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• W2076132445 abstract "The C2 domain of cytosolic phospholipase A2 (cPLA2) is involved in the Ca2+-dependent membrane binding of this protein. To identify protein residues in the C2 domain of cPLA2 essential for its Ca2+ and membrane binding, we selectively mutated Ca2+ ligands and putative membrane-binding residues of cPLA2 and measured the effects of mutations on its enzyme activity, membrane binding affinity, and monolayer penetration. The mutations of five Ca2+ ligands (D40N, D43N, N65A, D93N, N95A) show differential effects on the membrane binding and activation of cPLA2, indicating that two calcium ions bound to the C2 domain have differential roles. The mutations of hydrophobic residues (F35A, M38A, L39A, Y96A, Y97A, M98A) in the calcium binding loops show that the membrane binding of cPLA2 is largely driven by hydrophobic interactions resulting from the penetration of these residues into the hydrophobic core of the membrane. Leu39 and Val97 are fully inserted into the membrane, whereas Phe35 and Tyr96 are partially inserted. Finally, the mutations of four cationic residues in a β-strand (R57E/K58E/R59E/R61E) have modest and negligible effects on the binding of cPLA2 to zwitterionic and anionic membranes, respectively, indicating that they are not directly involved in membrane binding. In conjunction with our previous study on the C2 domain of protein kinase C-α (Medkova, M., and Cho, W. (1998) J. Biol. Chem. 273, 17544–17552), these results demonstrate that C2 domains are not only a membrane docking unit but also a module that triggers membrane penetration of protein and that individual Ca2+ ions bound to the calcium binding loops play differential roles in the membrane binding and activation of their parent proteins. The C2 domain of cytosolic phospholipase A2 (cPLA2) is involved in the Ca2+-dependent membrane binding of this protein. To identify protein residues in the C2 domain of cPLA2 essential for its Ca2+ and membrane binding, we selectively mutated Ca2+ ligands and putative membrane-binding residues of cPLA2 and measured the effects of mutations on its enzyme activity, membrane binding affinity, and monolayer penetration. The mutations of five Ca2+ ligands (D40N, D43N, N65A, D93N, N95A) show differential effects on the membrane binding and activation of cPLA2, indicating that two calcium ions bound to the C2 domain have differential roles. The mutations of hydrophobic residues (F35A, M38A, L39A, Y96A, Y97A, M98A) in the calcium binding loops show that the membrane binding of cPLA2 is largely driven by hydrophobic interactions resulting from the penetration of these residues into the hydrophobic core of the membrane. Leu39 and Val97 are fully inserted into the membrane, whereas Phe35 and Tyr96 are partially inserted. Finally, the mutations of four cationic residues in a β-strand (R57E/K58E/R59E/R61E) have modest and negligible effects on the binding of cPLA2 to zwitterionic and anionic membranes, respectively, indicating that they are not directly involved in membrane binding. In conjunction with our previous study on the C2 domain of protein kinase C-α (Medkova, M., and Cho, W. (1998) J. Biol. Chem. 273, 17544–17552), these results demonstrate that C2 domains are not only a membrane docking unit but also a module that triggers membrane penetration of protein and that individual Ca2+ ions bound to the calcium binding loops play differential roles in the membrane binding and activation of their parent proteins. phospholipase A2 cytosolic PLA2 bovine serum albumin calcium binding region 1,2-di-O-hexadecyl-sn-glycero-3-phosphocholine 1,2-di-O-hexadecyl-sn-glycero-3-phospholycerol phosphatidylcholine 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Phospholipases A2(PLA2s1; EC3.1.1.4) are a large family of lipolytic enzymes that catalyze the hydrolysis of the fatty acid ester at the sn-2-position of phospholipids (1Dennis E.A. J. Biol. Chem. 1994; 269: 13057-13060Abstract Full Text PDF PubMed Google Scholar, 2Murakami M. Nakatani Y. Atsumi G. Inoue K. Kudo I. Crit. Rev. Immunol. 1997; 17: 225-283Crossref PubMed Google Scholar). Among various mammalian PLA2s, 85-kDa cytosolic PLA2 (cPLA2) can selectively liberate arachidonic acid from membrane phospholipids, which can be converted to potent inflammatory lipid mediators, prostaglandins, thromboxanes, leukotrienes, and lipoxins, collectively known as eicosanoids (3Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar). Recent genetic studies showed that the deletion of the cPLA2 gene results in loss of lipid mediator biosynthesis (4Bonventre J.V. Huang Z. Taheri M.R. O'Leary E. Li E. Moskowitz M.A. Sapirstein A. Nature. 