Cardiac troponin I mutants. Phosphorylation by protein kinases C and A and regulation of Ca(2+)-stimulated MgATPase of reconstituted actomyosin S-1.

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 ␣ 1 -adrenergic (1)(2)(3)(4)(5), muscarinic (1,6), and purinergic (6) dynorphin A (7), endothelin-1 (8,9), and angiotensin II (10 -12) 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 Ca 2ϩ -dependent group of PKC 1 isozymes (␣, ␤ I , ␤ II , and ␥) and the novel Ca 2ϩ -independent group of PKC isozymes (␦, ⑀, , and ), whereas inositol trisphosphate, by increasing intracellular Ca 2ϩ , indirectly activates Ca 2ϩ -dependent PKC isozymes (for a review, see Ref. 13). It is worth noting that PKCand PKC-, atypical members of the Ca 2ϩ -independent group, are activated by neither diacylglycerol nor Ca 2ϩ (13). Several lines of recent evidence indicate involvement of PKC in cardiac function and development (14,15) as well as differential expression of the PKC isozymes in cardiac myocytes and tissue (14 -18). 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 (␣ 1 -adrenergic receptor agonist) elicited transient negative inotropy followed by sustained positive inotropy (3, 19 -21), endothelin-1 caused monotonic positive inotropy (22), whereas dynorphin A (-opioid receptor agonist) induced negative inotropy (7). 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 (23)(24)(25)(26)(27). 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 Ca 2ϩ transient, and the states and species of cellular proteins being phosphorylated.
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 (28), and some of the phosphorylation sites in these proteins have been determined (29,30). Phosphorylation of TnI and/or TnT by PKC resulted in an inhibition of Ca 2ϩ -stimulated MgATPase of the reconstituted actomyosin complex (31)(32)(33) or in native myofibril preparations (32,34), an effect associated with altered interactions among the contractile protein components (32,33). PKC also phosphorylated MLC2 (34 -36) and C-protein (34 -36) in myofibrillar and thick filament preparations. Phosphorylation of 1 The abbreviations used are: PKC, protein kinase C; TPA, 12-Otetradecanoylphorbal-13-acetate; Tn, troponin; TnI, troponin I; N32, cardiac TnI mutant with a deletion of the first 32 amino acid residues from the NH 2 terminus; TnT, troponin T; TnC, troponin C; Tm, tropomyosin; MLC, myosin light chain; PKA, protein kinase A; PCR, polymerase chain reaction; DTT, dithiothreitol; ET-18-OCH 3  MLC2 by PKC or MLC kinase has been shown to cause further activation of Ca 2ϩ -stimulated MgATPase of thick filamentsubstituted myofibrils (36). Because the overall effect of PKC phosphorylation of myofibrils (which caused phosphorylation of TnI, TnT, C-protein, and MLC2) was predominantly an inhibition of Ca 2ϩ -stimulated MgATPase, it was suggested that the inhibitory effect of TnI/TnT phosphorylation could override the stimulatory effect of MLC2 phosphorylation (35,36). Therefore, it appears that the actual activity (inhibition or activation) of Ca 2ϩ -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 ␣ 1 -adrenergic agonist (34,35).
Previously, we demonstrated that PKC phosphorylated bovine cardiac TnI at Ser-43/Ser-45, Ser-78, Thr-144, and other undetermined sites (29) and that phosphorylation of these multiple sites was associated with an inhibition of Ca 2ϩstimulated actomyosin MgATPase (31)(32). The effect of phosphorylation of the specific sites was unknown. We (33) 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 Ca 2ϩ sensitivity of the myofibrillar MgATPase. Furthermore, Swiderek et al. (30) 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 Ca 2ϩ -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 Ca 2ϩ 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 (39). It was suggested that the NH 2 -terminal extension functions primarily to provide a means, via phosphorylation at Ser-23/ Ser-24, for regulation of the Ca 2ϩ sensitivity of the contractile complex. Similarly, recent studies on deletion mutants of skeletal muscle TnI (40,41) suggested that the NH 2 -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.
Preparations of Enzymes and Bovine Heart Contractile Proteins-PKC (about 90% homogeneity and devoid of other contaminating protein kinases) was purified from pig brain extracts through the phenyl-Sepharose step (42). Immunoblot analysis using isozyme-specific antibodies (43) revealed that the PKC preparation primarily consisted of the ␣ isozyme and depended nearly entirely on phosphatidylserine and Ca 2ϩ 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 (44) and stored at Ϫ70°C in 50 mM Tris-HCl (pH 8.0) contain-ing 6 M urea, 1 mM EDTA, and 15 mM 2-mercaptoethanol. Tm was prepared by the method of Stull and Buss (45) and F-actin by the procedures of Pardee and Spudich (46). Myosin and its S-1 fragment were prepared according to the method of Siemankowski and White (47). 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 (48).
