Membrane Targeting by C1 and C2 Domains
2001; Elsevier BV; Volume: 276; Issue: 35 Linguagem: Inglês
10.1074/jbc.r100007200
ISSN1083-351X
Autores Tópico(s)Protein Kinase Regulation and GTPase Signaling
Resumoprotein kinase C phospholipase A2 cytosolic phospholipase A2 gastrin-releasing peptide fragment green fluorescent protein diacylglycerol phosphatidylcholine phosphatidylserine phorbol 12-myristate 13-acetate Many peripheral proteins involved in cell signaling translocate to different cell membranes in response to specific cell stimuli. Because cellular functions and regulation of these proteins depend on their specific subcellular localization (1Teruel M.N. Meyer T. Cell. 2000; 103: 181-184Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar), understanding the mechanisms of membrane targeting is of great importance. The membrane targeting of diverse peripheral proteins is mediated by a limited number of membrane-targeting domains, including protein kinase C (PKC)1conserved 1 (C1), PKC conserved2 (C2), and pleckstrin homology domains. Recent structural and functional studies of individual membrane targeting domains as well as the peripheral proteins harboring these domains have provided new insights into the molecular mechanisms underlying the specific subcellular targeting and activation of peripheral proteins. This review summarizes the recent progress in our understanding of the mechanisms of C1 and C2 domain-mediated membrane targeting, with an emphasis on the correlation between the membrane binding properties of the C1 and C2 domains and the peripheral proteins containing these domains and their subcellular targeting behaviors. There are several excellent reviews (2Nalefski E.A. Falke J.J. Protein Sci. 1996; 5: 2375-2390Crossref PubMed Scopus (687) Google Scholar, 3Rizo J. Sudhof T.C. J. Biol. Chem. 1998; 273: 15879-15882Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar, 4Ron D. Kazanietz M.G. FASEB J. 1999; 13: 1658-1676Crossref PubMed Scopus (552) Google Scholar, 5Rebecchi M.J. Scarlata S. Annu. Rev. Biophys. Biomol. Struct. 1998; 27: 503-528Crossref PubMed Scopus (249) Google Scholar, 6Hurley J.H. Misra S. Annu. Rev. Biophys. Biomol. Struct. 2000; 29: 49-79Crossref PubMed Scopus (225) Google Scholar) that contain more exhaustive surveys on the membrane targeting domains. The membrane binding of peripheral proteins involves different types of interactions (Fig. 1) that depend upon the physicochemical properties of both membrane and protein. Membranes of different cellular compartments have different compositions of bulk lipids that can modulate membrane targeting of proteins either by providing unique microenvironments or by producing specific lipid metabolites, such as diacylglycerol (DAG) and phosphoinositides, that function as second messengers. Extensive structural and mutational studies of phospholipases A2(PLA2) have shown that their membrane binding surfaces are composed of cationic, aliphatic, and aromatic residues (7Gelb M.H. Cho W. Wilton D.C. Curr. Opin. Struct. Biol. 1999; 9: 428-432Crossref PubMed Scopus (116) Google Scholar). A recent study by surface plasmon resonance analysis indicated that cationic residues primarily accelerate the association of protein to anionic membrane surfaces, whereas aliphatic residues mainly slow the membrane dissociation by penetrating into the hydrophobic core of the membrane (8Stahelin R.V. Cho W. Biochemistry. 2001; 40: 4672-4678Crossref PubMed Scopus (145) Google Scholar). Aromatic residues, particularly Trp, which has a preference for the water-lipid interface (9Yau W.M. Wimley W.C. Gawrisch K. White S.H. Biochemistry. 1998; 37: 14713-14718Crossref PubMed Scopus (824) Google Scholar), play a pivotal role in binding to zwitterionic PC membranes (7Gelb M.H. Cho W. Wilton D.C. Curr. Opin. Struct. Biol. 1999; 9: 428-432Crossref PubMed Scopus (116) Google Scholar, 10Han S.K. Kim K.P. Koduri R. Bittova L. Munoz N.M. Leff A.R. Wilton D.C. Gelb M.H. Cho W. J. Biol. Chem. 1999; 274: 11881-11888Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) by affecting both membrane association and dissociation steps (8Stahelin R.V. Cho W. Biochemistry. 2001; 40: 4672-4678Crossref PubMed Scopus (145) Google Scholar). A priori, the physicochemical principles learned from these in vitromembrane binding studies should allow the prediction of the subcellular targeting behaviors of peripheral proteins, provided that the subcellular targeting is driven mainly by membrane-protein interactions. The C1 domain (∼50 amino acids) is a cysteine-rich compact structure that contains five short β strands, a short α-helix, and two zinc ions (Fig. 2) (11Hommel U. Zurini M. Luyten M. Struct. Biol. 1994; 1: 383-387Crossref PubMed Scopus (137) Google Scholar, 12Zhang G. Kazanietz M.G. Blumberg P.M. Hurley J.H. Cell. 1995; 81: 917-924Abstract Full Text PDF PubMed Scopus (595) Google Scholar). The C1 domain was first identified as the interaction site for DAG and phorbol ester in PKCs (13Nishizuka Y. Nature. 