Regulation of PKCα Activity by C1-C2 Domain Interactions
2002; Elsevier BV; Volume: 277; Issue: 18 Linguagem: Inglês
10.1074/jbc.m112207200
ISSN1083-351X
AutoresSimon J. Slater, Jodie L. Seiz, Anthony Cook, Christopher J. Buzas, Steve A. Malinowski, Jennifer L. Kershner, Brigid A. Stagliano, Christopher D. Stubbs,
Tópico(s)Pharmacogenetics and Drug Metabolism
ResumoIn this study, the role of interdomain interactions involving the C1 and C2 domains in the mechanism of activation of PKC was investigated. Using an in vitro assay containing only purified recombinant proteins and the phorbol ester, 4β-12-O-tetradecanoylphorbol-13-acetate (TPA), but lacking lipids, it was found that PKCα bound specifically, and with high affinity, to a αC1A-C1B fusion protein of the same isozyme. The αC1A-C1B domain also potently activated the isozyme in a phorbol ester- and diacylglycerol-dependent manner. The level of this activity was comparable with that resulting from membrane association induced under maximally activating conditions. Furthermore, it was found that αC1A-C1B bound to a peptide containing the C2 domain of PKCα. The αC1A-C1B domain also activated conventional PKCβI, -βII, and -γ isoforms, but not novel PKCδ or -ɛ. PKCδ and -ɛ were each activated by their own C1 domains, whereas PKCα, -βI, -βII, or -γ activities were unaffected by the C1 domain of PKCδ and only slightly activated by that of PKCɛ. PKCζ activity was unaffected by its own C1 domain and those of the other PKC isozymes. Based on these findings, it is proposed that the activating conformational change in PKCα results from the dissociation ofintra-molecular interactions between the αC1A-C1B domain and the C2 domain. Furthermore, it is shown that PKCα forms dimers via inter-molecular interactions between the C1 and C2 domains of two neighboring molecules. These mechanisms may also apply for the activation of the other conventional and novel PKC isozymes. In this study, the role of interdomain interactions involving the C1 and C2 domains in the mechanism of activation of PKC was investigated. Using an in vitro assay containing only purified recombinant proteins and the phorbol ester, 4β-12-O-tetradecanoylphorbol-13-acetate (TPA), but lacking lipids, it was found that PKCα bound specifically, and with high affinity, to a αC1A-C1B fusion protein of the same isozyme. The αC1A-C1B domain also potently activated the isozyme in a phorbol ester- and diacylglycerol-dependent manner. The level of this activity was comparable with that resulting from membrane association induced under maximally activating conditions. Furthermore, it was found that αC1A-C1B bound to a peptide containing the C2 domain of PKCα. The αC1A-C1B domain also activated conventional PKCβI, -βII, and -γ isoforms, but not novel PKCδ or -ɛ. PKCδ and -ɛ were each activated by their own C1 domains, whereas PKCα, -βI, -βII, or -γ activities were unaffected by the C1 domain of PKCδ and only slightly activated by that of PKCɛ. PKCζ activity was unaffected by its own C1 domain and those of the other PKC isozymes. Based on these findings, it is proposed that the activating conformational change in PKCα results from the dissociation ofintra-molecular interactions between the αC1A-C1B domain and the C2 domain. Furthermore, it is shown that PKCα forms dimers via inter-molecular interactions between the C1 and C2 domains of two neighboring molecules. These mechanisms may also apply for the activation of the other conventional and novel PKC isozymes. protein kinase C bovine brain phosphatidylserine 1,2-dioctanoyl-sn-glycerol myelin basic protein peptide substrate peptide substrate based on the PKCɛ pseudosubstrate 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine 4β-12-O-tetradecanoylphorbol-13-acetate glutathione S-transferase surface plasmon resonance The 10 closely related isozymes that constitute the protein kinase C (PKC)1 family of serine/threonine kinases each occupy critical nodes in the complex cellular signal transduction networks that regulate diverse cellular processes, including: secretion, proliferation, differentiation, apoptosis, permeability, migration, and hypertrophy (1Mellor H. Parker P.J. Biochem. J. 1998; 332: 281-292Crossref PubMed Scopus (1357) Google Scholar, 2Nishizuka Y. FASEB J. 1995; 9: 484-496Crossref PubMed Scopus (2362) Google Scholar, 3Jaken S. Curr. Opin. Cell Biol. 1996; 8: 168-173Crossref PubMed Scopus (407) Google Scholar, 4Naruse K. King G.L. Circ. Res. 2000; 86: 1104-1106Crossref PubMed Scopus (75) Google Scholar, 5Clarke H. Marano C.W. Peralta Soler A. Mullin J.M. Adv. Drug Deliv. Rev. 2000; 41: 283-301Crossref PubMed Scopus (83) Google Scholar, 6Musashi M. Ota S. Shiroshita N. Int. J. Hematol. 