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• W2051228751 abstract "The significance of site-specific phosphorylation of cardiac troponin I (TnI) by protein kinase C and protein kinase A in the regulation of Ca2+-stimulated MgATPase of reconstituted actomyosin S-1 was investigated. The TnI mutants used were T144A, S43A/S45A, and S43A/S45A/T144A (in which the identified protein kinase C phosphorylation sites, Thr-144 and Ser-43/Ser-45, were, respectively, substituted by Ala) and S23A/S24A and N32 (in which the protein kinase A phosphorylation sites Ser-23/Ser-24 were either substituted by Ala or deleted). The mutations caused subtle changes in the kinetics of phosphorylation by protein kinase C, and all mutants were maximally phosphorylated to various extents (1.3-2.7 mol of phosphate/mol of protein). Protein kinase C could cross-phosphorylate protein kinase A sites but the reverse essentially could not occur. Compared to wild-type TnI and T144A, unphosphorylated S43A/S45A, S43A/S45A/T144, S23A/S24A, and N32 caused a decreased Ca2+ sensitivity of Ca2+-stimulated MgATPase of reconstituted actomyosin S-1. Phosphorylation by protein kinase C of wild-type and all mutants except S43A/S45A and S43A/S45A/T144A caused marked reductions in both the maximal activity of Ca2+-stimulated MgATPase and apparent affinity of myosin S-1 for reconstituted (regulated) actin. It was further noted that protein kinase C acted in an additive manner with protein kinase A by phosphorylating Ser-23/Ser-24 to bring about a decreased Ca2+ sensitivity of the myofilament.It is suggested that Ser-43/Ser-45 and Ser-23/Ser-24 in cardiac TnI are important for normal Ca2+ sensitivity of the myofilament, and that phosphorylation of Ser-43/Ser-45 and Ser-23/Ser-24 is primarily involved in the protein kinase C regulation of the activity and Ca2+ sensitivity, respectively, of actomyosin S-1 MgATPase. The significance of site-specific phosphorylation of cardiac troponin I (TnI) by protein kinase C and protein kinase A in the regulation of Ca2+-stimulated MgATPase of reconstituted actomyosin S-1 was investigated. The TnI mutants used were T144A, S43A/S45A, and S43A/S45A/T144A (in which the identified protein kinase C phosphorylation sites, Thr-144 and Ser-43/Ser-45, were, respectively, substituted by Ala) and S23A/S24A and N32 (in which the protein kinase A phosphorylation sites Ser-23/Ser-24 were either substituted by Ala or deleted). The mutations caused subtle changes in the kinetics of phosphorylation by protein kinase C, and all mutants were maximally phosphorylated to various extents (1.3-2.7 mol of phosphate/mol of protein). Protein kinase C could cross-phosphorylate protein kinase A sites but the reverse essentially could not occur. Compared to wild-type TnI and T144A, unphosphorylated S43A/S45A, S43A/S45A/T144, S23A/S24A, and N32 caused a decreased Ca2+ sensitivity of Ca2+-stimulated MgATPase of reconstituted actomyosin S-1. Phosphorylation by protein kinase C of wild-type and all mutants except S43A/S45A and S43A/S45A/T144A caused marked reductions in both the maximal activity of Ca2+-stimulated MgATPase and apparent affinity of myosin S-1 for reconstituted (regulated) actin. It was further noted that protein kinase C acted in an additive manner with protein kinase A by phosphorylating Ser-23/Ser-24 to bring about a decreased Ca2+ sensitivity of the myofilament. It is suggested that Ser-43/Ser-45 and Ser-23/Ser-24 in cardiac TnI are important for normal Ca2+ sensitivity of the myofilament, and that phosphorylation of Ser-43/Ser-45 and Ser-23/Ser-24 is primarily involved in the protein kinase C regulation of the activity and Ca2+ sensitivity, respectively, of