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THEJOURNAL

OF

Vol. 268,No 4. Issue of February 5. pp. 2705-2711.1993 Printed in U.S.A.

BIOLOGICAL CHEMISTRY

0 1993 by The American Society for Biochemistry and Molecular Biology, Inc

Protein Kinase C-mediatedPhosphorylation of Troponin I and Cprotein in Isolated Myocardial Cells IS Associated with Inhibition of Myofibrillar Actomyosin MgATPase* (Received for publication, July 6, 1992, and in revised form, September 8, 1992)

Richard C. VenemaS and J. F. Kuo From the Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322

Phosphorylation of cardiac myofibrillar proteins by protein kinase C (PKC) in isolated adult rat cardiomyocytes has been compared with that mediated by the CAMP-dependent protein kinase (PKA). PKA activation byB-adrenoreceptor (isoproterenol) stimulation results in stoichiometric phosphorylation of troponin I (TnI) and C-protein. PKC activation by either 12-0tetradecanoylphorbol-13-acetate (TPA)or bya-adrenoreceptor (phenylephrine plus propranolol) stimulation results in phosphorylation of the same two proteins to similar extents. Two-dimensional phosphopeptide mapping shows that the same sites in TnI are modified by PKC in vitro and in TPA- or a-agonist-stimulated cells. These sites are distinct from those phosphorylated in isoproterenol-stimulated cells orbyPKA in vitro. Phosphopeptide mapping analysis of C-protein shows that PKC and PKA phosphorylate identical residues in this protein in vitro and in situ. TPA-stimulated phosphorylation in myocytes is associated with a reduction in maximal activity of myofibrillar Ca2+dependent actomyosin MgATPase. Isoproterenol-stimulated phosphorylation has no effect on maximal activity but reduces the Ca2+sensitivity of the MgATPase. These data demonstrate that TnI and C-protein are phosphorylated in myocardial cells by both PKA and PKC, resulting in different functional consequences in each case.

channels (3) and the sarcoplasmic reticulum Ca2+ pump regulatory protein, phospholamban (4). The resulting increased availability of Ca2+ to the contractile apparatus causes an increase in theforce of cardiac contraction(positive inotropic effect). Direct phosphorylation of the contractile machinery itself is also recognized as having an important modulatory role in contraction (1, 2). Considerable effort, therefore, has gone into the study of PKA-catalyzedphosphorylation of cardiac contractile proteins and its functional consequences in mammalian heart cells. It is now well established that padrenergic stimulation of either intact heartsor isolated myocardial cells results in phosphorylation of C-protein and trorespectively, of ponin I (TnI) in the thick and thin filaments, cardiac myofilaments (1, 2, 5-9). Cardiac myofilaments, like those of other muscle cell types, contain an intrinsicactomyosin MgATPase activity, themolecular basis of contraction. Sensitivity of the actomyosin MgATPase to Ca2+ in striated muscle ismediated by the troponin complex inthethinfilament.Thismultiprotein complex is comprised of the Ca2+-binding TnC subunit, the MgATPase-inhibiting TnIsubunit,andthetropomyosinbinding T n T subunit. Phosphorylationof TnI by PKA results in a decreased myofibrillar Ca2+ sensitivity (8-10) because of areduced affinity of TnC for Ca2+ (11). Decreased Ca2+ responsiveness may function as a negative feedback mechanism or contribute to the increased relaxation rate of beating hearts under the influence of &adrenergic stimulation (12). Thefunction of C-protein in cardiac muscle is currently unknown as are theconsequences, if any, of its phosphorylaOver the past 20 years, manylaboratories have focused tion. Phosphorylation of C-protein does not appear, for extheir investigation on the p-adrenergic regulation of cardiac ample, to have a role in modulating myofibrillar Ca’+ sensicontractility through phosphorylation of cellular substrates tivity in mammalian heart cells (9). by the CAMP-dependent protein kinase (PKA)‘ (1, 2). Based Another importantsignaling protein kinase, protein kinase on these studies, there is now general agreement that PKAmediated protein phosphorylation has an important role in C (PKC) (13),also has a role in cardiac physiology. Although myocardial physiology. Stimulation of cardiac myocytes (the its precise function is not yet clear, several lines of evidence regulation. A number characteristic cell type of the heart) by catecholamines or p- implicate this protein kinase in cardiac of receptor systems in heart cells are coupled to PKC activaadrenergic agonists ( i e . isoproterenol) produces a n elevation tion, including the muscariniccholinergic (14), angiotensin I1 of intracellular Ca2+. This occurs primarily through PKAmediated phosphorylation and activation of sarcolemmal Ca2+ (15), and&,-adrenergic (14,16-19) receptor systems. Furthermore, phorbol esters (activators of cellular PKC) are known * This work was supported by National Institutes of Health Re- to have a pronounced negative inotropic effect on a variety of search Grants HL-15696 and CA-36777 (to J. F. K.) and National cardiac preparations, including the single cardiomyocyte (15, Research Service Award F32 HL08615 (to R. C. V.). The costs of 20, 21). It is assumed that the inhibitory effect is mediated publication of this article were defrayed in part by the payment of Very little is page charges. This article must therefore be hereby marked “aduer- via PKC-catalyzedproteinphosphorylation. known, however, with regard to which myocardial substrates tisement” in accordance with 18U.S.C. Section 1734 solelyto indicate are important in the contractile response. Also unclearis this fact. $ TOwhom all reprint requests and inquiries should be addressed: whetherthecontractilemachinery is affecteddirectly or Division of Cardiology, P.O. Drawer LL, Emory University School of whether it isonly indirectly affected through changes in Ca2+ Medicine, Atlanta, GA 30322. Fax: 404-727-3330. homeostasis. However, PKC target substrates are likely to be The abbreviations used are: PKA, CAMP-dependent protein kifound in the myofilament compartment of heart cells since nase; PKC, protein kinase C; TnI, troponin I; TnT, troponin T; TnC, troponin C; MLC, myosin light chain; TPA, 12-0-tetradecanoylphor- the enzyme is translocated to themyofibrils upon oc-adrenerbol-13-acetate. gic stimulation (17) andhas beenimmunocytochemically