1988; 334: 661-665Crossref PubMed Scopus (3532) Google Scholar). In conventional (α, βI, βII, and γ) and novel (δ, ε, θ, and η) PKCs, the C1 domain occurs in a tandem repeat (C1A and C1B). C1 domains have been subsequently found in other proteins with diverse functions, including protein kinase D (PKD/PKCµ), chimaerin, Ras-GRP, Unc-13, Munc13 isoforms, DAG kinases, and Raf (4Ron D. Kazanietz M.G. FASEB J. 1999; 13: 1658-1676Crossref PubMed Scopus (552) Google Scholar). In general, C1 domains show a high degree of amino acid sequence homology. Some C1 domains, including those found in atypical PKCs (ζ and ι/λ), however, do not bind lipids due to minor sequence variations and might be involved in protein-protein interactions (4Ron D. Kazanietz M.G. FASEB J. 1999; 13: 1658-1676Crossref PubMed Scopus (552) Google Scholar). This review will focus mainly on the C1 domains involved in DAG and phorbol ester binding. Structural (12Zhang G. Kazanietz M.G. Blumberg P.M. Hurley J.H. Cell. 1995; 81: 917-924Abstract Full Text PDF PubMed Scopus (595) Google Scholar) and mutation (14Kazanietz M.G. Bustelo X.R. Barbacid M. Kolch W. Mischak H. Wong G. Pettit G.R. Bruns J.D. Blumberg P.M. J. Biol. Chem. 1994; 269: 11590-11594Abstract Full Text PDF PubMed Google Scholar) studies of PKCδ-C1B have defined the phorbol ester/DAG binding pocket. The polar binding pocket is located at the tip of the molecule and is surrounded by aliphatic and aromatic residues, which are adjoined by a ring of cationic residues in the middle part of the molecule (Fig. 2). A NMR study of PKCγ-C1B (15Xu R.X. Pawelczyk T. Xia T.-H. Brown S.C. Biochemistry. 1997; 36: 10709-10717Crossref PubMed Scopus (121) Google Scholar) and a monolayer penetration study of PKCα (16Medkova M. Cho W. J. Biol. Chem. 1999; 274: 19852-19861Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar) showed that the hydrophobic residues of the C1 domain penetrate the membrane for DAG/phorbol ester binding. The phorbol ester binding seals the polar surface in the binding pocket and thereby generates a contiguous hydrophobic surface (12Zhang G. Kazanietz M.G. Blumberg P.M. Hurley J.H. Cell. 1995; 81: 917-924Abstract Full Text PDF PubMed Scopus (595) Google Scholar), which in turn greatly enhances the stability of the C1-membrane complex (17Mosior M. Newton A.C. J. Biol. Chem. 1995; 270: 25526-25533Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 18Medkova M. Cho W. Biochemistry. 1998; 37: 4892-4900Crossref PubMed Scopus (78) Google Scholar). Further mutational studies of PKCα showed that clustered cationic residues in the C1A domain are involved in nonspecific electrostatic interactions with anionic phospholipids (19Bittova L. Stahelin R.V. Cho W. J. Biol. Chem. 2001; 276: 4218-4226Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). In agreement with this finding, the isolated C1 domain repeat (i.e. C1A + C1B) of PKCα exhibits little head group specificity among anionic phospholipids while discriminating against PC (16Medkova M. Cho W. J. Biol. Chem. 1999; 274: 19852-19861Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Together these studies on PKC C1 domains have led to a model for C1 domain-membrane interactions illustrated in Fig. 2. In this model, cationic residues accelerate the initial adsorption of the C1 domain to the anionic membrane surfaces and properly position the C1 domain at the membrane surface. Then, the hydrophobic tip of the domain penetrates the membrane to bind DAG that is partially buried in the membrane because of its hydrophobic nature. Because C1 domains with the exposed hydrophobic patch are subject to protein aggregation in solution, a tightly controlled triggering mechanism would be necessary for the C1 domain-mediated membrane targeting. In the case of conventional PKCs, it has been shown that C1 domains are buried at the resting state and become accessible to DAG or phorbol esters only after Ca2+-dependent membrane binding (19Bittova L. Stahelin R.V. Cho W. J. Biol. Chem. 2001; 276: 4218-4226Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 20Oancea E. Meyer T. Cell. 1998; 95: 307-318Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar). The C1 domains of PKD/PKCµ (21Johannes F.J. Prestle J. Eis S. Oberhagemann P. Pfizenmaier K. J. Biol. Chem. 1994; 269: 6140-6148Abstract Full Text PDF PubMed Google Scholar, 22Valverde A.M. Sinnett-Smith J. Van Lint J. Rozengurt E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8572-8576Crossref PubMed Scopus (357) Google Scholar), Ras-GRP (23Tognon C.E. Kirk H.E. Passmore L.A. Whitehead I.P. Der C.J. Kay R.J. Mol. Cell. Biol. 1998; 18: 6995-7008Crossref PubMed Scopus (203) Google Scholar), and β2-chimaerin (24Caloca M.J. Garcia-Bermejo M.L. Blumberg P.M. Lewin N.E. Kremmer E. Mischak H. Wang S. Nacro K. Bienfait B. Marquez V.E. Kazanietz M.