Construction of the Plasmids Expressing Cardiac Wild-type TnI and Mutants-Mouse cardiac wild-type TnI was cloned by reverse transcriptase-PCR as described elsewhere (39). 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. (49). 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 (39). The two "inside" primers which direct Thr-144 to Ala-144 were 5Ј-TTA-AGCGGCCCGCTCTCCGAAGAG-3Ј and 5Ј-CTCTTCGGAGAGCGGGC-CGCTTAA-3Ј. The two "inside" primers converting Ser-43 and Ser-45 to Ala-43 and Ala-45 were 5Ј-AGAAAAAGTCTAAGATCGCCGCCGCCA-GAAAACTTCAGTTG-3Ј and 5Ј-CAACTGAAGTTTTCTTGGCGGCGGC-GATCTTAGACTTTTTCT-3Ј. Similarly, the primers converting Ser-23 and Ser-24 to Ala-23 and Ala-24 were 5Ј-TCCGACGCCGCCGCGCCGCT-GCCAACTAC-3Ј and 5Ј-GTAGTTGGCAGCGGCGCGGCGGCGTCGGA-3Ј. 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/BamHIdigested 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/NH 2 ) mutant of rat cardiac TnI, in which the first 32 amino acid residues in the NH 2 terminus were deleted, was generated by PCR mutagenesis as described previously (39).
Expression and Purification of Cardiac Wild-type TnI and Mutants-Expression and purification of wild-type TnI and mutants by CM-Sephadex cation-exchange chromatography were as described for wild-type TnI (39). 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 (39) 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 K 2 HPO 4 , 1 M KCl, and 1 mM DTT and applied to a hydroxylapatite (Bio-Rad HPT) column (2.5 ϫ 15 cm) which was equilibrated in the same buffer and eluted with a linear gradient of 1-50 mM K 2 HPO 4 (pH 7.0). TnI eluted from the column at 20 -30 mM K 2 HPO 4 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 (50) which begins with a NH 2 -terminal Ala, and from the amino acid sequence predicted by the nucleotide sequence of the cDNA clone of mouse cardiac wild-type TnI (39) which begins with a NH 2 -terminal Met.
Phosphorylation of Wild-type TnI and Mutants-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 (44). The conditions for phosphorylation by PKC and/or PKA were essentially the same as described previously (29,(31)(32)(33). Briefly, for phosphorylation kinetic studies, reaction mixtures (0. 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 32 P incorporation was analyzed as described elsewhere (29). 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 M 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 M final concentration) of the PKC inhibitor ET-18-OCH 3 (51). 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 (29), parallel reaction mixtures contained 0.4 mM [␥-32 P]ATP (4 -8 ϫ 10 6 cpm). The 32 P-TnI was separated using 6 -15% linear gradient SDS-polyacrylamide gels according to the method of Laemmli (52). 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 (29).  (44). The resulting complex was concentrated by ultrafiltration, and either stored at Ϫ70°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.

Reconstitution of Tn and Thin Filament-Reconstituted
Actomyosin S-1 MgATPase Assay and Thin Filament Binding to Myosin S-1-Prior to assay, actomyosin S-1 was reconstituted from 4 M regulated actin and 0.3-0.4 M 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 Ca 2ϩ -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 M), 10 mM MOPS-KOH (pH 7.0 or 6.5), 4.1 mM magnesium acetate, 2.1 mM [␥-32 P]ATP (5-8 ϫ 10 6 cpm), 1.0 mM DTT, 1.0 mM EGTA, and 1.05 mM CaCl 2 (the free Ca 2ϩ concentration was calculated to be 40 M). CaCl 2 , when present, was added as a mixture with EGTA and MOPS-KOH to give the calculated free Ca 2ϩ concentrations of 0.01-100 M 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 [␥-32 P]ATP and terminated by addition of 10% charcoal suspension, and the released 32 P i was determined as described previously (31,53). Stability constants used in the calculation of the total concentrations of reaction constituents required to give the necessary free Ca 2ϩ concentrations were determined using the procedures of Fabiato and Fabiato (54) as described previously (31). MgATPase activity, reported as mol of P i /mol of S-1/s (s Ϫ1 ), was linear as a function of time or amount of reconstituted actomyosin S-1 under the assay conditions. Kinetic data for Ca 2ϩ stimulation of actomyosin S-1 MgATPase (as a function of Ca 2ϩ concentration) were calculated by nonlinear least-squares regression analysis using a modified form of the A. V. Hill equation as described previously (31,32,36). Similarly, the kinetic data for Ca 2ϩ -stimulated actomyosin S-1 activity, as a function of thin filament concentration, were analyzed using the A. V. Hill equation where K app is defined as the concentration of thin filament producing 50% maximal Ca 2ϩ -stimulated MgATPase activity, and V max is the estimated maximal thin filament-stimulated activity. The binding of bovine cardiac myosin S-1 (in the presence of ATP) to reconstituted thin filaments containing various TnI preparations was assessed, as described previously (32), by separating free S-1 from actin-bound S-1 using the ultracentrifugation techniques described by Chalovich and Eisenberg (55), as modified by Tobacman and Adelstein (56).