actomyosin S-1 MgATPase. In cardiac myocytes, the activation of several types of receptors, such as α1-adrenergic(1Brown J.H. Buxton I.L. Brunton L.L. Circ. Res. 1985; 57: 532-537Crossref PubMed Google Scholar, 2Woodcock E.A. White B.S. Smith A.E. McLeod J.K. Circ. Res. 1987; 61: 625-631Crossref PubMed Scopus (63) Google Scholar, 3Otani H. Otani H. Das D.K. Circ. Res. 1988; 62: 8-17Crossref PubMed Scopus (182) Google Scholar, 4Steinberg S.F. Kaplan L.M. Imuoye T. Zhang J.I. Robinson R.B. J. Pharmacol. Exp. Ther. 1989; 250: 1141-1148PubMed Google Scholar, 5Kaku T. Lakatta E.G. Filburn C. Am. J. Physiol. 1991; 260: C635-C642Crossref PubMed Google Scholar), muscarinic(1Brown J.H. Buxton I.L. Brunton L.L. Circ. Res. 1985; 57: 532-537Crossref PubMed Google Scholar, 6Kohl C. Linck B. Schmitz W. Scholz J. Toth M. Br. J. Pharmacol. 1990; 101: 829-834Crossref PubMed Scopus (48) Google Scholar), and purinergic (6Kohl C. Linck B. Schmitz W. Scholz J. Toth M. Br. J. 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Ther. 1989; 251: 578-585PubMed Google Scholar) receptors, stimulates the hydrolysis of membrane phosphoinositides leading to the generation of two classes of second messengers, diacylglycerol and inositol trisphosphate. In many tissues diacylglycerol directly activates both the conventional Ca2+-dependent group of PKC 1The abbreviations used are: PKCprotein kinase CTPA12-O-tetradecanoylphorbal-13-acetateTntroponinTnItroponin IN32cardiac TnI mutant with a deletion of the first 32 amino acid residues from the NH2 terminusTnTtroponin TTnCtroponin CTmtropomyosinMLCmyosin light chainPKAprotein kinase APCRpolymerase chain reactionDTTdithiothreitolET-18-OCH31-O-octadecyl-2-O-methyl glycero-3-phosphateMOPS3-(N-morpholino)-2-hydroxypropanesulfonic acidMBPmyelin basic protein. isozymes (α, βI, βII, and γ) and the novel Ca2+-independent group of PKC isozymes (δ, ∊,η, and θ), whereas inositol trisphosphate, by increasing intracellular Ca2+, indirectly activates Ca2+-dependent PKC isozymes (for a review, see (13Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4230) Google Scholar)). It is worth noting that PKC-ζ and PKC-λ, atypical members of the Ca2+-independent group, are activated by neither diacylglycerol nor Ca2+(13Nishizuka Y. Science. 1992; 258: 607-614Crossref PubMed Scopus (4230) Google Scholar). Several lines of recent evidence indicate involvement of PKC in cardiac function and development (14Puceat M. Brown J.H. Kuo J.F. Protein Kinase C. Oxford University Press, New York1994: 249-268Google Scholar, 15Rybin V.O. Steinberg S.F. Circ. Res. 1994; 74: 299-309Crossref PubMed Scopus (226) Google Scholar) as well as differential expression of the PKC isozymes in cardiac myocytes and tissue(14Puceat M. Brown J.H. Kuo J.F. Protein Kinase C. Oxford University Press, New York1994: 249-268Google Scholar, 15Rybin V.O. Steinberg S.F. Circ. Res. 1994; 74: 299-309Crossref PubMed Scopus (226) Google Scholar, 16Disatnik M.-H. Buraggi G. Mochly-Rosen D. Exp. Cell. Res. 1994; 210: 287-297Crossref PubMed Scopus (313) Google Scholar, 17Bogoyevitch M.A. Parker P.J. Sugden P.H. Circ. Res. 1993; 72: 757-767Crossref PubMed Scopus (255) Google Scholar, 18Puceat M. Hilal-Dandan R. Strulovici B. Brunton L.L. Brown J.H. J. Biol. Chem. 1994; 269: 16938-16944Abstract Full Text PDF PubMed Google Scholar). However, the complex molecular events mediated by PKC (or more precisely, by the individual isozymes) that are responsible for cardiac contractility regulation, for example, remain largely unclear. It has been reported that phenylephrine (α1-adrenergic receptor agonist) elicited transient negative inotropy followed by sustained positive inotropy(3Otani H. Otani H. Das D.K. Circ. Res. 1988; 62: 8-17Crossref PubMed Scopus (182) Google Scholar, 19Gambassi G. Spurgeon H.A. Lakatta E.G. Blank P.S. Capogrossi M.C. Circ. Res. 1992; 71: 870-882Crossref PubMed Scopus (86) Google Scholar, 20Otani H. Uriu T. Hara M. Inoue M. Omori K. Cragoe E.J. Inagaki C. Br. J. Pharmacol. 