Title: The Origin of C1A-C2 Interdomain Interactions in Protein Kinase Cα
Abstract: The regulatory domain of protein kinase Cα (PKCα) contains three membrane-targeting modules, two C1 domains (C1A and C1B) that bind diacylglycerol and phorbol ester, and the C2 domain that is responsible for the Ca2+-dependent membrane binding. Accumulating evidence suggests that C1A and C2 domains of PKCα are tethered in the resting state and that the tethering is released upon binding to the membrane containing phosphatidylserine. The homology modeling and the docking analysis of C1A and C2 domains of PKCα revealed a highly complementary interface that comprises Asp55-Arg252 and Arg42-Glu282 ion pairs and a Phe72-Phe255 aromatic pair. Mutations of these residues in the predicted C1A-C2 interface showed large effects on in vitro membrane binding, enzyme activity, phosphatidylserine selectivity, and cellular membrane translocation of PKCα, supporting their involvement in interdomain interactions. In particular, D55A (or D55K) and R252A (or R252E) mutants showed much higher basal membrane affinity and enzyme activity and faster subcellular translocation than wild type, whereas a double charge-reversal mutant (D55K/R252E) behaved analogously to wild type, indicating that a direct electrostatic interaction between the two residues is essential for the C1A-C2 tethering. Collectively, these studies provide new structural insight into PKCα C1A-C2 interdomain interactions and the mechanism of lipid-mediated PKCα activation. The regulatory domain of protein kinase Cα (PKCα) contains three membrane-targeting modules, two C1 domains (C1A and C1B) that bind diacylglycerol and phorbol ester, and the C2 domain that is responsible for the Ca2+-dependent membrane binding. Accumulating evidence suggests that C1A and C2 domains of PKCα are tethered in the resting state and that the tethering is released upon binding to the membrane containing phosphatidylserine. The homology modeling and the docking analysis of C1A and C2 domains of PKCα revealed a highly complementary interface that comprises Asp55-Arg252 and Arg42-Glu282 ion pairs and a Phe72-Phe255 aromatic pair. Mutations of these residues in the predicted C1A-C2 interface showed large effects on in vitro membrane binding, enzyme activity, phosphatidylserine selectivity, and cellular membrane translocation of PKCα, supporting their involvement in interdomain interactions. In particular, D55A (or D55K) and R252A (or R252E) mutants showed much higher basal membrane affinity and enzyme activity and faster subcellular translocation than wild type, whereas a double charge-reversal mutant (D55K/R252E) behaved analogously to wild type, indicating that a direct electrostatic interaction between the two residues is essential for the C1A-C2 tethering. Collectively, these studies provide new structural insight into PKCα C1A-C2 interdomain interactions and the mechanism of lipid-mediated PKCα activation. The mammalian protein kinase C (PKC) 2The abbreviations used are:PKCprotein kinase CBAPTA-AM1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl esterCHAPS(3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonatecPKCconventional protein kinase CDAGsn-1,2-diacylglycerolOPG1-octanoyl-2-(8-pyrenyloctanoyl)-sn-3-glycerolDiC8sn-1,2-dioctanoylglycerolDiC18sn-1,2-dioleoylglycerolDMEMDulbecco's modified Eagle's mediumEGFPenhanced green fluorescent proteinHEKhuman embryonic kidneynPKCnovel protein kinase CPOPC1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholinePOPE1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolaminePOPG1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerolPOPI1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoinositolPOPS1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserinePGphosphatidylglycerolPSphosphatidylserineSPRsurface plasmon resonance family consists of at least 11 isoforms of serine/threonine kinases that mediate a wide variety of cellular processes such as proliferation, apoptosis, differentiation, migration, and neuronal signaling (1Parekh D.B. Ziegler W. Parker P.J. EMBO J. 2000; 19: 496-503Crossref PubMed Scopus (510) Google Scholar, 2Newton A.C. Chem. Rev. 2001; 101: 2353-2364Crossref PubMed Scopus (835) Google Scholar, 3Shirai Y. Saito N. J. Biochem. (Tokyo). 