Inhibition by Calmodulin of the CAMP-dependent Protein Kinase Activation of Phosphorylase Kinase*

Transcription

1 Inhibition by Calmodulin of the CAMP-dependent Protein Kinase Activation of Phosphorylase Kinase* (Received for publication, June 23, 1982) Daniel E. Cox$ and Ronald D. Edstrom From the Department of Biochemistry, University of Minnesota, Medical School, Minneapolis, Minnesota Calmodulin is shown to inhibit both the activation identical to calmodulin, a calcium-binding protein first idenand phosphorylation of phosphorylase kinase by tified in brain (4,8). This protein has been shown to activate CAMP-dependent protein kinase. Maximal inhibition of cyclic nucleotide phosphodiesterase (9-111, myosin light chain both processes was approximately 66% at the highest kinase (12), and Ca2+-dependent ATPase (13), among others. calmodulin concentration tested (5.5 p ~). It was found It is becoming increasingly evident that calmodulin is a prithat the inhibition of phosphorylation was calcium-de- mary intracellular calcium-binding protein. The biochemistry pendent, reversible by trifluoperazine, and specific for of calmodulin has been reviewed recently (14-17). the #I subunit of phosphorylase kinase with no signifi- The relationship of calcium -to phosphorylase kinase is cant inhibition of phosphorylation of the a subunit. complex. A calcium-dependent protease can irreversibly acti- This inhibitory activity of calmodulin appears to be due vate phosphorylase kinase by cleavage of a peptide from the toaninteractionbetweencalmodulin andthesubstrate, phosphorylase kinase. This finding implies protein (18). The level of calcium required for proteolysis is either that the site of exogenous calmodulin interaction on the order of 1 m ~ weli, above physiological range. Phoswith phosphorylase kinase is at the /3 subunit or that phorylase kinase has an absolute requirement for calcium at this interaction results in a conformational change of the micromolar level (19) presumably due to the calcium phosphorylase kinase that inhibits the interaction be- binding properties of the 6 subunit. tween CAMP-dependent protein kinase and the B sub- A second role of calcium in the regulation of phosphorylase unit of phosphorylase kinase. The #I subunit may con- kinase has been determined. This regulatory role is also due tain a regulatory site that is recognized by either pro- to the calcium-calmodulin complex. In this case, calmodulin tein kinase or calmodulin. These findings further sub- is not acting as the S subunit but in a looser association with stantiate the role of the #I subunits in the activation of phosphorylase kinase. This exogenous calmoddin has been phosphorylase kinase and provide an additional ex- shown to activate phosphorylase kinase beyond the level of ample of substrate-directed control of phosphorylation. calcium alone (4, 20, 21). It is apparent that phosphorylase Phosphorylase kinase (ATP:phosphorylase-b phosphotransferase, EC ) is a complex enzyme that holds a central position in the cascade of enzymatic reactions involved in the regulation of glycogenolysis. It acts on glycogen phosphorylase b, catalyzing a calcium-dependent phosphorylation of phosphorylase b to yield its activated form, phosphorylase a. The biochemistry of phosphorylase kinase and its role in metabolic control have been the subject of recent reviews (1, 2). Skeletal muscle phosphorylase kinase is now believed to be a hexadecamer, with four different types of subunits, designated a,,8, y, and 6 (3,4). The holoenzyme has a molecular weight of 1.34 X lo6, with subunit molecular weights of a, 145,000; p, 128,000; y, 45,000; and 6, 17,000 (3, 4). The a and fl subunits function as sites of regulation in that they each contain a unique amino acid sequence with a serine residue as phosphate acceptor (5). Phosphorylation of these sites is catalyzed by CAMP-dependent protein kinase. The,8 subunits are phosphorylated most readily and this action has been correlated with activation of phosphorylase kinase (3). With different techniques, catalytic activity has been associated with both the /3 and y subunits (6, 7). The recently detected smallmolecularweight subunit (6) has been shown to he * Supported by the American Diabetes Association, Minnesota Affiliate. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. +Present address, Department of Biology, The Johns Hopkins University, Baltimore, MD kinase possesses two types of calmoddin-binding sites. Picton et al. (22) have presented evidence obtained through the use of a cross-linking reagent that an association exists between the y and S subunits and between exogenous calmodulin and both the a and 0 sununits. Recently, Conti and Adelstein (23) have reported the interesting finding of an effect of calmodulin on the phosphorylation of myosin light chain kinase, a calmodulin-dependent enzyme. When this enzyme is utilized as a substrate for CAMPdependent protein kinase in the presence of bound calmodulin, there is an inhibition of phosphorylation of one of the two phosphate acceptor sites on the enzyme. We have examined the effect of exogenouscalmodulin on the activation and phosphorylation of phosphorylase kinase by the catalytic subunit of CAMP-dependent protein kinase. Calmodulin, in the presence of calcium, inhibits both effects of protein kinase by inhibition of the phosphorylation of one class of sites on phosphorylase kinase. EXPERIMENTAL PROCEDURES Materials-Phosphorylase kinase was prepared from rabbit muscle by the procedure of Cohen (3). The specific activity of this preparation was approximately units/mg, with one unit equivalent to that amount of enzyme catalyzing the incorporation of 1 pmol of phosphate into phosphorylase b/min at 30 C at ph 8.2. This enzyme was in its nonactivated form as judged by a ph 6.8/8.2 ratio of 0.1 or less. The ability of the enzyme to be activated by calmodulin varied somewhat among preparations. The observed extent of activation was up to 3- fold at ph 8.2 and up to 2-fold at ph 6.8. Calmoduhn was prepared from bovine brain by the method of Grand et al. (24) and by a modification of the procedure of Dedman et el. (25). SephadexC-100 was used instead of Ultragel AcA44. Minor

2 Calmodulin Inhibition of Phc mphorylase Activation Kinase impurities were removed by hydroxylapatite-cellulose chromatography as described by Wolff et al. (26). Calmoddin prepared by either method migrated as a single species on polyacrylamide gel electrophoresis under nondenaturing conditions as well as on SDSI-polyacrylamide gel electrophoresis in the presence of EGTA. In the presence of calcium, both preparations exhibited enhanced electrophoretic mobility as well as the appearance of multiple bands similar to the pattern described by Burgess et al. (27). The catalytic subunit of CAMP-dependent protein kinase was obtained from Sigma or as a gift from Dr. E. G. Krebs of the University of Washington, Seattle. All other enzyme and protein preparations were obtained from Sigma. The procedure of Walseth and Johnson (28) was followed to prepare [Y-~ P]ATP utilizing carrierfree 3zP obtained from New England Nuclear. The labeled nucleotide was generously provided by Dr. Walseth, Department of Phannacology, University of Minnesota. Trifluoperazine was from Smith, Kline, and French Pharmaceuticals, Philadelphia, PA. Buffer A contained 50 m~ a-glycerol phosphate, 2 mm EDTA, 1 mm P-mercaptoethanol, ph 6.8. Buffer B was 50 m~ a-glycerol phosphate, 2 mm EDTA, 1 m~ P-mercaptoethanol, ph 7.0. Enzyme Assays-Phosphorylase kinase was assayed by measurement of 3zP incorporation into phosphorylase b. Reaction mixtures contained 67 m~ Hepes, ph 6.8 or 8.2, 200 ~ L M Ca2+, 10 mm Mg2*, 2 mg/d of phosphorylase b, appropriate amounts of phosphorylase kinase, and 0.94 mm [Y-~~P]ATP in a total volume of Reactions were initiated by the addition of ATP and incubated at 30 C for 10 min. A 50-pl aliquot was removed and spotted on Whatman No. 3MM filter paper (2 cm ) and processed for measurement of protein-bound 32P as described by Reimann et al. (29). Protein kinase assays were performed by incubation of the catalytic subunit with the appropriate acceptor under the conditions described in the figure legends. Phosphate incorporation was determined as above in the phosphorylase kinase assay. Activation of phosphorylase kinase was measured by incubation of the enzyme with the catalytic subunit of protein kinase under the conditions described in the figure legends. Reactions were stopped by dilution of an aliquot of the reaction mixture in cold buffer containing an excess of bovine heart protein kinase inhibitor (30). SDS-polyacrylamide slab gel electrophoresis was performed according to the procedure of Laemmli (31). Protein concentrations were determined by the method of Lowry et al. (32) or in the case of calmodulin by absorbance measurements at 276 nm (El*. = 2.0). RESULTS Inhibition of Protein Kinase Activation of Phosphorylase Kinase by Calmodulin-Initial experiments were performed to determine the effect of simultaneous action of calmodulin and protein kinase on phosphorylase kinase activity. Table I