pathway, because JNK can be activated by other stimuli, e.g. UV irradiation and an alkylating agent N-methyl-N'-nitrosoguanidine in these immortalized or transformed cells. More importantly, the refractory JNK and JNKK activation response to TPA can be restored by treatment of the cells with a combination of TPA and a protein-tyrosine phosphatase inhibitor, sodium orthovanadate. Furthermore, pretreatment of cells with TPA partially inhibited UV- or N-methyl-N'-nitrosoguanidine-induced JNK activity. These results suggest that a TPA-inducible, orthovanadate-sensitive protein-tyrosine phosphatase may specifically down-regulate JNK signaling pathway in these immortalized/transformed epithelial cells. In contrast, ERK and p38/Mpk2 are not regulated by this TPA-induced phosphatase. This putative protein-tyrosine phosphatase appears to be JNK pathway-specific.
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PMID:12-O-tetradecanoylphorbol-13-acetate (TPA)-induced c-Jun N-terminal kinase (JNK) phosphatase renders immortalized or transformed epithelial cells refractory to TPA-inducible JNK activity. 1080 30

Angiotensin II (Ang II) binds to specific G protein-coupled receptors and is mitogenic in Chinese hamster ovary (CHO) cells stably expressing a rat vascular angiotensin II type 1A receptor (CHO-AT(1A)). Cyclin D1 protein expression is regulated by mitogens, and its assembly with the cyclin-dependent kinases induces phosphorylation of the retinoblastoma protein pRb, a critical step in G(1) to S phase cell cycle progression contributing to the proliferative responses. In the present study, we found that in CHO-AT(1A) cells, Ang II induced a rapid and reversible tyrosine phosphorylation of various intracellular proteins including the protein-tyrosine phosphatase SHP-2. Ang II also induced cyclin D1 protein expression in a phosphatidylinositol 3-kinase and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK)-dependent manner. Using a pharmacological and a co-transfection approach, we found that p21(ras), Raf-1, phosphatidylinositol 3-kinase and also the catalytic activity of SHP-2 and its Src homology 2 domains are required for cyclin D1 promoter/reporter gene activation by Ang II through the regulation of MAPK/ERK activity. Our findings suggest for the first time that SHP-2 could play an important role in the regulation of a gene involved in the control of cell cycle progression resulting from stimulation of a G protein-coupled receptor independently of epidermal growth factor receptor transactivation.
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PMID:The protein-tyrosine phosphatase SHP-2 is required during angiotensin II-mediated activation of cyclin D1 promoter in CHO-AT1A cells. 1084 91

The tissue inhibitors of metalloproteinases (TIMPs) block matrix metalloproteinase (MMP)-mediated increases in cell proliferation, migration, and invasion that are associated with extracellular matrix (ECM) turnover. Here we demonstrate a direct role for TIMP-2 in regulating tyrosine kinase-type growth factor receptor activation. We show that TIMP-2 suppresses the mitogenic response to tyrosine kinase-type receptor growth factors in a fashion that is independent of MMP inhibition. The TIMP-2 suppression of mitogenesis is reversed by the adenylate cyclase inhibitor SQ22536, and implicates cAMP as the second messenger in these effects. TIMP-2 neither altered the release of transforming growth factor alpha from the cell surface, nor epidermal growth factor (EGF) binding to the cognate receptor, EGFR. TIMP-2 binds to the surface of A549 cells in a specific and saturable fashion (K(d) = 147 pm), that is not competed by the synthetic MMP inhibitor BB-94 and is independent of MT-1-MMP. TIMP-2 induces a decrease in phosphorylation of EGFR and a concomitant reduction in Grb-2 association. TIMP-2 prevents SH2-protein-tyrosine phosphatase-1 (SHP-1) dissociation from immunoprecipitable EGFR complex and a selective increase in total SHP-1 activity. These studies represent a new functional paradigm for TIMP-2 in which TIMP suppresses EGF-mediated mitogenic signaling by short-circuiting EGFR activation.
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PMID:Tissue inhibitor of metalloproteinases-2 (TIMP-2) suppresses TKR-growth factor signaling independent of metalloproteinase inhibition. 1104 84

Prostaglandins play regulatory roles in a variety of physiological and pathological processes in immune response and inflammation. Epigallocatechin-3-gallate (EGCG) is known to potent antitumor agent with antioxidant property. We first investigated the effect of EGCG on the production of prostaglandin E(2) (PGE(2)) and the expression of cyclooxygenase-2 (COX-2), the rate-limiting enzyme in the synthesis of PGE(2), using macrophage cell line, Raw264.7. Our results showed that COX-2 expression and PGE(2) production are upregulated by EGCG treatment and that this induction of COX-2 is regulated in part at the transcriptional level. In addition, we demonstrated the signal transduction pathway of mitogen-activated protein kinase (MAP kinase) in EGCG-mediated COX-2 expression. The MEK inhibitor (PD098059) prevented EGCG-induced COX-2 expression, whereas sodium orthovanadate (protein-tyrosine phosphatase inhibitor) significantly enhanced COX-2 expression and PGE(2) production. These results suggest that EGCG mediated COX-2 expression and PGE(2) production is associated with the activation of both the ERK and protein-tyrosine phosphatase signaling pathways.
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PMID:Involvement of ERK and protein tyrosine phosphatase signaling pathways in EGCG-induced cyclooxygenase-2 expression in Raw 264.7 cells. 1152 57

SRC family kinases have been consistently and recurrently implicated in neurite extension events, yet the mechanism underlying their neuritogenic role has remained elusive. We report that epidermal growth factor (EGF) can be converted from a non-neuritogenic into a neuritogenic factor through moderate activation of endogenous SRC by receptor-protein-tyrosine phosphatase alpha (a physiological SRC activator). We show that such a qualitative change in the response to EGF is not accompanied by changes in the extent or kinetics of ERK induction in response to this factor. Instead, the pathway involved relies on increased tyrosine phosphorylation of, and recruitment of Crk to, the SRC substrate Sin/Efs. The latter is a scaffolding protein structurally similar to the SRC substrate Cas, tyrosine phosphorylation of which is critical for migration in fibroblasts and epithelial cells. Expression of a dominant negative version of Sin interfered with receptor-protein-tyrosine phosphatase alpha/EGF- as well as fibroblast growth factor-induced neurite outgrowth. These observations uncouple neuritogenic signaling in PC12 cells from sustained activation of ERK kinases and for the first time identify an effector of SRC function in neurite extension.
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PMID:c-SRC mediates neurite outgrowth through recruitment of Crk to the scaffolding protein Sin/Efs without altering the kinetics of ERK activation. 1186 27


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