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Cardiac MyofibrillarProtein Phosphorylation by PKC and PKA

localized at these sites in unstimulated myocytes (22). More- phosphate-free Dulbecco's minimum essential medium. The viability over, several myocardial contractile proteins are stoichiomet- of isolated cells was assessed by microscopic inspection. Cells were rically phosphorylated by PKC in vitro including C-protein considered viable if they had a rod-shaped appearance and showed no evidence of blebs or granulations. Only cellsuspensions of greater (23),TnI, and TnT(24-26).Phosphorylation of T n I or T n T that 70% viability were used in the experiments. by PKC in the troponin or troponin-tropomyosin complex Labeling of Myofibrils in Intact Cardiomyocytes with p2P]Orthoresults in a decreased Ca2+-dependent MgATPase activity in phosphate-Isolated myocytes from one rat heart were suspended in 10 ml of phosphate-free Dulbecco's minimum essential medium conreconstituted actomyosin (26). Whether this mechanism is involved in the negative inotropic effect of phorbol esters on taining [32P]orthophosphate(1.0 mCi/ml). Cells were incubated in a intact myocardial cellsis presentlyunclear. Also, the question culture flask for 2 h at 37 "C in a 5% CO, humidified incubator. After 2 h, aliquots (1 ml) of 32P-labeledcell suspension were pipetted into of whether these or other myofibril proteins are phosphoryl- tubes. 100 nM TPA, 100 nM isoproterenol, 50 p~ phenylephrine plus ated by PKC in adult heart cells has not been definitively 1 FM propranolol, or 100 nM calyculin A (in dimethyl sulfoxide, 10 answered. pl) was added, and further incubation at 37 "C was carried out for 10 Previous investigation in this laboratory hasprovided evi- min. Control incubations were performed by adding dimethyl sulfdence that TnT and, toa lesser extent, TnI are phosphoryl- oxide (solvent control) only. At the end on the incubation period, ated in neonatal ratcardiomyocytes upon activation of PKC cells were quickly pelleted in a microcentrifuge (30 s at 100 X g) and washed twice in ice-cold physiological saline containing protease by the phorbol ester, 12-0-tetradecanoylphorbol-13-acetate inhibitors (1 mM phenylmethylsulfonyl fluoride, 0.1 mg/mlleupeptin, (TPA) (22).Determination of the stoichiometry, sitespecific- 0.1 mg/ml antipain, 0.1 mg/ml pepstatin A and 5 mM EGTA) and ity, and functional consequences of these phosphorylation protein phosphatase inhibitors (0.1 mM sodium orthovanadate and reactions is now required to assess their physiological signif- 100 nM calyculin A). Myocyte pellets were then homogenized in icecold "inhibiting" buffer (32), modified to contain 1%Triton X-100 icance. Furthermore, it is important to determine whether plus the protease and phosphatase inhibitors indicated above, and these reactions occur in adult heart cells since neonatal and left on ice for 30 min. Detergent-extracted myofibrils were pelleted adult mammalian hearts express different isoforms of both at 5,000 X g. Myofibril proteins were then immediately solubilized in T n I (27)and TnT (28).Cloning and sequencing of rat neo- sodium dodecyl sulfate (SDS) sample buffer by heating for 5 min a t natal TnI hasrevealed that it lacks the preferred phosphoryl-100 "C. ation sitesfor both PKA- and PKC-mediated phosphorylation Phosphorylation of Isolated Myofibrils by PKC and PKA-Deter(27).This may explain (at least in part) the relative insensi- gent-extracted cardiac myofibrils were prepared from previously frotivity of the immature myocardium to the inotropiceffects of zen adult rat hearts by the method described by Stull and Buss (33) for preparation of myofibrils