1997; 390: 622-625Crossref PubMed Scopus (757) Google Scholar, 5Uozumi N. Kume K. Nagase T. Nakatani N. Ishii S. Tashiro F. Komagata Y. Maki K. Ikuta K. Ouchi Y. Miyazaki J. Shimizu T. Nature. 1997; 390: 618-622Crossref PubMed Scopus (636) Google Scholar). cPLA2 is therefore an attractive target for developing specific inhibitors that can be used as novel anti-inflammatory drugs. cPLA2 is translocated to endoplasmic reticulum membranes and nuclear envelopes in response to a rise in intracellular Ca2+ (6Glover S. de Carvalho M.S. Bayburt T. Jonas M. Chi E. Leslie C.C. Gelb M.H. J. Biol. Chem. 1995; 270: 15359-15367Crossref PubMed Scopus (314) Google Scholar, 7Schievella A.R. Regier M.K. Smith W.L. Lin L.L. J. Biol. Chem. 1995; 270: 30749-30754Crossref PubMed Scopus (421) Google Scholar). This membrane translocation of cPLA2 is mediated by its amino-terminal C2 domain, which contains calcium and membrane binding sites (8Clark J.D. Lin L.L. Kriz R.W. Ramesha C.S. Sultzman L.A. Lin A.Y. Milona N. Knopf J.L. Cell. 1991; 65: 1043-1051Abstract Full Text PDF PubMed Scopus (1454) Google Scholar, 9Nalefski E.A. Sultzman L.A. Martin D.M. Kriz R.W. Towler P.S. Knopf J.L. Clark J.D. J. Biol. Chem. 1994; 269: 18239-18249Abstract Full Text PDF PubMed Google Scholar). However, the mechanism by which the C2 domain of cPLA2drives its Ca2+-dependent membrane targeting is not fully understood. Our recent structure-function study of the C2 domain of protein kinase C-α showed that the C2 domain not only brings the protein to the membrane surface but also triggers the membrane penetration of protein (10Medkova M. Cho W. Biochemistry. 1998; 37: 4892-4900Crossref PubMed Scopus (78) Google Scholar). Also, the study revealed that individual Ca2+ ions bound to the C2 domain play differential roles in membrane binding and activation. Tertiary structures of the C2 domains of protein kinase C (11Sutton R.B. Sprang S.R. Structure. 1998; 6: 1395-1405Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar) and cPLA2 (12Perisic O. Fong S. Lynch D.E. Bycroft M. Williams R.L. J. Biol. Chem. 1998; 273: 1596-1604Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 13Xu G.Y. McDonagh T. Yu H.A. Nalefski E.A. Clark J.D. Cumming D.A. J. Mol. Biol. 1998; 280: 485-500Crossref PubMed Scopus (100) Google Scholar), despite their overall similarity, have noticeable differences; i.e. they have different β-strand connectivity (14Nalefski E.A. Falke J.J. Protein Sci. 1996; 5: 2375-2390Crossref PubMed Scopus (685) Google Scholar), and, more importantly, the C2 domain of cPLA2 contains two clusters of exposed hydrophobic residues in the calcium binding loops (see Fig. 1 A) (12Perisic O. Fong S. Lynch D.E. Bycroft M. Williams R.L. J. Biol. Chem. 1998; 273: 1596-1604Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Consistent with this unique structure, both cPLA2 (15Lichtenbergova L. Yoon E.T. Cho W. Biochemistry. 1998; 37: 14128-14136Crossref PubMed Scopus (40) Google Scholar) and its isolated C2 domain (16Davletov B. Perisic O. Williams R.L. J. Biol. Chem. 1998; 273: 19093-19096Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) have been shown to be able to penetrate into the membrane. In this study, we performed an extensive structure-function analysis of the C2 domain of cPLA2 to assess the roles of six hydrophobic residues and five Ca2+ligands located in the calcium binding loops in the membrane binding and activation of cPLA2. We also determined the role of a cluster of cationic residues in a β-strand. In conjunction with our previous study on protein kinase C-α, this study reveals interesting similarities and differences between the two C2 domains. Phospholipases A2(PLA2s1; EC3.1.1.4) are a large family of lipolytic enzymes that catalyze the hydrolysis of the fatty acid ester at the sn-2-position of phospholipids (1Dennis E.A. J. Biol. Chem. 1994; 269: 13057-13060Abstract Full Text PDF PubMed Google Scholar, 2Murakami M. Nakatani Y. Atsumi G. Inoue K. Kudo I. Crit. Rev. Immunol. 