2002; 132: 663-668Crossref PubMed Scopus (128) Google Scholar). All PKCs contain an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain. Based upon structural differences in the regulatory domain, PKCs are typically subdivided into three classes; conventional PKC (cPKC: α, βI, βII, and γ subtypes), novel PKC (nPKC: δ, ϵ, η, and θ subtypes), and atypical PKC (ζ and λ/ι subtypes). cPKCs and nPKCs have two types of membrane-targeting domains, a tandem repeat of C1 domains (C1A and C1B) and a C2 domain, in the regulatory domain. protein kinase C 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester (3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate conventional protein kinase C sn-1,2-diacylglycerol 1-octanoyl-2-(8-pyrenyloctanoyl)-sn-3-glycerol sn-1,2-dioctanoylglycerol sn-1,2-dioleoylglycerol Dulbecco's modified Eagle's medium enhanced green fluorescent protein human embryonic kidney novel protein kinase C 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoinositol 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine phosphatidylglycerol phosphatidylserine surface plasmon resonance The C1 domain (∼50 residues) is a highly conserved, cysteine-rich compact structure that was initially identified as the interaction site for 1,2-diacyl-sn-3-glycerol (DAG) and phorbol ester in PKCs (4Brose N. Rosenmund C. J. Cell Sci. 2002; 115: 4399-4411Crossref PubMed Scopus (305) Google Scholar, 5Yang C. Kazanietz M.G. Trends Pharmacol. Sci. 2003; 24: 602-608Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 6Cho W. Stahelin R.V. Annu. Rev. Biophys. Biomol. Struct. 2005; 34: 119-151Crossref PubMed Scopus (486) Google Scholar). Recent studies have shown, however, that the C1 domains of PKCs have a wide range of affinities for DAG and phorbol ester (7Irie 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, 8Ananthanarayanan B. Stahelin R.V. Digman M.A. Cho W. J. Biol. Chem. 2003; 278: 46886-46894Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 9Stahelin R.V. Digman M.A. Medkova M. Ananthanarayanan B. Rafter J.D. Melowic H.R. Cho W. J. Biol. Chem. 2004; 279: 29501-29512Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). All C1 domains characterized so far have a common structure with exposed hydrophobic residues surrounding the ligand-binding pocket and a cluster of cationic residues in the middle of the molecule (see Fig. 1) (10Hommel U. Zurini M. Luyten M. Struct. Biol. 1994; 1: 383-387Crossref PubMed Scopus (137) Google Scholar, 11Zhang G. Kazanietz M.G. Blumberg P.M. Hurley J.H. Cell. 1995; 81: 917-924Abstract Full Text PDF PubMed Scopus (598) Google Scholar, 12Xu R.X. Pawelczyk T. Xia T.-H. Brown S.C. Biochemistry. 1997; 36: 10709-10717Crossref PubMed Scopus (122) Google Scholar, 13Mott H.R. Carpenter J.W. Zhong S. Ghosh S. Bell R.M. Campbell S.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8312-8317Crossref PubMed Scopus (158) Google Scholar, 14Zhou M. Horita D.A. Waugh D.S. Byrd R.A. Morrison D.K. J. Mol. Biol. 2002; 315: 435-446Crossref PubMed Scopus (70) Google Scholar, 15Canagarajah B. Leskow F.C. Ho J.Y. Mischak H. Saidi L.F. Kazanietz M.G. Hurley J.H. Cell. 2004; 119: 407-418Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 16Shen N. Guryev O. Rizo J. Biochemistry. 2005; 44: 1089-1096Crossref PubMed Scopus (50) Google Scholar). Owing to this unique structural feature, C1 domains can effectively penetrate the membrane containing anionic lipids. Although it was reported that PKCβII C1B domain has stereospecificity for the phosphatidylserine (PS) headgroup (17Johnson J.E. Giorgione J. Newton A.C. Biochemistry. 2000; 39: 11360-11369Crossref PubMed Scopus (112) Google Scholar), other PKC C1 domains do not distinguish among anionic phospholipids (8Ananthanarayanan B. Stahelin R.V. Digman M.A. Cho W. J. Biol. Chem. 2003; 278: 46886-46894Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 9Stahelin R.V. Digman M.A. Medkova M. Ananthanarayanan B. Rafter J.D. Melowic H.R. Cho W. J. Biol. Chem. 2004; 279: 29501-29512Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). The C2 domain (∼130 residues) is an eight-stranded β sandwich protein that shows higher structural and functional diversity than the C1 domain (6Cho W. Stahelin R.V. Annu. Rev. Biophys. Biomol. Struct. 