from beefhearts. The protease inhibitors both a - and /3-adrenergic agonists (29).Finally, further inves- indicated above (but not the phosphatase inhibitors) were included tigation of PKC effects inadultheart cells iswarranted in all isolation buffers. PKC was purified from bovine brain through because, indisagreementwith our publishedresults with the phenyl-Sepharose step as described previously (34). Bovine brain neonatal myocytes (22),other studies haveconcluded that PKA was purified according to the method of Miyamoto et al. (35). PKC does not phosphorylate cardiac myofibrillar proteins in Isolated myofibrils were phosphorylated by PKC in the presence and of phospholipids, diolein, and Ca" under conditions deadult mammalian hearts (30,31).In the present study, there- absence scribed previously (36), except that l mM sodium orthovanadate was fore, we have examined and compared TPA-, a-adrenergic-, included in all incubations to prevent ATP depletion by the myofiand /3-adrenergic-stimulated phosphorylation of myofibrillar brillar MgATPase activity. Phosphorylation by PKA was carried out proteins in isolated adult rat cardiomyocytes. Phosphoryla- under the same conditions in the presence and absence of20 p~ tion of native myofibrils by PKC and PKA in vitro has also CAMP. Gel Electrophoresis and Autoradiography-One-dimensional SDSbeen investigated. PKC-mediated phosphorylation has been gel electrophoresis of 32P-labeledmyofibril proteins compared with PKA-mediated phosphorylation in terms of polyacrylamide was carried out on 5-15% acrylamide gradient gels. Following elecits differential substrate specificity, stoichiometry, site speci- trophoresis, proteins were stained with either silver nitrate or with ficity, and functionalconsequences. Coomassie Blue. Gels were then dried and subjected to autoradiogEXPERIMENTALPROCEDURES

Materials-Collagenase B was purchased from Boehringer Mannheim; phosphate-free Dulbecco's minimum essential medium was from GIBCO; calyculin A and TPA were from LC Services Corp.; forskolin, dibutyryl CAMP, (-)-isoproterenol HCl, phenylephrine HCI, propranolol HCI, trypsin (diphenylcarbamyl chloride-treated), phosphorylase b, phosphorylase kinase, phosphatidylserine, diolein, CAMP, 3-isobutyl-l-methylxanthine,phosphoserine, phosphothreonine, and calmodulin were from Sigma; [32P]orthophosphate and [y-32P]ATPwere from ICN Radiochemicals. Cardiac myosin light chainsand gizzard and skeletal myosin light chain kinase were generously provided by Dr. James T. Stull, University of Texas Southwestern Medical Center at Dallas. TnI and TnT,purified from bovine heart, were kindly supplied by Dr. Thomas A. Noland, Jr. of this laboratory. Isolation of Cardiomyocytes-Adult rat cardiac myocytes were prepared from 2-4-month-old Sprague-Dawley rat hearts essentially as described by Kaku et al. (19). Excised hearts were retrogradely perfused at 37 "C with Earle's balanced salt solution containing 15 mM glucose and 1 mMMgC1, which was continuously gassed with 95% O,, 5% CO,. Initial perfusion was for 3-5 min with Ca2+-freeEarle's balanced salt solution followed by a 20-25-min perfusion with recirculating Earle's balanced salt solution containing 50 p M CaCL and 1 mg/ml collagenase. The ventricles of the digested heart were minced with a scissors, and the liberated cells were filtered through 390-pm nylon mesh. Cells were allowed to settle in a 20mM Hepes (pH 7.4), containing 137 mM NaCI, 4.9 mM KC1,l.Z mMMgC12, 14 mM glucose, and 0.25 mM CaC1,. Cells were then resuspended and allowed to settle in the same medium containing 0.5 mM CaCI2 followed by a final settling step in 1 mM CaC1,. Myocytes were then resuspended in