1990; 100: 207-210Crossref PubMed Scopus (37) Google Scholar, 21Terzic A. Puceat M. Clement O. Scamps F. Vassort G. J. Physiol. 1992; 447: 275-292Crossref PubMed Scopus (131) Google Scholar), endothelin-1 caused monotonic positive inotropy(22Kramer B.K. Smith T.W. Kelly R.A. Circ. Res. 1991; 68: 269-279Crossref PubMed Scopus (264) Google Scholar), whereas dynorphin A (κ-opioid receptor agonist) induced negative inotropy(7Ventura C. Spurgeon H. Lakatta E.G. Guarnieri C. Capogrossi M.C. Circ. Res. 1992; 70: 66-81Crossref PubMed Scopus (189) Google Scholar). All three of these distinct receptor agonists are believed to act, at least in part, through PKC activation. Furthermore, phorbol esters (such as TPA), potent and long-acting PKC activators, produced predominantly negative inotropic effects in various cardiac preparations(23Capogrossi M.C. Kaku T. Filburn C.R. Pelto D.J. Hansford R.G. Spurgeon H.A. Lakatta E.G. Circ. Res. 1990; 66: 1143-1155Crossref PubMed Scopus (136) Google Scholar, 24Leatherman G.F. Kim D. Smith T.W. Am. J. Physiol. 1987; 253: H205-H209Crossref PubMed Google Scholar, 25Yuan S. Sunahara F.A. Sen A.K. Circ. Res. 1987; 61: 372-378Crossref PubMed Scopus (123) Google Scholar, 26Dosemeci A. Dhallan R.S. Cohen N.M. Lederer W.J. Rogers T.B. Circ. Res. 1988; 62: 347-357Crossref PubMed Scopus (215) Google Scholar, 27Rogers T.B. Gaa S.T. Massey C. Dosemeci A. J. Biol. Chem. 1990; 265: 4302-4308Abstract Full Text PDF PubMed Google Scholar). These seemingly paradoxical observations might reflect certain opposing factors of PKC activation which include the net effects of intracellular pH change, the size of intracellular Ca2+ transient, and the states and species of cellular proteins being phosphorylated. protein kinase C 12-O-tetradecanoylphorbal-13-acetate troponin troponin I cardiac TnI mutant with a deletion of the first 32 amino acid residues from the NH2 terminus troponin T troponin C tropomyosin myosin light chain protein kinase A polymerase chain reaction dithiothreitol 1-O-octadecyl-2-O-methyl glycero-3-phosphate 3-(N-morpholino)-2-hydroxypropanesulfonic acid myelin basic protein. One target for PKC in the heart is the contractile apparatus itself. Cardiac TnI and TnT from the thin filament have been shown to be effective substrates for PKC(28Katoh N. Wise B.C. Kuo J.F. Biochem. J. 1983; 209: 189-195Crossref PubMed Scopus (97) Google Scholar), and some of the phosphorylation sites in these proteins have been determined(29Noland Jr., T.A. Raynor R.L. Kuo J.F. J. Biol. Chem. 1989; 264: 20778-20786Abstract Full Text PDF PubMed Google Scholar, 30Swiderek K. Jaquet K. Meyer H.E. Schachtele C. Hofmann F. Heilmeyer L.M. Eur. J. Biochem. 1990; 190: 575-582Crossref PubMed Scopus (50) Google Scholar). Phosphorylation of TnI and/or TnT by PKC resulted in an inhibition of Ca2+-stimulated MgATPase of the reconstituted actomyosin complex (31Noland Jr., T.A. Kuo J.F. J. Biol. Chem. 1991; 266: 4974-4978Abstract Full Text PDF PubMed Google Scholar, 32Noland Jr., T.A. Kuo J.F. J. Mol. Cell. Cardiol. 1993; 25: 53-65Abstract Full Text PDF PubMed Scopus (74) Google Scholar, 33Noland Jr., T.A. Kuo J.F. Biochem. J. 1992; 288: 123-129Crossref PubMed Scopus (50) Google Scholar) or in native myofibril preparations(32Noland Jr., T.A. Kuo J.F. J. Mol. Cell. Cardiol. 