Publications

• Cloning and identification of amino acid residues of human phospholipase C delta 1 essential for catalysis.

  Cheng H.F., Jiang M.J., Chen C.L., Liu S.M., Wong L.P., Lomasney J.W., King K.

  In vitro single point mutagenesis, inositol phospholipid hydrolysis, and substrate protection experiments were used to identify catalytic residues of human phosphatidylinositide-specific phospholipase C delta 1 (PLC delta 1) isolated from a human aorta cDNA library. Invariant amino acid residues c ... >> More

  In vitro single point mutagenesis, inositol phospholipid hydrolysis, and substrate protection experiments were used to identify catalytic residues of human phosphatidylinositide-specific phospholipase C delta 1 (PLC delta 1) isolated from a human aorta cDNA library. Invariant amino acid residues containing a functional side chain in the highly conserved X region were changed by in vitro mutagenesis. Most of the mutant enzymes were still able to hydrolyze inositol phospholipid with activity ranging from 10 to 100% of levels in the wild type enzyme. Exceptions were mutants with the conversion of Arg338 to Leu (R338L), Glu341 to Gly (E341G), or His356 to Leu (H356L), which made the enzyme severely defective in hydrolyzing inositol phospholipid. Phospholipid vesicle binding experiments showed that these three cleavage-defective mutant forms of PLC delta 1 could specifically bind to phosphatidylinositol 4,5-bisphosphate (PIP2) with an affinity similar to that of wild type enzyme. Western blotting analysis of trypsin-treated enzyme-PIP2 complexes revealed that a 67-kDa major protein fragment survived trypsin digestion if the wild type enzyme, E341G, or H356L mutant PLC delta 1 was preincubated with 7.5 microM PIP2, whereas if it was preincubated with 80 microM PIP2, the size of major protein surviving was comparable to that of intact enzyme. However, mutant enzyme R338L was not protected from trypsin degradation by PIP2 binding. These observations suggest that PLC delta 1 can recognize PIP2 through a high affinity and a low affinity binding site and that residues Glu341 and His356 are not involved in either high affinity or low affinity PIP2 binding but rather are essential for the Ca(2+)-dependent cleavage activity of PLC. << Less

  J. Biol. Chem. 270:5495-5505(1995) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Cloning, sequencing, purification, and Gq-dependent activation of phospholipase C-beta 3.

  Jhon D.-Y., Lee H.-H., Park D., Lee C.-W., Lee K.-H., Yoo O.J., Rhee S.G.

  Six mammalian phospholipase C isozymes (PLC-beta 1, PLC-beta 2, PLC-gamma 1, PLC-gamma 2, PLC-delta 1, and PLC-delta 2) have been identified at both protein and DNA levels. Here, cDNAs corresponding to a previously unidentified PLC isozyme were isolated from a rat thyroid cell FRTL cDNA library. C ... >> More

  Six mammalian phospholipase C isozymes (PLC-beta 1, PLC-beta 2, PLC-gamma 1, PLC-gamma 2, PLC-delta 1, and PLC-delta 2) have been identified at both protein and DNA levels. Here, cDNAs corresponding to a previously unidentified PLC isozyme were isolated from a rat thyroid cell FRTL cDNA library. Comparison of the predicted amino acid sequence of this new PLC with other known PLC isozymes revealed a high degree of overall similarity with PLC-beta 1 and PLC-beta 2. Thus, the new PLC was named PLC-beta 3. Comparison with PLC-beta 1 and PLC-beta 2 also revealed that the deduced amino-terminal sequence of PLC-beta 3 was incomplete by 10-20 amino acids. With the use of antibodies raised against synthetic peptides corresponding to PLC-beta 3-specific amino acid sequences, we purified PLC-beta 3 from a rat brain particulate fraction. The purified enzyme exhibited an apparent molecular mass of 152 kDa on SDS-polyacrylamide gels, as compared with 150 and 140 kDa for PLC-beta 1 and PLC-beta 2, respectively. Studies of the activation of PLC-beta isozymes by three alpha subunits of Gq class G proteins, alpha q, alpha 11, and alpha 16 in the presence of guanosine 5-O-(3-thiotriphosphate) (GTP gamma S) revealed that the extent of activation decreased in the order of PLC-beta 1 > or = PLC-beta 3 >> PLC-beta 2 for all three alpha subunits, suggesting a certain degree of specificity in the interaction of Gq alpha subunits with different PLC-beta isozymes. << Less

  J. Biol. Chem. 268:6654-6661(1993) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Phospholipase C delta-4 overexpression upregulates ErbB1/2 expression, Erk signaling pathway, and proliferation in MCF-7 cells.

  Leung D.W., Tompkins C., Brewer J., Ball A., Coon M., Morris V., Waggoner D., Singer J.W.

