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:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Autophosphorylation of gp185erbB-2 in vivo is confined to its carboxy terminus and is required for optimal erbB-2 transforming activity under conditions of receptor overexpression. It remains unresolved, however, to what extent autophosphorylation regulates erbB-2 mitogenic signaling in normal cells, nor is the biochemical basis for such a regulatory function known. To address these issues, we utilized a chimeric molecule encompassing the extracellular domain of the epidermal growth factor (EGF) receptor (EGFR) fused to the transmembrane and intracellular domains of the erbB-2 product. In this EGFR/erbB-2 chimera, erbB-2 kinase activity is regulated by EGF binding. An EGFR/erbB-2 mutant bearing multiple Tyr----Phe substitutions at erbB-2 autophosphorylation sites (EGFR/erbB-2 5P) displayed markedly reduced phosphotyrosine content following EGF stimulation in comparison with the non-mutated chimera. When expressed in NR6 cells, the EGFR/erbB-2 5P mutant was unable to deliver a sizeable mitogenic signal when activated by EGF at physiological levels. In intact cells, the 5P mutant was still able to stimulate phosphorylation of the gamma isozyme of phospholipase C (PLC-gamma), a prototype erbB-2 substrate, although with a delayed time course, indicating that the 5P mutation decreased the affinity of the erbB-2 kinase for this substrate. This conclusion was further supported by the inability of the 5P mutant to associate with PLC-gamma in co-immunoprecipitation experiments. We infer that a major role of autophosphorylation is to increase the affinity of the erbB-2 kinase for its cellular substrates, so that, under physiological conditions, autophosphorylation is absolutely required for erbB-2 mitogenic signaling.
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PMID:erbB-2 autophosphorylation is required for mitogenic action and high-affinity substrate coupling. 135 97

The erbB-2 gene product, gp185erbB-2, unlike the structurally related epidermal growth factor (EGF) receptor (EGFR), exhibits constitutive kinase and transforming activity. We used a chimeric EGFR/erbB-2 expression vector to compare the mitogenic signaling pathway of the erbB-2 kinase with that of the EGFR, at similar levels of expression, in response to EGF stimulation. The EGFR/erbB-2 chimera was significantly more active in inducing DNA synthesis than the EGFR when either was expressed in NIH 3T3 cells. Analysis of biochemical pathways implicated in signal transduction by growth factor receptors indicated that both phospholipase C type gamma (PLC-gamma) and the p21ras GTPase-activating protein (GAP) are substrates for the erbB-2 kinase in NIH 3T3 fibroblasts. However, under conditions in which activation of the erbB-2 kinase induced DNA synthesis at least fivefold more efficiently than the EGFR, the levels of erbB-2- or EGFR-induced tyrosine phosphorylation of PLC-gamma and GAP were comparable. In addition, the stoichiometry of tyrosine phosphorylation of these putative substrates by erbB-2 appeared to be at least an order of magnitude lower than that induced by platelet-derived growth factor receptors at comparable levels of mitogenic potency. Thus, our results indicate that differences in tyrosine phosphorylation of PLC-gamma and GAP do not account for the differences in mitogenic activity of the erbB-2 kinase compared with either the EGFR or platelet-derived growth factor receptor in NIH 3T3 fibroblasts.
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PMID:The erbB-2 mitogenic signaling pathway: tyrosine phosphorylation of phospholipase C-gamma and GTPase-activating protein does not correlate with erbB-2 mitogenic potency. 167 40