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11854-11859Crossref PubMed Scopus (93) Google Scholar) have also been shown to drive the cellular membrane targeting of the corresponding peripheral proteins in response to DAG and phorbol esters. Because no detailed analysis of their interactions with membranes has been reported, it is not known whether the mechanisms of C1 domain-mediated membrane binding of these peripheral proteins are similar to that of PKCs. Conventional and novel PKCs contain two copies of C1 domains. Earlier studies on conventional PKC reported the one-to-one stoichiometry of PKC-DAG (25Hannun Y.A. Loomis C.R. Bell R.M. J. Biol. Chem. 1985; 260: 10039-10043Abstract Full Text PDF PubMed Google Scholar) and PKC-phorbol ester binding (26Kikkawa U. Takai Y. Tanaka Y. Miyake R. Nishizuka Y. J. Biol. Chem. 1983; 258: 11442-11445Abstract Full Text PDF PubMed Google Scholar, 27Hannun Y.A. Bell R.M. J. Biol. Chem. 1986; 261: 9341-9347Abstract Full Text PDF PubMed Google Scholar), suggesting that only one C1 domain is directly involved in DAG/phorbol ester binding and PKC activation. A recent study of isolated C1A and C1B domains of PKCs revealed that C1B domains have much higher affinities for phorbol 12,13-dibutyrate than C1A domains (28Irie K. Oie K. Nakahara A. Yanai Y. Ohigashi H. Wender P.A. Fukuda H. Konishi H. Kikkawa U. J. Am. Chem. Soc. 1998; 120: 9159-9167Crossref Scopus (141) Google Scholar). The only exception was PKCγ, the C1A and C1B domains of which show comparably high affinities. For PKCδ, good correlation was observed between the intrinsic phorbol ester affinities of C1A and C1B domains and their relative importance in phorbol ester-induced activation (29Szallasi Z. Bogi K. Gohari S. Biro T. Acs P. Blumberg P.M. J. Biol. Chem. 1996; 271: 18299-18301Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), supporting the notion that the disparate roles of C1 domains mainly derive from their different intrinsic affinities for phorbol esters. More recently, however, it was shown that C1A and C1B domains of PKCα play an equivalent role in cellular membrane translocation in response to phorbol 12-myristate 13-acetate (PMA) despite their distinct phorbol ester affinities (30Bogi K. Lorenzo P.S. Szallasi Z. Acs P. Wagner G.S. Blumberg P.M. Cancer Res. 1998; 58: 1423-1428PubMed Google Scholar). To date, correlation between the intrinsic DAG affinities of C1 domains and their relative importance in DAG-dependent PKC activation has not been documented. A series of spectroscopic vesicle binding studies of PKCα indicated that PKCα contains two phorbol ester binding sites with high and low affinities and that DAG and phorbol esters bind to the two sites with opposite affinity (31Slater S.J. Kelly M.B. Taddeo F.J. Ho C. Rubin E. Stubbs C.D. J. Biol. Chem. 1994; 269: 4866-4871Abstract Full Text PDF PubMed Google Scholar, 32Slater S.J. Ho C. Kelly M.B. Larkin J.D. Taddeo F.J. Yeager M.D. Stubbs C.D. J. Biol. Chem. 1996; 271: 4627-4631Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The notion that DAG and phorbol esters might have different C1A versus C1B selectivity was further supported by the finding that the C1A domain is exclusively involved in the DAG-induced vesicle binding and activation of PKCα (16Medkova M. Cho W. J. Biol. Chem. 1999; 274: 19852-19861Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Clearly, more studies are needed to fully understand these complex interactions of the C1 domains of PKC with their ligands. The subcellular targeting of isolated C1 domains and PKC holoenzymes in response to DAG and phorbol esters has been measured in various mammalian cells transfected with green fluorescent protein (GFP)-tagged proteins (20Oancea E. Meyer T. Cell. 1998; 95: 307-318Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar, 33Sakai N. Sasaki K. Ikegaki N. Shirai Y. Ono Y. Saito N. J. Cell Biol. 1997; 139: 1465-1476Crossref PubMed Scopus (198) Google Scholar, 34Ohmori S. Shirai Y. Sakai N. Fujii M. Konishi H. Kikkawa U. Saito N. Mol. Cell. Biol. 1998; 18: 5263-5271Crossref PubMed Google Scholar, 35Oancea E. Teruel M.N. Quest A.F. Meyer T. J. Cell Biol. 1998; 140: 485-498Crossref PubMed Scopus (291) Google Scholar, 36Wang Q.J. Bhattacharyya D. Garfield S. Nacro K. Marquez V.E. Blumberg P.M. J. Biol. Chem. 1999; 274: 37233-37239Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 37Wang Q.J. Fang T.W. Fenick D. Garfield S. Bienfait B. Marquez V.E. Blumberg P.M. J. Biol. Chem. 2000; 275: 12136-12146Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 38Vallentin A. Prevostel C. Fauquier T. Bonnefont X. Joubert D. J. Biol. Chem. 2000; 275: 6014-6021Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 39Almholt K. Arkhammar P.O. Thastrup O. Tullin S. Biochem. J. 1999; 337: 211-218Crossref PubMed Scopus (53) Google Scholar). PKCγ translocated to plasma membrane in response to PMA in COS-7 cells (33Sakai N. Sasaki K. Ikegaki N. Shirai Y. Ono Y. Saito N. J. Cell Biol. 1997; 139: 1465-1476Crossref PubMed Scopus (198) Google Scholar). In RBL cells, PKCγ and its C1A, C1B, and C1A-C1B domains all showed the translocation to plasma membrane in response to PMA or DAG (35Oancea E. Teruel M.N. Quest A.F. Meyer T. J. Cell Biol. 1998; 140: 485-498Crossref PubMed Scopus (291) Google Scholar). As is the case with PKCγ, PKCβII (40Feng X. Hannun Y.A. J. Biol. Chem. 1998; 273: 26870-26874Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 41Feng X. Becker K.P. Stribling S.D. Peters K.G. Hannun Y.A. J. Biol. Chem. 2000; 275: 17024-17034Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), PKCδ (34Ohmori S. Shirai