1356 Studies on Phosphorylation of Canine Cardiac Sarcoplasmic Reticulum by Calmodulin-Dependent Protein Kinase LOUISE M. BILEZIKJIAN, EVANGELIA G. KRANIAS, JAMES D. POTTER, AND ARNOLD SCHWARTZ SUMMARY Two endogenous protein kinase activities, cAMP-dependent and calmodulin-Ca'^-dependent, are associated with isolated cardiac sarcoplasmic reticulum (SR) vesicles. Both klnases phosphorylate an endogenous substrate of -22,000 daltons (phospholamban). The phosphorylation of phospholamban by either the intrinsic or by exogenous cAMP-dependent protein kinase is found to be Ca1+independent between 0.06 and 100 pM free Ca>+. Calmodulin-dependent phosphorylation, on the other hand, does not require cAMP and is absolutely dependent on the presence of free Ca1+ over a concentration range that corresponds to physiological levels (10~T to 10"* M). Phosphorylation of SR vesicles by both kinasee is additive and the extent of saturation of the cAMP-gpeciflc sites has no effect on the degree of stimulation by cahnodulin or its Cal+-dependence. Trifluoperarine, an inhibitor of calmodulin, inhibitg cahnodulin-dependent phosphorylation without affecting cAMP-dependent phosphorylation, indicating the presence of two types of kinases. This is made further evident by the selectivity of each kinase for exogenous substrates. Whereas cAMP-dependent protein kinase appears to phosphorylate histone QA (a basic protein) preferentially, cabnodulin-dependent protein kinase prefers phosvitin (an acidic protein). Ore Res 49:1356-1362, 1981 CARDIAC sarcoplasmic reticulum (SR) is phosphorylated by both endogenous and exogenous cAMP-dependent protein kinases (cAMP-protein kinase) (LaRaia and Morkin, 1974; Tada et al., 1975; Kirchberger and Tada, 1976; Schwartz et al., 1976; Wray and Gray, 1977). Cyclic AMP-dependent phosphorylation is associated with stimulation of both Ca2+ transport and Ca2+-dependent ATPase activities (Ca2+-ATPase) (LaRaia and Morkin, 1974; Tada et al., 1974; Wray and Gray, 1977) of SR vesicles and may represent an important regulatory mechanism. The substrate for these kinases has been shown to be a membrane component of Mr~22,000 as determined by dodecyl sulfate-polyacrylamide gel electrophoresis (Tada et al., 1975). This component, referred to as phospholamban (Kirchberger et al., 1975), was recently purified by Le Peuch et al. (1980) and by Collins et al. (1981) and it appears to be a proteolipid. Recently, another possible regulatory mechaFrom the Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio. This research was supported in part by U.S. Public Health Service Grants P01HL 22819-03 (III A, HI E, IV A), in part by a Grant-in-Aid from the American Heart Association (78-1167), and by a Grant-in-Aid from the Southwestern Ohio Chapter of the American Heart Association. Submitted by L. M. Bilezikjian in partial fulfillment of the Doctor of Philosophy degree. Dr. Bflezikjian was supported by National Institutes of Health Training Grant T32 HL 07382-02. Address for reprints: Dr. Arnold Schwarti, Department of Pharmacology and Cell Biophysics, University of Cincinnati, College of Medicine, 231 B«thesda Avenue, Cincinnati, Ohio 46267. Received December 4, 1380; accepted for publication September 1, 1981. nism of cardiac SR was also proposed. Katz and Remtulla (1978) reported that incubation of cardiac SR with calmodulin, in the presence or absence of cAMP and cAMP-protein kinase, results in an enhancement of both Ca2+ binding and Ca2+ transport. However, no mechanism was postulated. Le Peuch et aL (1979) and we (Bilezikjian et al., 1980) have since suggested that regulation of SR by calmodulin may involve activation of a Ca2+-dependent protein kinase. Calmodulin, an ubiquitous, multifunctional calcium-dependent regulator protein is now recognized to modulate various cellular functions (Wolff and Brostrom, 1979; Cheung, 1980). Le Peuch et al. (1979) report that, in the presence of 0.5 mM CaCU, calmodulin stimulates phosphorylation of phospholamban. This is totally inhibited in the presence of 5 mM EGTA but is unaffected by the