1993; 25: 53-65Abstract Full Text PDF PubMed Scopus (74) Google Scholar, 34Venema R.C. Kuo J.F. J. Biol. Chem. 1993; 268: 2705-2711Abstract Full Text PDF PubMed Google Scholar), an effect associated with altered interactions among the contractile protein components(32Noland Jr., T.A. Kuo J.F. J. Mol. Cell. Cardiol. 1993; 25: 53-65Abstract Full Text PDF PubMed Scopus (74) Google Scholar, 33Noland Jr., T.A. Kuo J.F. Biochem. J. 1992; 288: 123-129Crossref PubMed Scopus (50) Google Scholar). PKC also phosphorylated MLC2 (34Venema R.C. Kuo J.F. J. Biol. Chem. 1993; 268: 2705-2711Abstract Full Text PDF PubMed Google Scholar, 35Venema R.C. Raynor R.L. Noland Jr., T.A. Kuo J.F. Biochem. J. 1993; 294: 401-406Crossref PubMed Scopus (89) Google Scholar, 36Noland Jr., T.A. Kuo J.F. Biochem. Biophys. Res. Commun. 1993; 193: 254-260Crossref PubMed Scopus (63) Google Scholar) and C-protein (34Venema R.C. Kuo J.F. J. Biol. Chem. 1993; 268: 2705-2711Abstract Full Text PDF PubMed Google Scholar, 35Venema R.C. Raynor R.L. Noland Jr., T.A. Kuo J.F. Biochem. J. 1993; 294: 401-406Crossref PubMed Scopus (89) Google Scholar, 36Noland Jr., T.A. Kuo J.F. Biochem. Biophys. Res. Commun. 1993; 193: 254-260Crossref PubMed Scopus (63) Google Scholar) in myofibrillar and thick filament preparations. Phosphorylation of MLC2 by PKC or MLC kinase has been shown to cause further activation of Ca2+-stimulated MgATPase of thick filament-substituted myofibrils(36Noland Jr., T.A. Kuo J.F. Biochem. Biophys. Res. Commun. 1993; 193: 254-260Crossref PubMed Scopus (63) Google Scholar). Because the overall effect of PKC phosphorylation of myofibrils (which caused phosphorylation of TnI, TnT, C-protein, and MLC2) was predominantly an inhibition of Ca2+-stimulated MgATPase, it was suggested that the inhibitory effect of TnI/TnT phosphorylation could override the stimulatory effect of MLC2 phosphorylation(35Venema R.C. Raynor R.L. Noland Jr., T.A. Kuo J.F. Biochem. J. 1993; 294: 401-406Crossref PubMed Scopus (89) Google Scholar, 36Noland Jr., T.A. Kuo J.F. Biochem. Biophys. Res. Commun. 1993; 193: 254-260Crossref PubMed Scopus (63) Google Scholar). Therefore, it appears that the actual activity (inhibition or activation) of Ca2+-stimulated myofibrillar MgATPase could be regulated by the relative phosphorylation states of these proteins (i.e. TnI/TnT versus MLC2). The biological significance of PKC phosphorylation of contractile proteins has been substantiated by the findings that their in vitro phosphorylation sites were found to be the same as those in situ as determined by using living cardiomyocytes incubated with TPA or α1-adrenergic agonist(34Venema R.C. Kuo J.F. J. Biol. Chem. 1993; 268: 2705-2711Abstract Full Text PDF PubMed Google Scholar, 35Venema R.C. Raynor R.L. Noland Jr., T.A. Kuo J.F. Biochem. J. 1993; 294: 401-406Crossref PubMed Scopus (89) Google Scholar). Previously, we demonstrated that PKC phosphorylated bovine cardiac TnI at Ser-43/Ser-45, Ser-78, Thr-144, and other undetermined sites (29Noland Jr., T.A. Raynor R.L. Kuo J.F. J. Biol. Chem. 1989; 264: 20778-20786Abstract Full Text PDF PubMed Google Scholar) and that phosphorylation of these multiple sites was associated with an inhibition of Ca2+-stimulated actomyosin MgATPase(31Noland Jr., T.A. Kuo J.F. J. Biol. Chem. 1991; 266: 4974-4978Abstract Full Text PDF PubMed Google Scholar, 32Noland Jr., T.A. Kuo J.F. J. Mol. Cell. Cardiol. 1993; 25: 53-65Abstract Full Text PDF PubMed Scopus (74) Google Scholar). The effect of phosphorylation of the specific sites was unknown. We (33Noland Jr., T.A. Kuo J.F. Biochem. J. 1992; 288: 123-129Crossref PubMed Scopus (50) Google Scholar) and others (for reviews, see Refs. 37 and 38) have previously reported that phosphorylation of Ser-23/Ser-24 by PKA resulted in a decreased Ca2+ sensitivity of the myofibrillar MgATPase. Furthermore, Swiderek et al. (30Swiderek K. Jaquet K. Meyer H.E. Schachtele C. Hofmann F. Heilmeyer L.M. Eur. J. Biochem. 