RESULTS
The abilities of recombinant mouse cardiac wild-type TnI and various mutants, in which the identified phosphorylation sites for PKC and PKA were either substituted or deleted, compared to the native bovine cardiac TnI, MBP, and histone H1, to serve as substrates for PKC and PKA were examined, and the kinetic constants are summarized (Table I). It was found that the mutations caused subtle changes in the substrate activities (indicated by the V max /K m ratios) of the resulting proteins for PKC, i.e. in a decreasing order, wild-type Ͼ T144A Ͼ N32 ϳ S43A/S45A Ͼ S43A/S45A/T144A Ͼ S23A/ S24A. The mutations on PKC phosphorylation sites, in contrast, caused little or no change in substrate activities for PKA, i.e. wild-type ϳ T144A ϭ S43A/S45A ϳ S43A/S45A/ T144A. As expected, mutants S23A/S24A and N32, in which PKA phosphorylation sites were, respectively, substituted and deleted, were not significantly phosphorylated by PKA, indicating that PKA was essentially unable to phosphorylate the PKC sites. Bovine TnI was an inferior substrate, compared to mouse wild-type TnI, for both PKC and PKA. Although MBP and histone H1 were effective substrates for PKC in the presence of 0.3 M KCl (which was required for keeping TnI preparations in the soluble form), they were not appreciably phosphorylated by PKA under the same conditions. Because MBP and histone H1 were effectively phosphorylated by PKA in the absence of 0.3 M KCl (albeit lower than by PKC), it seemed that PKA was more sensitive to salt inhibition than PKC.
The time-dependent phosphorylation of TnI preparations and MBP by PKC indicated that the initial reaction rates for these proteins were somewhat different, with S23A/S24A being the least effective substrate (Fig. 1). If the reactions were carried out for an extended time (3 h) with high The V max (pmol/min) for PKC phosphorylation of wild-type was 13 Ϯ 1 which was taken as 100%. b The V max (pmol/min) for PKA phosphorylation of wild-type was 71 Ϯ 2 which was taken as 100%. c ND, not determined; activities at 10 M substrate were Ͻ2 pmol/min. amounts of PKC and ATP, higher phosphorylation extents of 1.3-2.7 mol of phosphate/mol of protein were obtained for all TnI preparations (Fig. 1). The initial phosphorylation rate by PKA was found to be similar for wild-type, S43A/S45A and T144A, but was slightly higher for S43A/S45A/T144A (Fig. 2). For these TnI preparations, phosphorylation of about 0.8 -1.4 mol of phosphate/mol of protein was obtained after prolonged incubation with high amounts of PKA and ATP. As expected, S23A/S24A and N32 were not appreciably phosphorylated by PKA. Two-dimensional tryptic peptide maps of various TnI preparations phosphorylated by PKC (Fig. 3) and PKA (Fig. 4) were examined. A pattern of five 32 P-phosphopeptides was readily detected for bovine TnI phosphorylated by PKC (Fig. 3). We previously determined, using bovine cardiac TnI, that the phosphorylated residues were Ser-78 in peptide 1, Ser-43/Ser-45 in peptide 2, and Thr-144 in peptide 3 (29). Our previous evidence also suggested that peptide 5 contains phosphorylated Ser-23/ Ser-24 (34). The identity of the phosphorylated residue in peptide 4 is unknown. A similar peptide map was obtained for mouse wild-type TnI phosphorylated by PKC. Phosphopeptide 1 was absent from maps of mouse TnI preparations because Ser-78 in the bovine and rat sequences (50) is replaced by a nonphosphorylatable Arg residue in the mouse sequence (39). Substitutions of Ala for the phosphorylatable residues in the mouse TnI mutants T144A, S43A/S45A, and S43A/S45A/ T144A were confirmed by the absence of corresponding phosphopeptides in their tryptic peptide maps (Fig. 3). For the mouse TnI mutant S23A/S24A, phosphopeptide 5 was absent while phosphorylation at Ser-43/Ser-45 (peptide 2) was predominant. Similarly, peptide 5 was absent while phosphopeptides 1, 2, 3, and 4 were present of the rat TnI mutant N32. Rat cardiac TnI (50) has Val at position 76 instead of Ala in the bovine sequence and thus the N32 mutant would produce a tryptic phosphopeptide (containing Ser-78) with a different mobility than that from bovine TnI (Fig. 3). In comparison to PKC, PKA almost exclusively and preferentially phosphorylated Ser-23/Ser-24 in all TnI preparations tested, as confirmed by the lack of phosphopeptide 5 in maps for N32 (Fig. 4) and S23A/S24A (data not shown). A very minor phosphorylation at Thr-144 (phosphopeptide 3) by PKA was also noted in all TnI preparations containing that amino acid residue (Fig. 4).