  <h4>Background</h4>The expression of the rodent phosphoinositide-specific phospholipase C delta-4 (PLCdelta4) has been found to be elevated upon mitogenic stimulation and expression analysis have linked the upregulation of PLCdelta4 expression with rapid proliferation in certain rat transformed ce ... >> More

  <h4>Background</h4>The expression of the rodent phosphoinositide-specific phospholipase C delta-4 (PLCdelta4) has been found to be elevated upon mitogenic stimulation and expression analysis have linked the upregulation of PLCdelta4 expression with rapid proliferation in certain rat transformed cell lines. The human homologue of PLCdelta4 has not been extensively characterized. Accordingly, we investigate the effects of overexpression of human PLCdelta4 on cell signaling and proliferation in this study.<h4>Results</h4>The cDNA for human PLCdelta4 has been isolated and expressed ectopically in breast cancer MCF-7 cells. Overexpression of PLCdelta4 selectively activates protein kinase C-phi and upregulates the expression of epidermal growth factor receptors EGFR/erbB1 and HER2/erbB2, leading to constitutive activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway in MCF-7 cells. MCF-7 cells stably expressing PLCdelta4 demonstrates several phenotypes of transformation, such as rapid proliferation in low serum, formation of colonies in soft agar, and capacity to form densely packed spheroids in low-attachment plates. The growth signaling responses induced by PLCdelta4 are not reversible by siRNA.<h4>Conclusion</h4>Overexpression or dysregulated expression of PLCdelta4 may initiate oncogenesis in certain tissues through upregulation of ErbB expression and activation of ERK pathway. Since the growth responses induced by PLCdelta4 are not reversible, PLCdelta4 itself is not a suitable drug target, but enzymes in pathways activated by PLCdelta4 are potential therapeutic targets for oncogenic intervention. << Less

  Mol. Cancer 3:15-15(2004) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Cloning, sequencing, expression, and Gq-independent activation of phospholipase C-beta 2.

  Park D., Jhon D.-Y., Kriz R., Knopf J., Rhee S.G.

  cDNAs corresponding to a previously uncharacterized phospholipase C were isolated from an HL-60 cell cDNA library. The cDNAs encodes a putative polypeptide of 1181 amino acids with a calculated molecular mass of 133,700 daltons. Comparison of the amino acid sequence of the predicted protein with t ... >> More

  cDNAs corresponding to a previously uncharacterized phospholipase C were isolated from an HL-60 cell cDNA library. The cDNAs encodes a putative polypeptide of 1181 amino acids with a calculated molecular mass of 133,700 daltons. Comparison of the amino acid sequence of the predicted protein with those of five mammalian phospholipase C isoforms (PLC-beta 1, PLC-gamma 1, PLC-gamma 2, PLC-delta 1, and PLC-delta 2) revealed that the new enzyme is most closely related to PLC-beta 1 with an overall amino acid sequence identity of 48%. Thus, the new phospholipase C was named PLC-beta 2. The least similarity between PLC-beta 1 and PLC-beta 2 is apparent in the carboxyl-terminal 450 amino acids. Both PLC-beta 1 and PLC-beta 2 were purified from extracts of HeLa cells that had been transfected with vaccinia virus containing the corresponding cDNAs. Like other mammalian PLC isoforms, including PLC-beta 1, the catalytic activity of PLC-beta 2 was entirely dependent on Ca2+, and PLC-beta 2 preferred phosphatidyl-inositol 4,5-bisphosphate to phosphatidylinositol as substrate. Recently, the alpha subunit of the pertussis toxin-insensitive G-protein alpha q has been shown to activate PLC-beta 1 but not PLC-gamma 1 and PLC-delta 1. When alpha q purified from bovine brain was reconstituted with PLC-beta 1 or PLC-beta 2, no stimulation of PLC-beta 2 was observed in the presence of either AlF4- or guanosine 5-O-(3-thiotriphosphate) (GTP gamma S), whereas PLC-beta 1 activity was enhanced markedly in the presence of AlF4- and less markedly but significantly in the presence of GTP gamma S. These results suggest that the receptor-dependent stimulation of PLC-beta 1 and that of PLC-beta 2 may require different G-protein alpha subunits. (see also accompanying article (Lee, C. H., Park, D., Wu, D., Rhee, S. G., and Simon, M. I. (1992) J. Biol. Chem. 267, 16044-16047). << Less

  J. Biol. Chem. 267:16048-16055(1992) [PubMed] [EuropePMC]

• src-homology 2 (SH2) domain ligation as an allosteric regulator: modulation of phosphoinositide-specific phospholipase C gamma 1 structure and activity.

  Koblan K.S., Schaber M.D., Edwards G., Gibbs J.B., Pompliano D.L.

