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)

The discovery of mutated, GTPase-deficient alpha subunits of Gs or Gi2 in certain human endocrine tumors has suggested that heterotrimeric G proteins play a role in the oncogenic process. Expression of these altered forms of G alpha s or G alpha i2 proteins in rodent fibroblasts activates or inhibits endogenous adenylyl cyclase, respectively, and causes certain alterations in cell growth. However, it is not clear whether growth abnormalities result from altered cyclic AMP synthesis. In the present study, we asked whether a recently discovered family of G proteins, Gq, which does not affect adenylyl cyclase activity, but instead mediates the activation of phosphatidylinositol-specific phospholipase C harbors transforming potential. We mutated the cDNA for the alpha subunit of murine Gq in codons corresponding to a region involved in binding and hydrolysis of GTP. Similar mutations unmask the transforming potential of p21ras or activate the alpha subunits of Gs or Gi2. Our results show that when expressed in NIH 3T3 cells, activating mutations convert G alpha q into a dominant acting oncogene.
Mol Cell Biol 1992 Oct
PMID:Mutated alpha subunit of the Gq protein induces malignant transformation in NIH 3T3 cells. 132 59

The rat neurotensin receptor cDNA sequence was transfected in Chinese hamster ovary cells and cellular clones which stably express the corresponding protein were isolated and characterized. The Scatchard analysis of the specific binding of [3H]neurotensin indicated a Kd value of 0.45 +/- 0.08 nM and a Bmax value of 3.27 +/- 0.29 pmol/mg of protein. Displacement experiments using peptidic analogs of neurotensin and levocabastine confirmed that the transfected receptor exhibits the binding properties of the neurotensin receptor characterized in the rat brain. Neurotensin stimulated the phosphoinositides hydrolysis in a time- and concentration-dependent manner and this effect was mimicked by neurotensin(8-13) and by neuromedin N. The stimulation of phosphoinositides hydrolysis was not inhibited by pertussis toxin. These results indicate that the transfected cells actively express the rat neurotensin receptor which is functionally coupled to phospholipase C through a pertussis toxin-insensitive GTP-binding protein, and that neuromedin N is able to induce the phosphoinositides turnover by interaction with the neurotensin receptor.
Brain Res Mol Brain Res 1992 Oct
PMID:Phospholipase C activation by neurotensin and neuromedin N in Chinese hamster ovary cells expressing the rat neurotensin receptor. 133 89

The 47-kDa protein coimmunoprecipitated with phospholipase C (PLC)-gamma 1 by anti-PLC-gamma 1 monoclonal antibodies is proved to be Nck, a protein composed almost exclusively of one SH2 and three SH3 domains. Nck and PLC-gamma 1 are recognized by certain anti-PLC-gamma 1 monoclonal antibodies because Nck and PLC-gamma 1 share an epitope that likely is located in their SH3 domains. Nck is widely distributed in rat tissues, with an especially high level of expression in testes. The expression levels of Nck remains unchanged during the development of rat brain, whereas PLC-gamma 1 decreases during the same developmental period. Stimulation of A431 cells with epidermal growth factor elicits the tight association of Nck with the epidermal growth factor receptor and phosphorylation of Nck on both serine and tyrosine residues. The phosphorylation of Nck is also enhanced in response to stimulation of the nerve growth factor receptor in PC12 cells, the T-cell receptor complex in Jurkat cells, the membrane immunoglobulin M in Daudi cells, and the low-affinity immunoglobulin G receptor (Fc gamma RII) in U937 cells. The phosphorylation of Nck was also enhanced following treatment of A431 cells with phorbol 12-myristate 13-acetate or forskolin. These results suggest that Nck is a target for a variety of protein kinases that might modulate the postulated role of Nck as an adaptor for the physical and functional coordination of signalling proteins.
Mol Cell Biol 1992 Dec
PMID:Phosphorylation of Nck in response to a variety of receptors, phorbol myristate acetate, and cyclic AMP. 133 46