We reported previously that PKC phosphorylation of bovine cardiac TnI at multiple sites resulted in a reduced maximal Ca 2ϩ -stimulated MgATPase activity of reconstituted actomyosin and actomyosin S-1 (31). With the use of the recombinant TnI mutants, we have now determined which of the phosphorylation sites were responsible for this effect. Since TnI has been shown to be an important regulator of the pH-dependent Ca 2ϩ sensitivity of cardiac myofilaments (39,57,58), phosphorylation of TnI might also affect this pH dependence. We have therefore examined the MgATPase activity of reconstituted actomyosin S-1 at pH 6.5 in addition to the standard pH 7.0 (Fig. 5), and the kinetic constants are summarized (Table II). Phosphorylation by PKC of wild-type resulted in 62 and 85% reductions in the maximal Ca 2ϩ stimulation when the MgATPase activity was assayed at pH 7.0 and 6.5, respectively. Similarly, PKC-phosphorylated T144A caused 78 and 80% reductions of the corresponding values, whereas phosphorylation of S43A/S45A led to much smaller reductions of 25 and 24%, suggesting that phosphorylation at Ser-43/Ser-45, but not Thr-144, was primarily responsible for the reduced Ca 2ϩ -stimulated activity. Phosphorylation of the S43A/S45A/T144A produced intermediate reductions, i.e. 34 and 48% at pH 7.0 and 6.5. As with Thr-144, phosphorylation at Ser-23/Ser-24 (the PKA-preferred sites) could not account for the reduced maximal Ca 2ϩ -stimulated activity, because PKC phosphorylation of S23A/S24A and N32 still resulted in marked reductions (51-68%) of the enzyme activity. PKC phosphorylation of bovine TnI reduced the maximal Ca 2ϩ stimulation to an extent similar to that reported previously (31), which was less than that for phosphorylated mouse wild-type TnI (Table II).
The mutations at the phosphorylation sites also affected the Ca 2ϩ sensitivity of the actomyosin MgATPase ( Fig. 5 and Table  II). All of the reconstituted actomyosin S-1 preparations, containing unphosphorylated TnI mutants as well as wild-type or bovine TnI, exhibited decreased Ca 2ϩ sensitivity at pH 6.5 compared to pH 7.0, although the extent of the decrease varied somewhat among the TnI preparations. However, when assayed at pH 7.0, higher EC 50 values for Ca 2ϩ (i.e. decreased Ca 2ϩ sensitivity) of 1.6 -2.7 M were noted for reconstituted actomyosin S-1 preparations containing unphosphorylated S43A/S45A, S43A/S45A/ T144A, S23A/S24A, and N32, but not T144A (1.1 M), compared to mouse wild-type (1.2 M) and bovine TnI (1.1 M). Qualitatively similar differences in Ca 2ϩ sensitivity were also observed when the preparations were assayed at pH 6.5. The findings suggested the importance of Ser-23/Ser-24 and Ser-43/Ser-45 in the regulation of the thin filament Ca 2ϩ sensitivity. PKC phosphorylation of wild-type, T144A, and bovine TnI caused up to 3-fold decreases in Ca 2ϩ sensitivity at pH 7.0 and 6.5, but the effect was less pronounced or even undetectable for other TnI mutants, particularly N32 and S43A/S45A, further supporting the importance of Ser-23/Ser-24 and Ser-43/Ser-45 in Ca 2ϩ sensitivity. No apparent differences were observed in the basal MgATPase activities (in the absence of added Ca 2ϩ ) among the actomyosin S-1 preparations containing the recombinant TnI proteins, whether phosphorylated or unphosphorylated (data not shown).