cAMP-protein kinase inhibitor. Our study was undertaken to characterize further the mechanism by which regulation of cardiac SR is achieved by calmodulin. Our results confirm those of Le Peuch et al. (1979) in that cahnodulin stimulates phosphorylation of cardiac SR. Calmodulin-dependent phosphorylation is totally Ca2+-dependent; however, unlike the high Ca2+ concentration used by Le Peuch et al. (1979), our data indicate that calmodulin-stimulated phosphorylation can occur within a range of Ca2+ concentrations compatible with levels postulated to occur during the contraction-relaxation cycle of the heart (Katz, 1977). Our results confirm previous reports (Tada et al., 1975) showing that phospholamban is ~22,000 daltons, although it is possible that this represents Downloaded from http://circres.ahajournals.org/ by guest on February 10, 2016 REGULATION OF CARDIAC SARCOPLASMIC RETICULUM/Btfezwfe/ian et al. an oligomer of a lower molecular weight component (Le Peuch et al., 1979). Methods Miscellaneous Methods All biochemical reagents, including beef heart cyclic AMP-dependent protein kinase, were purchased from Sigma Chemical Co. All chemicals were of "Chemical pure grade." Disodium ATP was purchased from Boehringer-Manheim and[7-32P]ATP, ammonium salt (10-40 Ci/mmol), was purchased from New England Nuclear. Trifluoperazine dihydrochloride was a generous gift from Smith Kline and French Laboratories. Calmodulin was prepared from bovine brain tissue by the method of Lin et al. (1974). The levels of contaminating calmodulin in our SR vesicles were determined in 1% Lubrol as described previously (Piascik et al., 1980). The concentrations of free Ca2+, at pH 7.0, were achieved by the addition of sufficient CaCl2 to 500 JIM EGTA as has been described previously (Potter and Gergely, 1975; Piascik et al., 1980). Sodium dodecyl sulfate (0.1%) polyacrylamide (12.5%) gel electrophoresis of ^P.-labeled SR was carried out according to the method of Laemmli (1970). Preparation of SR Vesicles Canine cardiac SR vesicles were prepared by a modification of the Harigaya and Schwartz procedure (Harigaya and Schwartz, 1969; Sumida et al., 1978). The isolation was carried out in the presence of 0.3 M sucrose in 20 mM Tris-maleate buffer (buffer A), pH 7.0. The microsomes enriched in SR were homogenized and washed in 0.6 M KC1 twice, followed by one wash in buffer A. The final pellet was suspended in 30 mM Tris-maleate, pH 7.0, containing 100 mM KC1 and 0.3 M sucrose at a protein concentration of 20 mg/ml. Protein was determined by the biuret procedure using bovine serum albumin as standard. The suspension was quick-frozen in liquid N2 and stored at —75°C. This preparation was found to be stable for several weeks, and no significant decrease in kinase activity was observed during storage. The final yield of SR protein ranged from 0.6 to 0.8 mg per gram of ventricular tissue. The levels of contaminating sarcolemmal and mitochondrial membranes were very low as assessed by [3H]-ouabain binding and cytochrome c oxidase activities, respectively (Sumida et aL, 1978). Phosphorylation of SR Phosphorylation of SR was carried out in a final volume of 0.2 ml at pH 7.0. The reaction medium contained 100 jig SR protein, 50 mM phosphate buffer, 10 mM MgCl2, 10 mM NaF, 1 /tM cAMP, and 500 IJM [ r - m P]ATP. Cyclic AMP-dependent phosphorylation was measured in the presence of 500 fiu EGTA catalysed either by endogenous cAMP- 1357 protein kinase of SR or by saturating concentrations of exogenous cAMP-protein kinase (0.02 mg/ reaction). Calmodulin-stimulated phosphorylation was carried out under identical conditions but in the presence of purified brain calmodulin and added CaCl2 to achieve the desired [Ca2+]. The reaction was carried out at 30°C and allowed to proceed for 2 minutes, at which time maximal phosphorylation was observed (Kranias et aL, 1980). It was then terminated by the addition of 2.5 ml of 7% cold perchloric acid containing 7% polyphosphoric acid. After addition of 0.5 mg of SR carrier protein, the samples were washed three times with the