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)

We have generated two groups of deletion mutants of the alpha PDGFR and one group of chimeras between alpha PDGFR and beta PDGFR to further investigate the structural requirements of the alpha PDGFR for binding to platelet-derived growth factor (PDGF)-AA and to a monoclonal antibody against alpha PDGFR (designated as mAb-alpha R1). The mAb-alpha R1 has recently been reported to block high affinity binding of PDGF-AA to alpha PDGFR. The first group of mutants were carboxyl-terminal deletion mutants encoding the first two immunoglobulin (Ig)-like domains (alpha R1-216), the first four Ig-like domains (alpha R1-415), or all five Ig-like domains (alpha R1-530) of the alpha PDGFR. Since these mutants lacked transmembrane domains, their expression in NIH/3T3 cells resulted in secretion of the truncated alpha PDGFRs. Using conditioned medium from NIH/3T3 transfectants, we showed that mAb-alpha R1 was able to immunoprecipitate each of these secreted form of alpha PDGFRs, suggesting that the epitope recognized by mAb-alpha R1 is located within Ig-like domains 1 and 2 of the alpha PDGFR. Furthermore, PDGF-AA exhibited detectable binding to alpha R1-415 or alpha R1-530 but failed to interact with alpha R1-216, suggesting that the first two Ig-like domains of the alpha PDGFR are not sufficient for PDGF-AA binding. The second group of alpha PDGFR mutants were internal deletion mutants lacking Ig-like loop 1 (alpha R delta 49-100), Ig-like loop 2 (alpha R delta 150-189), Ig-like loop 3 (alpha R delta 235-290), or part of Ig-like loops 4 and 5 (alpha R delta 375-450). The internal deletion mutants were transfected into 32D cells which lack both alpha PDGFR and beta PDGFR. PDGF-AA bound with high affinity to 32D cells expressing alpha R delta 375-450 but not to 32D cells expressing the other three internal deletion mutants, suggesting that the region required for PDGF-AA binding should be within the first three Ig-like domains of the alpha PDGFR. In addition, mAb-alpha R1 bound to 32D cells expressing alpha R delta 235-290 but failed to bind 32D cells transfected with alpha R delta 49-100 or alpha R delta 150-189.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Structural coincidence of alpha PDGFR epitopes binding to platelet-derived growth factor-AA and a potent neutralizing monoclonal antibody. 751 2

To delineate potential angiogenic roles of platelet-derived growth factor (PDGF), we have investigated PDGF and its receptors on bovine aortic endothelial cells that exhibit spontaneous angiogenesis in vitro (angiogenic endothelial cells). Initiation of cord/tube formation by angiogenic endothelial cells required bovine or human serum. Neutralization of PDGF-BB in human serum with a monoclonal anti-PDGF-BB antibody reduced cord/tube formation by 37 +/- 10%, whereas neutralizing anti-PDGF-AA and an IgG isotype-matched control antibody had no effect. DNA synthesis in response to PDGF-BB increased as the cords and tubes developed; furthermore, PDGF-BB induced the incorporation of BrdU in the nuclei of cells associated with these structures. PDGF beta-receptor (PDGF-beta) mRNA increased concomitantly with cord/tube formation, and PDGFR-beta were specifically localized by immunocytochemistry to developing and mature cords and tubes. However, PDGFR-beta transcripts and protein were undetectable in nonangiogenic endothelial cells, and PDGF alpha-receptor mRNA was not expressed in either endothelial cell strain. In contrast to nonangiogenic endothelial cells, angiogenic endothelial cells did not express the PDGF B-chain, the required ligand for the PDGFR-beta. We conclude that (a) PDGF-BB can contribute to angiogenesis in vitro, (b) PDGFR-beta are specific for cord/tube-forming endothelial cells and mediate endothelial proliferation and cord/tube formation, and (c) in angiogenic and nonangiogenic endothelial cells, the expression of PDGFR-beta and PDGF B-chain is inversely correlated. We therefore suggest that paracrine PDGF might amplify angiogenesis via direct action on endothelially expressed PDGFR-beta.
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PMID:PDGF-BB modulates endothelial proliferation and angiogenesis in vitro via PDGF beta-receptors. 751 7

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

The upstream regulatory region of the c-fos promoter contains two growth factor-regulated promoter elements: the serum response element, which binds a ternary complex comprising serum response factor (SRF) and a ternary complex factor (TCF); and the sis-inducible element (SIE) which binds STAT transcription factors. We used transient transfection of c-fos promoter mutants in NIH 3T3 cells to assess the contributions of these elements to activation by different extracellular stimuli. Colony-stimulating factor-1, platelet-derived growth factor and epidermal growth factor activate the c-fos promoter via cooperation of the SIE and the SRE; however, mutants that can bind SRF but not STATs or TCF remain inducible by whole serum. Activation by the SIE is context-dependent: interferons activate STAT DNA binding activity and transcription of SIE reporter genes, but not the c-fos promoter, which requires an additional ras-dependent signal. SRE activation by receptor tyrosine kinases requires TCF binding, and can be mediated by the TCF Elk-1. In contrast, SRE activation following activation of heterotrimeric G proteins by lysophosphatidic acid or aluminium fluoride ion requires SRF but is independent of TCF binding. These results suggest that heterotrimeric G proteins activate a signalling pathway distinct from those that activate the STATs and the TCFs, that controls SRF activity.
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PMID:Differential activation of c-fos promoter elements by serum, lysophosphatidic acid, G proteins and polypeptide growth factors. 758 32