shows the results of a typical experiment in which phosphorylase kinase was incubated with a saturating amount of calmodulin (calmodulin:phosphorylase kinase molar ratio = 94) and with the catalytic subunit of CAMP-dependent protein kinase. The presence of cahodulin in the reaction mixture caused a significant inhibition of the protein kinase-catalyzed activation of phosphorylase kinase. There was a small influence of calmoddin on the baseline activity of phosphorylase kinase under these conditions. The experiments described in Fig. 1 examined the effect of calmoddin concentration on the protein kinase activation of phosphorylase kinase as measured by the incorporation of phosphate into phosphorylase b. There was significant inhibition of activation at all levels of calmodulin tested. This inhibition was greater than 50% when the concentration of calmoddin was 32 times that of phosphorylase kinase (92 pg/ ml or 5.5 p~ calmoddin). The absolute level of activation could be increased by using a higher concentration of protein kinase. However, the shape of the curve of inhibition was similar in both cases. Calmodulin had no effect, on the autophosphorylation of phosphorylase kinase at ph 6.8 in the absence of protein kinase, The abbreviations used are: SDS, sodium dodecyl sulfate; EGTA, ethylene glycol bis(p-aminoethy1 ether)-n,n,n,n tetraacetic acid; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid. TABLE I The effect of calmodulin on the activation ofphosphorylase kinase by CAMP-dependent protein kinase Reaction mixtures containing 41 mm a-glycerol phosphate, ph 6.8, 0.86 m~ CaCL, 6.1 mm MgCL, 1.7 mm EDTA, 0.83 m~ P-mercaptoethanol, 0.12 mm ATP, 87 pg of phosphorylase kinase (PhK), 102 pg of calmoddin (CaM, where indicated) and 0.16 pg of catalytic subunit (PrK, where indicated) in a total volume of 175 pl. The reaction was initiated by the addition of ATP and incubated for 5 min at 30 C. An aliquot of each sample was diluted 1:25 in cold Buffer A containing 50 pg/ml of bovine heart protein kinase inhibitor. AU samples were then assayed for phosphorylase kinase at ph 6.8 as described under Experimental Procedures. Reaction mixture Phosphorylase kinase activity nmol/min mg PhK alone 85 PhK plus CaM 98 PhK plus PrK 176 PhK ulus PrK PIUS CaM 132 ml OO 8 I Holm CalmodullnlMole PhofIDhorylaBe Kinare FIG. 1. The effect of calmodulin concentration on the activation of phosphorylase kinase by CAMP-dependent protein kinase. Reaction mixtures contained 41 mm cy-glycerol phosphate, ph 7.0, 1.7m~ EDTA, 0.83 m~ p-mercaptoethanol, 0.22 mg/d (29 pmol) of phosphorylase kinase, 0.43 mm eaz+, 3.3 m~ Mg, 2.9 mg/ ml of dithiothreitol, 120 p~ ATP, the indicated molar ratios of calmodulin, and 2.3 pg/ml (A), 0.45 pg/d (o), or no added protein kinase (0) in a total volume of 175 p1. Calmodulin concentrations ranged from 2.7 to 91.6 pg/ml. Reactions were initiated by the simultaneous addition of protein kinase and ATP. Incubations were carried out at 30 C for 5 min, at which point 10 pl of each sample was diluted 1:20 in Buffer B containing 15 pg/ml of protein kinase inhibitor. Each sample was assayed for phosphorylase kinase activity at ph 6.8 by 32P incorporation into phosphorylase b as described under Experimental Procedures. Inhibition of Protein Kinase Catalyzed Phosphorylation of Phosphorylase Kinase by Added Calmodulin-Since calmodulin interferes with the protein kinase induced activation of phosphorylase kinase, it was important to determine whether this effect was due to an inhibition of the phosphorylation of the enzyme. Fig. 2 shows a time course of the effect of added calmodulin on the rate of protein kinase catalyzed phosphorylation of phosphorylase kinase. There is a signifi- cant inhibition of the incorporation of 32P into phosphorylase kinase in the presence of calmodulin. In this experiment, the concentration of calmoddin was 33 times that of phosphorylase kinase on a molar basis (92 pg/ml, 5.5 p~ calmodulin). This concentration of calmodulin inhibited P incorporation into phosphorylase kinase at all times points examined. Fig. 3 shows the effect of calmodulin Concentration on phosphorylase kinase phosphorylation. There was significant inhibition of phosphorylation at low levels of calmodulin, increasing