Src homology regions 2 (SH2) and 3 (SH3) are noncatalytic domains that are conserved among several proteins implicated in the regulation of cell proliferation. Using bacterially expressed fusion proteins containing the SH2 domain of the abl tyrosine kinase, we have quantitated the binding of these domains to the activated epidermal growth factor (EGF) receptor (EGFR). A 35S-labeled abl SH2 fusion protein binds to the human EGFR immunoprecipitated from EGF-treated NIH3T3 cells that overexpress the receptor. This binding is totally dependent on the pretreatment of cells with EGF. The interaction is rapid, reaching 50% of maximum within 1 min, and attaining apparent equilibrium by 10 min. Dissociation of the complex is biphasic with a rapidly dissociating component (t1/2 of less than 1 min), as well as a slowly dissociable component. The 35S-labeled abl SH2 fusion protein specifically binds to the EGFR in a saturable manner and is differentially inhibited by unlabeled fusion proteins containing SH2 domains from phospholipase C, the p85 subunit of phosphatidylinositol-3 kinase, and the GTPase activation protein of ras. To identify residues critical for abl SH2-EGFR binding, six point mutants were constructed in the highly conserved FLVRES motif. Three mutants (V170L, E172Q, and E174Q) display binding affinities similar to that of wild type. However, three other mutants (R171K, S173C, and S175C) have greatly reduced affinity. Interestingly, the binding affinity to the EGFR determined by the in vitro assay directly correlates with the transforming ability of the corresponding v-abl constructs in vivo (Mayer, B. J., Jackson, P. K., Etten, R. A. V., and Baltimore, D. (1992) Mol. Cell. Biol. 12, 609-618). These data indicate that the Arg-171, Ser-173, and Ser-175 are critical for both transformation and abl SH2 domain binding to phosphotyrosine-containing proteins.
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PMID:Direct analysis of the binding of the abl Src homology 2 domain to the activated epidermal growth factor receptor. 767 9

It is shown that in the A431 cells, EGFR is co-immunoprecipitated with a group of proteins recognized by antibodies to phospholipase C gamma. These are 145- and 47-kDa proteins corresponding to phospholipase C gamma and Nck, respectively, and an unidentified 66-kDa protein. The association of phosphoinositide-specific phospholipase C gamma and 66-kDa protein to EGFR was observed in the A431 cells with or without the EGF treatment. Trypsin peptide maps of these two proteins are similar so it is assumed that the 66-kDa protein is related to phospholipase C gamma.
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PMID:A 66-kDa protein associated with epidermal growth factor receptor is a proteolytic fragment of phosphoinositide-specific phospholipase C. 780 59

Mutation of the autophosphorylation sites of receptor protein-tyrosine kinases alters ligand dependent internalization and down-regulation, indicating a critical role for these sites in receptor processing. Currently, no differences in receptor processing based on an individual autophosphorylation site have been defined. By using a glutathione S-transferase fusion protein containing the src homology 2 domains of phospholipase C-gamma1 to specifically recognize tyrosine 992 on the EGF receptor (Tyr(P)992), we have found differences in this subpopulation of receptors. Following EGF stimulation, the number of Tyr(P)992 receptors increased 2-fold over receptors identified by an antibody that recognizes activated EGF receptors (alpha-Act. EGFR) in A431 cells. Confocal fluorescence microscopy showed that Tyr(P)992 receptors underwent endocytosis at a slower rate and did not rapidly concentrate in juxtanuclear bodies. Tyr(P)992 receptors were associated with more SOS, Ras-GTPase activating protein, phosphatidylinositol 3-kinase, and SHPTP2/syp, but less Grb2, than receptors in the general population, and these receptors were more heavily phosphorylated than the general population of active receptors. These findings suggest that autophosphorylation status is relevant to the endocytosis, degradation, and effector molecule interaction of individual EGF receptors. Further investigations based on phosphorylation status should provide new insights into how receptor protein-tyrosine kinase signaling is regulated.
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PMID:Subsets of epidermal growth factor receptors during activation and endocytosis. 902 Jan 17

Extracts of human liver were found to contain activities which copurified and coeluted with the two major subtypes of mediators (type A and type P) isolated from insulin-stimulated rat liver. The putative type A mediator from human liver inhibited cAMP-dependent protein kinase from bovine heart, decreased phosphoenolypyruvate carboxykinase mRNA levels in rat hepatoma cells, and stimulated lipogenesis in rat adipocytes. The putative type P mediator stimulated bovine heart pyruvate dehydrogenase phosphatase. Both fractions were able to stimulate proliferation of EGFR T17 fibroblasts and the type A was able to support growth in organotypic cultures of chicken embryo cochleovestibular ganglia. Both activities were resistant to Pronase treatment and the presence of carbohydrates, phosphate, and free-amino groups were confirmed in the two fractions. These properties are consistent with the structure/ function characteristics of the type A and P inositolphosphoglycans (IPG) previously characterized from rat liver. Further, the ability of the human-derived mediators to interact with rat adipocytes and bovine-derived metabolic enzymes suggests similarity in structure between the mediators purified from different species. Galactose oxidase-susceptible membrane-associated glycosylphosphatidylinositols (GPI) have been proposed to be the precursors of IPG. GPI was purified from human liver membranes followed by treatment with galactose oxidase and reduction with NaB3H4. Serial t.l.c. revealed three radiolabeled bands which comigrated with the putative GPI precursors found in rat liver. These galactose-oxidase-reactive lipidic compounds, however, were only partially susceptible to hydrolysis with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis and were resistant to glycosylphosphatidylinositol-specific phospholipase C from Trypanosoma brucei. These data indicate that IPG molecules with insulin-like biological activities are present in human liver.
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PMID:Isolation and partial characterisation of insulin-mimetic inositol phosphoglycans from human liver. 925 87