Signalling proteins such as phospholipase C-gamma (PLC-gamma) or GTPase-activating protein (GAP) of ras contain conserved regions of approximately 100 amino acids termed src homology 2 (SH2) domains. SH2 domains were shown to be responsible for mediating association between signalling proteins and tyrosine-phosphorylated proteins, including growth factor receptors. Nck is an ubiquitously expressed protein consisting exclusively of one SH2 and three SH3 domains. Here we show that epidermal growth factor or platelet-derived growth factor stimulation of intact human or murine cells leads to phosphorylation of Nck protein on tyrosine, serine, and threonine residues. Similar stimulation of Nck phosphorylation was detected upon activation of rat basophilic leukemia RBL-2H3 cells by cross-linking of the high-affinity immunoglobulin E receptors (Fc epsilon RI). Ligand-activated, tyrosine-autophosphorylated platelet-derived growth factor or epidermal growth factor receptors were coimmunoprecipitated with anti-Nck antibodies, and the association with either receptor molecule was mediated by the SH2 domain of Nck. Addition of phorbol ester was also able to stimulate Nck phosphorylation on serine residues. However, growth factor-induced serine/threonine phosphorylation of Nck was not mediated by protein kinase C. Interestingly, approximately fivefold overexpression of Nck in NIH 3T3 cells resulted in formation of oncogenic foci. These results show that Nck is an oncogenic protein and a common target for the action of different surface receptors. Nck probably functions as an adaptor protein which links surface receptors with tyrosine kinase activity to downstream signalling pathways involved in the control of cell proliferation.
Mol Cell Biol 1992 Dec
PMID:The SH2 and SH3 domain-containing Nck protein is oncogenic and a common target for phosphorylation by different surface receptors. 133 47

Acrosomal reaction is an essential prerequisite to fertilization. The changes in lipid composition of sperm membranes cause fusion of the plasma and outer acrosomal membranes that results in the exocytosis of acrosomal contents. We report that both bull and rabbit spermatozoa contain a phosphatidylcholine-specific phospholipase C (PC-PLC) that hydrolyzes L-alpha-dipalmitoyl-(choline-methyl-14C-153.0 Ci/mmol and a phosphatidylinositol-specific phospholipase C (PI-PLC) that hydrolyzes L-alpha-(Myo-Inositol-2-3H (N)-5.2 Ci mmol. PI-PLC from bull sperm acrosome has been purified 568 x fold with a specific activity 6.25 +/- 0.6 nmol/min/mg protein, km 0.004 mM, and Vmax 12 nmol/min/mg protein. Both enzymes had optimum at pH 7.5. The activity of PC-PLC remained unaffected by varying concentrations of Ca2+, whereas PI-PLC activity was significantly increased. The bulk of PI-PLC was found to be associated with inner acrosomal membrane of bull and rabbit sperm, while PC-PLC was found in the outer acrosomal membranes in the bull sperm and the plasma membrane of the rabbit sperm. Both enzymes are compartmentalized in sperm cell.
Mol Reprod Dev 1992 Nov
PMID:Phosphatidylcholine and phosphatidylinositol-specific phospholipases C of bull and rabbit spermatozoa. 133 40

The receptor that interacts with the mammalian bombesin-related peptide neuromedin B (NMB) is ubiquitous in the gastrointestinal tract and central nervous system. However, little is known regarding its cellular mechanisms of action. This receptor has been recently cloned, sequenced, and stably transfected into BALB 3T3 fibroblasts, permitting detailed study of the pharmacology and coupled biological activities of this receptor. In the present study, we compare the ability of transfected receptors to alter cell function with that of receptors natively expressed in small numbers by the rat glioblastoma cell line C6. NMB inhibited binding of 125I-[D-Tyro]NMB with high affinity in transfected cells (Ki = 3.08 +/- 0.14 nM) and in C6 cells (Ki = 1.90 +/- 1.10 nM), whereas the bombesin-related agonists gastrin-releasing peptide (GRP) and [D-Phe6, D-Ala11, Leu14]bombesin(6-16) (GRP analogue) had 100- and 300-fold lower affinities, respectively, for NMB receptors in either cell type. For both cell systems, maximal binding was observed between 5 and 15 min at 22 degrees. Both cell types internalized NMB at similar rates, with > 70% of bound ligand being internalized by 60 min at 22 degrees. The nonhydrolyzable guanosine analogue guanosine 5'-(beta,gamma-imido)triphosphate was equipotent in causing a decrease in binding of 125I-[D-Tyro]NMB due to decreased receptor affinity in both cell types, without a change in receptor number, demonstrating that the NMB receptor remained coupled to a guanine nucleotide-binding protein in both native and transfected cells. In both cell systems, NMB increased inositol monophosphate, inositol bisphosphate, and inositol trisphosphate in a time-dependent fashion. Inositol phosphates were increased in a dose-dependent fashion, with similar half-maximal values being obtained for NMB in both cell types (transfected, 1.01 +/- 0.09 nM; C6, 2.09 +/- 0.15 nM) and for the GRP analogue (transfected, 1855 +/- 140 nM; C6, 2129 +/- 250 nM). NMB mobilized intracellular Ca2+ in both cell systems, and the dose-response curves were superimposible (EC50 for transfected, 0.10 +/- 0.08 nM; C6, 0.11 +/- 0.02 nM). These data demonstrate that activation of the receptor for NMB stimulates phospholipase C and increases intracellular Ca2+. These results also demonstrate that transfected and native NMB receptors behave similarly, suggesting that the transfected cell line will be useful in future studies investigating ligand-receptor interactions, as well as in molecular biological studies of the structure-function relationship of the receptor.
Mol Pharmacol 1992 Dec
PMID:Neuromedin B receptors retain functional expression when transfected into BALB 3T3 fibroblasts: analysis of binding, kinetics, stoichiometry, modulation by guanine nucleotide-binding proteins, and signal transduction and comparison with natively expressed receptors. 133 12

