Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

Binding of platelet-derived growth factor (PDGF) to the PDGF receptor (PDGFR) beta subunit triggers receptor tyrosine phosphorylation and the stable association of a number of signal transduction molecules, including phospholipase C gamma (PLC gamma), the GTPase activating protein of ras (GAP), and phosphatidylinositol-3 kinase (PI3K). Previous reports have identified three PDGFR tyrosine phosphorylation sites in the kinase insert domain that are important for stable association of GAP and PI3K. Two of them, tyrosine (Y) 740, and Y-751 are required for the stable association of PI3K, while Y-771 is required for binding of GAP. Here we present data for two additional tyrosine phosphorylation sites, Y-1009 and Y-1021, that are both in the carboxy-terminal region of the PDGFR. Characterization of PDGFR mutants in which these phosphorylation sites are substituted with phenylalanine (F) indicated that Y-1021 and Y-1009 were required for the stable association of PLC gamma and a 64-kDa protein, respectively. An F-1009/F-1021 double mutant selectively failed to bind both PLC gamma and the 64-kDa protein, whereas all of the carboxy-terminal mutants bound wild-type levels of GAP and PI3K. The carboxy terminus encodes the complete binding site for PLC gamma, since a phosphorylated carboxy-terminal fusion protein selectively bound PLC gamma. To determine the biological consequences of failure to associate with PLC gamma, we measured PDGF-dependent inositol phosphate production and initiation of DNA synthesis. The PDGFR mutants that failed to associate with PLC gamma were not able to mediate the PDGF-dependent production of inositol phosphates. Since tyrosine phosphorylation of PLC gamma enhances its enzymatic activity, we speculated that PDGFR mutants that failed to activate PLC gamma were unable to mediate its tyrosine phosphorylation. Surprisingly, the F-1021 receptor mediated readily detectable levels of PDGF-dependent PLC gamma tyrosine phosphorylation. Thus, the production of inositol phosphates requires not only PLC gamma tyrosine phosphorylation but also its association with the PDGFR. Comparison of the mutant PDGFRs' abilities to initiate PDGF-dependent DNA synthesis indicated that failure to associate with PLC gamma and produce inositol phosphates diminished the mitogenic response by 30%. In contrast, preventing the PDGFR from binding the 64-kDa protein did not compromise PDGF-triggered DNA synthesis at saturating concentrations of PDGF. Thus, it appears that phosphorylation of the PDGFR at Y-1021 is required for the stable association of PLC gamma to the receptor's carboxy terminus, the production of inositol phosphates, and initiation of the maximal mitogenic response.
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PMID:Tyrosines 1021 and 1009 are phosphorylation sites in the carboxy terminus of the platelet-derived growth factor receptor beta subunit and are required for binding of phospholipase C gamma and a 64-kilodalton protein, respectively. 767 51

In order to examine the mechanisms underlying smooth-muscle cell proliferation, we investigated effect of platelet-derived growth factor (PDGF) dimers on proliferation of rabbit vascular smooth-muscle cells (VSMCs) and also involvement of phospholipase C (PLC) isoforms in the signal transduction. PDGF-BB and -AB, but not -AA, stimulated cell proliferation and intracellular production of inositol trisphosphate. Northern and Western analyses demonstrated that VSMCs mainly expressed PLC-gamma 2 and PLC-delta 1 among four PLC isoforms tested. A number of cellular proteins, including PLC-gamma 2, but not PLC-delta 1, were phosphorylated on a tyrosine residue by the stimulation of either PDGF-BB or -AB. These results suggest a functional association of PDGF receptor and PLC-gamma 2 that might be responsible for PDGF-dependent VSMC growth. In addition, the expression of PLC-gamma 2 was extremely low in the primary VSMC cultures and was induced during further cultivation of the primary cultures, indicating that an acquisition of PDGF-signal-transducing components, including PLC-gamma 2, may be an important step for proliferation of smooth-muscle cells.
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PMID:Evidence for involvement of phospholipase C-gamma 2 in signal transduction of platelet-derived growth factor in vascular smooth-muscle cells. 768 Dec 81

Human platelets contain phospholipase C (PLC)-gamma 2, a distinct isoform closely related to PLC-gamma 1. Both inositol phospholipid-specific phospholipases C contain the src-related SH2 regions. Stimulation of platelets with the potent agonist, thrombin, led to a rapid and transient phosphorylation of PLC-gamma 2 on tyrosine residues. Activated platelets lysed in the absence of sodium orthovanadate had levels of tyrosine-phosphorylated PLC-gamma 2 paralleling those seen in unstimulated platelets. Previously, it had been shown that PLC-gamma 1 was phosphorylated on tyrosine residues by the agonist-occupied platelet-derived growth factor (PDGF) receptor and epidermal growth factor (EGF) receptor in cells other than platelets. In addition, more recent data have indicated that PLC-gamma 2 is also capable of being tyrosine-phosphorylated in cells of hematopoietic origin, such as B cells and natural killer (NK) cells. Here we report that PLC-gamma 2 expressed in a terminally-differentiated hematopoietic cell is also tyrosine-phosphorylated in response to an agonist.
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PMID:Thrombin activation of human platelets causes tyrosine phosphorylation of PLC-gamma 2. 768 59