raphy. In some experiments, radiolabeled proteins were excised from the gel, and 32Pincorporated into protein was quantitated by liquid scintillation counting. Determination of Stoichiometry of Phosphate Incorporation into Myofibrillar Proteins in Cardiomyocytes or Isolated Myofibrils-Mol of phosphate incorporated into myofibril proteins in vitro was calculated based on the amount of 32Ppresent in excised protein bands and the known specific radioactivity of the [y-32P]ATPused. Mol of phosphate incorporated into proteins in cardiomyocytes in response to various treatments was determined by quantitating the increase (over basal) in the amount of 32Pin excised bands of individual proteins and determining the specific radioactivity of intracellular ATP by the method of England and Walsh (37). Mol of protein in gel bands was determined by densitometric scanning of Coomassiestained gels and comparison with standards of pure, isolated Cprotein, TnT, TnI, andmyosin light chain 2 (MLC2).Isolated TnT, TnI, andMLC2 preparations were those described under "Materials". C-protein was purified from rat heart by the method of Hartzell and Glass (38). The amount of protein in standards was quantitated by the method of Bradford (39). Calculation of mol of protein was based on a molecular mass of 150, 43, 30, and 19 kDa for C-protein, TnT, TnI, andMLCP, respectively. Two-dimensional Phosphopeptide Mapping and Phosphoamino Acid Analysis-Two-dimensional phosphopeptide mapping of radiolabeled proteins excised from gels was performed essentially as described by Noland et al. (25). Electrophoresis in the first dimension was for 2 h at 450 V in pyridine:acetic acidwater (1:10:89, v/v). Ascending chromatography in the second dimension was carried out in butan0l:pyridine:acetic acidwater (125:100:3:125, v/v). Phosphopeptides were visualized by autoradiography. Autoradiograms of proteins phosphorylated under different conditions in situ were exposed

Cardiac Myofibrillar Protein Phosphorylation to x-ray film for equal lengths of time. Proteins were phosphorylated in vitro with a higher specific radioactivity of ATP and, therefore, were exposed to film for shorter times. Two-dimensional phosphoamino acid analysis was performed according to Cooper et al. (40). Hydrolyzed samples were electophoresed for 45 min a t 1,250 V in pyridine:acetic acidwater (0.5:5:94.5, v/v) followedby ascending chromatography in isobutyric acid, 0.5 M ammonium hydroxide (5:3, v/ v). Phosphoamino acids were visualized by ninhydrin spraying and autoradiography. Actomyosin MgATPase Assay-The effects of TPA and isoproterenol on myofibrillar actomyosin MgATPase activity were determined in experiments similar to those used to analyze phosphorylation. Myocytes from three rat hearts were pooled and incubated in 30 ml of Dulbecco's minimum essential medium for 2 h. The cell suspension was divided into 3 aliquots of 10 ml each and incubated for a further 0.2 100 nM TPA, or0.6 100 nM 10 min with either0.2no addition (control), isoproterenol. Myofibrils were then prepared as described by Murphy and Solaro (41) except that 100 nM calyculin A was included in all isolation buffers to prevent dephosphorylation. Protein concentrations of the myofibrillar preparations were determined by the method of Bradford (39). Ca"-dependent actomyosin MgATPase activities of equal quantities (60 pg) of myofibrillar protein from each condition were measured at different concentrations of free Ca2+,varied through the use of Ca2+/EGTAbuffers (26).