3 _c_ Calmodulin Inhibition of Phosphorylase Kinase Activation Incubation Time (minutes) FIG. 2. The effect of added calmodulin on the rate of protein kinase catalyzed phosphorylation of phosphorylase kinase. Reaction mixtures contained 41 m~ a-glycerol phosphate, ph 6.8,1.7 m~ EDTA, 0.84 m~ P-mercaptoethanol, 0.22 mg/d of phosphorylase kinase (86 pmol), 0.43 m~ Ca2+, 3.3 mm Mg", 120 p~ [y-32p]atp, 0.8 pg/ml of protein kinase and with (A) or without (0) 92 pg/ml of calmodulin (33-fold molar excess) in a total volume of 525 pl. Reactions were initiated by the simultaneous addition of ATP and protein kinase and incubated at 30 "C. At the times indicated, 50-pl aliquots were removed and spotted on filter paper squares. Phosphate incorporation was determined as described under "Experimental Procedures." nonspecific interactions. To show this, a sample of calmodulin was dialyzed into Buffer B to remove bound calcium and then tested for its ability to inhibit phosphorylation reactions. These data are shown in Table 11. It is clear that in the absence of calcium the inhibitory effect of calmodulin is completely abolished. The effect of trifluoperazine, an inhibitor of calmodulin action, is also shown in Table 11. In the presence of this compound, the inhibitory activity is eliminated. These data in combination with those showing calcium dependence clearly demonstrate that this is a calmodulin effect. Table I11 shows the results of an experiment designed to test the ability of calmodulin to inhibit the phosphorylation of standard alternative substrates for CAMP-dependent protein kinase. There was no inhibition of the autophosphorylation of phosphorylase kinase in the absence of protein kinase. A slight activation was in fact observed. This effect has been previously documented (21). There was a calcium-dependent TABLE I1 The effect of calmodulin on the phosphorylation of phosphorylase kinase by CAMP-dependent protein kinase Reaction mixtures were as described in Table I with the following exceptions: (A) 32 pg of calmodulin (CaM) added where indicated, 77 pg of phosphorylase kinase, 0.28 pg of catalytic subunit, and 0.43 mm calcium, where indicated in a total volume of 0.35 ml, (8) 30 pg of phosphorylase kinase, 0.09 pg of catalytic subunit, 71 p~ trifluoperazine (TFP), where indicated, and 8.5 pgof calmodulin in 175 pl. Reactions were initiated by the simultaneous addition of catalytic subunit and ATP and incubated for 5 min at 30 "C. Experiment Phosphate incorporated pmol/rncn A. Requirement for calcium PhK, Ca PhK, CaM 9.9 PhK, CaM, Caz+ 5.3 B. Effect of trifluoperazine CaZ+ PhK, 10.5 Ca2+, PhK, CaM 5.9 Ca2+, PhK, CaM, TFP " ' 16 1 i 24 " 32 Mole* CalmodullnlMole PhO8DhOIyla8e Kina89 FIG. 3. The effect of calmodulin concentration on phosphorylation of phosphorylase kinase by protein kinase. Reaction mixtures were as described in Fig. 1 with 2.3 pgiml of protein kinase and 201 pm [Y-~~P~ATP and the indicated concentrations of calmodulin in a total volume of 175 p1. Reactions were initiated by the simultaneous addition of protein kinase and ATP and incubated for 5 min at 30 "C. A 5O-pl aliquot of each sample was then spotted on filter paper and phosphate incorporation was determined as described under ''Experimental Procedures." Phosphate incorporation is indicated as total pmol incorporated into 29 pmol of phosphorylase kinase. to 66% inhibition at a molar ratio of 32:l. There was no significant autophosphorylation of phosphorylase kinase under these conditions. This inhibition curve correlates very well with the inhibition of activation shown in Fig. 1. It is also important, when ascribing an effect to calmodulin, to show a dependence on calcium, i.e. that the effect is due to the biological properties of calmodulin rather than due to TABLE I11 The effect of calmodulin on thephosphorylation of alternative substrates for CAMP-dependent protein kinase Reaction mixture contained in 175 pl, 41 mm a-glycerol phosphate, ph m~ p-mercaptoethanol, 1.7 mm EDTA, 0.86 mm CaC12 or 0.86 m~ EGTA (for experiments in the absence ofca"), 6.1 mm MgCL, 9.0 pg/d of catalytic subunit (PrK), 120 p~ [y3'p]atp, 57 pg/d of calmoddin (where indicated) and 0.5 mg/d of phosphorylase kinase, 7.1 mg/d of casein, 2.9 mg/ml of histone mixture (Sigma type 11-A) or 0.29 mg/ml of histone fzb. All protein solutions were dialyzed against Buffer A to remove calcium. Reactions were initiated by the simultaneous addition of catalytic subunit and ATP. After 5 min at 30 "C, a 5O-pl aliquot was spotted on filter paper for the determination of phosphate incorporation. Autophosphorylation of phosphorylase kinase was determined in the absence of catalytic subunit. The degree of phosphorylation of phosphorylase kinase represents the net value after this baseline autophosphorylation reaction is subtracted. Phosphate incorporated Substrate -Ca" +Ct+ -CaM +CaM -CaM +CaM pmollmin Phosphorylase kinase (auto, -PrK) Phosphorylase kinase Casein Histone mixture Histone f2b