2000; 72: 12-19PubMed Google Scholar, 7Dempsey E.C. Newton A.C. Mochly-Rosen D. Fields A.P. Reyland M.E. Insel P.A. Messing R.O. Am. J. Physiol. Lung Cell. Mol. Physiol. 2000; 279: L429-L438Crossref PubMed Google Scholar). In common with many signaling proteins, the structure of PKC is modular, consisting of a C-terminal catalytic region containing the active site, and a regulatory region with conserved domains that mediate membrane association and activation. PKC isozymes are classified according to the structural and functional differences in these conserved domains (8Newton A.C. J. Biol. Chem. 1996; 48: 28495-28498Google Scholar, 9Newton A.C. Curr. Opin. Cell Biol. 1997; 9: 161-167Crossref PubMed Scopus (850) Google Scholar). In the case of the "conventional" PKCα, -βI/βII, and -γ isozymes, these include the activator-binding C1 domains, and the Ca2+-binding C2 domain. The C1 domains consist of a tandem C1A and C1B arrangement, each of which can potentially bind the endogenous activator, diacylglycerol and exogenous activators including phorbol esters. The "novel" PKCδ, -ɛ, -η, -θ, and -μ isozymes, contain C2 domains that lack Ca2+ binding ability, while retaining functional C1A and C1B domains. The "atypical" PKCζ, -ι, and -λ regulatory domains also lack a functional C2 domain and contain a single C1 domain that lacks the ability to bind activators, the function of which remains obscure. Each isozyme becomes catalytically competent by undergoing multiple serine/threonine and tyrosine phosphorylations that are either autocatalytic, or catalyzed by "PKC kinases" such as the phosphoinositide-dependent kinase, PDK1 (7Dempsey E.C. Newton A.C. Mochly-Rosen D. Fields A.P. Reyland M.E. Insel P.A. Messing R.O. Am. J. Physiol. Lung Cell. Mol. Physiol. 2000; 279: L429-L438Crossref PubMed Google Scholar, 10Parekh D.B. Ziegler W. Parker P.J. EMBO J. 2000; 19: 496-503Crossref PubMed Scopus (509) Google Scholar, 11Li W. Yu H. Mischak J.C. Wang L.M. Mushinski J.F. Heidaran M.A. Pierce J.H. J. Biol. Chem. 1994; 269: 2349-2352Abstract Full Text PDF PubMed Google Scholar). The mechanism by which the conventional PKC isozymes become membrane associated, and thus activated, involves two sequential steps. The first involves an initial Ca2+- and anionic phospholipid-dependent interaction of the C2 domain with the membrane, which is then followed by binding of diacylglycerol or phorbol esters to the C1 domains (12Newton A.C. Johnson J.E. Biochim. Biophys. Acta. 1998; 1376: 155-172Crossref PubMed Scopus (244) Google Scholar, 13Johnson J.E. Giorgione J. Newton A.C. Biochemistry. 2000; 39: 11360-11369Crossref PubMed Scopus (112) Google Scholar, 14Oancea E. Meyer T. Cell. 1998; 95: 307-318Abstract Full Text Full Text PDF PubMed Scopus (546) Google Scholar, 15Cho W. J. Biol. Chem. 2001; 276: 32407-32410Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Mutagenesis studies have identified a number of critical hydrophobic residues within the C1 domains of PKCδ and PKCα that appear to have distinct roles in ligand binding and membrane association (16Medkova M. Cho W. J. Biol. Chem. 1999; 274: 19852-19861Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 17Wang Q.J. Fang T.W. Nacro K. Marquez V.E. Wang S. Blumberg P.M. J. Biol. Chem. 2001; 276: 19580-19587Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 18Kazanietz M.G. Wang S. Milne G.W. Lewin N.E. Liu H.L. Blumberg P.M. J. Biol. Chem. 1995; 270: 21852-21859Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Based on the x-ray crystallographic structure of the C1B domain of PKCδ, it has been suggested that activator binding to the C1 domain facilitates membrane-insertion by "capping" a hydrophilic groove to form a contiguous