Some growth factors transduce positive growth signals, while others can act as growth inhibitors. Nuclear signaling events of previously quiescent cells stimulated with various growth factors have been studied by isolating the complexed chromatin-associated proteins and chromatin-associated proteins. Signals from the plasma membrane are integrated within the cells and quickly transduced to the nucleus. It is clear that several growth factors, such as epidermal growth factor, transforming growth factor alpha (but not transforming growth factor beta), and platelet-derived growth factor, utilize similar intracellular signaling biochemistries to modulate nucleosomal characteristics. The very rapid and consistent phosphorylation of nuclear p33, p54, and low molecular mass proteins in the range of 15-18 kDa after growth factor stimulation implies that there is a coordination and integration of the cellular signaling processes. Additionally, phosphorylation of p33 and some low molecular mass histones has been found to occur within 5 min of growth factor treatment and to reach a maximum by 30 min. In this study, we report that Neu receptor activating factor also utilizes the same signaling mechanism and causes p33 to become phosphorylated. In addition, both the tumor promoter okadaic acid (which inhibits protein phosphatases 1 and 2A) and phorbol ester (phorbol 12-tetradecanoate 13-acetate) stimulate phosphorylation of p33, p54, and low molecular mass histones. However, transforming growth factor beta, which is a growth inhibitor for fibroblasts, fails to increase p33 phosphorylation. In general, p33 phosphorylation patterns correspond to positive and negative mitogenic signal transduction. p33 isolated from the complexed chromatin-associated protein fraction appears to be a kinase, or tightly associated with a kinase, and shares antigenicity with the cell division cycle-dependent Cdk2 kinase as determined by antibody-dependent analysis. The rapid phosphorylation of nucleosomal proteins may influence sets of early genes needed for the induction and progression of the cell cycle.
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PMID:A kinase associated with chromatin that can be activated by ligand-p185c-Neu or epidermal growth factor-receptor interactions. 760 37

Adhesion to extracellular matrix mediates cell cycle progression in mid-late G1; this effect involves an integrin-dependent organization of the cytoskeleton and a consequent change in cell shape. In an effort to identify potential signal-transducing agents that are associated with integrin-dependent shape changes, we looked for kinase activities that were stimulated by long-term adhesion of G0-synchronized NIH-3T3 cells to fibronectin-coated dishes. Several kinase activities were stimulated by this procedure, two of which migrated at 42 and 44 kDa and phosphorylated myelin basic protein in vitro. Blotting with anti-phosphotyrosine and anti-mitogen-activated protein (MAP) kinase antibodies identified these enzymes as ERK 1 and ERK 2. In contrast to the rapid and transient activation of these MAP kinases by platelet-derived growth factor, stimulation of MAP kinase activity by fibronectin was gradual, persistent, and associated with cell spreading rather than cell attachment itself. Cytochalasin D blocked the activation of MAP kinase activity that was induced by the binding of cells to fibronectin. Moreover, MAP kinase was also activated by adhesion of cells to vitronectin and type IV collagen; these effects were also associated with cell spreading. These results distinguish the regulation of G1 phase MAP kinase activity by soluble mitogens and extracellular matrix. They also implicate MAP kinase in shape-dependent cell cycle progression.
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PMID:Integrin-dependent activation of MAP kinase: a link to shape-dependent cell proliferation. 761 63

We have fused the cytoplasmic domain of the p55 tumor necrosis factor (TNF) receptor to the extracellular and transmembrane domain of the mouse platelet-derived growth factor (PDGF) receptor. Mouse mammary gland epithelial (NMuMG) cells were stably transfected with the PDGFR-TR55 chimeric receptor. These cells lack endogenous PDGF receptor expression and do not respond to PDGF. In the PDGFR-TR55 transfectants, PDGF elicited a cytotoxic response, which is indistinguishable from that induced by the wild type p55 TNF receptor. In addition, PDGF-induced activation of the PDGFR-TR55 chimeric receptor resulted in nuclear translocation of NF-kappa B. The data presented suggest that cross-linking of the p55 TNF receptor cytoplasmic domain by a dimeric ligand such as PDGF is sufficient to generate cellular responses that do not differ from those observed with the trimeric ligand TNF.
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PMID:Cross-linking of the p55 tumor necrosis factor receptor cytoplasmic domain by a dimeric ligand induces nuclear factor-kappa B and mediates cell death. 761 52