Overexpression of surrogate receptors [epidermal growth factor (EGF) receptor (EGFR) and platelet-derived growth factor receptor] in adipocytes has demonstrated that multiple signaling pathways may lead to GLUT4-mediated glucose uptake. These implicated pathways function independently of IRS-1 phosphorylation and PI3-kinase activation. In addition, we previously demonstrated that EGFR tyrosyl autophosphorylation is required to stimulate GLUT4-mediated glucose transport in 3T3-L1 adipocytes. This observation suggests that signaling molecules that are dependent on EGFR autophosphorylation, such as phospholipase C (PLC), may lie in the signaling pathway to glucose transport. As PLC has been implicated in glucose transport by several clinical and basic mechanistic studies, we investigated whether EGFR signaling may promote glucose transport via modulation of PLC activity. Activation of EGFR overexpressing 3T3-L1 adipocytes leads to a 3.4 +/- 1.2-fold stimulation of PLC activity over basal levels vs. only 1.06 +/- 0.01-fold stimulation by insulin. Pharmacological inhibition of PLC by 50 microM U73122 reduced phosphoinositide accumulation by 79.2 +/- 16.9% and resulted in a concomitant 56.0 +/- 12.7% decrease in EGF-induced glucose transport. This inhibition of glucose transport by U73122 was specific, because the inactive congener, U73343, failed to block EGF-induced glucose transport. Despite the low levels of insulin-induced PLC activity, insulin-stimulated glucose transport activity was similarly inhibited by U73122 (55.9 +/- 13.1% inhibition). Inhibition of PLC activation did not impair either EGF- or insulin-induced activation of glycogen synthase or incorporation of glucose into lipid, supporting the hypothesis that both EGF- and insulin-induced glucose disposal can be independent of GLUT4-mediated glucose transport. The diminution of glucose transport secondary to inhibition of PLC activity was reflected by a decrease in GLUT4 translocation to the plasma membrane upon either EGF or insulin stimulation. These results are consistent with either a permissive or an active role for PLC activity in the translocation of GLUT4 to the plasma membrane.
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PMID:A role for phospholipase C activity in GLUT4-mediated glucose transport. 938 97

A recently reported new member of the Vav family proteins, Vav3 has been identified as a Ros receptor protein tyrosine kinase (RPTK) interacting protein by yeast two-hybrid screening. Northern analysis shows that Vav3 has a broad tissue expression profile that is distinct from those of Vav and Vav2. Two species of Vav3 transcripts, 3.4 and 5.4 kb, were detected with a differential expression pattern in various tissues. Transient expression of Vav in 293T and NIH 3T3 cells demonstrated that ligand stimulation of several RPTKs (epidermal growth factor receptor [EGFR], Ros, insulin receptor [IR], and insulin-like growth factor I receptor [IGFR]) led to tyrosine phosphorylation of Vav3 and its association with the receptors as well as their downstream signaling molecules, including Shc, Grb2, phospholipase C (PLC-gamma), and phosphatidylinositol 3 kinase. In vitro binding assays using glutathione S-transferase-fusion polypeptides containing the GTPase-binding domains of Rok-alpha, Pak, or Ack revealed that overexpression of Vav3 in NIH 3T3 cells resulted in the activation of Rac-1 and Cdc42 whereas a deletion mutant lacking the N-terminal calponin homology and acidic region domains activated RhoA and Rac-1 but lost the ability to activate Cdc42. Vav3 induced marked membrane ruffles and microspikes in NIH 3T3 cells, while the N-terminal truncation mutants of Vav3 significantly enhanced membrane ruffle formation but had a reduced ability to induce microspikes. Activation of IR further enhanced the ability of Vav3 to induce membrane ruffles, but IGFR activation specifically promoted Vav3-mediated microspike formation. N-terminal truncation of Vav3 activated its transforming potential, as measured by focus-formation assays. We conclude that Vav3 mediates RPTK signaling and regulates GTPase activity, its native and mutant forms are able to modulate cell morphology, and it has the potential to induce cell transformation.
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PMID:Vav3 mediates receptor protein tyrosine kinase signaling, regulates GTPase activity, modulates cell morphology, and induces cell transformation. 1109 73