  Phosphoinositide-specific phospholipase C gamma 1 (PI-PLC gamma 1) catalyses the hydrolysis of PtdIns(4,5)P2 to generate the second messengers diacylglycerol and Ins(1,4,5)P3. PI-PLC gamma 1, an src-homology 2/3 (SH2/SH3)-domain-containing enzyme, is activated in response to growth-factor-induced ... >> More

  Phosphoinositide-specific phospholipase C gamma 1 (PI-PLC gamma 1) catalyses the hydrolysis of PtdIns(4,5)P2 to generate the second messengers diacylglycerol and Ins(1,4,5)P3. PI-PLC gamma 1, an src-homology 2/3 (SH2/SH3)-domain-containing enzyme, is activated in response to growth-factor-induced tyrosine phosphorylation, and, in vivo, is translocated from the cytosol to the particulate cell fraction. Here we report the bacterial expression of rat brain PI-PLC gamma 1 under the control of the T7 promoter. Production of the active enzyme in amounts suitable for structure-function analysis depended on coupling the translation of PLC gamma 1 to the expression of the phage-phi 10 coat protein. Purification of the enzyme was facilitated by the presence of a three-amino-acid C-terminal antibody epitope tag (Glu-Glu-Phe) engineered into the cloned PLC gamma 1. Examination of the specific activity, pH-rate profile, [Ca2+]-dependence and substrate specificity of bacterially expressed PLC gamma indicated that it had kinetic properties similar to those of PLC gamma isolated from bovine brain. The substrate specificity was dependent on [Ca2+]: at low [Ca2+] (1-10 microM) PtdIns(4,5)P2 was a better substrate than PtdIns. Addition of phosphotyrosine-containing peptides (12-mers) with the cognate sequence of the high-affinity binding site for PLC gamma 1 on the activated epidermal-growth-factor (EGF) receptor (Tyr-992) increased enzyme activity (up to 85%) in vitro. Cognate non-phosphorylated peptides had no effect on activity. When c.d. spectroscopy was used to monitor the effect of added phosphotyrosine-containing peptide on the structure of recombinant PLC gamma 1, significant spectral shifts, indicative of a conformational change, were observed upon complexation with the EGF-receptor phosphotyrosine-containing 12-residue peptide (Tyr*-992). How SH2 domains from PLC gamma 1 can mediate structural rearrangements and modulate enzymic activity on their ligation by growth-factor receptors is discussed. << Less

  Biochem. J. 305:745-751(1995) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Purification, molecular cloning, and sequencing of phospholipase C-beta 4.

  Lee C.-W., Park D.J., Lee K.-H., Kim C.G., Rhee S.G.

  We have characterized inositol phospholipid-specific phospholipase C (PLC) isozymes in bovine retina. Chromatography of a retinal homogenate on a heparin column partially resolved six peaks of PLC activity, which differed in their relative selectivities for the substrates phosphatidyl 4,5-bisphosp ... >> More

  We have characterized inositol phospholipid-specific phospholipase C (PLC) isozymes in bovine retina. Chromatography of a retinal homogenate on a heparin column partially resolved six peaks of PLC activity, which differed in their relative selectivities for the substrates phosphatidyl 4,5-bisphosphate (PIP2) and phosphatidylinositol (PI). Five of the peaks were shown to correspond to the known PLC isozymes PLC-beta 1, PLC-beta 3, PLC-gamma 1, PLC-delta 1, and PLC-delta 2. PLC-beta 1, PLC-beta 3, PLC-gamma 1, and PLC-delta 1 in the retinal fractions were identified by immunoblotting with isozyme-specific antibodies, and PLC-delta 2 was identified by direct sequencing of tryptic peptides. PLC-gamma 2 and PLC-beta 2 were not detectable by immunoblot analysis. In addition to five of the seven mammalian PLC isozymes identified to date, bovine retina contained a previously unidentified PLC, which exhibited the highest selectivity for PIP2 over PI. The new PLC was purified from a retinal particulate fraction to yield a preparation that contained a major protein band with an apparent molecular mass of 130 kDa on SDS-polyacrylamide gels. Sequence analysis of 12 tryptic peptides derived from the 130-kDa protein suggested that the primary structure of the new PLC is similar to those members of beta-type PLC isozymes, especially to that of PLC-norpA, which was originally identified in Drosophila eye. The new enzyme was thus named PLC-beta 4. A search of a rat brain cDNA library with the polymerase chain reaction and oligonucleotide primers based on common PLC amino acid sequences resulted in the cloning of a rat brain cDNA corresponding to a previously uncharacterized PLC. The cDNA encoded a putative polypeptide of 1176 amino acids, with a calculated molecular mass of 134,532 daltons, that contained the sequences of all 12 tryptic peptides of PLC-beta 4. Furthermore, the deduced amino acid sequence of the encoded protein was more related to PLC-norpA than to any of the three mammalian PLC-beta isozymes. These results suggest that the brain cDNA encodes PLC-beta 4, which is likely a mammalian homolog of PLC-norpA. << Less

  J. Biol. Chem. 268:21318-21327(1993) [PubMed] [EuropePMC]

• Nonspecific phospholipase C of Listeria monocytogenes: activity on phospholipids in Triton X-100-mixed micelles and in biological membranes.