1997; 17: 225-283Crossref PubMed Google Scholar). Among various mammalian PLA2s, 85-kDa cytosolic PLA2 (cPLA2) can selectively liberate arachidonic acid from membrane phospholipids, which can be converted to potent inflammatory lipid mediators, prostaglandins, thromboxanes, leukotrienes, and lipoxins, collectively known as eicosanoids (3Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar). Recent genetic studies showed that the deletion of the cPLA2 gene results in loss of lipid mediator biosynthesis (4Bonventre J.V. Huang Z. Taheri M.R. O'Leary E. Li E. Moskowitz M.A. Sapirstein A. Nature. 1997; 390: 622-625Crossref PubMed Scopus (757) Google Scholar, 5Uozumi N. Kume K. Nagase T. Nakatani N. Ishii S. Tashiro F. Komagata Y. Maki K. Ikuta K. Ouchi Y. Miyazaki J. Shimizu T. Nature. 1997; 390: 618-622Crossref PubMed Scopus (636) Google Scholar). cPLA2 is therefore an attractive target for developing specific inhibitors that can be used as novel anti-inflammatory drugs. cPLA2 is translocated to endoplasmic reticulum membranes and nuclear envelopes in response to a rise in intracellular Ca2+ (6Glover S. de Carvalho M.S. Bayburt T. Jonas M. Chi E. Leslie C.C. Gelb M.H. J. Biol. Chem. 1995; 270: 15359-15367Crossref PubMed Scopus (314) Google Scholar, 7Schievella A.R. Regier M.K. Smith W.L. Lin L.L. J. Biol. Chem. 1995; 270: 30749-30754Crossref PubMed Scopus (421) Google Scholar). This membrane translocation of cPLA2 is mediated by its amino-terminal C2 domain, which contains calcium and membrane binding sites (8Clark J.D. Lin L.L. Kriz R.W. Ramesha C.S. Sultzman L.A. Lin A.Y. Milona N. Knopf J.L. Cell. 1991; 65: 1043-1051Abstract Full Text PDF PubMed Scopus (1454) Google Scholar, 9Nalefski E.A. Sultzman L.A. Martin D.M. Kriz R.W. Towler P.S. Knopf J.L. Clark J.D. J. Biol. Chem. 1994; 269: 18239-18249Abstract Full Text PDF PubMed Google Scholar). However, the mechanism by which the C2 domain of cPLA2drives its Ca2+-dependent membrane targeting is not fully understood. Our recent structure-function study of the C2 domain of protein kinase C-α showed that the C2 domain not only brings the protein to the membrane surface but also triggers the membrane penetration of protein (10Medkova M. Cho W. Biochemistry. 1998; 37: 4892-4900Crossref PubMed Scopus (78) Google Scholar). Also, the study revealed that individual Ca2+ ions bound to the C2 domain play differential roles in membrane binding and activation. Tertiary structures of the C2 domains of protein kinase C (11Sutton R.B. Sprang S.R. Structure. 1998; 6: 1395-1405Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar) and cPLA2 (12Perisic O. Fong S. Lynch D.E. Bycroft M. Williams R.L. J. Biol. Chem. 1998; 273: 1596-1604Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 13Xu G.Y. McDonagh T. Yu H.A. Nalefski E.A. Clark J.D. Cumming D.A. J. Mol. Biol. 1998; 280: 485-500Crossref PubMed Scopus (100) Google Scholar), despite their overall similarity, have noticeable differences; i.e. they have different β-strand connectivity (14Nalefski E.A. Falke J.J. Protein Sci. 1996; 5: 2375-2390Crossref PubMed Scopus (685) Google Scholar), and, more importantly, the C2 domain of cPLA2 contains two clusters of exposed hydrophobic residues in the calcium binding loops (see Fig. 1 A) (12Perisic O. Fong S. Lynch D.E. Bycroft M. Williams R.L. J. Biol. Chem. 1998; 273: 1596-1604Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Consistent with this unique structure, both cPLA2 (15Lichtenbergova L. Yoon E.T. Cho W. Biochemistry. 1998; 37: 14128-14136Crossref PubMed Scopus (40) Google Scholar) and its isolated C2 domain (16Davletov B. Perisic O. Williams R.L. J. Biol. Chem. 1998; 273: 19093-19096Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) have been shown to be able to penetrate into the membrane. In this study, we performed an extensive structure-function analysis of the C2 domain of cPLA2 to assess the roles of six hydrophobic residues and five Ca2+ligands located in the calcium binding loops in the membrane binding and activation of cPLA2. We also determined the role of a cluster of cationic residues in a β-strand. In conjunction with our previous study on protein kinase C-α, this study reveals interesting similarities and differences between the two C2 domains." @default.
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• W2076132445 date "1999-04-01" @default.
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• W2076132445 title "A Structure-Function Study of the C2 Domain of Cytosolic Phospholipase A2" @default.
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• W2076132445 doi "https://doi.org/10.1074/jbc.274.14.9665" @default.
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