Because PKC cross-phosphorylated PKA sites (Ser-23/Ser-24) in TnI (Fig. 3) and phosphorylation at these sites led to a decreased Ca 2ϩ sensitivity of MgATPase ( Fig. 5 and Table II), we directly compared the effects of PKC and/or PKA phosphorylation of wild-type TnI (Fig. 6). The kinetic data are summarized (Table III). A short (15-min) exposure of TnI to PKC caused the incorporation of 1.1 mol of phosphate/mol of TnI and phosphorylation of all PKC sites, but only minor phosphorylation of PKA sites Ser-23/Ser-24 (phosphopeptide 5) was noted. This short exposure to PKC produced a 32% decrease in maximal Ca 2ϩ stimulation and a 2.2-fold increase in the EC 50 for Ca 2ϩ . In contrast, a brief exposure of TnI to PKA, which allowed exclusive phosphorylation (1.3 mol of phosphate/mol of protein) at Ser-23/Ser24, resulted in a 2.9-fold increase in the EC 50 for Ca 2ϩ without appreciably affecting the maximal Ca 2ϩ activation. When all sites were phosphorylated (2.3 mol of phosphate/mol of TnI) due to a short exposure to both PKA and PKC, there was 55% reduction in this activation, accompanied by a 3.9-fold increase in the EC 50 for Ca 2ϩ . The combined effects of the two enzymes were similar to those produced by a long (2-h) incubation with PKC, yielding phosphorylation (2.3 mol of phosphate/mol of TnI) at all sites, a 56% reduction of the maximal Ca 2ϩ -stimulated activity, and a 6-fold increase in the EC 50 for Ca 2ϩ . These findings are consistent with the idea that PKC decreased Ca 2ϩ sensitivity by cross-phosphorylating the typical PKA sites (Ser-23/Ser-24) or by phosphorylating certain PKC sites. Moreover, the actions of the two enzymes could be additive under certain conditions.

FIG. 5. Effects of PKC phosphorylation of wild-type TnI and mutants on the Ca 2ϩ -dependent stimulation of MgATPase of reconstituted actomyosin S-1.
The unphosphorylated and phosphorylated (1.8 -2.3 mol/mol) TnI preparations were used for reconstitution, and the enzyme activity was assayed in the presence of varying Ca 2ϩ concentrations at pH 6.5 or 7.0. Actomyosin S-1 MgATPase activity in the absence of Ca 2ϩ was subtracted from values obtained in the presence of Ca 2ϩ and was less than 15% of the total MgATPase activity. The maximal Ca 2ϩ stimulated MgATPase activities for reconstituted actomyosin S-1 containing respective unphosphorylated TnI preparations assayed at pH 7.0 and 6.5 were taken as 100%. The findings were confirmed in three or four experiments. See "Experimental Procedures" for further details. The curves drawn are the "best fits" of the data to the Hill equation using nonlinear regression. The results from the above experiments (Figs. 5 and 6) suggested that PKC phosphorylation of TnI affected its interactions with other components of the thin filament, preventing full Ca 2ϩ -dependent activation of the complex. We were also interested in determining if phosphorylation altered interactions of the thick and thin filaments. Therefore, we next examined the concentration-dependent effects of the thin filament (regulated actin), containing various TnI preparations, on the Ca 2ϩ -stimulated MgATPase activity of myosin S-1 (Fig. 7). The kinetic data are summarized (Table IV). PKC phosphorylation of all of the TnI preparations, except S43A/S45A and S43A/ S45A/T144A, markedly increased (up to 4-fold) the K app (i.e decreased apparent affinity) for the thin filament. Phosphorylation of all of the TnI preparations except N32 and S43A/S45A also caused a reduction in the V max , with T144A and S23A/ S24A providing the greater (55 and 43%, respectively) reductions compared to other TnI preparations (2-33%). This evidence suggested that, while phosphorylation at Ser-43/Ser-45 was primarily responsible for the majority of the apparent decreased V max and affinity of myosin S1 for regulated actin, phosphorylation of TnI at other sites (excluding Thr-144) may also affect these parameters. We also noted that the K app of myosin S-1 for thin filaments containing either unphosphorylated S43A/S45A, S43A/S45A/T144A, S23A/S24A, or N32 was greater than that containing wild-type or T144A. These findings suggested that substitution of Ser-43/Ser-45 or Ser-23/ Ser-24 by Ala residues or deletion of the NH 2 -terminal sequence containing Ser-23/Ser-24 caused a decreased affinity of S-1 for the thin filament. In agreement with the observations of Tobacman and Adelstein (56), we also observed that, for all preparations of regulated actin studied, the addition of Ca 2ϩ altered primarily the V max of the MgATPase activity and not the K app of myosin S-1 for regulated actin (data not shown). In additional experiments, we found that the binding of 0.3 M myosin S-1 to 20 M regulated actin, in the presence of 2.1 mM ATP, was reduced 26 -30% in preparations containing PKCphosphorylated bovine TnI, wild-type TnI, and T144A, compared to a 10% reduction for the phosphorylated S43A/S45A (data not shown). These findings underscored the importance of