perchloric acid solution, dissolved in 1 ml of 10 mM NaOH and transferred to 9 ml of scintillation counting fluid (Aquasol II, New England Nuclear). M P, incorporation was determined in a model 3320 Packard Tri-Carb liquid scintillation spectrometer. The majority of phosphorylation measured is due to the formation of phosphoester bonds in phosphoLamban of —22,000 daltons, is insensitive to hydroxylamine, and is due to cAMP-dependent or calmodulin-dependent protein-kinases. A small amount (about 12%) of phosphorylation occurs in a high molecular (~ 100,000 dalton) protein and is hydroxylamine sensitive. Results Sarcoplasmic retdculum vesicles isolated as described above contain an endogenous cAMP-protein kinase as previously reported (LaRaia and Morkin, 1974; Wray and Gray, 1977). Incubation of cardiac SR vesicles with 1 /XM C A M P and 500 fifa EGTA resulted in phosphorylation of these vesicles catalyzed by the endogenous cAMP-protein kinase. The extent of phosphorylation, measured by K P, incorporation, varied from one SR preparation to the other with values ranging from 200 to 350 pmols Pi/mg SR. In the absence of cAMP, incorporation of -100 to 200 pmol Pi/mg SR occurred due to the presence of free catalytic subunits of cAMP-protein kinase, since this could be inhibited up to 80% in the presence of the heat-stable inhibitor (Kranias et al., 1980). Addition of cAMP and exogenous cAMP-protein kinase enhanced SR phosphorylation up to 2.5-fold. Since the main purpose of this study was to characterize the role of calmodulin, which is a Ca2+-dependent protein, on SR phosphorylation, it was first essential to determine the effects of calcium on cAMP-dependent phosphorylation. Phosphorylation of SR vesicles, catalyzed by either the endogenous or the exogenous cAMP-protein kinase in the presence of 1 /iM cAMP, was Ca2+independent over the range of 0.05 to 100 MM free Ca2+ (Fig. 1). Under identical conditions (1 /tM cAMP, 500 /tM EGTA), addition of 10~7 M calmodulin did not affect the levels of cAMP-dependent phosphorylation by either the endogenous or exogenous cAMP-protein kinase (Fig. 1). However, as the free Ca2+ concen- Downloaded from http://circres.ahajournals.org/ by guest on February 10, 2016 CIRCULATION RESEARCH 1358 0 pCa 1 The Ca2'*-dependence of cAMP-dependent and calmodulin-dependent phosphorylation of SR vesicles. All reactions were carried out at 30°C in a volume of 0.2 ml as described under Methods. The desired [Ca1*] was achieved by adding appropriate amounts of CaCl? to 500 HM EGTA in the reaction medium (pH 7.0). Cyclic AMP-dependent phosphory lation was catalyzed by the endogenous cAMP-protein kinase of SR (O- - -O, • - - -%), or by exogenous cAMP-protein kinase (0.02 mg/ reaction) (O O, • %) in the absence of (open circles) or presence (closed circles) of 10~7M calmodulin as described under Methods. For each experiment, values were normalized to their maximum, and each point represents the arithmetic mean of 10 determinations (SE values are within 5-10% of the mean). The maximal level (100%) represents 3.5 nmol phosphate/mg. FIGURE tration was raised up to 10 * M, a concentrationdependent increase in ^P, incorporation was observed in the presence of calmodulin (Fig. 1). The extent of stimulation by calmodulin, at any given [Ca2+], was independent of whether cAMP-dependent phosphorylation was simultaneously catalyzed by either the endogenous or exogenous cAMP-protein kinases. Furthermore, the enhancement of SR phosphorylation by calmodulin did not require the activation of cAMP-protein kinase because calmodulin-stimulated phosphorylation occurred to the same extent in the absence of cAMP. These data confirm previously published results (Le Peuch et al., 1979) and further suggest that calmodulin-stimulated phosphorylation may in fact be regulated by concentrations of free Ca + occurring in the cell during the contraction-relaxation cycle (Potter and Gergely, 1975). For calmodulin-stimulated phosphorylation of cardiac SR, the estimated KD'S for Ca2+ were 0.6 and 0.5 /XM in the presence of endogenous and exogenous