2005; 34: 119-151Crossref PubMed Scopus (486) Google Scholar, 18Nalefski E.A. Falke J.J. Protein Sci. 1996; 5: 2375-2390Crossref PubMed Scopus (689) Google Scholar, 19Rizo J. Sudhof T.C. J. Biol. Chem. 1998; 273: 15879-15882Abstract Full Text Full Text PDF PubMed Scopus (708) Google Scholar, 20Cho W. J. Biol. Chem. 2001; 276: 32407-32410Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). The C2 domains of cPKCs bind multiple Ca2+ ions in their Ca2+-binding loops and thereby play a key role in Ca2+-dependent membrane binding of cPKCs (6Cho W. Stahelin R.V. Annu. Rev. Biophys. Biomol. Struct. 2005; 34: 119-151Crossref PubMed Scopus (486) Google Scholar, 18Nalefski E.A. Falke J.J. Protein Sci. 1996; 5: 2375-2390Crossref PubMed Scopus (689) Google Scholar, 19Rizo J. Sudhof T.C. J. Biol. Chem. 1998; 273: 15879-15882Abstract Full Text Full Text PDF PubMed Scopus (708) Google Scholar, 20Cho W. J. Biol. Chem. 2001; 276: 32407-32410Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). The C2 domains of cPKCs have been shown to have definite PS specificity. In particular, recent structural (21Verdaguer N. Corbalan-Garcia S. Ochoa W.F. Fita I. Gomez-Fernandez J.C. EMBO J. 1999; 18: 6329-6338Crossref PubMed Scopus (287) Google Scholar) and mutational (22Conesa-Zamora P. Lopez-Andreo M.J. Gomez-Fernandez J.C. Corbalan-Garcia S. Biochemistry. 2001; 40: 13898-13905Crossref PubMed Scopus (55) Google Scholar, 23Stahelin R.V. Rafter J.D. Das S. Cho W. J. Biol. Chem. 2003; 278: 12452-12460Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) studies have demonstrated that the PS headgroup directly interacts with Ca2+ and including Asn189 several residues, in the Ca2+ binding loops of PKCα (see Fig. 1). All nPKCs contain a Ca2+-independent C2 domain in the amino terminus, but its role in membrane binding and activation of nPKCs is not fully understood. Membrane binding and activation of cPKCs and nPKCs require DAG and PKC phosphorylation (and Ca2+ for cPKCs) and may also depend on binding to other lipids and proteins (1Parekh D.B. Ziegler W. Parker P.J. EMBO J. 2000; 19: 496-503Crossref PubMed Scopus (510) Google Scholar, 2Newton A.C. Chem. Rev. 2001; 101: 2353-2364Crossref PubMed Scopus (835) Google Scholar, 3Shirai Y. Saito N. J. Biochem. (Tokyo). 2002; 132: 663-668Crossref PubMed Scopus (128) Google Scholar). Earlier in vitro membrane binding and activity assays indicated that PS is an essential activator of PKCs (24Orr J.W. Newton A.C. Biochemistry. 1992; 31: 4667-4673Crossref PubMed Scopus (107) Google Scholar, 25Newton A.C. Keranen L.M. Biochemistry. 1994; 33: 6651-6658Crossref PubMed Scopus (121) Google Scholar). More recent studies have shown, however, that PS dependence (or selectivity) in membrane binding and activation varies among PKC isoforms: e.g. PKCα and PKCδ show high PS selectivity, whereas PKCγ and PKCϵ exhibit little PS selectivity (8Ananthanarayanan B. Stahelin R.V. Digman M.A. Cho W. J. Biol. Chem. 2003; 278: 46886-46894Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 9Stahelin R.V. Digman M.A. Medkova M. Ananthanarayanan B. Rafter J.D. Melowic H.R. Cho W. J. Biol. Chem. 2004; 279: 29501-29512Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 26Medkova M. Cho W. Biochemistry. 1998; 37: 4892-4900Crossref PubMed Scopus (78) Google Scholar, 27Stahelin R.V. Digman M.A. Medkova M. Ananthanarayanan B. Melowic H.R. Rafter J.D. Cho W. J. Biol. Chem. 2005; 280: 19784-19793Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Thus, it appears that PS selectivity of the intact PKC molecules is a complex phenomenon that does not simply reflect the PS specificity of isolated membrane targeting domains. Extensive biochemical studies have been performed to elucidate the mechanisms by which cPKC and nPKC isoforms bind to the membrane and get activated. Our recent studies on PKCα (28Medkova M. Cho W. J. Biol. Chem. 1999; 274: 