by PKC and PKA

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TABLE I Stoichiometry of phosphorylation of cardiac myofibrilproteins Stoichiometry of phosphorylation was determined as described under "Experimental Procedures." Values represent the average of two experiments. In uitro Protein

In situ phosphorylation in response to

by

TPAPKAPKC

C-protein 1.5 Troponin T ITroponin MLCZ

Isoproterenol

mol phosphutelmol protein 1.1 1.3

1.5 1.3 0.8

0.9

0.8

1.0

\

.o

RESULTS

Phosphorylation of Cardiac Myofibrils by PKC and P K A in Vitro-Isolated cardiac myofibrils from adult rat heart contained proteins identified by Coomassie staining of SDSpolyacrylamide gels as myosin heavy chain (205 kDa), Cprotein (150 kDa), actin (45 kDa),T n T (43 kDa), tropomyosin (35 kDa), TnI (30 kDa), myosin light chain 1 (MLC1) (27 kDa), and myosin light chain 2 (MLC2) (19 kDa) (Fig. 1). Myofibrils were examined as substrate for PKC and PKA in vitro (Fig. 1 and Table I). The PKA holoenzyme selectively phosphorylated C-protein and TnI in myofibrils in a CAMPdependent manner. Athird phosphorylated proteinvisible on autoradiograms represented autophosphorylationof the PKA regulatory subunit. Phosphorylation of the two myofibrillar proteins was maximal after a 60-min incubation a t 30 "C and reached a stoichiometry of 1.5 and 0.9 mol of phosphate/mol FIG.2. Phosphorylation (in situ)of myofibrillar proteins in of C-protein and TnI,respectively. Phosphorylation of intact isolated cardiomyocytes in response to TPA and isoprotereAUTOSTAINRADIOGRAM

C-prot

M L C 2 M

I

-

. }MLC2

~-

PKC

pSIDO/Ca PKA CAMP

+ + - --- +-- -+- -++

FIG.1. Phosphorylation of isolated adult rat cardiac myofibrils in vitroby PKC and PKA. Myofibrils were incubated with [y3*P]ATP and either PKC or PKA for 60 min a t 30 "C.Incubations with PKC were in the absence or presence of phosphatidylserine/ diolein/Ca*+ (PS/DO/Ca), and incubations with PKA were in the absence or presence of 20 p~ CAMP. Phosphorylated proteins were analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography. Major myofibril proteins were identified by Coomassie staining as myosin heavy chain (MHC), C-protein (C-prot), actin, TnT, tropomyosin, TnI, MLC1, and MLC2. The results shown are representative of at least three experiments.

nol. Isolated cardiomyocytes were incubated for 2 h with "Pi and for an additional 10 min with either no addition (control), 100 nM TPA, or 100 nM isoproterenol (Isop.). 32P incorporation into proteins of detergent-extracted myofibrils was analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. A typical autoradiogram is shown. Five major 32P-labeledproteins were identified by Coomassie staining (data notshown) as C-protein, TnT, tropomyosin, TnI, and MLC2. The results shown are representative of at least 12 experiments.

myofibrils by PKC in uitro was more extensive than that by PKA. PKC incorporated phosphate stoichiometrically into four myofibrillar proteins identified as C-protein (1.5 mol/ mol), T n T (1.3 mol/mol), TnI (0.8 mol/mol), and MLCZ (0.6 mol/mol). Phosphorylation was maximal within 60 min and was completely dependent on the presence of phospholipid, diolein, and Ca2+. Phosphorylation of Cardiac Myofibrils by PKC and PKA in Cardiomyocytes-The PKC- and PKA-mediated phosphorylation of cardiac myofibrillar proteins was also investigated in isolated adult rat cardiomyocytes (Fig. 2 and Table I). Myofibrils prepared from "P-labeled control myocytes contained phosphate label in five different myofibrillar proteins identified by Coomassie and silver staining ofgels as C-protein, TnT, tropomyosin, TnI, and MLC2. Identities of C-protein, TnT, TnI, and MLCZ were confirmed by comigration of these 32P-labeledproteins on polyacrylamide gels with the single, isolated cardiac proteins (data not shown). MLC2 was additionally identified as theonly myofibrillar protein phosphorylated in vitro by myosin light chain kinase at sites identical to