1990; 190: 575-582Crossref PubMed Scopus (50) Google Scholar) found that PKC also phosphorylated bovine cardiac TnI at Ser-23/Ser-24. In the present study we have systematically investigated the functional consequences of site-specific phosphorylation with the use of TnI point mutants in which the phosphorylation sites were substituted by Ala residues and a truncated mutant in which phosphorylation sites were deleted. The findings indicated that PKC phosphorylation of Ser-43/Ser-45 was critical for the inhibition of maximal Ca2+-stimulated actomyosin S-1 MgATPase, an effect associated with an apparent decreased affinity of S-1 for the thin filament. Furthermore, we found that PKC and/or PKA phosphorylation at Ser-23/Ser-24 also resulted in decreased Ca2+ sensitivity of the reconstituted actomyosin complex. The present study is of interest in view of a recent report from one of our laboratories on a deletion mutant that lacks the first 32 amino acids specific to cardiac TnI(39Guo X. Wattanapermpool J. Palmiter K.A. Murphy A.M. Solaro R.J. J. Biol. Chem. 1994; 269: 15210-15216Abstract Full Text PDF PubMed Google Scholar). It was suggested that the NH2-terminal extension functions primarily to provide a means, via phosphorylation at Ser-23/Ser-24, for regulation of the Ca2+ sensitivity of the contractile complex. Similarly, recent studies on deletion mutants of skeletal muscle TnI (40Sheng Z. Pan B.-S. Miller T.E. Potter J.D. J. Biol. Chem. 1992; 267: 25407-25413Abstract Full Text PDF PubMed Google Scholar, 41Farah C.S. Miyamoto C.A. Ramos C.H.I. da Silva A.C.R. Quaggio R.B. Fujimori K. Smillie L.B. Reinach F.C. J. Biol. Chem. 1994; 269: 5230-5240Abstract Full Text PDF PubMed Google Scholar) suggested that the NH2-terminal domain surrounding Ser-43/Ser-45 (of the cardiac sequence) functions to anchor TnI to TnC and other components of the thin filament and thus represents an important site for possible regulation by phosphorylation. Phosphatidylserine, sn-1,2-diolein, and TPA were purchased from Sigma; [γ-32P]ATP was from Amersham Corp., ET-18-OCH3 was from Calbiochem; fresh bovine hearts and porcine brains were from local slaughterhouses. PKC (about 90% homogeneity and devoid of other contaminating protein kinases) was purified from pig brain extracts through the phenyl-Sepharose step(42Girard P.R. Mazzei G.J. Kuo J.F. J. Biol. Chem. 1986; 261: 370-375Abstract Full Text PDF PubMed Google Scholar). Immunoblot analysis using isozyme-specific antibodies (43Zheng B. Chambers T.C. Raynor R.L. Markham P.N. Gebel H.M. Vogler W.R. Kuo J.F. J. Biol. Chem. 1994; 269: 12332-12338Abstract Full Text PDF PubMed Google Scholar) revealed that the PKC preparation primarily consisted of the α isozyme and depended nearly entirely on phosphatidylserine and Ca2+ for its activity as assayed using histone H1 and cardiac TnI as substrates. Bovine heart ventricles were used for the purification of all of the nonrecombinant (native) contractile proteins. TnC, TnI, and TnT were purified according to the method of Potter (44Potter J.D. Methods Enzymol. 1982; 85: 164-181Crossref PubMed Scopus (911) Google Scholar) and stored at −70°C in 50 mM Tris-HCl (pH 8.0) containing 6 M urea, 1 mM EDTA, and 15 mM 2-mercaptoethanol. Tm was prepared by the method of Stull and Buss (45Stull J.T. Buss J.E. J. Biol. Chem. 1977; 252: 851-857Abstract Full Text PDF PubMed Google Scholar) and F-actin by the procedures of Pardee and Spudich(46Pardee J.D. Spudich J.A. Methods Enzymol. 