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. 1997 Oct 15;327(Pt 2):431–437. doi: 10.1042/bj3270431

Membrane-binding properties of phospholipase C-beta1 and phospholipaseC-beta2: role of the C-terminus and effects of polyphosphoinositides, G-proteins and Ca2+.

J M Jenco 1, K P Becker 1, A J Morris 1
PMCID: PMC1218812  PMID: 9359412

Abstract

We have studied the binding of two G-protein-regulated phospholipase C (PLC) enzymes, PLCs-beta1 and -beta2, to membrane surfaces using sucrose-loaded bilayer phospholipid vesicles of varying compositions. Neither enzyme binds appreciably to pure phosphatidylcholine vesicles at lipid concentrations up to 10(-3) M. PLC-beta1 and PLC-beta2 bind vesicles composed of phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine (molar ratio 1:1:1) with an approximate Kd of 10(-5) M. Inclusion of 2% PtdIns(4,5)P2 in these vesicles had no effect on the affinity of this interaction. As reported by others, removal of the C-terminus of PLC-beta1 and PLC-beta2 produces catalytically active fragments. The affinity of these truncated proteins for phospholipid vesicles is dramatically reduced suggesting that this region of the proteins contains residues important for membrane binding. Inclusion of G-protein alpha- and betagamma-subunit activators in the phospholipid vesicles does not increase the binding of PLC-beta1 or PLC-beta2, and the magnitude of G-protein-mediated PLC activation observed at low phospholipid concentrations (10(-6) M) is comparable to that observed at concentrations at which the enzymes are predominantly membrane-bound (10(-3) M). PLC-beta1 and -beta2 contain C2 domains but Ca2+ does not enhance binding to the vesicles. Our results indicate that binding of these enzymes to membranes involves the C-temini of the proteins and suggest that activation of these enzymes by G-proteins results from a regulated interaction between the membrane-bound proteins rather than G-protein-dependent recruitment of soluble enzymes to a substrate-containing phospholipid surface.