cAMP-protein kinase, respectively. These values are not significantly different. Inclusion of A23187 (10 /IM) in the reaction to eliminate Ca2+ sequestration into the vesicles had no effect on these KD values. The high [Ca2+] (0.5 mM) used by Le Peuch et al. (1979) is unphysiological and far above the saturation point for the reaction. VOL.49, NO. 6, DECEMBER 1981 The concentration dependence of phosphorylation of SR vesicles by calmodulin in the presence of near optimal [Ca2+] is shown in Figure 2. At a free Ca2+ concentration of 1 [iM, half maximal stimulation of 32Pi incorporation occurred at 45 nM calmodulin. Interestingly, this value is slightly lower than the 70 nM value reported by Le Peuch et al. (1979) measured in the presence of 0.5 mM Ca2+. The basal level of phosphorylation seen in Figure 2, at no added calmodulin, represents MP, incorporation by the endogenous cAMP-protein kinase in the presence of 1 ftM cAMP. It is unlikely that the endogenous calmodulin present in our SR preparation would contribute to this value. Under our experimental conditions, only a maximum of 10 nM contaminating calmodulin would be introduced to the assay since the endogenous calmodulin levels were less than 0.3 jug/mg SR as determined by the method of Piascik et al. (1980). This amount is far less than the concentration of the activator protein required for any effect (Fig. 2). Similar Km values for calmodulin were obtained when calmodulin-dependent phosphorylation was measured in the presence of exogenous cAMP-protein kinase. To determine the specificity of calmodulin in its ability to stimulate phosphorylation, independent of cAMP-protein kinase, the effects of trifluoperazine (TFP), a specific inhibitor of calmodulin (Levin and Weiss, 1977) were determined. In the presence of IO-^M free Ca2+ and 10"7M calmodulin, TFP specifically inhibited the calmodulin-stimulated phosphorylation in a dose-dependent manner with an IC50 of approximately 1.8 X 10"fi M TFP (Fig. 3). Complete inhibition by TFP reduced the levels of SR phosphorylation to those obtained by cAMPprotein kinase, indicating that this drug does not 100 - 0 10"° 10"-7 [Calmodulin] M 10- 6 FIGURE 2 The effect of increasing concentrations of calmodulin on phosphorylation of SR vesicles. The reaction was carried out in the presence of 1 /IM cAMP, 500 IIM EGTA, and 370 /uw CaCk (1 IIM free Cai+). SR vesicles (0.1 mg/reaction) were incubated for 2 minutes at 30°C with increasing amounts of calmodulin as described under Methods. Downloaded from http://circres.ahajournals.org/ by guest on February 10, 2016 REGULATION OP CARDIAC SARCOPLASMIC RETICULUM/BiZeziJb/ia/i et al. 100 10 -4 [Trifluoperazine] M 3 The inhibition of calmodulin-stimulated phosphorylation by trifluoperazine (TFP). Calmodulin was preincubated with varying amounts of TFP for 30 minutes at room temperature. The mixture then was added to the phosphorylation reaction mixture to give a final concentration of 10~7M calmodulin. The reaction conditions were: 1 yM cAMP, 500 pM EGTA (O- - -O); 1 fiM cAMP, 500 fiM EGTA, and 370 pM CaCk (1 p.M free Ca2+) in the absence (O O) or presence ( • 9) of 10~7M calmodulin. FIGURE dependent phosphorylation of SR vesicles implies that two classes of sites are available for each process (Fig. 1). It would be reasonable to speculate from these data that site specificity is a consequence of the occurrence of two different types of protein kinases associated with SR. We have previously reported that the endogenous cAMP-protein kinase of SR preferentially uses basic polypeptides as substrates (Kranias et al., 1980). As shown in Table 1, the endogenous cAMP-protein kinase (in the presence of 1 ftM cAMP, 500 fiM EGTA) preferentially phosphorylated histones (a basic polypeptide) much less than did phosvitin (an acidic substrate). Calmodulin-protein kinase (in the presence of 0.1 /XM calmodulin and 1 /IM free Ca2+), on the other hand, was most effective with phosvitin (an acidic protein) as a substrate. The inhibition of SR phosphorylation catalyzed by the calmodulin-protein kinase, in