1982; 85: 164-181Crossref PubMed Scopus (982) Google Scholar). Myosin and its S-1 fragment were prepared according to the method of Siemankowski and White(47Siemankowski R.F. White H.D. J. Biol. Chem. 1984; 259: 5045-5053Abstract Full Text PDF PubMed Google Scholar). In order to prevent oxidation of TnI and Tm, 1.0 mM DTT or 15 mM 2-mercaptoethanol were added to all solutions throughout the preparation and reconstitution procedures. Concentrations of contractile proteins were determined using molecular weights and extinction coefficients provided by Tobacman(48Tobacman L.S. J. Biol. Chem. 1988; 263: 2668-2672Abstract Full Text PDF PubMed Google Scholar). Mouse cardiac wild-type TnI was cloned by reverse transcriptase-PCR as described elsewhere(39Guo X. Wattanapermpool J. Palmiter K.A. Murphy A.M. Solaro R.J. J. Biol. Chem. 1994; 269: 15210-15216Abstract Full Text PDF PubMed Google Scholar). Construction of the T144A, S43A/S45A, and S23A/S24A mutants of mouse cardiac TnI was based on the method of recombinant PCR described by Higuchi et al. (49Higuchi R. Krummel B. Saiki R.K. Nucleic Acids Res. 1988; 16: 7351-7367Crossref PubMed Scopus (2102) Google Scholar). The DNA template for recombinant PCR reactions was the mouse wild-type cardiac TnI cDNA cloned into the pET-3d vector at NcoI and BamHI sites(39Guo X. Wattanapermpool J. Palmiter K.A. Murphy A.M. Solaro R.J. J. Biol. Chem. 1994; 269: 15210-15216Abstract Full Text PDF PubMed Google Scholar). The two “inside” primers which direct Thr-144 to Ala-144 were 5′-TTAAGCGGCCCGCTCTCCGAAGAG-3′ and 5′-CTCTTCGGAGAGCGGGCCGCTTAA-3′. The two “inside” primers converting Ser-43 and Ser-45 to Ala-43 and Ala-45 were 5′-AGAAAAAGTCTAAGATCGCCGCCGCCAGAAAACTTCAGTTG-3′ and 5′-CAACTGAAGTTTTCTTGGCGGCGGCGATCTTAGACTTTTTCT-3′. Similarly, the primers converting Ser-23 and Ser-24 to Ala-23 and Ala-24 were 5′-TCCGACGCCGCCGCGCCGCTGCCAACTAC-3′ and 5′-GTAGTTGGCAGCGGCGCGGCGGCGTCGGA-3′. The PCR products were gel-purified and cloned into pET-3d at NcoI and BamHI sites. Several clones were selected for DNA sequence analysis, and one clone from each construct was used to transform BL21(DE3) (Novagen) for expression. To construct the S43A/S45A/T144A triple mutation, clone S43A/S45A was digested by NcoI and PstI (a unique site in the gene) restriction enzymes and the T144A clone was digested by PstI and BamHI. The NcoI/PstI fragment of S43A/S45A and the PstI/BamHI fragment of T144A were gel-purified and ligated into the NcoI/BamHI-digested pET-3d vector. The resulting clone containing the triple mutation was analyzed by nucleotide sequencing and was used to transform BL21(DE3) for expression. The N32 (formerly referred to as TnI/NH2) mutant of rat cardiac TnI, in which the first 32 amino acid residues in the NH2 terminus were deleted, was generated by PCR mutagenesis as described previously (39Guo X. Wattanapermpool J. Palmiter K.A. Murphy A.M. Solaro R.J. J. Biol. Chem. 1994; 269: 15210-15216Abstract Full Text PDF PubMed Google Scholar). Expression and purification of wild-type TnI and mutants by CM-Sephadex cation-exchange chromatography were as described for wild-type TnI(39Guo X. Wattanapermpool J. Palmiter K.A. Murphy A.M. Solaro R.J. J. Biol. Chem. 1994; 269: 15210-15216Abstract Full Text PDF PubMed Google Scholar). Following chromatography on CM-Sephadex, the recombinant proteins were further purified by either affinity chromatography using an Affi-Gel 15 column (Bio-Rad) to which TnC had been covalently attached (39Guo X. Wattanapermpool J. Palmiter K.A. Murphy A.M. Solaro R.J. J. Biol. Chem. 1994; 269: 15210-15216Abstract Full Text PDF PubMed Google Scholar) or by hydroxylapatite chromatography. For