hydrophobic surface that can interact with the membrane interior (19Zhang G. Kazanietz M.G. Blumberg P.M. Hurley J.H. Cell. 1995; 81: 917-924Abstract Full Text PDF PubMed Scopus (598) Google Scholar). However, it would appear from our studies that the interaction of the phorbol ester induces a conformational change in the C1 domain, the extent of which is reflected in the activity of the enzyme, implying a more active role for phorbol ester-C1 domain interactions beyond that of presenting a hydrophobic surface to the interior of the membrane (20Ho C. Slater S.J. Stagliano B.A. Stubbs C.D. Biochem. J. 1999; 344: 451-460Crossref PubMed Scopus (18) Google Scholar, 21Ho C. Slater S.J. Stagliano B. Stubbs C.D. Biochemistry. 2001; 40: 10334-10341Crossref PubMed Scopus (27) Google Scholar). The interaction of diacylglycerol with the C1 domains also results in an increased stereo- and regiospecificity of both membrane association and activation for PS (13Johnson J.E. Giorgione J. Newton A.C. Biochemistry. 2000; 39: 11360-11369Crossref PubMed Scopus (112) Google Scholar, 22Newton A.C. Keranen L.M. Biochemistry. 1994; 33: 6651-6658Crossref PubMed Scopus (121) Google Scholar, 23Orr J.W. Newton A.C. Biochemistry. 1992; 31: 4667-4673Crossref PubMed Scopus (107) Google Scholar, 24Bittova L. Stahelin R.V. Cho W. J. Biol. Chem. 2000; 11: 11Google Scholar). The combined interactions of the C1 and C2 domains with the membrane provides the free energy required for structural rearrangements that lead to the dissociation of the N-terminal pseudosubstrate from the active site to allow substrate binding (25House C. Kemp B.E. Science. 1987; 238: 1726-1728Crossref PubMed Scopus (789) Google Scholar, 26Orr J.W. Keranen L.M. Newton A.C. J. Biol. Chem. 1992; 267: 15263-15266Abstract Full Text PDF PubMed Google Scholar, 27Makowske M. Rosen O.M. J. Biol. Chem. 1989; 264: 16155-16159Abstract Full Text PDF PubMed Google Scholar). This process is thought to be further facilitated by a weak interaction of the released pseudosubstrate with anionic head groups at the membrane surface (28Mosior M. McLaughlin S. Biophys. J. 1991; 60: 149-159Abstract Full Text PDF PubMed Scopus (110) Google Scholar). There is increasing evidence supporting the notion that the existence of two C1 domains affords a complex modulatory role in the regulation of PKC activity. Phorbol esters have been shown to interact with both of the C1A and C1B domains, with distinct low and high affinities (29Slater S.J. Kelly M.B. Ho J.D. Larkin C. Mazurek A. Taddeo F.J. Yeager M.D. Stubbs C.D. J. Biol. Chem. 1997; 272: 6167-6173Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 30Slater S.J. Kelly M.B. Taddeo F.J. Rubin E. Stubbs C.D. J. Biol. Chem. 1994; 269: 17160-17165Abstract Full Text PDF PubMed Google Scholar, 31Ho S.J. Slater 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, 32Slater S.J. Taddeo F.J. Mazurek A. Stagliano B.A. Milano S.K. Ho M.B. Kelly C. Stubbs C.D. J. Biol. Chem. 1998; 273: 23160-23168Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 33Janoshazi A. de Barry J. Biochemistry. 1999; 38: 13316-13327Crossref PubMed Scopus (9) Google Scholar). Furthermore, we have shown that diacylglycerol inhibits the low affinity phorbol ester interaction while enhancing high affinity phorbol ester binding, indicating that the diacylglycerol has a higher affinity for the low affinity phorbol ester-binding site than does phorbol ester itself (30Slater S.J. Kelly M.B. Taddeo F.J. Rubin E. Stubbs C.D. J. Biol. Chem. 1994; 269: 17160-17165Abstract Full Text PDF PubMed Google Scholar,31Ho S.J. Slater 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). Additional support for the non-equivalence of the interaction of diacylglycerol, phorbol esters, and also other activators with the two C1 domains has been provided by other studies that have observed non-equivalent roles of the domains with respect to membrane association and activation (34Shieh H.L. Hansen H. Zhu J. Riedel H. Mol. Carcinogen. 