phosphorylation at Ser-43/Ser-45 in regulating the interactions of TnI with other components of the thin filament and ultimately interactions of the thick and thin filaments. DISCUSSION Point mutations of PKC and PKA phosphorylation sites and a deletion of PKA phosphorylation sites in cardiac TnI did not bring about gross changes in the general properties of the mutants. The mutants retained their abilities to reconstitute into functional actomyosin S-1, which demonstrated typical activation by Ca 2ϩ and pH-dependent Ca 2ϩ sensitivity of MgATPase. Certain observations concerning phosphorylation of the mutants by PKC seemed noteworthy. Although mutations altered phosphorylation kinetics as expected, the extents of phosphorylation that we observed for the mutants were quite surprising. If one assumed Ser-23/Ser-24, Ser-43/Ser-45, Thr-144, and the unidentified site in phosphopeptide 4 in mouse wild-type TnI could be exhaustively phosphorylated, a stoichiometry of six could be expected. However, we failed to obtain a maximal value that was higher than three. On the other hand, a stoichiometry similar to that for wild-type was noted for the triple mutant S43A/S45A/T144A (e.g. Fig. 1). These findings seemed to support a hypothesis that phosphorylation of multi-TABLE II Summary of kinetic constants for Ca 2ϩ -stimulated MgATPase of reconstituted actomyosin S-1, containing PKC phosphorylated or unphosphorylated TnI preparations and assayed at pH 7.0 and 6.5 The values (ϮS.E.) were calculated using nonlinear least-squares regression analysis using a modified form of the Hill equation, based upon the data from three or four independent experiments such as shown in Fig. 5 (in which the data for bovine TnI are not shown for reasons of brevity). See "Experimental Procedures" for details. ple sites in TnI might be mutually regulating. It is conceivable, therefore, that phosphorylation of certain sites in wild-type could negatively regulate or even restrict further phosphorylation of other sites, resulting in phosphorylation that was much lower than the theoretical value. In contrast, absence of preferred sites such as in mutant S43A/S45A/T144A could lead to an extensive and rapid cross-phosphorylation of the PKA sites (Ser-23/Ser-24) (e.g. Fig. 3), resulting in phosphorylation that was higher than expected. This "plasticity" concerning multisite phosphorylation might be of special significance, in light of the complex roles played by TnI (and perhaps TnT as well) in the regulation of contractile activity. The PKC phosphorylation sites in cardiac TnI are located in specific functional domains of the protein. Studies on fast skeletal muscle TnI have indicated that two functional domains exist in the protein (40, 41, 59 -71). The NH 2 -terminal domain of TnI binds strongly to the COOH-terminal domain of TnC and serves to anchor TnI to the other components of the Tn complex. The COOH-terminal half of TnI contains a region, residues 96 -116, corresponding to residues 130 -150 in the bovine and mouse cardiac sequences (39,50), that can bind to Tmactin and inhibit the actomyosin MgATPase activity, as well as bind to TnC (40, 41, 59 -71). Talbot and Hodges (61) identified the minimal "inhibitory" sequence within this region to be comprised of residues 105-114, corresponding to residues 139 -148 in the bovine cardiac sequence (50). In the absence of Ca 2ϩ , this inhibitory domain of TnI remains bound to Tm-actin. When Ca 2ϩ binds to TnC, a conformational change in the Tn complex occurs causing the inhibitory region, along with the remainder of the COOH-terminal region of TnI, to bind to certain sites in the NH 2 -and COOH-terminal domains of TnC, leading to removal of inhibition by TnI (40,41,67). Within this inhibitory sequence, cardiac TnI has Thr at position 144 in place of Pro in the skeletal muscle sequences. Using synthetic peptides corresponding to residues 104 -115 of the skeletal muscle sequence and residues 138 -149 of the cardiac sequence, Talbot and Hodges (62) showed that Pro or Thr substitutions had no effect on the inhibitory activity of the peptides. In contrast, Van Eyk and Hodges (64) demonstrated that replacement of these polar residues with the neutral amino acid Gly caused a 38% loss of inhibitory activity for the synthetic peptide. We, however, mutated this site in cardiac TnI to Ala (T144A) and found little or no effects on MgATPase activity. Because the inhibitory region of TnI contains several basic amino acids, we suspected that adding a negatively charged phosphate group on Thr-144 would affect TnI-thin filament interactions sufficiently to account for the PKC-mediated inhibition of the Ca 2ϩ -stimulated MgATPase activity. This, however, was not the case in that a full inhibitory effect due to phosphorylation by PKC was retained with the T144A mutant. Because none of our mutants could be phosphorylated only at Thr-144, we were not able to ascertain the direct effects of phosphorylation at this site.