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Phosphorylation by PKC and PKA

those modified in situ? Activation of PKA in myocytes by isoproterenol treatment (100 nM for 10 min) resulted in the stoichiometric phosphorylation of two proteins in isolated myofibrils, C-protein (1.3 mol/mol) and TnI (1.0 mol/mol). In addition, a small increase in phosphate content of MLC2 (0.2 mol/mol) was consistently observed. PKA activation by forskolin (20 PM), dibutyryl CAMP (1 mM), or 3-isobutyl-lmethylxanthine (100 PM) gave qualitatively andquantitatively similar results (data not shown). The effect of TPA (100 nM for 10 min) on the stateof phosphorylation of isolated myofibrils was also strikingly similar to that of isoproterenol in terms of its substrate specificity and phosphorylation stoichiometry. Activation of endogenous PKC by the phorbol ester resulted in stoichiometric phosphorylation of both Cprotein (1.1 mol/mol) and TnI (0.8 mol/mol). Also, as observed with @-adrenergicstimulation, MLC2 was phosphorylated to a small extent (0.2 mol/mol) in response to TPA. In addition to the experiments in which 32P-labeled myofibrils were isolated and analyzed, other experiments were carried out in which 32P-labeledmyocytes were incubated for 10 min with either no addition (control), 100 nM TPA, or 100 nM isoproterenol and then directly solubilized in SDS sample buffer prior to analysis of proteins by gel electrophoresis and autoradiography. The purpose of these experiments was to determine whether the phosphorylation state of isolated myofibril preparations accurately reflected that of the intact myocyte. Direct solubilization excludes the involvement of phosphorylation/dephosphorylationevents that might occur during the washing, homogenization, and extraction procedures used to isolate myofibrils. The results of direct solubilization experiments were very similar to those obtained with detergent-extracted myofibrils except for a higher background of unidentified 32P-labeledproteins on gels (data notshown). The dose dependence and time dependence of TPA- and isoproterenol-stimulated phosphorylation were also investigated. TPA-stimulated phosphorylation was detected at 1nM and was maximal at 10 nM. Isoproterenol effects were detected at 1 nM and were maximal at 100 nM. Phosphorylation in response to isoproterenol reached its maximum within 2 min of exposure to the agonist, In contrast, TPA stimulation required 10 min to reach a maximum. Two-dimensional Phosphopeptide Mapping of TnI and Cprotein Phosphorylated in Situ and in Vitro-The site specificity of phosphorylation of TnI in response to TPA- and@adrenergic stimulation was examined by two-dimensional tryptic phosphopeptide mapping. A pattern of six tryptic phosphopeptides was obtained upon mapping of TnI phosphorylated under basal (control) conditionsin myocytes (Fig. 3A). The number of tryptic phosphopeptides may be greater than thenumber of phosphorylation sites because of alternate cleavages by trypsin adjacent to certain charged amino acid or phosphoserine residues. TPA treatment resulted in an increase in 32Pcontent of three phosphopeptides (spots 5, 6, and 8) and the appearance of a new phosphopeptide (spot 7 ) (Fig. 3B). Isoproterenol treatment stimulated phosphorylation of clearly different sites in TnI, as revealed by a very different pattern of phosphorylated peptides (Fig. 3C). No increase in labeling of phosphopeptides 5, 6, and 8 was observed in this condition. Instead, one very large and diffuse spot appeared (labeled as 9 on the map). Poor resolution of this spot may relate to thefact that PKA phosphorylatestwo adjacent residues (Ser-22 and Ser-23) in adult ratcardiac TnI (27). The large spot on the map probably represents a mixture of monophosphorylated and biphosphorylated derivatives of the same tryptic peptide. Sites in TnI that were phosphoryl*R. C. Venema, R. L. Raynor, and J. F. Kuo, manuscript in preparation.