Y. Sakai N. Fujii M. Konishi H. Kikkawa U. Saito N. Mol. Cell. Biol. 1998; 18: 5263-5271Crossref PubMed Google Scholar), PKCα (39Almholt K. Arkhammar P.O. Thastrup O. Tullin S. Biochem. J. 1999; 337: 211-218Crossref PubMed Scopus (53) Google Scholar), and PKCα-C1A-C1B (38Vallentin A. Prevostel C. Fauquier T. Bonnefont X. Joubert D. J. Biol. Chem. 2000; 275: 6014-6021Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) were shown to translocate to plasma membrane in response to PMA or DAG. In the case of PKC holoenzymes, Ca2+ enhanced the rate of translocation of proteins but did not affect their localization. Later studies showed that whereas PMA and other hydrophobic phorbol esters induced the initial translocation of PKCδ to the plasma membrane, less hydrophobic DAG and PDB caused PKCδ to translocate to the perinuclear region (36Wang Q.J. Bhattacharyya D. Garfield S. Nacro K. Marquez V.E. Blumberg P.M. J. Biol. Chem. 1999; 274: 37233-37239Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 37Wang Q.J. Fang T.W. Fenick D. Garfield S. Bienfait B. Marquez V.E. Blumberg P.M. J. Biol. Chem. 2000; 275: 12136-12146Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Our cell study 2J. Rafter and W. Cho, manuscript in preparation. of GFP-PKCα with a fluorescent phorbol ester analog, sapintoxin D, supported the notion that the differential subcellular localization of C1 ligands is a main determinant of the differential subcellular targeting of PKCs. This in turn suggests that the in vitromembrane binding and the subcellular targeting of C1 domain are driven by the same forces. Binding to neither adapter proteins nor cytoskeleton appears to contribute significantly to the subcellular targeting of PKC-C1 domains because the putative adapter binding site is not located in the C1 domains (42Mochly-Rosen D. Gordon A.S. FASEB J. 1998; 12: 35-42Crossref PubMed Scopus (509) Google Scholar) and the cytoskeleton inhibitors show little effect on C1 domain translocation (35Oancea E. Teruel M.N. Quest A.F. Meyer T. J. Cell Biol. 1998; 140: 485-498Crossref PubMed Scopus (291) Google Scholar, 39Almholt K. Arkhammar P.O. Thastrup O. Tullin S. Biochem. J. 1999; 337: 211-218Crossref PubMed Scopus (53) Google Scholar). The C2 domain (∼130 residues) was first discovered as the Ca2+-binding site in conventional PKCs (15Xu R.X. Pawelczyk T. Xia T.-H. Brown S.C. Biochemistry. 1997; 36: 10709-10717Crossref PubMed Scopus (121) Google Scholar). A great number of proteins containing the C2 domain have been identified since, and most of them are involved in signal transduction (e.g., PKC, cytosolic PLA2 (cPLA2), phospholipases C, plant phospholipase D, and phosphatidylinositol 3-kinase) or membrane trafficking (e.g., synaptotagmins, rabphilin-3A, and Unc-13) (2Nalefski E.A. Falke J.J. Protein Sci. 1996; 5: 2375-2390Crossref PubMed Scopus (687) Google Scholar, 3Rizo J. Sudhof T.C. J. Biol. Chem. 1998; 273: 15879-15882Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar). Structural analyses of multiple C2 domains have indicated that all C2 domains share a common fold of eight-stranded antiparallel β-sandwich connected by variable loops, with the Ca2+-binding sites located at one side of the domain (Fig.2) (43Shao X. Davletov B.A. Sutton R.B. Sudhof T.C. Rizo J. Science. 1996; 273: 248-251Crossref PubMed Scopus (294) Google Scholar, 44Sutton R.B. Davletov B.A. Berghuis A.M. Sudhof T.C. Sprang S.R. Cell. 1995; 80: 929-938Abstract Full Text PDF PubMed Scopus (604) Google Scholar, 45Essen L.O. Perisic O. Cheung R. Katan M. Williams R.L. Nature. 1996; 380: 595-602Crossref PubMed Scopus (514) Google Scholar, 46Verdaguer N. Corbalan-Garcia S. Ochoa W.F. Fita I. Gomez- Fernandez J.C. EMBO J. 1999; 18: 6329-6338Crossref PubMed Scopus (284) Google Scholar, 47Perisic 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). When compared with C1 domains, C2 domains show a larger degree of variation in amino acid sequence, particularly in the loop regions (2Nalefski E.A. Falke J.J. Protein Sci. 1996; 5: 2375-2390Crossref PubMed Scopus (687) Google Scholar, 3Rizo J. Sudhof T.C. J. Biol. Chem. 1998; 273: 15879-15882Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar). Consistent with this finding, C2 domains show greater functional diversities. Most Ca2+-dependent membrane-binding C2 domains prefer anionic membranes to zwitterionic ones; however, cPLA2-C2 strongly favors PC membranes (48Nalefski E.A. McDonagh T. Somers W. Seehra J. Falke J.J. Clark J.D. J. Biol. Chem. 1998; 273: 1365-1372Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Among anionic lipid-selective C2 domains, PKCα-C2 (16Medkova M. Cho W. J. Biol. Chem. 1999; 274: 19852-19861Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar) and PLCδ1-C2 (49Lomasney J.W. Cheng H.F. Roffler S.R. King K. J. Biol. Chem. 1999; 274: 21995-22001Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) exhibit PS selectivity. Furthermore, there are many non-Ca2+-binding C2 domains; some of them, such as PTEN-C2 (50Lee J.O. Yang H. Georgescu M.M. Di Cristofano A. Maehama T. Shi Y. Dixon J.E. Pandolfi P. Pavletich N.P. Cell. 1999; 99: 323-334Abstract Full Text Full Text PDF PubMed Scopus (868) Google Scholar), still bind the membrane, and others might be involved in protein-protein interactions (2Nalefski E.A. Falke J.J. Protein Sci. 1996; 5: 2375-2390Crossref PubMed Scopus (687) Google Scholar, 3Rizo J. Sudhof T.C. J. Biol. Chem. 1998; 273: 15879-15882Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar). This review will mainly deal with the C2 domains that bind phospholipids in a Ca2+-dependent manner. The Ca2+-binding sites of C2 domains are composed of three variable loops that contain ligands for multiple Ca2+ions. Structural (43Shao X. Davletov B.A. Sutton R.B. Sudhof T.C. Rizo J. Science. 