19852-19861Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 29Bittova L. Stahelin R.V. Cho W. J. Biol. Chem. 2001; 276: 4218-4226Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), PKCγ (8Ananthanarayanan B. Stahelin R.V. Digman M.A. Cho W. J. Biol. Chem. 2003; 278: 46886-46894Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), PKCδ (9Stahelin R.V. Digman M.A. Medkova M. Ananthanarayanan B. Rafter J.D. Melowic H.R. Cho W. J. Biol. Chem. 2004; 279: 29501-29512Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), and PKCϵ (27Stahelin R.V. Digman M.A. Medkova M. Ananthanarayanan B. Melowic H.R. Rafter J.D. Cho W. J. Biol. Chem. 2005; 280: 19784-19793Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) have revealed that individual PKC isoforms follow distinct membrane-binding mechanisms and show different PS selectivity due in part to the differences in the accessibility and DAG affinity of their C1 domains. In the case of PKCα, initial Ca2+- and PS-dependent membrane binding by the C2 domain is followed by the subsequent membrane penetration and DAG binding by the C1A domain, which eventually leads to enzyme activation (26Medkova M. Cho W. Biochemistry. 1998; 37: 4892-4900Crossref PubMed Scopus (78) Google Scholar, 28Medkova M. Cho W. J. Biol. Chem. 1999; 274: 19852-19861Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 29Bittova L. Stahelin R.V. Cho W. J. Biol. Chem. 2001; 276: 4218-4226Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 30Medkova M. Cho W. J. Biol. Chem. 1998; 273: 17544-17552Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). In the resting state, the C1A domain is not available for DAG binding, because it is tethered to the C2 domain (or other parts of the molecule) via conserved Asp55 in the C1A domain. It has been proposed that PS specifically unleashes this interdomain tethering, because its carboxyl group effectively replaces that of Asp55; hence the PS selectivity (29Bittova L. Stahelin R.V. Cho W. J. Biol. Chem. 2001; 276: 4218-4226Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) (see Fig. 6). The C1B domain is not directly involved in membrane binding and activation because of its extremely low DAG affinity (8Ananthanarayanan B. Stahelin R.V. Digman M.A. Cho W. J. Biol. Chem. 2003; 278: 46886-46894Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). In contrast to PKCα, PKCγ has high membrane binding and enzymatic activities without PS, because its C1A and C1B domains are readily accessible and have comparably high DAG affinities (8Ananthanarayanan B. Stahelin R.V. Digman M.A. Cho W. J. Biol. Chem. 2003; 278: 46886-46894Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Among nPKCs, PKCδ behaves like PKCα, and PKCϵ is similar to PKCγ with respect to PS selectivity and the DAG affinity and accessibility of their C1A and C1B domains (9Stahelin R.V. Digman M.A. Medkova M. Ananthanarayanan B. Rafter J.D. Melowic H.R. Cho W. J. Biol. Chem. 2004; 279: 29501-29512Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 27Stahelin R.V. Digman M.A. Medkova M. Ananthanarayanan B. Melowic H.R. Rafter J.D. Cho W. J. Biol. Chem. 2005; 280: 19784-19793Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The notion of the intramolecular tethering (or masking) of C1 domains has been also supported by reports from other laboratories (31Oancea E. Meyer T. Cell. 1998; 95: 307-318Abstract Full Text Full Text PDF PubMed Scopus (547) Google Scholar, 32Conesa-Zamora P. Gomez-Fernandez J.C. Corbalan-Garcia S. Biochim. Biophys. Acta. 2000; 1487: 246-254Crossref PubMed Scopus (30) Google Scholar, 33Slater S.J. Seiz J.L. Cook A.C. Buzas C.J. Malinowski S.A. Kershner J.L. Stagliano B.A. Stubbs C.D. J. Biol. Chem. 2002; 277: 15277-15285Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 34Ochoa W.F. Corbalan-Garcia S. Eritja R. Rodriguez-Alfaro J.A. Gomez-Fernandez J.C. Fita I. Verdaguer N. J. Mol. Biol. 2002; 320: 277-291Crossref PubMed Scopus (69) Google Scholar, 35Rodriguez-Alfaro J.A. Gomez-Fernandez J.C. Corbalan-Garcia S. J. Mol. Biol. 2004; 335: 1117-1129Crossref PubMed Scopus (37) Google Scholar). For example, Conesa-Zamora (32Conesa-Zamora P. Gomez-Fernandez J.C. Corbalan-Garcia