In order to verify the role of activation of phosphatidylcholine (PC) hydrolysis by phospholipase D (PLD) in the initiation of mitogenic process of retinal capillary pericytes, platelet-derived growth factor (PDGF), a known PC hydrolysis stimulator, and exogenous PLD have been used to stimulate pericytes. Exogenous PLD (Streptomyces chromofuscus PLD) or PDGF BB homodimer (PDGF) was added to a medium of quiescent pericytes prelabeled with [32P]orthophosphate. In the presence of ethanol (300 mM), phosphatidic acid (PA) and its stable transphosphatidylated product, phosphatidylethanol (PEt), were determined. In parallel, [3H]thymidine incorporation was measured. Downregulation of PKC was achieved by long-term treatment with a phorbol ester. The addition of exogenous PLD or PDGF stimulated both [3H]thymidine incorporation and [32P]PEt formation in a similar kinetic fashion, suggesting that PC hydrolysis is involved in PDGF-mitogenic signaling pathway. PDGF-stimulated [3H]PA formation was significantly higher in the presence than in the absence of PA phosphohydrolase (PAP) inhibitor, indicating the activation of PLD/PAP pathway. In the presence of ethanol, a substantial level of PA at the steady state can be abolished by an inhibitor of diacylglycerol (DAG) kinase. This phenomenon indicates the existence of PC-phospholipase C (PLC)/DAG kinase pathway in PC hydrolysis. Insulin potentiated both PLD- and PDGF-induced DNA synthesis. Though similarities occur in the induction of DNA synthesis and PC hydrolysis by exogenous PLD and PDGF, the maximum extent of DNA synthesis of exogenous PLD was only approximately 43% of that induced by PDGF. Moreover, exogenous PLD-induced DNA synthesis was not blunted, while PDGF-elicited DNA synthesis was markedly reduced, by PKC downregulation. In addition, PDGF-induced PC hydrolysis was attenuated by a tyrosine kinase inhibitor, whereas exogenous PLD-induced PC hydrolysis was unchanged. Taken together, exogenous PLD may mimic PDGF action and partially account for the efficacy on DNA synthesis elicited by PDGF. The signal transduction initiated by exogenous PLD is able to bypass the PKC- and PTK-dependent activation of endogenous PLD. These findings provide evidence for the importance of PLD-mediated PC hydrolysis in pericyte DNA synthesis stimulated by PDGF.
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PMID:Phosphatidylcholine hydrolysis and DNA synthesis in cultured retinal capillary pericytes. 764 54

Expression of platelet-derived growth factor (PDGF) and PDGF receptors was examined in cultured human pancreatic cancer cells, in the normal human pancreas and in pancreatic adenocarcinomas. mRNA transcripts encoding PDGF A and B chains, and PDGF receptor beta (PDGFR beta) were present in PANC-I and HPAF human pancreatic cancer cells. Transforming growth factor beta I (TGF-beta I), but not PDGF-AA or -BB, enhanced PDGF A and B chain mRNA levels in both cell lines. In the normal human pancreas PDGF A chain mRNA levels were relatively abundant, whereas PDGF B chain mRNA levels were not detected and PDGF receptor alpha (PDGFR alpha) and beta mRNA transcripts were present at low levels. PDGF immunoreactivity was present in islet cells, and PDGFR alpha was present in acinar cells, whereas PDGFR beta was present in acinar cells and in the connective tissue. In the pancreatic cancers, PDGF A chain mRNA transcripts were also abundant, and 6 of 13 samples exhibited the PDGF B chain mRNA transcript. In addition, there was a 7-fold increase in the levels of PDGFR alpha and PDGFR beta in the cancer samples by comparison with the normal pancreas. By immunohistochemistry, PDGF and both PDGF receptors were present in the cancer cells, and PDGFR beta was abundant in fibroblasts and endothelial cells within the connective tissue.
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PMID:Induction of platelet-derived growth factor A and B chains and over-expression of their receptors in human pancreatic cancer. 766 22

Regulation of cell proliferation, differentiation, and metabolic homeostasis is associated with the phosphorylation and dephosphorylation of specific tyrosine residues of key regulatory proteins. The phosphotyrosine phosphatase 1D (PTP 1D) contains two amino terminally located Src homology 2 (SH2) domains and is similar to the Drosophila corkscrew gene product, which positively regulates the torso tyrosine kinase signal transduction pathway. PTP activity was found to be regulated by physical interaction with a protein tyrosine kinase. PTP 1D did not dephosphorylate receptor tyrosine kinases, despite the fact that it associated with the epidermal growth factor receptor and chimeric receptors containing the extracellular domain of the epidermal growth factor receptor and the cytoplasmic domain of either the HER2-neu, kit-SCF, or platelet-derived growth factor beta (beta PDGF) receptors. PTP 1D was phosphorylated on tyrosine in cells overexpressing the beta PDGF receptor kinase and this tyrosine phosphorylation correlated with an enhancement of its catalytic activity. Thus, protein tyrosine kinases and phosphatases do not simply oppose each other's action; rather, they may work in concert to maintain a fine balance of effector activation needed for the regulation of cell growth and differentiation.
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PMID:Activation of a phosphotyrosine phosphatase by tyrosine phosphorylation. 768 Dec 17


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