Cardiac TnI differs from fast and slow skeletal muscle TnI primarily in the NH 2 -terminal region. The presence of a cardiac-specific extension of 24 -36 amino acids at the NH 2 terminus suggests that an additional functional domain exists in cardiac TnI (50,72). However, deletion of this region (the N32 mutant) conferred little or no changes to the inhibition by TnI of actomyosin MgATPase activity or to overall regulation by Ca 2ϩ or pH of myofilament function (39) (Table II). Reconstituted actomyosin S-1 containing the unphosphorylated S23A/S24A or N32 mutants did, however, display decreased Ca 2ϩ sensitivity (Fig. 5, Table II  important for normal Ca 2ϩ sensitivity of the actomyosin complex. A similar effect was apparent when wild-type TnI was replaced with the N32 mutant in cardiac myofibril preparations (39). Furthermore, phosphorylation by PKA of Ser-23/ Ser-24 produced a decreased Ca 2ϩ -sensitivity of reconstituted actomyosin and native myofibrils (34,37,38) (Fig. 6). These results suggest that phosphorylation of Ser-23/Ser-24 alters the interaction of the NH 2 -terminal extension with other functional domains of TnI which regulate the TnI-TnC or TnI-actin interactions. Although PKA is considered the primary protein kinase for phosphorylation at these Ser residues, our studies indicated that PKC can cross-phosphorylate Ser-23/Ser-24 and decrease the Ca 2ϩ sensitivity of the resulting reconstituted actomyosin complex (Figs. 5 and 6, Tables II and III). Since both PKA and PKC can phosphorylate TnI at Ser-23/Ser-24, it was suggested that PKC, in addition to PKA, could regulate the Ca 2ϩ sensitivity of the myofilaments. Simply put, PKA appeared to regulate primarily the Ca 2ϩ sensitivity, whereas PKC modulated both the Ca 2ϩ sensitivity and activity of the myofilament MgATPase. It appeared that PKC phosphorylated more readily Ser-23/Ser-24 (phosphopeptide 5) in mouse TnI than in bovine TnI (Fig. 3). This difference may account for the more pronounced decrease in Ca 2ϩ sensitivity of MgATPase of actomyosin-containing mouse TnI, compared to bovine TnI, phosphorylated by PKC when the enzyme was assayed at pH 7.0 (Table II), and our earlier findings that PKC phosphorylation of bovine TnI resulted primarily in inhibition of the activity without consistently affecting the Ca 2ϩ sensitivity of actomyosin MgATPase (31)(32)(33).