1996; 273: 248-251Crossref PubMed Scopus (294) Google Scholar, 44Sutton R.B. Davletov B.A. Berghuis A.M. Sudhof T.C. Sprang S.R. Cell. 1995; 80: 929-938Abstract Full Text PDF PubMed Scopus (604) Google Scholar, 45Essen L.O. Perisic O. Cheung R. Katan M. Williams R.L. Nature. 1996; 380: 595-602Crossref PubMed Scopus (514) Google Scholar, 46Verdaguer N. Corbalan-Garcia S. Ochoa W.F. Fita I. Gomez- Fernandez J.C. EMBO J. 1999; 18: 6329-6338Crossref PubMed Scopus (284) Google Scholar, 47Perisic 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) and binding analyses (51Fukuda M. Kojima T. Mikoshiba K. J. Biol. Chem. 1996; 271: 8430-8434Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 52Nalefski E.A. Slazas M.M. Falke J.J. Biochemistry. 1997; 36: 12011-12018Crossref PubMed Scopus (113) Google Scholar, 53Zheng L. Krishnamoorthi R. Zolkiewski M. Wang X. J. Biol. Chem. 2000; 275: 19700-19706Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) have determined the calcium binding stoichiometry, geometry, and affinity for several C2 domains. Two major roles of Ca2+ ions in the membrane targeting of the C2 domain have been experimentally demonstrated. The first role of Ca2+ ions is to provide a bridge between the C2 domain and anionic phospholipids. This Ca2+ bridge model is supported by an x-ray structure of the PKCα-C2-(Ca2+)2-PS complex (46Verdaguer N. Corbalan-Garcia S. Ochoa W.F. Fita I. Gomez- Fernandez J.C. EMBO J. 1999; 18: 6329-6338Crossref PubMed Scopus (284) Google Scholar), showing that a short-chain PS molecule is specifically coordinated to a Ca2+ ion and other residues in the Ca2+-binding loops. This structure also accounts for the PS selectivity of PKCα-C2 (16Medkova M. Cho W. J. Biol. Chem. 1999; 274: 19852-19861Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). The second role of Ca2+ ions is to induce intra- or interdomain conformational changes, which in turn trigger membrane-protein interactions. Despite earlier negative reports (43Shao X. Davletov B.A. Sutton R.B. Sudhof T.C. Rizo J. Science. 1996; 273: 248-251Crossref PubMed Scopus (294) Google Scholar), accumulating evidence has supported the occurrence of Ca2+-induced conformational changes in the C2 domain (44Sutton R.B. Davletov B.A. Berghuis A.M. Sudhof T.C. Sprang S.R. Cell. 1995; 80: 929-938Abstract Full Text PDF PubMed Scopus (604) Google Scholar,52Nalefski E.A. Slazas M.M. Falke J.J. Biochemistry. 1997; 36: 12011-12018Crossref PubMed Scopus (113) Google Scholar, 53Zheng L. Krishnamoorthi R. Zolkiewski M. Wang X. J. Biol. Chem. 2000; 275: 19700-19706Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 54Grobler J.A. Essen L.O. Williams R.L. Hurley J.H. Nat. Struct. Biol. 1996; 3: 788-795Crossref PubMed Scopus (102) Google Scholar, 55Chapman E.R. Davis A.F. J. Biol. Chem. 1998; 273: 13995-14001Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Recent structure-function studies of the C2 domains of PKCα (18Medkova M. Cho W. Biochemistry. 1998; 37: 4892-4900Crossref PubMed Scopus (78) Google Scholar) and cPLA2 (56Bittova L. Sumandea M. Cho W. J. Biol. Chem. 1999; 274: 9665-9672Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), both of which bind two Ca2+ ions, showed that two Ca2+ ions play distinct roles, with one primarily involved in inducing the conformational changes and the other in Ca2+ bridging. The main difference is that Ca2+-induced conformational changes are critical for the membrane binding of cPLA2-C2, whereas Ca2+ bridging is a relatively more important role of PKCα-C2. Non-Ca2+-coordinating protein residues in the Ca2+-binding loops also play important roles in the membrane binding and phospholipid selectivity of the C2 domain. A large degree of structural variations have been found in the Ca2+-binding loops of C2 domains in terms of both primary and tertiary structures (43Shao X. Davletov B.A. Sutton R.B. Sudhof T.C. Rizo J. Science. 1996; 273: 248-251Crossref PubMed Scopus (294) Google Scholar, 44Sutton R.B. Davletov B.A. Berghuis A.M. Sudhof T.C. Sprang S.R. Cell. 1995; 80: 929-938Abstract Full Text PDF PubMed Scopus (604) Google Scholar, 45Essen L.O. Perisic O. Cheung R. Katan M. Williams R.L. Nature. 1996; 380: 595-602Crossref PubMed Scopus (514) Google Scholar, 46Verdaguer N. Corbalan-Garcia S. Ochoa W.F. Fita I. Gomez- Fernandez J.C. EMBO J. 1999; 18: 6329-6338Crossref PubMed Scopus (284) Google Scholar, 47Perisic 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). Mutational (56Bittova L. Sumandea M. Cho W. J. Biol. Chem. 1999; 274: 9665-9672Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 57Medkova M. Cho W. J. Biol. Chem. 1998; 273: 17544-17552Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) and labeling (58Nalefski E.A. Falke J.J. Biochemistry. 