A. Control (in situ)

B. TPA (in situ)

C. Isop. (in situ) I

D. CL-A (in situ)

E. PKC (in vitro)

F. PKA (in vitro)

-

FIG.3. Identification of sites phosphorylated in cardiac TnI in response to TPA, isoproterenol, and calyculin A (in situ) or by PKC and PKA (in vitro).Myofibrils were phosphorylated in situ in 32P-labeledcardiomyocytes by incubation for 2 h with 32Pi followedby afurther 10-min incubation with either no addition (control), 100 nM TPA, 100 IIM isoproterenol (hop.), or 100 nM calyculin A (CL-A). Myofibrils were also phosphorylated in vitro by incubation with [T-~'P]ATP and either PKCor PKA. Equal amounts of myofibrillar protein from each condition were analyzed by SDSpolyacrylamide gel electrophoresis followed by autoradiography. TnI wasexcisedfrom the gel, trypsin digested, and subjected to twodimensional tryptic phosphopeptide mapping. The origins are indicated by arrows. The radioactivity in each sample was: A, control (1,238 cpm); B, TPA (4,255 cpm); C, isoproterenol (4,821 cprn); D, calyculin A (4,561 cpm); E , PKC in oitro (8,222 cpm); F, PKA in vitro (9,560 cpm). Similar results have been obtained in a t least four experiments. ated in situ in response to TPA (Fig. 3B) (phosphopeptides 5, 6, 7, and 8) were also modified in vitro by PKC (Fig. 3E), indicating that direct phosphorylation by PKC occurs in myocytes, as opposed to indirect phosphorylation by a "downstream" kinase (42). Furthermore, the sites in TnI that were phosphorylated in situ in response to isoproterenol (Fig. 3C) (phosphopeptide 9) were also modified in vitro by PKA (Fig. 3F). Phosphopeptide mapping also revealed that minor amounts of nonspecific phosphorylation occurred in vitro (for example, spot 10 in the case of PKC and spots5, 6, 7, and 8 in the case of PKA). Interestingly, nonspecific phosphorylation by PKA in vitro occurred at sites that were phosphorylated only by PKC (and notby PKA) in intact cells. Phosphopeptide mapping was further utilized to investigate the sitespecific phosphorylation of TnI in cardiomyocytes treated with calyculin A, a highly potent inhibitor of phosphoprotein phosphatases 1and 2A (43). Calyculin A (100 nM) stimulated an increase in the TnIphosphate content of 0.9 mol/mol after only 2 min of exposure to the inhibitor (data not shown). Incorporation of phosphate in the presence of calyculin A occurred at both PKC andPKA sites (Fig. 30), indicating a rapid turnoverof phosphate in these sites even in theabsence of hormonal stimulation. One additional phosphopeptide was detected with calyculin A that was not detected with either TPA or isoproterenol (spot 3). This suggests that a third protein kinase may exist which also phosphorylates TnI in myocytes. C-protein phosphorylation was also analyzed by two-dimensional tryptic phosphopeptide mapping. A single tryptic phosphopeptide (spot 5 ) was obtained for C-protein phosphorylated in control myocytes (Fig. 4.4). Mapping of C-protein from isoproterenol-stimulated cells showed an increase in

Cardiac Myofibrillar Protein Phosphorylation

by PKC and PKA

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myofibrillar MgATPase with no change in the Ca2+concentration required for activation (Fig. 5). In contrast, myofibrils from isoproterenol-treated cells showed a decreased Ca2+sensitivity of the enzyme (Ca2+ concentrationrequired for halfmaximal stimulation increased from 0.8 p~ in control to 3.0 p~ in treated cells). No change, however,wasobserved in maximal MgATPase activity (Fig. 5). Phosphorylation of Cardiac Myofibrils in Response to a:.5 Adrenergic Stimulation-The potential involvement of PKCD. CL-A (in situ) E. PKC (In vitro) F. PKA (In vltro) mediated phosphorylation of myofibrillar proteinsin hormonal regulation of myocardial cells was also investigated. aAdrenergic stimulation of cardiomyocytes with 50 p~ phenylephrine and 1 p~ propranolol (10 min) produced a pattern of myofibrillar protein phosphorylation that was qualitatively and quantitatively similar to that observed with TPA-stimulation (data notshown). TnI andC-protein were stoichiometrically phosphorylated in response to thea-adrenergic agonist, whereas MLC2 was phosphorylated to a lesser extent. Furthermore, phosphorylation of TnI following a-adrenergic Electrophorerlr stimulation occurred at sites in the protein identical to those FIG. 4. Identification of sites phosphorylated in cardiac Cprotein in response to TPA, isoproterenol, and calyculin A (in phosphorylated in response to TPAor phosphorylated in vitro situ) or by PKC and PKA (in vitro). Myofibrils were phosphoryl- by PKC (Fig. 6).

