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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the present study, we investigated possible mechanisms behind exogenous phospholipase C-induced glycerol production in irreversibly damaged myocytes. Rat ventricular myocytes were preincubated for 60 min in substrate-free Krebs-Henseleit bicarbonate buffer equilibrated with 95% N2-5% CO2 (37 degrees C, pH = 7.4), resulting in exhaustion of cellular high energy phosphates and loss of rod-shaped morphology. At the end of the preincubation period, the incubation vials were divided into two groups; one receiving 10 mU/ml phospholipase C (PC-PLC), whereas the other received an equivalent volume of buffer (control incubations). Incubation was then continued for another 60 min under 95% air-5% CO2 atmosphere. Samples for measurement of metabolite levels were taken immediately after cell isolation, at the end of the preincubation period and at the end of the normoxic incubation period. During the 60 min incubation period following reoxygenation, glycerol output was markedly higher from PC-PLC treated than from control myocytes. However, the elevated glycerol output from these cells was not accompanied by a simultaneous rise in glycerol-3-phosphate, nor was it inhibited by inclusion of pyruvate in the incubation buffer. On the other hand, glycerol output from PC-PLC treated myocytes was effectively inhibited by a diacylglycerol lipase inhibitor (U-57908, The Upjohn Company). Analysis of cellular lipids revealed a 22% reduction of phospholipid in PC-PLC treated myocytes (P < 0.02), while the content of triacylglycerol, diacylglycerol and unesterified fatty acids increased by 76, 261 and 103%, respectively (P < 0.02). No significant changes were observed for these parameters in control myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
J Mol Cell Cardiol 1995 Mar
PMID:Phospholipid degradation in hypoxic/reoxygenated cardiomyocytes in response to phospholipase C from Bacillus cereus. 760 7

The contributions of phosphoinositide (PI)- and phosphatidylcholine (PC)-specific phospholipases [PI-specific phospholipase C (PI-PLC), PC-specific phospholipase C (PC-PLC), and phospholipase D (PLD)] to diacylglycerol (DAG) formation and regulation of the enzymes by G proteins, Ca2+, and protein kinase C (PKC) were examined in dispersed intestinal circular and longitudinal muscle cells. DAG formation induced by cholecystokinin was biphasic and paralleled by PKC activity. The initial phase (approximately 1 min) was mediated by PI-PLC in circular muscle cells and by both PI- and PC-PLC in longitudinal muscle cells, whereas the sustained phase was mediated by PC-PLC and PLD in both cell types. PC-PLC activity during the initial phase was identified by rapid formation of the initial products [3H]phosphocholine (5 sec) and [3H]myristate-labeled DAG (approximately 15 sec). PLD activity did not contribute to DAG formation during the initial phase, and PI hydrolysis had no effect on PC-PLC or PLD activity during the initial or sustained phases. PLD activity during the sustained phase was evident by the formation of [3H]phosphatidylethanol, a PLD-specific transphosphatidylation product. Dephosphorylation of phosphatidic acid (PA) by phosphatidate phosphohydrolase (PPH) accounted for about 50% of DAG formation; inhibition of PPH activity by propranolol or suppression of PA formation by ethanol inhibited DAG formation by 59-69% and 57-62%, respectively. Residual DAG in the presence of ethanol was augmented 55-57% by DAG kinase inhibitor, whereas residual PA was inhibited by 60-67%, implying that PA was derived from DAG, and DAG from PLC-mediated PC hydrolysis. In the presence of ethanol, calphostin C inhibited phosphatidylethanol formation but had no effect on PA or DAG levels, implying that only PLD activity was modulated by PKC. Maintenance of resting intracellular Ca2+ concentrations, rather than an agonist-induced increase in the intracellular Ca2+ concentration, was required for optimal PC-PLC and PLD activity. Guanosine-5'-O-(beta-thio)diphosphate abolished DAG and PA formation in reversibly permeabilized muscle cells. We conclude that DAG formation in intestinal muscle is mediated by time-dependent activation of three phospholipases (PI-PLC, PC-PLC, and PLD) and two converting enzymes (DAG kinase and PPH). PC-PLC and PLD are Ca2+ dependent and appear to be G protein coupled; only PLD is PKC sensitive.
Mol Pharmacol 1995 Aug
PMID:Agonist-mediated activation of phosphatidylcholine-specific phospholipase C and D in intestinal smooth muscle. 765 63