S. Biochim. Biophys. Acta. 2000; 1487: 246-254Crossref PubMed Scopus (30) Google Scholar) reported that the C2 domain of PKCα is directly involved in the DAG-dependent binding of the C1 domain to membrane. Stubbs and coworkers (33Slater S.J. Seiz J.L. Cook A.C. Buzas C.J. Malinowski S.A. Kershner J.L. Stagliano B.A. Stubbs C.D. J. Biol. Chem. 2002; 277: 15277-15285Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) subsequently showed that the C2 domain of PKCα was able to associate with the C1 domain. More recently, it has been reported that a cluster of Lys residues on the concave surface of the C2 domain interact with Asp55 (34Ochoa W.F. Corbalan-Garcia S. Eritja R. Rodriguez-Alfaro J.A. Gomez-Fernandez J.C. Fita I. Verdaguer N. J. Mol. Biol. 2002; 320: 277-291Crossref PubMed Scopus (69) Google Scholar, 35Rodriguez-Alfaro J.A. Gomez-Fernandez J.C. Corbalan-Garcia S. J. Mol. Biol. 2004; 335: 1117-1129Crossref PubMed Scopus (37) Google Scholar). Despite this mounting evidence, little is known about the nature of C1-C2 interdomain interactions and the residues involved in these interactions. In the present study, we performed computational modeling, in vitro membrane binding and activity measurements, and cellular membrane translocation measurements of PKCα and mutants to identify the residues directly involved in the C1A-C2 interdomain interactions in PKCα. The results not only provide new structural insight into PKCα C1A-C2 interdomain interactions but also lend further support on our proposed mechanism of PKCα activation. Materials—1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoinositol (POPI), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), 1,2-dioctanoyl-sn-3-glycerol (DiC8), and 1,2-dioleoyl-sn-3-glycerol (DiC18) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL) and used without further purification. A fluorescent DAG analog, 1-octanoyl-2-(8-pyrenenyloctanoyl)-sn-glycerol (OPG) was synthesized as described previously (27Stahelin R.V. Digman M.A. Medkova M. Ananthanarayanan B. Melowic H.R. Rafter J.D. Cho W. J. Biol. Chem. 2005; 280: 19784-19793Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Phorbol 12,13-dibutyrate, phorbol 12-myristate-13-acetate, cholesterol, fatty acid-free bovine serum albumin, Triton X-100, ATP, octyl glucoside, and CHAPS were from Sigma. Phospholipids concentrations were determined by a modified Bartlett analysis (36Kates M. Techniques of Lipidology. Elsevier, Amsterdam1986: 114-115Google Scholar). [γ-32P]ATP (3 Ci/mmol) was from Amersham Biosciences. Restriction endonucleases and enzymes for molecular biology were obtained from New England Biolabs (Beverly, MA). Pioneer L1 sensor chip was from Biacore AB(Piscataway, NJ). Dulbecco's modified Eagle's medium (DMEM) and Lipofectamine™ were from Invitrogen. Human embryonic kidney(HEK) 293 cell line, Zeocin, and ponasterone A were from Invitrogen (San Diego, CA). Molecular Modeling of C1-C2 Domain Interactions—The homology model of the C1A domain of PKCα was built (see Fig. 1) with the Nest (37Petrey D. Xiang Z. Tang C.L. Xie L. Gimpelev M. Mitros T. Soto C.S. Goldsmith-Fischman S. Kernytsky A. Schlessinger A. Koh I.Y. Alexov E. Honig B. Proteins. 2003; 53: 430-435Crossref PubMed Scopus (272) Google Scholar) and Modeler (38Eswar N. John B. Mirkovic N. Fiser A. Ilyin V.A. Pieper U. Stuart A.C. Marti-Renom M.A. Madhusudhan M.S. Yerkovich B. Sali A. Nucleic Acids Res. 2003; 31: 3375-3380Crossref PubMed Scopus (383) Google Scholar) programs using the crystal structure of PKCδ C1B (11Zhang G. Kazanietz M.G. Blumberg P.M. Hurley J.H. Cell. 1995; 81: 917-924Abstract Full Text PDF PubMed Scopus (598) Google Scholar) (PDB id: 1PTR) as the template and the alignment obtained with BLAST as a guide. The sequence identity between these two C1 domains is very high (42%). Zinc was docked into the homology model based on structure superposition with the C1B structure. The quality of the model is good based on the results from Verify3D (39Luthy R. Bowie J.U. Eisenberg D. Nature. 