1998; 37: 17642-17650Crossref PubMed Scopus (80) Google Scholar,59Ball A. Nielsen R. Gelb M.H. Robinson B.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6637-6642Crossref PubMed Scopus (85) Google Scholar) studies have identified the residues in the Ca2+-binding loops that play a key role in membrane binding. In general, cationic residues on the surface of Ca2+-binding loops are important for anionic lipid-selective C2 domains (57Medkova M. Cho W. J. Biol. Chem. 1998; 273: 17544-17552Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 60Zhang X. Rizo J. Sudhof T.C. Biochemistry. 1998; 37: 12395-12403Crossref PubMed Scopus (165) Google Scholar), whereas aliphatic and aromatic residues are essential for PC-selective cPLA2-C2 (56Bittova L. Sumandea M. Cho W. J. Biol. Chem. 1999; 274: 9665-9672Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 58Nalefski E.A. Falke J.J. Biochemistry. 1998; 37: 17642-17650Crossref PubMed Scopus (80) Google Scholar,59Ball A. Nielsen R. Gelb M.H. Robinson B.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6637-6642Crossref PubMed Scopus (85) Google Scholar). A predominant cationic cluster in the β-sandwich region has been implicated in inositol polyphosphate binding of synaptotagmin II (61Fukuda M. Kojima T. Aruga J. Niinobe M. Mikoshiba K. J. Biol. Chem. 1995; 270: 26523-26527Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar) but does not significantly affect the membrane binding of conventional PKCs (62Johnson J.E. Edwards A.S. Newton A.C. J. Biol. Chem. 1997; 272: 30787-30792Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) and cPLA2 (56Bittova L. Sumandea M. Cho W. J. Biol. Chem. 1999; 274: 9665-9672Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Together, thesein vitro studies have indicated that anionic lipid-selective C2 domains and cPLA2-C2 have distinct membrane binding modes. As shown in Fig. 2, PKCα-C2 binds to the membrane in an orientation that optimizes its electrostatic interactions with the anionic membrane (46Verdaguer N. Corbalan-Garcia S. Ochoa W.F. Fita I. Gomez- Fernandez J.C. EMBO J. 1999; 18: 6329-6338Crossref PubMed Scopus (284) Google Scholar, 57Medkova M. Cho W. J. Biol. Chem. 1998; 273: 17544-17552Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 60Zhang X. Rizo J. Sudhof T.C. Biochemistry. 1998; 37: 12395-12403Crossref PubMed Scopus (165) Google Scholar), whereas cPLA2-C2 binds to the membrane in an orientation that optimizes the membrane penetration of its hydrophobic residues (56Bittova L. Sumandea M. Cho W. J. Biol. Chem. 1999; 274: 9665-9672Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 58Nalefski E.A. Falke J.J. Biochemistry. 1998; 37: 17642-17650Crossref PubMed Scopus (80) Google Scholar, 59Ball A. Nielsen R. Gelb M.H. Robinson B.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6637-6642Crossref PubMed Scopus (85) Google Scholar, 63Davletov B. Perisic O. Williams R.L. J. Biol. Chem. 1998; 273: 19093-19096Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Interestingly, membrane binding properties of cPLA2 and its C2 domain are similar (56Bittova L. Sumandea M. Cho W. J. Biol. Chem. 1999; 274: 9665-9672Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 64Lichtenbergova L. Yoon E.T. Cho W. Biochemistry. 1998; 37: 14128-14136Crossref PubMed Scopus (40) Google Scholar), whereas those of PKCα and its C2 domain have noticeable differences (16Medkova M. Cho W. J. Biol. Chem. 1999; 274: 19852-19861Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 18Medkova M. Cho W. Biochemistry. 1998; 37: 4892-4900Crossref PubMed Scopus (78) Google Scholar). This discrepancy implies that the relative contribution of a C2 domain to the membrane binding of a peripheral protein depends on the structural context of the protein, particularly on the presence of other membrane targeting domains in the same molecule. The subcellular targeting of GFP-tagged C2 domains and C2-containing peripheral proteins, most notably conventional PKCs and cPLA2, has been studied in different mammalian cells. In general, the subcellular localization behaviors of C2 domains are consistent with their in vitro membrane binding properties. For instance, C2 domains of conventional PKCs that prefer anionic phospholipids rapidly translocate to plasma membrane (20Oancea E. Meyer T. Cell. 1998; 95: 307-318Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar) and PC-selective cPLA2-C2 to the perinuclear region in response to Ca2+ import (65Perisic O. Paterson H.F. Mosedale G. Lara-Gonzalez S. Williams R.L. J. Biol. Chem. 1999; 274: 14979-14987Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 66Gijon M.A. Spencer D.M. Kaiser A.L. Leslie C.C. J. Cell Biol. 1999; 145: 1219-1232Crossref PubMed Scopus (184) Google Scholar). Also this subcellular localization pattern of isolated C2 domains correlates with that of peripheral proteins harboring the C2 domains: i.e. conventional PKCs translocate