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.
Mol Cell Biol 1993 Jan
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

The two forms of angiotensin II (Ang II) receptors, AT1 and AT2 subtypes, have been demonstrated in many other cells beside the anterior pituitary cells. Attempting to investigate the subtype(s) of Ang II receptors implicated in the multiple transduction mechanisms involved in Ang II stimulation of prolactin (PRL) release by lactotropes, we studied the effect of selective nonpeptidergic Ang II antagonists on the PRL release, adenylate cyclase (AC), and phospholipase C activities. In intact cells, the AT1 antagonist DuP753 blocked Ang II-induced PRL release, reversed in a dose dependent manner Ang II-evoked inositol phosphates production, and inhibited completely the PLC and protein kinase C (PKC) dependent cAMP accumulation induced by Ang II. In membrane preparations, the Ang II receptors were negatively coupled to AC. The AT1 antagonist blocked in a dose dependent manner the inhibitory effect of Ang II on cAMP production. In intact cells, the negative coupling of Ang II receptor with AC was observed only when PKC was down regulated by long term 12-O-tetradecanolylphorbol-13-acetate pretreatment. Ang II was able to inhibit vasoactive intestinal peptide-induced cAMP accumulation, a response which was also prevented by DuP753. The different coupling of Ang II receptor described above implicated only the AT1 type receptor since the AT2 antagonists (PD123177 and PD123319) were ineffective at any doses tested (10(-8) to 10(-5) M). The obtained results indicate that the regulation of PRL secretion involves the AT1 receptor subtype and that this receptor might be coupled to multiple effectors.
Mol Cell Neurosci 1994 Dec
PMID:Angiotensin II effects on second messengers involved in prolactin secretion are mediated by AT1 receptor in anterior pituitary cells. 770 34

We have characterized a membrane-bound phosphatidylcholine (PC) specific phospholipase C (PC-PLC) in plasma membranes from rat cardiac muscle, and have investigated the role of PC-PLC and PC-specific phospholipase D (PC-PLD) activities in the mechanism of action of atrial natriuretic factor (ANF). In purified sarcolemma, ANF stimulated over a wide range of concentrations with a maximum at 10(-11) M the hydrolysis of phosphatidylcholine through PC-PLD giving phosphatidate and choline, whereas higher concentrations of ANF (10(-10) M) preferentially stimulated PC breakdown through PC-PLC to form diacylglycerol and phosphocholine. To confirm the involvement of the PC-PLD in the mechanism of ANF action, we measured the transphosphatidylation reaction, a specific assay for this phospholipase which in the presence of ethanol catalyses the phosphatidylethanol formation from PC. ANF stimulated phosphatidylethanol formation with the same dose-response behavior as phosphatidate formation. The significant diacylglycerol increase at 10(-10) M ANF, in the presence of propranolol, a potent inhibitor of phosphatidate phosphatase which can hydrolyse phosphatidate to give diacylglycerol, suggested a direct involvement of PC-PLC. The use of GTP-gamma-S, a non hydrolysable analog of GTP, and of pertussis toxin showed the involvement of a pertussis toxin insensitive G protein in PC-PLC mediated ANF signal transduction. We suggest a differential effect of ANF on PC breakdown by phospholipases C and D depending on the concentration of the peptide.
J Mol Cell Cardiol 1994 Dec
PMID:Selective activation by atrial natriuretic factor of phosphatidylcholine-specific phospholipase activities in purified heart muscle plasma membranes. 773 Oct 62