In rat cardiac sarcolemmal membranes a phosphoinositide-specific phospholipase C (PLC) was found to be present. The enzyme hydrolysed exogenous [3H-]phosphatidylinositol 4,5-biphosphate ([3H-]PtdIns(4,5)P2) in an optimized assay mixture containing 15 micrograms SL protein, 100 mM NaCl, 1 mM free Ca2+, 14 mM Na-cholate and 20 microM [3H-]PtdIns-(4,5)P2 (400-500 dpm/microliter) in 30 mM HEPES-Tris buffer (pH 7.0). The average specific activity was 9.14 +/- 0.55 nmol.mg-1.2.5 min-1. The addition of Mg2+ to the assay mixture did not change PLC activity but increased the relative amounts of dephosphorylated inositol products. In the absence of Na+ and at a low Ca2+ concentration (0.3 microM), Mg2+ also enhanced the intraSL levels of PtdIns4P and PtdIns, and, moreover, inhibited PLC activity (IC50-0.07 mM). PtdIns4P seemed to be a good substrate for th rat SL PLC (23.07 +/- 1.57 nmol.mg-1.2.5 min-1) whereas PtdIns was hydrolysed at a very low rate (0.36 +/- 0.08 nmol.mg-1.2.5 min-1). Unlike PtdIns(4,5)P2, PLC-dependent PtdIns4P and PtdIns hydrolysis was not inhibited by Ca2+ concentrations over 1 mM. The possibility of distinct isozymes being responsible for the different hydrolytic activities is discussed.
Mol Cell Biochem 1992 Oct 21
PMID:The substrate specificity of phosphoinositide-phospholipase C in rat heart sarcolemma. 133 20

Phosphatidylinositol (PtdIns) kinase and phosphatidylinositol 4-phosphate (PtdIns4P) kinase have been studied in a purified sarcolemmal fraction isolated from rat heart. Both enzymes were Mg(2+)-dependent and their activities were maximal at 2.5 mM Mg2+ and pH 7.5. Kinetic analysis of endogenous substrate phosphorylation by ATP showed that the apparent Km and Vmax values for PtdIns kinase were 292 +/- 17 microM and 1390 +/- 80 pmol.mg-1.min-1, respectively, while the apparent Km and Vmax values for PtdIns4P kinase were 398 +/- 25 microM and 382 +/- 24 pmol.mg-1.min-1. Under normal conditions, the activity of PtdIns4P kinase was lower than that of PtdIns kinase; however, the former activity increased several fold in the presence of PtdIns4P as an exogenous substrate. The enzymatic synthesis of intramembranal PtdIns4P and phosphatidylinositol 4,5-bisphosphate (PtdIns (4,5)P2) was maximally enhanced by 0.1% Triton X-100 and inhibited by micromolar concentrations of Ca2+. Inhibition of PtdIns and PtdIns4P kinase showed IC50 values for Ca2+ of 20 and 6 microM, respectively, and was independent of either Ca(2+)-induced activation of phospholipase C and polyphosphoinositide monophosphoesterases or low ATP concentrations. The results indicate that purified rat heart sarcolemmal membranes contain a very active phosphoinositide phosphorylation system which is regulated by micromolar levels of Ca2+. The Ca2+ effect may contribute to the feedback inhibition of the receptor-activated formation of inositol 1,4,5-trisphosphate.
Mol Cell Biochem 1992 Nov 18
PMID:Phosphoinositide kinases in rat heart sarcolemma: biochemical properties and regulation by calcium. 133 11