  Goldfine H., Johnston N.C., Knob C.

  Listeria monocytogenes secretes a phospholipase C (PLC) which has 39% amino acid sequence identity with the broad-specificity PLC from Bacillus cereus. Recent work indicates that the L. monocytogenes enzyme plays a role during infections of mammalian cells (J.-A. Vazquez-Boland, C. Kocks, S. Drams ... >> More

  Listeria monocytogenes secretes a phospholipase C (PLC) which has 39% amino acid sequence identity with the broad-specificity PLC from Bacillus cereus. Recent work indicates that the L. monocytogenes enzyme plays a role during infections of mammalian cells (J.-A. Vazquez-Boland, C. Kocks, S. Dramsi, H. Ohayon, C. Geoffroy, J. Mengaud, and P. Cossart, Infect. Immun. 60:219-230, 1992). The homogeneous enzyme has a specific activity of 230 mumol/min/mg when phosphatidylcholine (PC) is dispersed in sodium deoxycholate. With phospholipid-Triton X-100 mixed micelles, the enzyme had a broad pH optimum between 5.5 and 8.0, and the rates of lipid hydrolysis were in the following order: PC > phosphatidylethanolamine (PE) > phosphatidylserine > sphingomyelin >> phosphatidylinositol (PI). Activity on PC was stimulated 35% by 0.5 M NaCl and 60% by 0.05 mM ZnSO4. When Escherichia coli phospholipids were dispersed in Triton X-100, PE and phosphatidylglycerol, but not cardiolipin, were hydrolyzed. The enzyme was active on all phospholipids of vesiculated human erythrocytes including PI, which was rapidly hydrolyzed at pH 7.0. PI was also hydrolyzed in PI-PC-cholesterol liposomes by the nonspecific PLC from L. monocytogenes and by the homologous enzyme from B. cereus. The water-soluble hydrolysis product was identified as inositol-1-phosphate. For the hydrolysis of human erythrocyte ghost phospholipids, a broad pH optimum was also observed. 32P-labelled Clostridium butyricum protoplasts, which are rich in ether lipids, were treated with PLC. The enzyme hydrolyzed the plasmalogen form of PE, its glycerol acetal, and cardiolipin, in addition to PE. I-, Cl- and F-stimulated activity on either PC-Triton X-100 mixed micelles or human erythrocyte ghosts, unlike the enzyme from B. cereus which is strongly inhibited by halides. Tris-HCl, phosphate, and calcium nitrate had similar inhibitory effects on the enzyme on the enzymes from L. monocytogenes and B. cereus. << Less

  J. Bacteriol. 175:4298-4306(1993) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.

• Cloning, tissue distribution and sub-cellular localisation of phospholipase C X-domain containing protein (PLCXD) isoforms.

  Gellatly S.A., Kalujnaia S., Cramb G.

  Phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes comprise a small family of receptor-regulated phosphodiesterases that control many cellular processes by the regulation of cytosolic calcium and/or the activity of several protein kinases. To date, six distinct classes of PI-PLC are kn ... >> More

  Phosphatidylinositol-specific phospholipase C (PI-PLC) enzymes comprise a small family of receptor-regulated phosphodiesterases that control many cellular processes by the regulation of cytosolic calcium and/or the activity of several protein kinases. To date, six distinct classes of PI-PLC are known to exist in mammals. Here we characterise a seventh class of PI-PLC, which contains only the catalytic X domain in its structure, termed phospholipase C X-domain containing protein (PLCXD). At least three tissue-specific PLCXD isoforms exist in humans, comprising hPLCXD-1, hPLCXD-2 and hPLCXD-3, with hPLCXD-2 exhibiting three C-terminal spliceforms (2.1, 2.2 and 2.3). Specific amino acids known to be essential for the catalytic function of PI-PLCs were found to be conserved in all three human PLCXDs and over-expression of hPLCXD-1, 2.1 and 3 in the HeLa cell line increased endogenous PI-PLC activity. Human PLCXD isoforms exhibited tissue-specific expression profiles in mice and humans and immunocytochemistry revealed distinct sub-cellular localisations when over-expressed in human cultured cell lines. These novel proteins may therefore possess fundamental, and as yet uncharacterised roles in cell physiology. << Less

  Biochem. Biophys. Res. Commun. 424:651-656(2012) [PubMed] [EuropePMC]