the latter step, the TnI preparations were dialyzed against 10 mM imidazole-HCl (pH 7.0) containing 1.0 mM K2HPO4, 1 M KCl, and 1 mM DTT and applied to a hydroxylapatite (Bio-Rad HPT) column (2.5 × 15 cm) which was equilibrated in the same buffer and eluted with a linear gradient of 1-50 mM K2HPO4 (pH 7.0). TnI eluted from the column at 20-30 mM K2HPO4 and was devoid of any proteolytic fragments. The purified preparations of native and recombinant TnI preparations were examined by 12% SDS-polyacrylamide gel electrophoresis and determined to be 99% homogenous. It should be noted that the precise positions of the amino acid residues referred to in this report are derived from the published amino acid sequence of bovine TnI (50Leszyk J. Dumaswala R. Potter J.D. Collins J.H. Biochemistry. 1988; 27: 2821-2827Crossref PubMed Scopus (71) Google Scholar) which begins with a NH2-terminal Ala, and from the amino acid sequence predicted by the nucleotide sequence of the cDNA clone of mouse cardiac wild-type TnI (39Guo X. Wattanapermpool J. Palmiter K.A. Murphy A.M. Solaro R.J. J. Biol. Chem. 1994; 269: 15210-15216Abstract Full Text PDF PubMed Google Scholar) which begins with a NH2-terminal Met. Prior to phosphorylation, all TnI preparations were dialyzed against dialysis buffer containing 10 mM Tris-HCl (pH 7.5) and 1 mM DTT with sequential KCl concentrations of 1, 0.7, and 0.3 M, according to the method of Potter (44Potter J.D. Methods Enzymol. 1982; 85: 164-181Crossref PubMed Scopus (911) Google Scholar). The conditions for phosphorylation by PKC and/or PKA were essentially the same as described previously(29Noland Jr., T.A. Raynor R.L. Kuo J.F. J. Biol. Chem. 1989; 264: 20778-20786Abstract Full Text PDF PubMed Google Scholar, 31Noland Jr., T.A. Kuo J.F. J. Biol. Chem. 1991; 266: 4974-4978Abstract Full Text PDF PubMed Google Scholar, 32Noland Jr., T.A. Kuo J.F. J. Mol. Cell. Cardiol. 1993; 25: 53-65Abstract Full Text PDF PubMed Scopus (74) Google Scholar, 33Noland Jr., T.A. Kuo J.F. Biochem. J. 1992; 288: 123-129Crossref PubMed Scopus (50) Google Scholar). Briefly, for phosphorylation kinetic studies, reaction mixtures (0.2 ml) for PKC contained 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1.0 mM DTT, 0.9 mM CaCl2, 0.5 mM EGTA, 0.3 M KCl, 5 μM [γ-32P]ATP (2-4 × 106 cpm), phosphatidylserine (38 μg/ml), diolein (5 μg/ml), 100 nM TPA, and 1.5 μM (or various concentrations up to 40 μM) of substrate. The reaction mixtures (0.2 ml) for PKA contained, 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 20 μM cyclic AMP, and 5 μM [γ-32P]ATP (2-4 × 106 cpm). Reactions were carried out for 10 min (or various times up to 180 min) at 30°C, terminated by addition of 5% trichloroacetic acid-tungstate, and the 32P incorporation was analyzed as described elsewhere(29Noland Jr., T.A. Raynor R.L. Kuo J.F. J. Biol. Chem. 1989; 264: 20778-20786Abstract Full Text PDF PubMed Google Scholar). The phosphorylation conditions, for TnI preparations used in reconstitution of actomyosin, were the same as described above except that reaction mixtures (2.0 ml) contained 0.4 mM nonradioactive ATP and 6.0 μM TnI. Reactions were initiated by addition of ATP, carried out for up to 3-4 h at 30°C for maximal phosphorylation, and terminated by addition (20 μM final concentration) of the PKC inhibitor ET-18-OCH3(51Helfman D.M. Barnes K.C. Kinkade Jr., J.M. Vogler W.R. Shoji M. Kuo J.F. Cancer Res. 1983; 43: 2955-2961PubMed Google Scholar). Control incubations were conducted in parallel experiments using heat-inactivated PKC. The reaction mixtures were then dialyzed against 10 mM imidazole-HCl (pH 7.0) containing 6 M urea, 20 mM NaF, and 1.0 mM DTT. To determine