1995; 12: 166-176Crossref PubMed Scopus (33) Google Scholar, 35Oancea E. Teruel M.N. Quest A.F. Meyer T. J. Cell Biol. 1998; 140: 485-498Crossref PubMed Scopus (291) Google Scholar, 36Bogi K. Lorenzo P.S. Szallasi Z. Acs P. Wagner G.S. Blumberg P.M. Cancer Res. 1998; 58: 1423-1428PubMed Google Scholar, 37Bogi K. Lorenzo P.S. Acs P. Szallasi Z. Wagner G.S. Blumberg P.M. FEBS Lett. 1999; 456: 27-30Crossref PubMed Scopus (31) Google Scholar). Studies from this laboratory showed that the increased level of phorbol ester binding that results from interaction of diacylglycerol with the low affinity phorbol ester-binding site on conventional PKC isozymes corresponded to an elevated level of enzyme activity that was greater than that induced by either phorbol ester or diacylglycerol alone (30Slater S.J. Kelly M.B. Taddeo F.J. Rubin E. Stubbs C.D. J. Biol. Chem. 1994; 269: 17160-17165Abstract Full Text PDF PubMed Google Scholar, 31Ho S.J. Slater 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, 38Slater S.J. Milano S.K. Stagliano B.A. Ho K.J. Gergich C. Mazurek A. Taddeo F.J. Kelly M.B. Yeager M.D. Stubbs C.D. Biochemistry. 1999; 38: 3804-3815Crossref PubMed Scopus (25) Google Scholar). The above, and recent data suggesting that the C1A of PKCα is the diacylglycerol-binding site, while the C1B domain binds phorbol ester (16Medkova M. Cho W. J. Biol. Chem. 1999; 274: 19852-19861Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 24Bittova L. Stahelin R.V. Cho W. J. Biol. Chem. 2000; 11: 11Google Scholar), are compatible with the C1A and C1B domains containing the low and high affinity phorbol ester-binding sites, respectively. Inter-domain interactions are important in PKC regulation, although detailed features of these interactions remain obscure. The conformational change that leads to the displacement of the pseudosubstrate of membrane-associated PKC isozymes involves pronounced rearrangements of the individual domains that constitute the enzyme molecule. In a recent study, it was shown that the rate of translocation of GFP-tagged PKCγ in RBL cells was markedly slower when compared with that of a truncation mutant lacking the N-terminal V1 region (14Oancea E. Meyer T. Cell. 1998; 95: 307-318Abstract Full Text Full Text PDF PubMed Scopus (546) Google Scholar). Based on this, it was suggested that membrane association and activation resulting from binding of diacylglycerol to the C1 domains of this isozyme first requires the release of a "V1 clamp," which holds the C1 domain between the catalytic and V1 region, due to an interaction of the pseudosubstrate with the active site. In another study, the mutation of a single aspartate (Asp55) residue in the C1A domain of PKCα was shown to result in a marked reduction in both the PS and Ca2+ concentration requirements for membrane association and activation, implicating a tethering of the C1A domain to a neighboring region within the PKCα molecule (15Cho W. J. Biol. Chem. 2001; 276: 32407-32410Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 24Bittova L. Stahelin R.V. Cho W. J. Biol. Chem. 2000; 11: 11Google Scholar). It was suggested that such an interaction might retain the isozyme in an inactive conformation by preventing the penetration of the C1A domain into the membrane and thus its interaction with diacylglycerol (24Bittova L. Stahelin R.V. Cho W. J. Biol. Chem. 2000; 11: 11Google Scholar). The aim of the present study was to determine the role of interdomain interactions in the mechanism of activation of PKCα. Using in vitro activity and binding assays, it was found that PKCα engaged in a phorbol ester-dependent, high affinity and specific interaction with a fusion protein that contained its own C1A and C1B domains (αC1A-C1B). The level of activity induced by interaction with the αC1A-C1B domain was found to be comparable with that resulting from membrane association induced under maximally activating conditions. Also, the αC1A-C1B domain interacted with a fusion protein containing the C2 domain of PKCα. Taken together, these findings provide evidence for the existence of an interdomain interaction between the C1 and C2 domains, the dissociation of which is a rate-determining step in the mechanism of activation of PKCα. Adenosine 5′-triphosphate (ATP) was from Roche Molecular Biochemicals (Indianapolis, IN). [γ-32P]ATP was from PerkinElmer Life Science (Boston, MA). 