To determine the molecular basis for the transforming function of platelet-derived growth factor (PDGF)-A in NIH/3T3 cells, we have constructed chimerae consisting of the extracellular domain of the human CSF-1R (fms) linked to the cytoplasmic domain of the alpha PDGF receptor (alpha R) containing a series of deletion or point mutations. The ability of fms/alpha R chimerae to mediate CSF-1-dependent anchorage-independent growth, focus formation, and chemotaxis of NIH/3T3 cells was then examined. Our results provide evidence that a domain encompassing amino acid residues 977-1024 of the alpha PDGFR is required for ligand-dependent focus formation, but not chemotaxis or anchorage-independent growth, and that tyrosine residues within this domain constitute the major binding site for phospholipase C gamma. Therefore, our findings suggest that: (i) the focus forming function of alpha PDGFR correlates well with the ability of the receptor to bind phospholipase C gamma, and (ii) the mechanism of focus formation mediated by alpha PDGFR may be distinguished from that required for chemotaxis or anchorage-independent growth.
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PMID:Differential requirement of a motif within the carboxyl-terminal domain of alpha-platelet-derived growth factor (alpha PDGF) receptor for PDGF focus forming activity chemotaxis, or growth. 770 38

The transforming protein of simian sarcoma virus is homologous to the platelet-derived growth factor (PDGF) B-chain. Fibroblasts transformed with simian sarcoma virus constitutively produce a growth factor that stimulates the endogenous tyrosine kinase of PDGF receptors in an autocrine manner. Autophosphorylation of PDGF receptors upon ligand stimulation provides binding sites for Src homology 2 domains of intracellular signaling molecules, which thereby become activated. We have characterized the PDGF receptor-mediated signal transduction in NIH 3T3 cells transformed with a PDGF B-chain cDNA (Sis 3T3 cells) in the absence and presence of suramin, a polyanionic compound that quenches PDGF-induced mitogenicity and reverts the transformed phenotype of the Sis 3T3 cells. Our data show that in the presence of suramin the general level of tyrosine phosphorylation was decreased. Nevertheless, autophosphorylated receptors complexed with substrates persisted in the cells. Suramin had no effect on activation of phosphatidylinositol 3'-kinase or on tyrosine phosphorylation of phospholipase C-gamma and GTPase-activating protein of Ras. On the other hand, kinase activation of Src and Raf-1, phosphorylation of protein-tyrosine phosphatase 1D/Syp and Shc, and complex formation with Grb2 were greatly diminished by suramin. A possible explanation for our findings is that different PDGF receptor-coupled signaling pathways are active in different structural or functional compartments in the cell. Those pathways that are not affected by suramin might elicit distinct cellular responses, which are not sufficient for growth and transformation.
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PMID:Compartmentalization of autocrine signal transduction pathways in Sis-transformed NIH 3T3 cells. 773 Mar 19

The intracellular signal transduction pathways that mediate the stimulatory effects of platelet-derived growth factor (PDGF)-BB and transforming growth factor (TGF)-beta on hyaluronan biosynthesis in human fibroblasts were investigated. The stimulatory effects of both PDGF-BB and TGF-beta 1 were dependent on protein kinase C (PKC), since the PKC inhibitor calphostin C inhibited the stimulation by the growth factors. Direct activation of PKC by phorbol 12-myristate 13-acetate (PMA) also stimulated hyaluronan production, and the combination of either PDGF-BB or TGF-beta 1 and PMA gave an increased effect. One possible mechanism for activation of PKC is via induction of phospholipase C (PLC) activity; U-17322, an inhibitor of PLC-gamma, was found to inhibit partially PDGF-BB-stimulated hyaluronan synthesis. PDGF-BB is known to activate PLC-gamma through tyrosine phosphorylation; however, a PDGF beta-receptor mutant unable to interact with and activate PLC-gamma was still able to mediate induction of hyaluronan biosynthesis, indicating that PDGF-mediated stimulation is not entirely dependent on PLC-gamma. The stimulations by PDGF-BB and TGF-beta 1 were partly dependent on protein synthesis, since parts of the effects were inhibited by cycloheximide; in contrast, the effects mediated by PMA were not. Our results indicate that PKC is involved in the transduction of the effects of growth factors on hyaluronan biosynthesis, and that the effects involve direct or indirect activation of existing hyaluronan synthetase molecules, as well as induction of new enzyme molecules.
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PMID:Stimulation of hyaluronan biosynthesis by platelet-derived growth factor-BB and transforming growth factor-beta 1 involves activation of protein kinase C. 774 13