I

ated in situ in :'2P-labeledcardiomyocytes by incubation for 2 h with 32Pi followed by a further 10-min incubation with either no addition (control), 100 nM TPA, 100 nM isoproterenol (Zsop.), or 100 nM calyculin A (CL-A).Myofibrils were also phosphorylated in vitro by incubation with [y3*P]ATP and either PKC or PKA. Equal amounts of myofibrillar protein from each condition were analyzed by SDSpolyacrylamide gel electrophoresis followed by autoradiography. Cprotein was excised from the gel, trypsin digested, and subjected to two-dimensional tryptic phosphopeptide mapping. The origins are indicated by arrows. The radioactivity in each sample was: A, control (456 cprn); B, TPA (1,530 cprn); C, isoproterenol (1,690cpm); D, calyculin A (487cprn); E, PKC in vitro (3,360cprn); F, PKA in vitro (3,422cpm). Similar results have been obtained in four separate experiments.

labeling of this phosphopeptide plus labeling of one other major phosphopeptide (spot 3) and three minor phosphorylated peptides (spots 1, 2, and 4 ) (Fig. 4C). TPA treatment produced a pattern of phosphorylated peptides (Fig. 4B)that was indistinguishable from that obtained for the isoproterenol-treated sample. Thus, in contrast to TnI, TPA and isoproterenol stimulated phosphorylationof identical sitesin Cprotein. Furthermore, these same sites were phosphorylated by both PKC andPKA in vitro (Fig. 3, E and F ) . In addition, a small amount of phosphorylation occurred in vitro at nonspecific sites (for example, spots 6, 7,8, and 9 in the case of PKC and spots 7 and 8 in the case of PKA). Calyculin A treatment was not associated with any increased phosphorylation of C-protein (Fig. 4 0 ) suggesting that, in the absence of hormonal stimulation, phosphate turnover in this protein is slow. Additional information about the site specificity of phosphorylation of TnI and C-protein was obtained from phosphoamino acid analysis. TnI and C-protein isolated from control, TPA-, orisoproterenol-stimulated myocytes or phosphorylated in vitro by either PKC or PKA contained phosphate exclusively in phosphoserine. No phosphothreonine was detected for either protein phosphorylated under any of the conditions examined (data notshown). Effectof Phosphorylation in Situ on Myofibrillar Actomyosin MgATPase Activity Assayed in Vitro-The functional significance of myofibrillar protein phosphorylation in cardiomyocytes was also investigated. Myofibrils were prepared from control, TPA-and isoproterenol-stimulated myocytes and assayed for Ca2+-dependentactomyosin MgATPase activity in vitro.TPA-stimulated phosphorylation was associated with a 25 f 2% (mean & S.E.) decrease in maximal activity of

DISCUSSION

In this study, we have identified TnI and C-proteinas physiological substrates for PKC in intact myocardial cells. Previous studies have shown that TnI (24-26) and C-protein (23) are effective substrates for PKC in vitro. It has been important to confirm that these reactions also occur in situ since protein kinases can phosphorylate proteins in cell-free systems that arenot necessarily physiological substrates (44).

Ca "(#"

FIG. 5. Effects of in situphosphorylation of myofibrillar proteins on Ca2+-dependentactomyosin MgATPase activity. Isolated cardiomyocytes were preincubated for 2 h (as described in Fig. 2, except that 32P;was omitted) followedby a further 10-min incubation with either no addition (control), 100 nM TPA, or 100 nM isoproterenol (Zsop.). Myofibrils were then prepared from myocytes, and equal quantities of myofibril protein (60 pg) from eachcondition were assayed for MgATPase activity at various concentrations of calculated free Ca2+.The maximum ATPase activity refers to the totalATPase activity (106 nmol/mg/min) of the control sample assayed a t 100 p~ free Ca2+which was taken as 100%. Each point and bar represent the mean f S.E. for four experiments. Upper panel, control versus TPA. Lower panel,control versus isoproterenol.

Cardiac Myofibrillar Protein Phosphorylation PKC by

2710 A. Control (in

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C. TPA situ) (in

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