1-Palmitoyl-2-oleoylphosphatidylcholine (POPC) and bovine brain phosphatidylserine (BPS) were each from Avanti Polar Lipids, Inc. (Alabaster, AL). Peptide substrates were custom synthesized by the Kimmel Cancer Center peptide synthesis facility of Thomas Jefferson University. 4β-12-O-Tetradecanoylphorbol-13-acetate (TPA) and the soluble diacylglycerol, 1,2-dioctanoyl-sn-glycerol (DiC8), were each obtained from Sigma. A mixed preparation of the catalytic subunits of the conventional PKC isozymes was purchased from Calbiochem (La Jolla, Ca). All other chemicals were of analytical grade and obtained from Fisher Scientific (Pittsburgh, PA). Recombinant PKCα, -βI, -βII, -γ, and -ɛ (rat brain) were prepared using the baculovirusSpodoptera frugiperda (Sf9) insect cell expression system as originally described (39Stabel S. Schaap D. Parker P.J. Methods Enzymol. 1991; 200: 670-673Crossref PubMed Scopus (44) Google Scholar), with modifications (40Taddeo F.J. Cloning, Expression, and Purification of Protein Kinase C: A Comparative Study of the Modes of Activation of Protein Kinase CPh.D. Thesis. Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, PA1998Google Scholar). Purification procedures were as previously described (30Slater S.J. Kelly M.B. Taddeo F.J. Rubin E. Stubbs C.D. J. Biol. Chem. 1994; 269: 17160-17165Abstract Full Text PDF PubMed Google Scholar, 40Taddeo F.J. Cloning, Expression, and Purification of Protein Kinase C: A Comparative Study of the Modes of Activation of Protein Kinase CPh.D. Thesis. Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, PA1998Google Scholar). Baculovirus encoding the PKCα mutant, D55A, in which the aspartate 55 of the C1A domain was mutated to an alanine (24Bittova L. Stahelin R.V. Cho W. J. Biol. Chem. 2000; 11: 11Google Scholar), was a kind gift from Dr. Wonhwa Cho, and was purified using the same procedure as that used for wild-type PKCα (30Slater S.J. Kelly M.B. Taddeo F.J. Rubin E. Stubbs C.D. J. Biol. Chem. 1994; 269: 17160-17165Abstract Full Text PDF PubMed Google Scholar, 40Taddeo F.J. Cloning, Expression, and Purification of Protein Kinase C: A Comparative Study of the Modes of Activation of Protein Kinase CPh.D. Thesis. Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, PA1998Google Scholar). The isoforms PKCδ, PKCɛ, and PKCζ were overexpressed in Sf9 cells as fusion proteins containing a (His)6 attached to the C terminus (40Taddeo F.J. Cloning, Expression, and Purification of Protein Kinase C: A Comparative Study of the Modes of Activation of Protein Kinase CPh.D. Thesis. Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, PA1998Google Scholar) and were purified as described (32Slater S.J. Taddeo F.J. Mazurek A. Stagliano B.A. Milano S.K. Ho M.B. Kelly C. Stubbs C.D. J. Biol. Chem. 1998; 273: 23160-23168Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 40Taddeo F.J. Cloning, Expression, and Purification of Protein Kinase C: A Comparative Study of the Modes of Activation of Protein Kinase CPh.D. Thesis. Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, PA1998Google Scholar). Fusion proteins containing fragments of PKCα encompassing the C1A, C1B, and C2 (αC1A-C1B-C2), the C1A and C1B (αC1A-C1B), the separate C1A (αC1A) and C1B (αC1B) domains, and also the C1A and C1B domains of PKCɛ (ɛC1A-C1B), were each prepared as described previously (32Slater S.J. Taddeo F.J. Mazurek A. Stagliano B.A. Milano S.K. Ho M.B. Kelly C. Stubbs C.D. J. Biol. Chem. 1998; 273: 23160-23168Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 40Taddeo F.J. Cloning, Expression, and Purification of Protein Kinase C: A Comparative Study of the Modes of Activation of Protein Kinase CPh.D. Thesis. Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, PA1998Google Scholar) (and see Fig. 1). To provide structural stability, solubility, and to aid purification, fusions peptides were tagged with glutathione S-transferase (GST) at the N terminus and with (His)6 at the C terminus. An expression vector containing the GST-tagged C1A and C1B domains of PKCδ (δC1A-C1B) was a kind gift from Dr. Peter M. Blumberg. The isolation and purification of each tagged protein was performed previously described (32Slater S.J. Taddeo F.J. Mazurek A. Stagliano B.A. Milano S.K. Ho M.B. Kelly C. Stubbs C.D. J. Biol. Chem. 1998; 273: 23160-23168Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 40Taddeo F.J. Cloning, Expression, and Purification of Protein Kinase C: A Comparative Study of the Modes of Activation of Protein Kinase CPh.D. Thesis. Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, PA1998Google Scholar). PKC isozyme activities were assayed by measuring the rate of phosphate incorporation into a peptide substrate as previously described (30Slater S.J. Kelly M.B. Taddeo F.J. Rubin E. Stubbs C.D. J. Biol. Chem. 1994; 269: 17160-17165Abstract Full Text PDF PubMed Google Scholar). For the "conventional" PKC isoforms and the their catalytic subunits, a peptide corresponding to the phosphorylation site domain of myelin basic protein (QKRPSQRSKYL, MBP4–14) was used as the substrate, whereas assays of novel PKC and atypical PKCζ activity used a peptide corresponding to the pseudosubstrate region of novel PKCɛ (ɛ-peptide), in which the single alanine residue was replaced by serine (25House C. Kemp B.E. Science. 1987; 238: 1726-1728Crossref PubMed Scopus (789) Google Scholar, 41Saido T.C. Mizuno K. Konno Y. Osada S.I. Ohno S. Suzuki K. Biochemistry. 1992; 31: 482-490Crossref PubMed Scopus (66) Google Scholar, 42Ohno S. Akita Y. Konno Y. Imajoh S. Suzuki K. Cell. 1988; 53: 731-741Abstract Full Text PDF PubMed Scopus (298) Google Scholar). Briefly, the assay (75 μl) consisted of 50 mm Tris-HCl (pH 7.40), 0.1 mm EGTA or CaCl2, 50 μm MBP4–14, or 50 μm ɛ-peptide, TPA (500 nm or as indicated) or varying levels of DiC8, and fusion proteins containing the appropriate PKC domains present at a fixed concentration of 10 nm unless otherwise indicated. Where added, POPC and BPS were present at a total concentration of 150 μm as large unilamellar vesicles, prepared as described previously (43MacDonald R.C. MacDonald R.I. Menco B.P. Takeshita K. Subbarao N.K. Hu L.R. Biochim. Biophys. Acta. 1991; 1061: 297-303Crossref PubMed Scopus (1382) Google Scholar). After thermal equilibration to 30 °C, assays were initiated by the simultaneous addition of the required PKC isoform (0.3 nm) along with 5 mm Mg2+, 15 μm ATP, 0.3 μCi of [γ-32P]ATP (3000 Ci/mmol) and terminated after 30 min with 100 μl of 175 mm phosphoric acid. Following this, 100 μl was transferred to P81 filter papers, which were washed three times in 75 mm phosphoric acid. Phosphorylated peptide was determined by scintillation counting. Binding of C1 domain peptides to PKC isozymes was determined using SPR from measurements of the accompanying increase in refractive index as a function of time using a BiacoreTM2000 (Biacore, Inc., Piscataway, NJ). All measurements were performed at 25 °C. Initially, the αC1A-C1B or ɛC1A-C1B domains were captured through the respective (His)6 tag to the nickel/NTA surface of an NTA sensor chip, prepared according to the manufacturers instructions (Biacore, Inc., Piscataway, NJ), to a level of 75 response units. Solutions containing either PKCα isozymes or fusion peptides at the required concentrations, in the presence or absence of 500 nm TPA were then injected over this surface and the response was measured as a function of time. The surface was regenerated after each injection by two 10-s injections of 100 mm NaOH, followed by a single 10-s injection of 10 mm HCl. After subtraction of the contribution of bulk refractive index changes and nonspecific interactions of PKC isozymes with the nickel-NTA surface, which were typically less than 1% of the total signal, the individual association (ka) and dissociation (kd) rate constants were obtained by global fitting of data to a 1:1 Langmuir binding model using BIAevaluationTM (Biacore, Inc.). These values were then used to calculate the dissociation constant (KD). The values of average squared residual (χ2) obtained were not found to be significantly improved by fitting data to models that assumed bivalent or heterogeneous interactions between PKC isozymes and C1 domain peptides. In a separate control experiment (results not shown), it was found that the contribution of mass transport to the observed values of KD was negligible, based on the observation that these values were independent of flow rate within a range