the extent of phosphorylation or for two-dimensional tryptic-phosphopeptide analysis (29Noland Jr., T.A. Raynor R.L. Kuo J.F. J. Biol. Chem. 1989; 264: 20778-20786Abstract Full Text PDF PubMed Google Scholar), parallel reaction mixtures contained 0.4 mM [γ-32P]ATP (4-8 × 106 cpm). The 32P-TnI was separated using 6-15% linear gradient SDS-polyacrylamide gels according to the method of Laemmli(52Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207211) Google Scholar). The gels were stained for protein with Coomassie Blue R-250, dried, and autoradiographed on Kodax X-Omat AR film. Tryptic digestion of samples exised from the gels and subsequent phosphopeptide mapping were conducted as described elsewhere(29Noland Jr., T.A. Raynor R.L. Kuo J.F. J. Biol. Chem. 1989; 264: 20778-20786Abstract Full Text PDF PubMed Google Scholar). Reconstituted Tn (30Swiderek K. Jaquet K. Meyer H.E. Schachtele C. Hofmann F. Heilmeyer L.M. Eur. J. Biochem. 1990; 190: 575-582Crossref PubMed Scopus (50) Google Scholar) was prepared by mixing (in 10 mM imidazole-HCl, pH 7.0, containing 6 M urea, 1.0 mM CaCl2, and 1.0 mM DTT) cardiac TnT and TnC and phosphorylated or unphosphorylated TnI at a molar ratio of 1:1:1. The mixture was then dialyzed (48 h, 4°C) against 10 mM imidazole (pH 7.0) containing 1.0 mM magnesium acetate, and 1.0 mM DTT with sequential concentrations of KCl of 1.0, 0.7, 0.3, and 0.1 M(44Potter J.D. Methods Enzymol. 1982; 85: 164-181Crossref PubMed Scopus (911) Google Scholar). The resulting complex was concentrated by ultrafiltration, and either stored at −70°C or used immediately to reconstitute the thin filament. Formation of the Tn complex was verified by gel filtration chromatography on Bio-Gel A-0.5m (Bio-Rad), or by co-sedimenting with Tm-F-actin at a 1:1:7 molar ratio of Tn:Tm:F-actin. The thin filament (referred to as “regulated actin”) was reconstituted by mixing Tn, bovine cardiac Tm, and F-actin at a molar ratio of 1:1:5, and the mixture was dialyzed against 10 mM imidazole-HCl (pH 7.0) containing 10 mM KCl, 1.0 mM ATP, 1.0 mM magnesium acetate, and 1.0 mM DTT for 16 h at 4°C. The resulting complex was stored at 4°C and used within 48 h. Prior to assay, actomyosin S-1 was reconstituted from 4 μM regulated actin and 0.3-0.4 μM bovine cardiac myosin S-1. The resulting actomyosin S-1 complex was incubated at 4°C for 0.5-2 h in the reaction mixtures (see below) without ATP. The Ca2+-stimulated MgATPase activity was assayed at 30°C for 10 min in reaction mixtures (0.5 ml) containing actomyosin S-1 (0.3-0.4 μM), 10 mM MOPS-KOH (pH 7.0 or 6.5), 4.1 mM magnesium acetate, 2.1 mM [γ-32P]ATP (5-8 × 106 cpm), 1.0 mM DTT, 1.0 mM EGTA, and 1.05 mM CaCl2 (the free Ca2+ concentration was calculated to be 40 μM). CaCl2, when present, was added as a mixture with EGTA and MOPS-KOH to give the calculated free Ca2+ concentrations of 0.01-100 μM in the assay mixtures, and appropriate volumes of 5 M KOH were added to adjust the pH. KCl was added to bring the final ionic strength to 18 mM. The reactions were initiated by addition of [γ-32P]ATP and terminated by addition of 10% charcoal suspension, and the released 32Pi was determined as described previously(31Noland Jr., T.A. Kuo J.F. J. Biol. Chem. 1991; 266: 4974-4978Abstract Full Text PDF PubMed Google Scholar, 53Oishi K. Zheng B. Kuo J.F. J. Biol. Chem. 1990; 265: 70-75Abstract Full Text PDF PubMed Google Scholar). Stability cons" @default.
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• W2051228751 date "1995-10-01" @default.
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• W2051228751 title "Cardiac Troponin I Mutants" @default.
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