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Selected References

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  1. Berridge M. J. Inositol trisphosphate and diacylglycerol: two interacting second messengers. Annu Rev Biochem. 1987;56:159–193. doi: 10.1146/annurev.bi.56.070187.001111. [DOI] [PubMed] [Google Scholar]
  2. Camps M., Hou C., Sidiropoulos D., Stock J. B., Jakobs K. H., Gierschik P. Stimulation of phospholipase C by guanine-nucleotide-binding protein beta gamma subunits. Eur J Biochem. 1992 Jun 15;206(3):821–831. doi: 10.1111/j.1432-1033.1992.tb16990.x. [DOI] [PubMed] [Google Scholar]
  3. Divecha N., Rhee S. G., Letcher A. J., Irvine R. F. Phosphoinositide signalling enzymes in rat liver nuclei: phosphoinositidase C isoform beta 1 is specifically, but not predominantly, located in the nucleus. Biochem J. 1993 Feb 1;289(Pt 3):617–620. doi: 10.1042/bj2890617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Essen L. O., Perisic O., Cheung R., Katan M., Williams R. L. Crystal structure of a mammalian phosphoinositide-specific phospholipase C delta. Nature. 1996 Apr 18;380(6575):595–602. doi: 10.1038/380595a0. [DOI] [PubMed] [Google Scholar]
  5. Ferguson K. M., Lemmon M. A., Schlessinger J., Sigler P. B. Structure of the high affinity complex of inositol trisphosphate with a phospholipase C pleckstrin homology domain. Cell. 1995 Dec 15;83(6):1037–1046. doi: 10.1016/0092-8674(95)90219-8. [DOI] [PubMed] [Google Scholar]
  6. Hepler J. R., Gilman A. G. G proteins. Trends Biochem Sci. 1992 Oct;17(10):383–387. doi: 10.1016/0968-0004(92)90005-t. [DOI] [PubMed] [Google Scholar]
  7. Hepler J. R., Kozasa T., Smrcka A. V., Simon M. I., Rhee S. G., Sternweis P. C., Gilman A. G. Purification from Sf9 cells and characterization of recombinant Gq alpha and G11 alpha. Activation of purified phospholipase C isozymes by G alpha subunits. J Biol Chem. 1993 Jul 5;268(19):14367–14375. [PubMed] [Google Scholar]
  8. James S. R., Paterson A., Harden T. K., Downes C. P. Kinetic analysis of phospholipase C beta isoforms using phospholipid-detergent mixed micelles. Evidence for interfacial catalysis involving distinct micelle binding and catalytic steps. J Biol Chem. 1995 May 19;270(20):11872–11881. doi: 10.1074/jbc.270.20.11872. [DOI] [PubMed] [Google Scholar]
  9. Jhon D. Y., Lee H. H., Park D., Lee C. W., Lee K. H., Yoo O. J., Rhee S. G. Cloning, sequencing, purification, and Gq-dependent activation of phospholipase C-beta 3. J Biol Chem. 1993 Mar 25;268(9):6654–6661. [PubMed] [Google Scholar]
  10. Katan M., Kriz R. W., Totty N., Philp R., Meldrum E., Aldape R. A., Knopf J. L., Parker P. J. Determination of the primary structure of PLC-154 demonstrates diversity of phosphoinositide-specific phospholipase C activities. Cell. 1988 Jul 15;54(2):171–177. doi: 10.1016/0092-8674(88)90549-1. [DOI] [PubMed] [Google Scholar]
  11. Kim C. G., Park D., Rhee S. G. The role of carboxyl-terminal basic amino acids in Gqalpha-dependent activation, particulate association, and nuclear localization of phospholipase C-beta1. J Biol Chem. 1996 Aug 30;271(35):21187–21192. doi: 10.1074/jbc.271.35.21187. [DOI] [PubMed] [Google Scholar]
  12. Kozasa T., Gilman A. G. Purification of recombinant G proteins from Sf9 cells by hexahistidine tagging of associated subunits. Characterization of alpha 12 and inhibition of adenylyl cyclase by alpha z. J Biol Chem. 1995 Jan 27;270(4):1734–1741. doi: 10.1074/jbc.270.4.1734. [DOI] [PubMed] [Google Scholar]
  13. Kuang Y., Wu Y., Smrcka A., Jiang H., Wu D. Identification of a phospholipase C beta2 region that interacts with Gbeta-gamma. Proc Natl Acad Sci U S A. 1996 Apr 2;93(7):2964–2968. doi: 10.1073/pnas.93.7.2964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lee S. B., Rhee S. G. Molecular cloning, splice variants, expression, and purification of phospholipase C-delta 4. J Biol Chem. 1996 Jan 5;271(1):25–31. doi: 10.1074/jbc.271.1.25. [DOI] [PubMed] [Google Scholar]
  15. Lee S. B., Shin S. H., Hepler J. R., Gilman A. G., Rhee S. G. Activation of phospholipase C-beta 2 mutants by G protein alpha q and beta gamma subunits. J Biol Chem. 1993 Dec 5;268(34):25952–25957. [PubMed] [Google Scholar]
  16. Lemmon M. A., Ferguson K. M., Schlessinger J. PH domains: diverse sequences with a common fold recruit signaling molecules to the cell surface. Cell. 1996 May 31;85(5):621–624. doi: 10.1016/s0092-8674(00)81022-3. [DOI] [PubMed] [Google Scholar]
  17. Maurice D. H., Waldo G. L., Morris A. J., Nicholas R. A., Harden T. K. Identification of G alpha 11 as the phospholipase C-activating G-protein of turkey erythrocytes. Biochem J. 1993 Mar 15;290(Pt 3):765–770. doi: 10.1042/bj2900765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Morris A. J., Rudge S. A., Mahlum C. E., Jenco J. M. Regulation of phosphoinositide-3-kinase by G protein beta gamma subunits in a rat osteosarcoma cell line. Mol Pharmacol. 1995 Sep;48(3):532–539. [PubMed] [Google Scholar]
  19. Morris A. J., Scarlata S. Regulation of effectors by G-protein alpha- and beta gamma-subunits. Recent insights from studies of the phospholipase c-beta isoenzymes. Biochem Pharmacol. 1997 Aug 15;54(4):429–435. doi: 10.1016/s0006-2952(97)00032-4. [DOI] [PubMed] [Google Scholar]
  20. Nalefski E. A., Sultzman L. A., Martin D. M., Kriz R. W., Towler P. S., Knopf J. L., Clark J. D. Delineation of two functionally distinct domains of cytosolic phospholipase A2, a regulatory Ca(2+)-dependent lipid-binding domain and a Ca(2+)-independent catalytic domain. J Biol Chem. 1994 Jul 8;269(27):18239–18249. [PubMed] [Google Scholar]