Further analysis of hybrid clones from an experimental cross of Trypanosoma brucei rhodesiense 058 and T. b. brucei 196 shows 2 of the hybrid clones to have DNA contents about 1.5 times parental values. This represents over 40,000 kb of extra DNA. Comparison of the molecular karyotypes of parental and progeny trypanosomes shows that the bulk of the extra DNA constitutes chromosomes greater than 1 Mb in size, although a small proportion can be accounted for by an increased number of mini-chromosomes. The 2 hybrid clones have 3 alleles at several loci for housekeeping genes as shown by RFLP and isoenzyme analysis. Trisomy of the chromosome carrying phosphoglycerate kinase and tubulin genes and that carrying the phospholipase C gene was demonstrated by analysis of molecular karyotypes. These chromosomes appear prone to substantial size alterations associated with genetic exchange. Our results for one of the hybrid clones are completely consistent with it being triploid and the product of fusion of haploid and diploid nuclei.
Mol Biochem Parasitol 1992 Apr
PMID:Trisomy and chromosome size changes in hybrid trypanosomes from a genetic cross between Trypanosoma brucei rhodesiense and T. b. brucei. 134 22

Previous studies have demonstrated that the Dictyostelium G alpha subunit G alpha 2 is essential for the cAMP-activation of adenylyl cyclase and guanylyl cyclase and that g alpha 2 null mutants do not aggregate. In this manuscript, we extend the analysis of the function of G alpha 2 in regulating downstream effectors by examining the in vivo developmental and physiological phenotypes of both wild-type and g alpha 2 null cells carrying a series of mutant G alpha 2 subunits expressed from the cloned G alpha 2 promoter. Our results show that wild-type cells expressing G alpha 2 subunits carrying mutations G40V and Q208L in the highly conserved GAGESG (residues 38-43) and GGQRS (residues 206-210) domains, which are expected to reduce the intrinsic GTPase activity, are blocked in multicellular development. Analysis of down-stream effector pathways essential for mediating aggregation indicates that cAMP-mediated activation of guanylyl cyclase and phosphatidylinositol-phospholipase C (PI-PLC) is almost completely inhibited and that there is a substantial reduction of cAMP-mediated activation of adenylyl cyclase. Moreover, neither mutant G alpha 2 subunit can complement g alpha 2 null mutants. Expression of G alpha 2(G43V) and G alpha 2(G207V) have little or no effect on the effector pathways and can partially complement g alpha 2 null cells. Our results suggest a model in which the dominant negative phenotypes resulting from the expression of G alpha 2(G40V) and G alpha 2(Q208L) are due to a constitutive adaptation of the effectors through a G alpha 2-mediated pathway. Analysis of PI-PLC in g alpha 2 null mutants and in cell lines expressing mutant G alpha 2 proteins also strongly suggests that G alpha 2 is the G alpha subunit that directly activates PI-PLC during aggregation. Moreover, overexpression of wild-type G alpha 2 results in the ability to precociously activate guanylyl cyclase by cAMP in vegetative cells, suggesting that G alpha 2 may be rate limiting in the developmental regulation of guanylyl cyclase activation. In agreement with previous results, the activation of adenylyl cyclase, while requiring G alpha 2 function in vivo, does not appear to be directly carried out by the G alpha 2 subunit. Our data are consistent with adenylyl cyclase being directly activated by either another G alpha subunit or by beta gamma subunits released on activation of the G protein containing G alpha 2.
Mol Biol Cell 1992 Jul
PMID:Amino acid substitutions in the Dictyostelium G alpha subunit G alpha 2 produce dominant negative phenotypes and inhibit the activation of adenylyl cyclase, guanylyl cyclase, and phospholipase C. 135 76


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