the presence of histone, may be explained I >- -36 -24 -20 - 18 - 16 - 14 TABLE 1 Phosphorylation* of Endogenous and Protein kinase activity (pmol Pi/mg)t Added substrate None His tone Protamine Casein Phosvitin cAMP-PK CAL-PK 247 540 237 274 340 0 -69 interfere with cAMP-dependent phosphorylation. Similarly, skeletal Troponin I (Tnl), which has been shown to bind cabnodulin specifically, thus preventing its effects (Kerrick et al., 1980), also caused inhibition of the calmodulin-stimulated phosphorylation without affecting the cAMP-dependent process. Furthermore, Troponin C, another calcium-binding protein structurally homologous to calmodulin (Potter et al., 1977), was ineffective and could not be substituted for calmodulin within the same concentration range where calmodulin is effective. The additive nature of cAMP and calmodulin- Exogenous Substrates by Cardiac SR cAMPDependent and Calmodulin-Deptndent Protein Kinases 1359 -DF b a Activity cAMP-PK CAL-PK 717 100 513 712 668 1100 219 96 111 138 100 72 99 93 153 cAMP-PK = cAMP-dependent protein kinese, CAL-PK - calmodulin-dependent protein kinase. * Phosphorylation of SR was carried out as described previously. Protein substrates were added at a concentration of 0 2 mg per reaction and assayed in the presence of either cAWP (1 JIM) alone or calmodulin (0 1 IXM) and CaJ* (1 jiM-fre*). f Values represent total *P, incorporation (SR and exogenous substrates) and they are the arithmetic means of three determinations each. i Values represent "Pi incorporation relative to control (100%). FIGURE 4 Autoradiogram of a 0.1% SDS-I2.5% acrylamide one-dimensional slab-gel. SR vesicles were phosphorylated with fy-^PJATP at 30°Cos described under Methods. Samples then were dialyzed against 1% SDS and 1% fi-mercaptoethanol before they were loaded on the gel. Conditions of phosphorylation were: a — 1 \XM cAMP and 500 fiM EGTA; b - I jiM cAMP, 500 pM EGTA 10'7M calmodulin; c = same as b, plus 370 \IM CaCh (1 /AM free Cai+). O = origin, DF = dye front. Molecular weight markers were: human serum albumin (M, 69,000); purified skeletal whole troponin (Mr 36,000, 24,000, 18,000); purified cardiac myosin light chains (Mw 20,700,19,000,16,500); lysozyme(M, 14,400). The numbers represent (Mr x 1CT*). Downloaded from http://circres.ahajournals.org/ by guest on February 10, 2016 CIRCULATION RESEARCH 1360 by the ability of histones to bind calmodulin (Itano et al., 1980). Such selective ability of SR protein kinases to phosphorylate exogenous substrates is an indication for the presence of two types of protein kinases, cAMP-dependent and calmodulin-dependent. T h e substrate for endogenous cAMP and calmodulin protein kinases was determined by autoradiography of one-dimensional 0.1% SDS-12.5% polyacrylamide slab gels. As shown in Figure 4, ^ P , is associated with a single low molecular weight protein band of ~22,000 daltons in agreement with previous reports (Tada et al., 1975). The same protein is phosphorylated by both cAMP- and calmodulin-protein kinases. Similar results were obtained with a linear gradient gel (0.1% SDS, 7-30% polyacrylamide). The small amount of ^ P , associated with a high molecular weight protein (presumably the Ca 2+ -Mg 2+ -ATPase) is accounted for by the formation of a small amount of phosphorylated intermediate of the Ca 2+ -Mg 2+ -ATPase (E~P) when free calcium is present. Under these conditions, 12% of the 32 P, incorporated was hydroxylamine sensitive, confirming the presence of some E ~ P (Wray and Gray, 1977; Kranias et al., 1980). Contrary to the report by Le Peuch et al. (1979), we did not detect an 11,000 dalton phosphorylated protein presumably representing the monomer of phospholamban. Our attempts to separate the dimer either with a combination of Triton X-100 and S D S or other methods were not successful. Recently, however, we have found that boiling the preparation for five minutes in SDS seems to convert the 22,000-dalton protein to an 11,000-dalton polypeptide. Discussion The results presented here confirm the presence of two types of phosphorylation