During induced cell motility the actin cytoskeleton at the leading edge must undergo constant reorganization. Recently, phosphoinositides have been shown to be central to cytoskeleton-membrane linkages and actin organization and turnover. Epidermal growth factor (EGF) receptor (EGFR)-mediated cell motility requires phospholipase C-gamma (PLCgamma), hydrolysis of phosphoinsotide 4,5-bisphosphate (PIP(2)) and subsequent release of gelsolin. We hypothesized this led to the mobilization of PIP(2)-binding proteins which modify the actin cytoskeleton and thus sought to determine whether the leading edge was a site of active PIP(2) hydrolysis and gelsolin redistribution to cytoskeleton. Herein, we report that during EGF-induced motility, the leading edge's submembranous region constitutes a distinct subcellular locale. The relevant phosphoinositide composition of this space was determined by probing with an antibody to PIP(2) and a green fluorescence protein (GFP)-tagged pleckstrin homology (PH) domain of PLCdelta (GFP-PH) that recognizes both PIP(2) and inositol 1,4,5-trisphosphate (IP(3)). PIP(2) was absent from leading lamellipodia despite an increase in IP(3) generation, suggesting an increase in PIP(2) hydrolysis at the leading edge. Visualized with immunofluorescence, gelsolin preferentially concentrated near the leading edge in a punctate fashion. Examining the Triton X-insoluble actin cytoskeleton fractions, we observe a PLCgamma-dependent increase of gelsolin incorporation upon EGF stimulation. At a molecular level, field emission scanning electron microscopy (FE-SEM) shows that gelsolin incorporates preferentially into the submembranous actin arcs at the leading edge of the lamellipodia. Together these data suggest a model of PIP(2) hydrolysis at the leading edge causing a localized release of PIP(2)-binding proteins-particularly gelsolin-that drives cytoskeletal rearrangement and protrusion.
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PMID:Distribution of gelsolin and phosphoinositol 4,5-bisphosphate in lamellipodia during EGF-induced motility. 1195 May 94

Epidermal growth factor (EGF) receptor (EGFR) regulates development of cell-cell communication in fetal lung, but the signal transduction mechanisms involved are unknown. We hypothesized that, in late-gestation fetal rat lung, phospholipase C-gamma (PLC-gamma) expression and activation by EGF is cell specific and developmentally regulated. PLC-gamma immunolocalized to cuboidal epithelium and mesenchymal clusters underlying developing saccules. PLC-gamma protein increased from day 17 to day 19 and then decreased. In cultured fetal lung fibroblasts, EGF stimulated PLC-gamma phosphorylation 2.6-fold (day 17), 10.8-fold (day 19), and 4.2-fold (day 21). EGF stimulated (3)H-labeled diacylglycerol production in fibroblasts (beginning on day 18 in female and on day 19 in male rats), but not in type II cells at any time during gestation. EGFR blockade abrogated the observed stimulation of PLC-gamma phosphorylation by EGF. In conclusion, PLC-gamma expression and activation by EGF in fetal lung are cell specific, corresponding to the development of EGFR expression. EGF induces diacylglycerol production in a cell- and gestation-specific manner. PLC-gamma activation by EGFR in fetal lung fibroblasts may be involved in EGF control of lung development.
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PMID:Cell-specific and developmental expression of phospholipase C-gamma and diacylglycerol in fetal lung. 1250 68


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