to the plasma membrane (33Sakai N. Sasaki K. Ikegaki N. Shirai Y. Ono Y. Saito N. J. Cell Biol. 1997; 139: 1465-1476Crossref PubMed Scopus (198) Google Scholar, 34Ohmori S. Shirai Y. Sakai N. Fujii M. Konishi H. Kikkawa U. Saito N. Mol. Cell. Biol. 1998; 18: 5263-5271Crossref PubMed Google Scholar, 35Oancea E. Teruel M.N. Quest A.F. Meyer T. J. Cell Biol. 1998; 140: 485-498Crossref PubMed Scopus (291) Google Scholar, 39Almholt K. Arkhammar P.O. Thastrup O. Tullin S. Biochem. J. 1999; 337: 211-218Crossref PubMed Scopus (53) Google Scholar) whereas cPLA2 moves to the perinuclear region (67Glover S. de Carvalho M.S. Bayburt T. Jonas M. Chi E. Leslie C.C. Gelb M.H. J. Biol. Chem. 1995; 270: 15359-15367Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). Furthermore, chimera proteins of PKCα and cPLA2 containing the C2 domains of cPLA2 and PKCα, respectively, are localized to the perinuclear region and plasma membrane, respectively. 3R. Stahelin, J. Rafter, and W. Cho, manuscript in preparation. Thus, it is expected that anionic lipid-selective C2 domains (and C2-bearing proteins) will translocate to plasma membrane and that PC-selective C2 domains (and C2-bearing proteins) will translocate to the perinuclear region. When conventional PKCs are activated by both C1 ligand and Ca2+ ionophore, however, their subcellular localization is governed primarily by the subcellular location of C1 ligands although the C2 domain also contributes to the kinetics and energetics of translocation (20Oancea E. Meyer T. Cell. 1998; 95: 307-318Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar). Our recent study3 of PKCα-C2, cPLA2-C2, and their mutants demonstrated quantitative correlation between the subcellular targeting and in vitromembrane binding: i.e. PKCα-C2 rapidly but transiently translocates to plasma membrane, but cPLA2-C2 slowly but irreversibly translocates to the perinuclear region in response to Ca2+ import. Also subcellular translocation kinetics of mutants exhibited good correlation with their altered in vitro membrane binding kinetics. Thus, it appears that the subcellular targeting of these C2 domains is primarily driven by forces that govern their membrane binding. Some Ca2+-dependent membrane-binding proteins, including the amino-terminal β-barrel domain of 5-lipoxygenase (68Hammarberg T. Provost P. Persson B. Radmark O. J. Biol. Chem. 2000; 275: 38787-38793Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar) and the domain III of calpains (69Hosfield C.M. Elce J.S. Davies P.L. Jia Z. EMBO J. 1999; 18: 6880-6889Crossref PubMed Scopus (289) Google Scholar, 70Strobl S. Fernandez-Catalan C. Braun M. Huber R. Masumoto H. Nakagawa K. Irie A. Sorimachi H. Bourenkow G. Bartunik H. Suzuki K. Bode W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 588-592Crossref PubMed Scopus (314) Google Scholar), have C2 domain-like structures. In particular, the C2-like domain of 5-lipoxygenase has been shown to have specific Ca2+ ligands (68Hammarberg T. Provost P. Persson B. Radmark O. J. Biol. Chem. 2000; 275: 38787-38793Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar) and to be responsible for the Ca2+-dependent nuclear translocation of 5-lipoxygenase (71Chen X.S. Funk C.D. J. Biol. Chem. 2001; 276: 811-818Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). The C2-like domain of 5-lipoxygenase has been shown to have PC selectiv-ity because of the presence of aromatic residues in the putative Ca2+-binding loops. 4S. Kulkarni and W. Cho, manuscript in preparation. The majority of peripheral proteins have two (or more) membrane targeting domains although the presence of extra domains is not absolutely required for their membrane translocation. This suggests that their membrane targeting and activation might involve a synergistic and/or regulatory interplay of the membrane targeting domains. A synergistic action of C1 and C2 domains in the prolonged membrane localization of PKC was demonstrated for PKCγ (20Oancea E. Meyer T. Cell. 1998; 95: 307-318Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar) and PKCβII (41Feng X. Becker K.P. Stribling S.D. Peters K.G. Hannun Y.A. J. Biol. Chem. 2000; 275: 17024-17034Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The regulatory interactions between C1 and C2 domains have also been suggested for the targeting and regulation of different PKC isoforms. In the case of PKCα, a single anionic residue in the C1A domain is implicated in the tethering of C1A to the other part of the molecule, most likely the C2 domain (19Bittova L. Stahelin R.V. Cho W. J. Biol. Chem. 2001; 276: 4218-4226Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The putative tethering keeps the protein in an inactive conformation at the resting state and is disrupted specifically by the Ca2+-dependent binding of the C2 domain to PS in the membrane, which in turn leads to the membrane penetration and DAG binding of the C1A domain and PKC activation. Similarly, it was suggested that the C2-like domain of novel PKC interacts with the C1 domain to regulate the enzyme activity (72Pepio A.M. Sossin W.S. J. Biol. Chem. 2001; 276: 3846-3855Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Further studies will reveal more examples of interdomain interactions in the membrane targeting of peripheral domains. In both protein-protein and membrane-protein interactions, the initial formation of nonspecific collisional complexes, driven by diffusion and electrostatic forces, is followed by the formation of tightly bound complexes, which are stabilized by specific interactions (8Stahelin R.V. Cho W. Biochemistry. 