  21. Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science. 1992 Oct 23;258(5082):607–614. doi: 10.1126/science.1411571. [DOI] [PubMed] [Google Scholar]
  22. Panayotou G., Waterfield M. D. The assembly of signalling complexes by receptor tyrosine kinases. Bioessays. 1993 Mar;15(3):171–177. doi: 10.1002/bies.950150305. [DOI] [PubMed] [Google Scholar]
  23. Park D., Jhon D. Y., Kriz R., Knopf J., Rhee S. G. Cloning, sequencing, expression, and Gq-independent activation of phospholipase C-beta 2. J Biol Chem. 1992 Aug 15;267(23):16048–16055. [PubMed] [Google Scholar]
  24. Park D., Jhon D. Y., Lee C. W., Lee K. H., Rhee S. G. Activation of phospholipase C isozymes by G protein beta gamma subunits. J Biol Chem. 1993 Mar 5;268(7):4573–4576. [PubMed] [Google Scholar]
  25. Park D., Jhon D. Y., Lee C. W., Ryu S. H., Rhee S. G. Removal of the carboxyl-terminal region of phospholipase C-beta 1 by calpain abolishes activation by G alpha q. J Biol Chem. 1993 Feb 15;268(5):3710–3714. [PubMed] [Google Scholar]
  26. Ponting C. P., Parker P. J. Extending the C2 domain family: C2s in PKCs delta, epsilon, eta, theta, phospholipases, GAPs, and perforin. Protein Sci. 1996 Jan;5(1):162–166. doi: 10.1002/pro.5560050120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rebecchi M., Peterson A., McLaughlin S. Phosphoinositide-specific phospholipase C-delta 1 binds with high affinity to phospholipid vesicles containing phosphatidylinositol 4,5-bisphosphate. Biochemistry. 1992 Dec 29;31(51):12742–12747. doi: 10.1021/bi00166a005. [DOI] [PubMed] [Google Scholar]
  28. Rhee S. G., Suh P. G., Ryu S. H., Lee S. Y. Studies of inositol phospholipid-specific phospholipase C. Science. 1989 May 5;244(4904):546–550. doi: 10.1126/science.2541501. [DOI] [PubMed] [Google Scholar]
  29. Romoser V., Ball R., Smrcka A. V. Phospholipase C beta2 association with phospholipid interfaces assessed by fluorescence resonance energy transfer. G protein betagamma subunit-mediated translocation is not required for enzyme activation. J Biol Chem. 1996 Oct 11;271(41):25071–25078. doi: 10.1074/jbc.271.41.25071. [DOI] [PubMed] [Google Scholar]
  30. Runnels L. W., Jenco J., Morris A., Scarlata S. Membrane binding of phospholipases C-beta 1 and C-beta 2 is independent of phosphatidylinositol 4,5-bisphosphate and the alpha and beta gamma subunits of G proteins. Biochemistry. 1996 Dec 24;35(51):16824–16832. doi: 10.1021/bi961606w. [DOI] [PubMed] [Google Scholar]
  31. Ryu S. H., Suh P. G., Cho K. S., Lee K. Y., Rhee S. G. Bovine brain cytosol contains three immunologically distinct forms of inositolphospholipid-specific phospholipase C. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6649–6653. doi: 10.1073/pnas.84.19.6649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Smrcka A. V., Hepler J. R., Brown K. O., Sternweis P. C. Regulation of polyphosphoinositide-specific phospholipase C activity by purified Gq. Science. 1991 Feb 15;251(4995):804–807. doi: 10.1126/science.1846707. [DOI] [PubMed] [Google Scholar]
  33. Stahl M. L., Ferenz C. R., Kelleher K. L., Kriz R. W., Knopf J. L. Sequence similarity of phospholipase C with the non-catalytic region of src. Nature. 1988 Mar 17;332(6161):269–272. doi: 10.1038/332269a0. [DOI] [PubMed] [Google Scholar]
  34. Sternweis P. C., Robishaw J. D. Isolation of two proteins with high affinity for guanine nucleotides from membranes of bovine brain. J Biol Chem. 1984 Nov 25;259(22):13806–13813. [PubMed] [Google Scholar]
  35. Suh P. G., Ryu S. H., Moon K. H., Suh H. W., Rhee S. G. Cloning and sequence of multiple forms of phospholipase C. Cell. 1988 Jul 15;54(2):161–169. doi: 10.1016/0092-8674(88)90548-x. [DOI] [PubMed] [Google Scholar]
  36. Suh P. G., Ryu S. H., Moon K. H., Suh H. W., Rhee S. G. Inositol phospholipid-specific phospholipase C: complete cDNA and protein sequences and sequence homology to tyrosine kinase-related oncogene products. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5419–5423. doi: 10.1073/pnas.85.15.5419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Taylor S. J., Chae H. Z., Rhee S. G., Exton J. H. Activation of the beta 1 isozyme of phospholipase C by alpha subunits of the Gq class of G proteins. Nature. 1991 Apr 11;350(6318):516–518. doi: 10.1038/350516a0. [DOI] [PubMed] [Google Scholar]
  38. Todderud G., Wahl M. I., Rhee S. G., Carpenter G. Stimulation of phospholipase C-gamma 1 membrane association by epidermal growth factor. Science. 1990 Jul 20;249(4966):296–298. doi: 10.1126/science.2374928. [DOI] [PubMed] [Google Scholar]
  39. Waldo G. L., Paterson A., Boyer J. L., Nicholas R. A., Harden T. K. Molecular cloning, expression and regulatory activity of G alpha 11- and beta gamma-subunit-stimulated phospholipase C-beta from avian erythrocytes. Biochem J. 1996 Jun 1;316(Pt 2):559–568. doi: 10.1042/bj3160559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wedegaertner P. B., Chu D. H., Wilson P. T., Levis M. J., Bourne H. R. Palmitoylation is required for signaling functions and membrane attachment of Gq alpha and Gs alpha. J Biol Chem. 1993 Nov 25;268(33):25001–25008. [PubMed] [Google Scholar]
  41. Wu D., Jiang H., Katz A., Simon M. I. Identification of critical regions on phospholipase C-beta 1 required for activation by G-proteins. J Biol Chem. 1993 Feb 15;268(5):3704–3709. [PubMed] [Google Scholar]

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