1992; 356: 83-85Crossref PubMed Scopus (2581) Google Scholar) (data not shown), where Verify 3D scores are plotted as a function of residue number in the model; the scores are consistently positive and high (>0.15) indicative of a good structural fit for the C1A sequence. The protein docking programs Global Range Molecular Matching (GRAMM) (40Vakser I.A. Jiang S. Methods Enzymol. 2002; 343: 313-328Crossref PubMed Scopus (22) Google Scholar) and DOCK4 (41Kuntz I.D. Science. 1992; 257: 1078-1082Crossref PubMed Scopus (893) Google Scholar) were used to dock the C1A homology model and the crystal structure of the PKCα C2 domain (21Verdaguer N. Corbalan-Garcia S. Ochoa W.F. Fita I. Gomez-Fernandez J.C. EMBO J. 1999; 18: 6329-6338Crossref PubMed Scopus (287) Google Scholar) (PDB id: 1dsy). GRAMM searches for geometric and hydrophobic complementarity between two proteins, whereas DOCK evaluates geometric and, more generally, chemical complementarity, although the user is required to define, as an input to the program, the docking surface on one protein, which is designated the "receptor" (here, the C2 domain). The parameters used for GRAMM are as follows: the matching mode was generic; the grid step was 6.8 Å; the repulsion was 6.5; the attraction double range was 0.0; the potential range type was grid-step; the projection was gray; the number of matches to output was 1000; and the angle of rotations was 20°. For DOCK4, we followed the recommended protocol, which defines how the system and information describing it is represented on the computational grid. Following docking, the results were corroborated by computational analysis of the predicted docked interface. Expression Vector Construction and Mutagenesis—Baculovirus transfer vectors encoding the cDNA of PKCα with appropriate C1 or C2 domain mutations were generated by the overlap extension PCR (42Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene. 1989; 77: 51-59Crossref PubMed Scopus (6832) Google Scholar) using pVL1392-PKC-α plasmid as a template (30Medkova M. Cho W. J. Biol. Chem. 1998; 273: 17544-17552Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). The PCR product was purified on an agarose gel, and the PKCα gene was digested with NotI and EcoRI and subcloned into the pVL1392 vector digested with the same restriction enzymes. The mutagenesis was verified by DNA sequencing. Mammalian expression vectors for PKCα and mutants with carboxyl-terminal enhanced green fluorescence protein (EGFP) tags were generated by subcloning the respective genes into the pIND (Invitrogen) with the spacer sequence, GGNSGG, as described previously (8Ananthanarayanan B. Stahelin R.V. Digman M.A. Cho W. J. Biol. Chem. 2003; 278: 46886-46894Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Protein Expression and Purification—Full-length PKCα and mutants were expressed in baculovirus-infected Sf9 cells. The transfection of Sf9 cells with pVL1392-PKCα constructs was performed using a BaculoGold™ transfection kit from BD Pharmingen. The plasmid DNA for transfection was prepared by using an EndoFree Plasmid Maxi kit (Qiagen) to avoid potential endotoxin contamination. Cells were incubated for 4 days at 27 °C, and the supernatant was collected and used to infect more cells for the amplification of virus. After three cycles of amplification, high-titer virus stock solution was obtained. Sf9 cells were maintained as monolayer cultures in TMN-FH medium (Invitrogen) containing 10% fetal bovine serum (Invitrogen). For protein expression, cells were grown to 2 × 106 cells/ml in 350-ml suspension cultures and infected with the multiplicity of infection of 10. The cells were then incubated for 60 h at 27 °C. PKCα wild type and mutants were purified as described previously (30Medkova M. Cho W. J. Biol. Chem. 1998; 273: 17544-17552Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Determination of PKC Activity—Activity of PKCα was assayed by measuring the initial rate of [32P]phosphate incorporation [γ-32 from P]ATP (50 μm, 0.6 μCi/tube) into the histone III-SS (400 μg/ml, Sigma). The reaction mixture contained large unilamellar vesicles (0.2 mm of total lipid concentration), 0.16 m KCl, 0.1 mm CaCl2, and 5 mm MgCl2 in 50 ml of 20 mm HEPES, pH 7.4. For experiments with different [Ca2+], the free calcium concentration was adjusted using the mixture of EGTA and CaCl2 according to the method of Bers (43Bers D.M. Am. J. Physiol. 