2001; 40: 4672-4678Crossref PubMed Scopus (145) Google Scholar, 73Cunningham B.C. Wells J.A. J. Mol. Biol. 1993; 234: 554-563Crossref PubMed Scopus (485) Google Scholar). The former interactions mainly enhance the association rate whereas the latter interactions primarily decrease the dissociation rate. For C1 domains, the initial binding is driven by nonspecific electrostatic interactions between cationic residues with bulk anionic lipids, but the tight complex formation is achieved by both hydrophobic interactions between hydrophobic C1 residues and the membrane core and hydrogen bonds between polar C1 residues and DAG (Fig. 2). Accordingly, the subcellular localization of the C1 domain is determined primarily by the location of DAG and phorbol ester, although the bulk lipid composition of the targeting membrane can affect the kinetics of translocation (20Oancea E. Meyer T. Cell. 1998; 95: 307-318Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar). For anionic lipid-selective C2 domains, the initial binding is driven by electrostatic interactions via C2-bound Ca2+, cationic residues, or a combination of both. However, the paucity of multiple specific interactions required for tight complex formation renders the binding relatively weak and transient (20Oancea E. Meyer T. Cell. 1998; 95: 307-318Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar). On the other hand, the membrane binding of PC-selective cPLA2-C2 is slowly driven by interactions between aromatic residues and PC molecules (8Stahelin R.V. Cho W. Biochemistry. 2001; 40: 4672-4678Crossref PubMed Scopus (145) Google Scholar), but the binding is tight due to membrane penetration and the resulting hydrophobic interactions. In accordance with these membrane binding properties, cPLA2-C2 alone can achieve the membrane localization of cPLA2 in the cell, whereas both C1 and C2 domains are required for the prolonged membrane localization of conventional PKC (20Oancea E. Meyer T. Cell. 1998; 95: 307-318Abstract Full Text Full Text PDF PubMed Scopus (545) Google Scholar, 41Feng X. Becker K.P. Stribling S.D. Peters K.G. Hannun Y.A. J. Biol. Chem. 2000; 275: 17024-17034Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Unlike the case of the C1 domain, the bulk lipid composition of the membrane is an important determinant of Ca2+-dependent localization of the C2 domains. For the reliable prediction of subcellular localization of C2 domains based on their lipid selectivity, the lipid compositions of various cellular membranes of different cells need to be fully characterized. Results summarized in this review show that much of the spatiotemporal dynamics of the peripheral proteins harboring membrane-binding C1 and C2 domains can be accounted for by the physicochemical principles that govern their in vitromembrane binding properties. The subcellular targeting of peripheral proteins containing a single targeting domain (e.g.cPLA2) usually reflects the membrane binding properties of the domain, whereas that of peripheral proteins with multiple C1 and C2 domains is determined by the delicate interplay of the domains and the synergistic actions of their agonists (e.g. Ca2+and DAG). The principles learned from these studies should help in understanding the subcellular targeting of peripheral proteins containing other membrane targeting domains. Because C1 and C2 domains can also interact with other proteins (42Mochly-Rosen D. Gordon A.S. FASEB J. 1998; 12: 35-42Crossref PubMed Scopus (509) Google Scholar), the subcellular targeting of some C1- and C2-containing proteins might involve both membrane-protein and protein-protein interactions. Moreover, protein phosphorylation might play an important regulatory role in the subcellular targeting of peripheral proteins (40Feng X. Hannun Y.A. J. Biol. Chem. 1998; 273: 26870-26874Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 41Feng X. Becker K.P. Stribling S.D. Peters K.G. Hannun Y.A. J. Biol. Chem. 2000; 275: 17024-17034Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Further systematic studies are required to comprehensively address these important issues. Finally, cellular translocation studies of peripheral proteins have been focused mainly on membrane translocation so far. To fully understand spatiotemporal dynamics and regulation of signaling peripheral proteins, however, it is necessary to simultaneously monitor the spatiotemporal dynamics and activities of peripheral proteins as well as the spatiotemporal dynamics of their stimuli in living cells. Recent technological advances in light microscopy and cell biology should greatly facilitate this challenging endeavor.
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