of cardiac sarcoplasmic reticulum (SR) (Le Peuch et al., 1979). We, as well as others, have previously demonstrated the association of cAMP-protein kinase with SR vesicles (Wray et al., 1973; LaRaia and Morkin, 1974; Kranias et al., 1980). This report provides evidence for the additional presence of a calmodulin-dependent protein kinase (calmodulin-protein kinase) which remains tightly associated with SR membranes. This is the first report showing the prsence of two endogenous kinases in cardiac SR. Le Peuch et al. (1979), in their report, failed to show any endogenous cAMP-protein kinase activity thus cAMP-dependent phosphorylation could only be obtained with the addition of exogenous free catalytic subunit of cAMP-protein kinase. The activation of the calmodulin-protein kinase is independent of cAMP and is totally dependent on the presence of free Ca2+ and calmodulin. The two activities appear to be additive in agreement with a previous publication (Le Peuch et al., 1979). VOL. 49, No. 6, DECEMBER 1981 Our data suggest that calmodulin and cAMP represent two independent pathways sharing a common substrate of 22,000 dalton (phospholamban). However, each kinase appears to phosphorylate distinct sites on phospholamban. At this time, however, it is not clear what the actual molecular weight of phospholamban is. Our data as well as previously published data (Kirchberger et al., 1975; Tada et al., 1975) suggest that phospholamban is a 22,000 dalton protein. The Mr -11,000 for this substrate reported by Le Peuch et al. (1979) is of interest but needs further investigation. The level of saturation of cAMP-dependent sites does not seem to affect the ability of calmodulin to stimulate ^Pi incorporation. These results are similar to those reported by Le Peuch et al. (1979), who also showed the presence of two classes of phosphorylatable sites on phospholamban. Assuming that phospholamban constitutes approximately 3% of the SR protein, our results show that up to 1.1 mol phosphate/phospholamban molecule (Mr ~22,000) is incorporated by exogenous cAMP-protein kinase within 2 minutes of incubation at 30°C. Simultaneous phosphorylation of phospholamban by cAMP- and calmodulin-dependent protein kinases increases this number up to 2.06 mol phosphate/phospholamban molecule. The presence of two distinct kinases is substantiated further by the selective inhibition of calmodulin-stimulated phosphorylation of SR by trifluoperazine (TFP) and skeletal troponin I (Tnl). Both TFP and Tnl prevent calmodulin-stimulated phosphorylation but they have no effect on cAMP-dependent phosphorylation. The absolute dependence of the membrane-bound calmodulin-dependent kinase on calmodulin distinguishes it from previously reported Ca2+-dependent kinases in skeletal SR and cardiac SR (Schwartz et al., 1976; Horl and Heilmeyer, 1978) which were postulated to be phosphorylase kinase. The preferential phosphorylation of histone by cAMP-protein kinase, in contrast to phosvitin by calmodulin-protein kinase, also suggests that two kinases are in fact associated with SR vesicles. The significance of calmodulin-stimulated phosphorylation is implicated by its Ca2+-dependence. Although Le Peuch et al. (1979) have presented evidence for cahnodulin-Ca2+-dependent phosphorylation of SR, the high [Ca2+] (0.5 HIM) used in their experiments precludes a physiological interpretation of their data. Since calmodulin is not a limiting factor in cardiac tissue (Grand et al., 1979), the trigger for activation of the calmodulin-dependent protein kinase must be fluctuations of myoplasmic [Ca2+]. As we have shown here, the levels of [Ca2+] required for calmodulin-stimulated phosphorylation are within a physiological range (10~7 to 10~5 M). Thus, it is entirely possible that calmodulin is an important regulator in cardiac muscle. The role of calmodulin as a regulator of Ca2+- Downloaded from http://circres.ahajournals.org/ by guest on February 10, 2016 REGULATION OF CARDIAC SARCOPLASMIC RETICULUM/Bilezikjian et al. transport of SR has been suggested (Katz and Remtulla, 1978; Le