The gene for phosphatidylinositol-specific phospholipase C (PI-PLC) of Listeria monocytogenes has been cloned and shown to be expressed in Escherichia coli cells from own as well as from the lactose gene promoter. The recombinant plasmid has been constructed on the basis of pRIT2T vector and carries the hybrid gone. 3-end of which is a fragment of protein A gene of Staphylococcus aureus. 3-end is a gene for phospholipase plcA, both in the same reading frame. The resultant construction is shown to code in Escherichia coli cells for the hybrid recombinant protein A:Pl-PLC. Purified preparation of the hybrid protein and polyclonal rabbit antiserum to it were obtained. The obtained antiserum to the hybrid protein containing phospholipase as en C-end domain has been shown to react specifically to phospholipase in Escherichia coli recombinant strain harbouring the constructed recombinant plasmid as well as the one in the culture fluid of listeria.
Mol Gen Mikrobiol Virusol
PMID:[Cloning and expression of the phosphatidylinositol-specific phospholipase C gene from Listeria monocytogenes]. 773 95

The beta receptor for platelet-derived growth factor (beta PDGFR) is activated by binding of PDGF and undergoes phosphorylation at multiple tyrosine residues. The tyrosine-phosphorylated receptor associates with numerous SH2-domain-containing proteins which include phospholipase C-gamma 1 (PLC gamma), the GTPase-activating protein of Ras (GAP), the p85 subunit of phosphatidylinositol 3 kinase (PI3K), the phosphotyrosine phosphatase Syp, and several other proteins. Our previous studies indicated that PI3K and PLC gamma were required for relay of the mitogenic signal of beta PDGFR, whereas GAP and Syp did not appear to be required for this response. In this study, we further investigated the role of GAP and Syp in mitogenic signaling by beta PDGFR. Focusing on the PLC gamma-dependent branch of beta PDGFR signaling, we constructed a series of mutant beta PDGFRs that contained the binding sites for pairs of the receptor-associated proteins: PLC gamma and PI3K, PLC gamma and GAP, or PLC gamma and Syp. Characterization of these mutants showed that while all receptors were catalytically active and bound similar amounts of PLC gamma, they differed dramatically in their ability to initiate DNA synthesis. This signaling deficiency related to an inability to efficiently tyrosine phosphorylate and activate PLC gamma. Surprisingly, the crippled receptor was the one that recruited PLC gamma and GAP. Thus, GAP functions to suppress signal relay by the beta PDGFR, and it does so by silencing PLC gamma. These findings demonstrate that the biological response to PDGF depends not only on the ability of the beta PDGFR to recruit signal relay enzymes but also on the blend of these receptor-associated proteins.
Mol Cell Biol 1995 Jun
PMID:The GTPase-activating protein of Ras suppresses platelet-derived growth factor beta receptor signaling by silencing phospholipase C-gamma 1. 776 Aug 2

Screening of a Drosophila genomic DNA library at reduced stringency hybridization conditions using a rat PLC alpha cDNA probe yielded a gene which encodes a member of the protein disulfide isomerase/PLC alpha family. The gene has been localized to band 74C on the left arm of the third chromosome and has been designated dpdi. Northern analysis shows that the dpdi gene encodes a transcript that is 2.3 kb in length and is present throughout development as well as in both heads and bodies of adults. The deduced dpdi protein is 496 amino acids in length and contains two domains exhibiting high similarity to thioredoxin, two regions that are similar to the hormone binding domain of human estrogen receptor, and a sequence of four amino acids (KDEL) at the C-terminus which has been described by others as being responsible for retention of proteins in the endoplasmic reticulum. Overall, dpdi contains a higher similarity to rat protein disulfide isomerase (53% identical) than to rat PLC alpha (30% identical). However, it is unclear whether dpdi functions in vivo as a PDI or as a PLC, or both. Drosophila, with its well characterized genetics and the ability to generate mutants in a gene that has been cloned, provides an excellent system in which to resolve this issue.
Insect Biochem Mol Biol 1995 May
PMID:A Drosophila gene that encodes a member of the protein disulfide isomerase/phospholipase C-alpha family. 778 47