1982; 242: C404-C408Crossref PubMed Google Scholar). Reactions were started by adding 50 mm MgCl2 to the mixture, incubating the mixture for 10 min at room temperature, and quenching by the addition of 50 μl of 5% phosphoric acid. 75 μl of quenched reaction mixtures were spotted on P-81 ion-exchange paper, washed 4 times with a 5% solution of phosphoric acid, followed by 1 wash in 95% ethanol. Papers were transferred into scintillation vials containing 4 ml of scintillation fluid (Fisher Scientific), and radioactivity was measured by liquid scintillation counting. Monolayer Measurements—Surface pressure (π) of solution in a circular Teflon trough (4-cm diameter × 1-cm deep) was measured using a Wilhelmy plate attached to a computer-controlled Cahn electrobalance (Model C-32) as described previously (30Medkova M. Cho W. J. Biol. Chem. 1998; 273: 17544-17552Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). 5-10 μl of phospholipid solution in ethanol/hexane (1:9 (v/v)) was spread onto 10 ml of subphase (20 mm HEPES, pH 7.4, containing 0.16 m KCl and either 0.1 mm EGTA or 0.1 mm CaCl2) to form a monolayer with a given initial surface pressure (π0). Once the surface pressure reading of monolayer had been stabilized (after ∼5 min), the protein solution (typically 40 μl) was injected into the subphase through a small hole drilled at an angle through the wall of the trough, and the change in surface pressure (Δπ) was measured as a function of time. Typically, the Δπ value reached a maximum after 20 min. The maximal Δπ value at a given π0 depended on the protein concentration and reached a saturation value (i.e. [PKCα] ≥ 1.5 μg/ml). Protein concentrations in the subphase were therefore maintained above such values to ensure that the observed Δπ represented a maximal value. The critical surface pressure (πc) was determined by extrapolating the Δπ versus π0 plot to the x-axis (44Cho W. Bittova L. Stahelin R.V. Anal. Biochem. 2001; 296: 153-161Crossref PubMed Scopus (114) Google Scholar). Surface Plasmon Resonance Analysis—Kinetics of vesicle-PKC binding was determined by the SPR analysis using a BIAcore X biosensor system (Biacore AB) and the L1 chip as described previously (29Bittova L. Stahelin R.V. Cho W. J. Biol. Chem. 2001; 276: 4218-4226Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 45Stahelin R.V. Cho W. Biochemistry. 2001; 40: 4672-4678Crossref PubMed Scopus (145) Google Scholar). The first flow cell was used as a control cell and was coated with 5400 resonance units of POPC. The second flow cell contained the surface coated with vesicles with varying lipid compositions (e.g. POPC/POPS/DiC18 = 79:20:1) at 5400 resonance units. After lipid coating, 30 μ l of 50 mm NaOH was injected at 100 μl/min three times to wash out loosely bound lipids. Typically, no further decrease in SPR signal was observed after one wash cycle. After coating, the drift in signal was allowed to stabilize below 0.3 resonance unit/min before any binding measurements, which were performed at 25 °C and at a flow rate of 30 μl/min. 90 μl of protein sample was injected for an association time of 3 min, and the dissociation was then monitored for 10 min in running buffer. After each measurement, the lipid surface was typically regenerated with a 10-μl pulse of 50 mm NaOH. The regeneration solution was passed over the immobilized vesicle surface until the SPR signal reached the initial background value before protein injection. For data acquisition, five or more different concentrations (typically within a 10-fold range above or below the Kd) of each enzyme were used, and data sets were repeated three or more times. When needed, the entire lipid surface was removed with a 5-min injection of 40 mm CHAPS followed by a 5-min injection of 40 mm octyl glucoside at 5 μl/min, and the sensor chip was recoated for the next set of measurements. All data were analyzed using BIAevaluation 3.0 software (Biacore) to determine the rate constants of association (ka) and dissociation (kd) as described prev