Peuch et al., 1979). Katz and Remtulla (1978) have shown that Ca2+-transport as well as Ca2+-binding are stimulated significantly by calmodulin above that seen by cAMP-protein kinase. Ca2+-ATPase also appears to be stimulated (Lopaschuk et al., 1980). However, stimulation is suggested to be mediated by a direct effect of calmodulin on SR membranes and not by phospholamban phosphorylation. This is in contrast to data presented by Le Peuch et al. (1979), who claim that the wellcharacterized stimulation of Ca2+ transport by cAMP-protein kinase (Kirchberger et al, 1979; Tada et aL, 1974; Wray and Gray, 1977) is in fact mediated by calmodulin. Our preliminary results show that Ca2+ transport is enhanced in SR vesicles prephosphorylated in the presence of either cAMP or calmodulin. Further investigation of the role of calmodulin in SR function is necessary to provide an understanding of the mechanism by which calmodulin may regulate cardiac SR function. Acknowledgments We are grateful to Dr. Michael Piascik for the determination of the calmodulin content of our SR preparation. References Bilezikjian LM, Kranias EG, Potter JD, Schwartz A (1980) Calmodulin-stimulated phosphorylation of cardiac sarcoplaamic reticulum (abstr). Fed Proc 39: 1663 Cheung WY (1980) Calmodulin plays a pivotal role in cellular regulation. Science 207: 19-27 Collins JH, Kranias EG, Reeves AS, Bilezikjian LM, Schwartz A (1981) Isolation of phospholamban and a second proteolipid component from canine cardiac sarcoplasmic reticulum Biochem. Biophys. Res. Commun. 99: 796-803 Dabrowska R, Sherry JMF, Aromatorio DK, Hartshorne DJ (1978) Modulator protein as a component of the myosin light chain kinase from chicken gizzard. Biochemistry 17: 253-268 Grand RJA, Perry SV, Weeks RA (1979) Troponin C-like proteins (calmodulins) from mammalian smooth muscle and other tissues. Biochem J 177: 521-629 Harigaya S, Schwartz A (1969) Rate of calcium binding and uptake in normal animal and failing human cardiac muscle Circ Res 26: 781-794 Horl WH, Heilmeyer HC Jr (1978) Evidence for the participation of a Ca1+-dependent protein kinase and protein phosphatase in the regulation of the Ca1+ transport ATPase of the sarcoplasmic reticulum. 2. Effect of phosphorylase kinase and phosphorylase phosphastase. Biochemistry 17: 766-772 Itano T, Itano R, Penniston JT (1980) Interactions of basic polypeptides and protein* with calmodulin. Biochem J 189: 455-^59 Katz AM (1977) Contractile proteins. In Physiology of the Heart New York, Raven Press, pp 89-106 Katz AM (1979) Role of the contractile proteins and sarcoplasmic reticulum in the response of the heart to catecholamines: A historical review. Adv Cyclic Nucleotide Res 11: 303-343 Katz S, Remtulla MA (1978) Phosphodiesterase protein activator stimulates calcium transport in cardiac microsomal preparations enriched in sarcoplasmic reticulum. Biochem Biophys Res Commun 83: 1373-1379 1361 Kerrick WGL, Belles L, Casaidy P, Coby RL, Razarik S, Hoar PE, Malencik DA (1930) The use of ATP-y-S, calmodulin, trifluoperazine, catalaytic subunit of protein kinase and Tnl to distinguish between Ca2+ control mechanisms in skinned fibers. Fed Proc 59: 2042 Kirchberger MA, Chu G (1976) Correlation between protein kinase-mediated stimulation of calcium transport by cardiac sarcoplasmic reticulum and phosphorylation of a 22,000 dalton protein. Biochim Biophys Acta 419: 559-562 Kirchberger MA, Tada M (1976) Effects of adenosine 3':5'-monophosphate-dependent protein kinase on aarcoplasmic reticulum isolated from cardiac and slow and fast contracting skeletal muscle. J Biol Chem 251: 725-729 Kirchberger, MA, Tada M, Katz AW (1974) Adenosine 3:5monophosphate dependent protein kinase-catalyzed phosphorylation reaction and its relationship to calcium-transport in cardiac sarcoplasmic reticulum. J Biol Chem 249: 6166-6173 Kirchberger MA, Tada M, Katz AM (1975) Phospholamban: A regulatory protein of the cardiac sarcoplasmic reticulum. In Recent Advances in Studies on Cardiac Structure and Metabolism, vol 5, edited by A Fleckenstein, NS Dhalla. Baltimore, University