Signal transduction induced by generations of second messengers from membrane phospholipids is a major regulatory mechanism in the control of cell proliferation. Indeed, oncogenic p21ras alters the intracellular levels of phospholipid metabolites in both mammalian cells and Xenopus oocytes. However, it is still controversial whether this alteration it is biologically significant. We have analyzed the ras-induced signal transduction pathway in Xenopus oocytes and have correlated its mechanism of activation with that of the three most relevant phospholipases (PLs). After microinjection, ras-p21 induces a rapid PLD activation followed by a late PLA2 activation. By contrast, phosphatidylcholine-specific PLC was not activated under similar conditions. When each of these PLs was studied for its ability to activate intracellular signalling kinases, all of them were found to activate maturation-promoting factor efficiently. However, only PLD was able to activate MAP kinase and S6 kinase II, a similar pattern to that induced by p21ras proteins. Thus, the comparison of activated enzymes after microinjection of p21ras or PLs indicated that only PLD microinjection mimetized p21ras signalling. Finally, inhibition of the endogenous PLD activity by neomycin substantially reduced the biological activity of p21ras. All these results suggest that PLD activation may constitute a relevant step in ras-induced germinal vesicle breakdown in Xenopus oocytes.
Mol Cell Biol 1995 Feb
PMID:Activation of intracellular kinases in Xenopus oocytes by p21ras and phospholipases: a comparative study. 782 25

The fission yeast Schizosaccharomyces pombe has no detectable endogenous receptor tyrosine kinases or associated signalling apparatus, and we have used this cell system to reconstitute mammalian platelet-derived growth factor beta (PDGF beta) receptor-linked activation of phospholipase C gamma 2 (PLC gamma 2). The PDGF beta receptor migrates as a glycosylated protein of 165 kDa associated exclusively with membrane fractions. No tyrosine autophosphorylation was detected when PDGF beta was expressed alone. PLC gamma 2 appears as a 140-kDa protein distributed between particulate and soluble fractions which exhibits characteristic selectivity for phosphatidylinositol 4,5-bisphosphate and is sensitive to powerful activation by Ca2+. When coexpressed, both PDGF beta and PLC gamma 2 undergo tyrosine phosphorylation, and this is accompanied by a > 26-fold increase in [3H]inositol 4,5-biphosphate ([3H]IP2) and [3H]inositol 1,4,5-triphosphate [3H]IP3 production. Treatment with the tyrosine phosphatase inhibitor pervanadate further increased PLC gamma 2 tyrosine phosphorylation as well as [3H]IP2 and [3H]IP3 generation. Phosphorylated PLC gamma 2 was found predominantly in membrane fractions. To test a nonreceptor tyrosine kinase, we then expressed the human proto-oncogene c-src together with its negative regulator Csk. These were immunodetectable as bands at 60 kDa (c-Src) and 50 kDa (Csk) and distributed between membrane and cytosolic fractions. When yeast coexpressing c-Src, Csk, and PLC gamma 2 was incubated with pervanadate, PLC gamma 2 was tyrosine phosphorylated and [3H]IP2 and [3H]IP3 production increased 11.0- and 7.0-fold, respectively. Csk expressed alone with PLC gamma 2 was ineffective. Similar PLC gamma 2 activation was observed upon in vitro mixing with the extracts expressing either c-Src or the PDGF beta receptor. In summary, this is the first report of a reconstitution of mammalian tyrosine kinase-linked effector activation in yeast cells and also the first demonstration of direct PLC gamma 2 activation by the proto-oncogene c-src. These observations indicate that S. pombe provides a powerful cell system in which to study critical molecular interactions and activities underlying receptor and nonreceptor tyrosine kinase-dependent cell signaling.
Mol Cell Biol 1995 Mar
PMID:Activation of phospholipase C gamma in Schizosaccharomyces pombe by coexpression of receptor or nonreceptor tyrosine kinases. 786 36


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