As suggested from studies on skeletal muscle TnI (40 -41, 59, 65-69), Ser-43/Ser-45 are located in the "anchor" region of cardiac TnI, residues 33-80 (50), that binds to TnC. Furthermore, these residues are located in the highly basic segment comprising residues 37-51 (50) that probably directly interacts with the acidic protein TnC (59,68,69). The NH 2 -terminal region of skeletal muscle TnI also interacts with TnT (70,71), implying that this region of TnI may not be accessible to PKC. However, by using reconstituted Tn, we demonstrated that these Ser residues were phosphorylated by PKC, even when TnI was complexed with TnC (29). Moreover, analysis of tryptic phosphopeptide maps of TnI from 32 P-labeled rat cardiac myocytes treated with phorbol ester or phenylephrine revealed the presence of a phosphopeptide corresponding to that containing Ser-43/Ser-45 (34). These results suggested that Ser-43/Ser-45 could be phosphorylated in intact myocytes upon activation of PKC. Due to the location of these amino acids, it was not surprising to find that phosphorylation at Ser-43/Ser-45 was responsible for the majority of the PKC-mediated inhibition of the maximal Ca 2ϩ -stimulated MgATPase activity of reconstituted actomyosin S-1 complex (Figs. 5 and 6, Tables III and IV). Furthermore, simple substitution of these Ser residues with FIG. 7. Activation of Ca 2ϩ -stimulated MgATPase of reconstituted actomyosin S-1 by regulated actin. The unphosphorylated and phosphorylated (1.8 -2.3 mol/mol) TnI preparations were used to reconstitute regulated actin, and MgATPase of actomyosin S-1 was assayed at pH 7.0 in the presence of varying concentrations of regulated actin. The enzyme activity in the absence of Ca 2ϩ was subtracted from the activity in the presence of Ca 2ϩ (40 M) and was less than 10% of the total enzyme activity. The findings were confirmed in two other experiments. See "Experimental Procedures" for further details. The curves drawn are the "best fits" of the data to the Hill equation using nonlinear regression. Ala residues resulted in a significant decrease in the maximal Ca 2ϩ -stimulated MgATPase activity (Table II). We can therefore hypothesize that Ser-43/Ser-45 appeared to be critical for maintaining the maximal Ca 2ϩ -stimulated actomyosin MgATPase activity and that phosphorylation of these residues represents an important mechanism for regulation (inhibition) of the enzyme activity and, hence, the function of the contractile apparatus. In addition, the Ala for Ser substitutions resulted in a decreased Ca 2ϩ sensitivity for the reconstituted actomyosin, and phosphorylation of Ser-23/Ser-24 in S43A/ S45A could not further decrease the Ca 2ϩ sensitivity (Fig. 5 and Table II). This suggested that Ser-43/Ser-45 may be in the region of TnI which interacts with the phosphorylated Ser-23/ Ser-24 and ultimately, through interactions with either TnC or TnT, may influence the Ca 2ϩ sensitivity of the contractile complex. With the use of additional mutants, we are currently investigating the more discrete effects of phosphorylation at Ser-43/Ser-45 on interactions of TnI with other components of the thin filament, such as binding of TnI to F-actin, TnT and TnC.
Phosphorylation of TnI at Ser-43/Ser-45 also caused a decrease in the apparent affinity (increased K app ) of S-1 for the thin filament, leading to MgATPase inactivation ( Fig. 7 and Table III) and a decrease in the actual binding of S-1⅐ATP to the thin filament (data not shown). It seemed that phosphorylation at Ser-43/Ser-45 would cause a conformational change in TnI such that TnC⅐Ca 2ϩ is prevented from completely "pulling" the inhibitory TnI away from certain binding sites on F-actin, with which myosin interacts. According to the model of Hill et al. (73), as modified by Lehrer (74), we hypothesize that phosphorylated TnI would then continue to stabilize a portion of the thin filament in the "off" state, preventing the binding of additional S-1⅐ATP (i.e. weak binding cross-bridges), thereby reducing the number of cross-bridges able to isomerize to "strongly bound" cross-bridges, and ultimately preventing the complete myosin head-induced "off to on" transition of Tm. Under the low ionic strength conditions (18 mM KCl) used in the present study, the binding of S-1⅐ATP to regulated actin is only modestly sensitive to Ca 2ϩ (56,75) and not inhibited by unphosphorylated TnI (75). We would predict, however, that the effects of phosphorylated TnI would be more pronounced under conditions of physiological ionic strength (120 mM KCl) where even unphosphorylated TnI has been shown to affect the binding of S-1⅐ATP to the thin filament (76). We are currently investigating such possibilities as well as other effects of phosphorylated TnI on the interactions of the thick and thin filaments.
Adult cardiac tissue is uniquely sensitive to the effects of pH. Acidotic conditions cause greater reductions in the maximal force, shortening velocity, and Ca 2ϩ sensitivity of fibers from adult cardiac muscle, compared to fibers from neonatal cardiac and slow and fast skeletal muscles (39,(57)(58)(77)(78)(79). These functional effects have been proposed to be the consequence of pH-sensitive alterations in the binding of Ca 2ϩ to TnC, and the differential transmission of the Ca 2ϩ binding signal has been suggested to be dependent upon the isoforms of TnI and TnC (57,58,78,79). In the present studies, we found that at pH 6.5, compared to pH 7.0, TnI phosphorylated by PKC caused greater reductions in the Ca 2ϩ -stimulated MgATPase activity of reconstituted actomyosin S-1 ( Fig. 5 and Table II). This suggested that effects on myocardial contractility due to phosphorylation of TnI by PKC may be more pronounced during acidosis.