Park Press, pp 103-115 Kranias EG, Bilezikjian LM, Potter JC, Piascik MT, Schwartz A (1980) The role of calmodulin in regulation of cardiac sarcoplasmic reticulum phosphorylation. Ann New York Acad Sci 368: 279-291 Kranias EG, Mandel F, Schwartz A (1980) Involvement of cAMP-dependent protein kinase and pH in the regulation of cardiac sarcoplasmic reticulum. Biochem Biophys Res Commun 92: 1370-1376 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680685 LaRaia PJ, Morkin E (1974) Adenosine 3':6'-monopho8phatedependent membrane phosphorylation. Circ Res 35: 298-306 Le Peuch CJ, Haiech J, Demaille JG (1979) Concerted regulation of cardiac sarcoplasmic reticuluin calcium transport by cyclic adenosine monophosphate dependent and calcium-calmodulin-dependent phosphorylation. Biochemistry 18: 5150-5157 Le Peuch CJ, Le Peuch, DAM, Demaille JG (1980) Phospholamban, activator of the cardiac sarcoplasmic reticulum calcium pump. Physicochemical properties and diagonal purification. Biochemistry 19: 3368-3373 Levin RM, Weiss B (1977) Binding of trifluoperazine to the calcium-dependent activator cyclic nucleotide phosphodieaterase. Mol Pharm 13: 690-697 Lin YM, Liu YP, Cheung WY (1974) Cyclic 3':5'-nudeotide phosphodiesterase. J Biol Chem 249: 4943-4954 Lopaschuk G, Richter B, K*tz S, (1980) Characterization of calmodulin effects on calcium transport in cardiac microsomes enriched in sarcoplasmic reticulum. Biochemistry 19: 56035607 Piascik MT, Wisler PL, Johnson CL, Potter JD (1980) Ca2*dependent regulation of guinea pig brain adenylate cyclase. J Biol Chem 285: 4176-4181 Potter JD, Gergely J (1975) The calcium and magnesium binding sites on troponin and their role in the regulation of myofibrillar adenosine triphosphatase. J Biol Chem 250: 4628-^4633 Potter JD, Johnson JD, Dedman JR, Schreiber WE, Mandel F, Jackson RL, Means AR (1977) Calcium-binding proteins; relationship of binding, structure, conformation and biological function. In Calcium-binding Protein and Calcium Function edited by RH Wasserman, RA Confldino, E CarofoJi, RH Kretainger, DH MacLennan, FL SiegeL New York, North Holland, pp 239-250 Schwartz A (1976) Cell membrane Na+,K+-ATPase and sarcoplasmic reticulum: possible regulators of intracellular ion activity. Fed Proc 38: 1279-1282 Schwarti A, Entman ML, Kaniike K, Lane LK, Van Winkle WB, Bornet EP (1976) The rate of calcium uptake into sarcoplasmic reticulum of cardiac muscle and skeletal muscle; Effects of cAMP-dependent protein kinase and phosphorylase kinase. Biochim Biophys Acta 426: 57-72 Downloaded from http://circres.ahajournals.org/ by guest on February 10, 2016 1362 CIRCULATION RESEARCH Sumida M, Wang T, Mandel F, FroehHch JP, Schwartz A (1978) Transient kinetics of Ca2+ transport of sarcoplasmic reticulum. J Biol Chem 253: 8772-8777 Tada M, Kirchberger MA, Repke DL, Katz AM (1974) The stimulation of calcium transport in cardiac sarcoplasrruc reticulum by 3'-5'-monosphophate-dependent protein kinase. J Biol Chem 249: 6174-6180 Tada M, Kirchberger MA, Katz AM (1975) Phosphorylation of a 22,000 dalton component of the cardiac sarcoplasmic reticulum by adenosine 3'.5'-monophosphate-dependent protein V O L . 4 9 , No 6, DECEMBER 1981 kinase. J Biol Chem 250; 2640-2647 Wray HL, Gray RR (1977) Cyclic AMP stimulation of membrane phosphorylation and Ca2+-activated, Mg^-dependent ATPase in cardiac sarcoplasmic reticulum. Biochim Biophys Acta 461: 441-459 Wray HL, Gray RR, Olsson RA (1973) Cyclic adenosine 3'-5'monophosphate-stimulated protein kinase and a substrate associated with cardiac sarcoplasmic reticulum J Biol Chem 248: 1496-1498 Downloaded from http://circres.ahajournals.org/ by guest on February 10, 2016 Studies on phosphorylation of canine cardiac sarcoplasmic reticulum by calmodulin-dependent protein kinase. L M Bilezikjian, E G Kranias, J D Potter and A Schwartz Circ Res. 1981;49:1356-1362 doi: 10.1161/01.RES.49.6.1356 Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 1981 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7330. 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