encompassing that used (10 to 50 μl min−1). To investigate the role of domain-domain intramolecular interactions in the mechanism of activation of PKC, the assumption was made that if such interactions occur within the isozyme molecule, and are involved in the activating conformational change, then peptides corresponding to the isolated domains might compete for these interactions and modulate the activity of the isozyme. To address this, various fusion proteins containing regions of the regulatory domain spanning the C1A and C1B domains were prepared, as shown in Fig.1. Binding of these domains to PKC, and the effects on PKC isozyme activities, were determined using assay systems that contained only purified proteins along with the required peptide substrates, cofactors, and activators. By excluding membranes from the assay systems, effects could be unambiguously ascribed to direct protein-protein interactions between these domains and PKC. Based on measurements of SPR, it was found that, in the presence of TPA, PKCα bound to an immobilized fusion peptide containing the isolated αC1A-C1B domain in a reversible, concentration-dependent manner (Fig. 2A). The interaction was phorbol ester-dependent since it was found that the level of binding in the absence of TPA was negligible. The value of KD, calculated from the ratio of the association and dissociation rate constants, indicated a high affinity interaction (Table I). Furthermore, the value of the maximal level of PKCα binding at equilibrium (Rmax) is consistent with a PKCα-αC1A-C1B binding stoichiometry of 1:1, assuming maximal occupancy of ligand-binding sites. PKCα was also found to interact with the ɛC1A-C1B domain (Fig. 2B), although with a markedly reduced affinity, as shown by the ∼500-fold increase in the value ofKD (Table I).Table IKinetic analysis of the interaction of intact PKCα or the αC1A-C1B-C2 peptide with the αC1A-C1B or the ɛC1A-C1B domains using SPRInteractionkakdKDχ2m−1s−1s−1mPKCα → αC1A-C1B(9.15 ± 0.60) × 105(4.74 ± 0.18) × 10−3(5.18 ± 0.54) × 10−91.91PKCα → ɛC1A-C1B(3.06 ± 0.28) × 103(8.23 ± 0.16) × 10−3(2.69 ± 0.29) × 10−62.41αC1A-C1B-C2 → αC1A-C1B(4.30 ± 0.11) × 105(5.31 ± 0.08) × 10−3(12.4 ± 1.2) × 10−90.69αC1A-C1B-C2 → ɛC1A-C1B(2.87 ± 0.80) × 103(1.28 ± 0.02) × 10−2(4.40 ± 1.20) × 10−61.10The αC1A-C1B and ɛC1A-C1B domains were initially immobilized on the surface of a nickel/NTA sensor chip and PKCα, or the αC1A-C1B-C2 peptide, were then injected over these surfaces in the presence of 500 nm TPA. The dissociation constants (KD) were obtained from the ratio of the association (ka) and dissociation (kd) rate constants, derived from global fits of response against time data to a 1:1 Langmuir binding model. Further details are given under "Experimental Procedures" and in the legend to Fig. 2. Open table in a new tab The αC1A-C1B and ɛC1A-C1B domains were initially immobilized on the surface of a nickel/NTA sensor chip and PKCα, or the αC1A-C1B-C2 peptide, were then injected over these surfaces in the presence of 500 nm TPA. The dissociation constants (KD) were obtained from the ratio of the association (ka) and dissociation (kd) rate constants, derived from global fits of response against time data to a 1:1 Langmuir binding model. Further details are given under "Experimental Procedures" and in the legend to Fig. 2. It has been suggested in a previous study that the C1A of PKCα may interact with residues within the C2 domain, and thereby impede the activating conformational change (24Bittova L. Stahelin R.V. Cho W. J. Biol. Chem. 2000; 11: 11Google Scholar). Consistent with this, the results shown in Fig. 2C indicate that in the presence of TPA, the αC1A-C1B domain binds to a fusion protein containing the C2 domain of PKCα (αC1A-C1B-C2). Similar to the interaction of PKCα with the αC1A-C1B, the binding of the αC1A-C1B-C2 peptide to the αC1A-C1B domain was found to be TPA-dependent. Note that in this experiment the αC1A-C1B domain was initially bound to the sensor chip surface through the (His)6 tag to a level corresponding to saturation. Under these conditions, any interactions of the αC1A-C1B with itself were also saturated,
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