We have previously reported that platelet-derived growth factor (PDGF) receptor mutants that lack high affinity binding sites for phosphatidylinositol 3-kinase (PI 3-kinase) fail to concentrate in juxtanuclear vesicular structures after activation with PDGF. We have now identified the point in the endocytic pathway at which PI 3-kinase binding sites are required. Receptor internalization from the plasma membrane, measured as the acquisition of acid resistance of prebound 125I-PDGF, was only slightly decreased in cells expressing a PDGF receptor mutant (F5) lacking PI 3-kinase, GTPase-activating protein (GAP), phospholipase C gamma, and Syp binding sites but not expressing mutants where any of these individual sites were restored nor expressing a mutant lacking exclusively PI 3-kinase binding sites. In contrast, the extent of down-regulation of PDGF binding sites from the cell surface after prolonged incubation with PDGF as well as the degradation of [35S]methionine-labeled receptor were markedly reduced in cells expressing the F5 mutant, mutants restored in GAP, phospholipase C gamma, or Syp binding sites or expressing the mutant exclusively lacking PI 3-kinase binding sites but not in cells expressing the mutant where PI 3-kinase binding sites were restored. Inhibition of PI 3-kinase activity with wortmannin caused a dramatic decrease in the rates of down-regulation and degradation of wild-type receptors. These results suggest that PI 3-kinase binding sites are not required for internalization of PDGF receptor but are required to divert the PDGF receptor to a degradative pathway. Furthermore, the requirement for PI 3-kinase binding sites on the receptor appears to be due to a requirement for PI 3-kinase catalytic activity.
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PMID:Phosphatidylinositol 3-kinase activity is required at a postendocytic step in platelet-derived growth factor receptor trafficking. 776 21

Insulin is the only known hormone which rapidly stimulates glucose uptake in target tissues, mainly by translocation to the cell surface of the intracellular insulin-regulatable glucose transporter (glucose transporter type 4, GLUT4). We have developed a cell line for direct, sensitive detection of GLUT4 on the cell surface. We have suggested that insulin-activated phosphatidylinositol (PI) 3-kinase may be involved in the signaling pathway of insulin-stimulated GLUT4 translocation. We report that platelet-derived growth factor (PDGF), which stimulates PI 3-kinase activity, triggers GLUT4 translocation in Chinese hamster ovary (CHO) cells stably overexpressing the PDGF receptor and in 3T3-L1 mouse adipocytes. Using mutant PDGF receptors that cannot bind to Ras-GTPase-activating protein, phospholipase C-gamma, and PI 3-kinase, respectively, we obtained evidence that PI 3-kinase binding sites play a key role in the signaling pathway of PDGF-stimulated GLUT4 translocation in the CHO cell system.
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PMID:Platelet-derived growth factor triggers translocation of the insulin-regulatable glucose transporter (type 4) predominantly through phosphatidylinositol 3-kinase binding sites on the receptor. 786 37

SH2 domains bind to specific phosphotyrosine-containing sites in a fashion dictated by the amino acids flanking the phosphotyrosine. Attention has focused on the role of the three COOH-terminal positions (+1 to +3) in generating specificity. Autophosphorylation of Tyr1021 in the tail of the beta-receptor for platelet-derived growth factor creates a specific binding site for the COOH-terminal SH2 domain of phospholipase C (PLC)-gamma 1. We show that the residues 4 and 5 amino acids COOH-terminal to Tyr1021 (+4 Leu and +5 Pro) are required for efficient PLC-gamma 1 binding, and that their replacement with the corresponding residues from a phosphatidylinositol 3'-kinase binding site abrogates stable association with PLC-gamma 1. In contrast, replacement of the +3 Pro with Met produces a Tyr1021 site with mixed specificity that binds both PLC-gamma 1 and phosphatidylinositol 3'-kinase. This motif is rendered specific for phosphatidylinositol 3'-kinase by further substitution of the +4 Leu. These results indicate that the +4 and +5 residues are important for the selective binding of specific SH2 domains. This study suggests that phosphotyrosine sites can be tailored to bind one or more SH2 domains with high affinity, depending on the combination of residues in the +1 to +5 positions.
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PMID:Construction of an SH2 domain-binding site with mixed specificity. 787 30


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