4 Calmodulin Inhibition of Phosphorylase Kinase Activation inhibition of phosphorylation of phosphorylase kinase, casein, and mixed histone. However, this effect was most pronounced when phosphorylase kinase was the substrate. There was a slight inhibition of phosphorylation of histone f2b that appears to be independent of calcium. Determination of the Site of Inhibition of Phosphorylation-Since protein kinase phosphorylates sites on both a and /3 subunits of phosphorylase kinase, it was of interest to determine if the inhibition caused by calmodulin was specific for either of these sites. Phosphorylated a and /3 subunits were separated by SDS-polyacrylamide slab gel electrophoresis. The location of the phosphorylated bands was determined by autoradiography and the respective bands were excised and 32 P incorporation determined by liquid scintillation. Fig. 4 shows the effect of calmodulin concentration on the 32P labeling of the a and p subunits of phosphorylase kinase. In this instance, there was minimal autophosphorylation in the absence of catalytic subunit and no effect of calmodulin on this autocatalytic reaction. This low level of autophosphorylation was subtracted to obtain protein kinase specitic phosphorylation. In the presence of increasing concentrations of calmodulin, there was no significant inhibition of phosphorylation of the a subunit while there was a progressive increase in the inhibition of the /3 subunit phosphorylation. This inhibition was nearly 50% at a calmodu1in:phosphorylase kinase molar ratio of 32:l. A time course of this reaction is illustrated in Fig. 5. In the absence of added calmoddin, protein kinase catalyzes a rapid phosphorylation of the &subunits with a slower phosphorylation of the a subunits. This confirms previous findings (3). In the presence of calmodulin, there is a strong inhibition of the /3 subunit phosphorylation reaction, while the rate of phosphorylation of the a subunit remains relatively unaffected. The apparently linear rates of phosphorylation over the first 5 min were and pmol min" for the LY subunit with 't Moles CalmodulinlMole Phosphorylase Kinase FIG. 4. Determination of the site of inhibition of phosphorylation by calmodulin. Reaction mixtures were prepared as described for Fig. l. Protein kinase concentration was 2.3 pg/ml and [y-"'piatp was 120,UM. Reactions were initiated by the addition of protein kinase and ATP. All samples were incubated for 5 min at 30 "C. A 40-pl aliquot of each sample was then brought to 1% in SDS and heated in a boiling water bath for 3 min. The samples were then applied to a 7.5% acrylamide slab gel (10.5 X 14.0 X 0.15 cm) and run at 20 ma until bromphenol blue dye front was 0.5 cm from the bottom. The gel was then fixed, dried, and the location of the cr and p bands determined by autoradiography. The bands were then excised from the dried gel and radioactive phosphate incorporation in the (Y subunit (0) and the /3 subunit (A) was determined by liquid scintillation counting. OO,5 IO Incubation Time (minutes) FIG. 5. The effect of added calmodulin on the rate of phosphorylation of the a and fl subunits of phosphorylase kinase. Reac-ion mixtures were prepared as described in Fig. 1, except that the total volume was increased to 0.35 ml. Reactions were initiated by the simultaneous addition of protein kinase and ATP. Samples were incubated at 30 "C and at the times indicated, a 40-p1 aliquot was removed and processed as in Fig. 4. Reactions without added calmodulin: (O),/3 subunit; (O), a subunit. Reactions with added calmodulin: (A), subunit; (O), cr subunit. and without added calmodulin, respectively. For the /3 subunit, the rate of 0.12 pmol min" fell to in the presence of 5.5 IJM calmodulin. (Linear regression correlation coefficients were all greater than 0.995). DISCUSSION Phosphorylase kinase which contains calmodulin as an intrinsic subunit (8) has also been shown to bind additional molecules of calmodulin in a reversible, calcium-dependent manner. There are two lines of evidence that demonstrate the existence of a second calmodulin binding site. First, phosphorylase kinase containing a stoichiometric amount of 6 subunit has been shown to bind to calmodulin-sepharose in a calciumdependent manner (4, 20). Secondly, the addition of calcium and calmodulin to phosphorylase kinase has been shown to increase the activity of the enzyme above the level of calcium alone. The degree of this activation reported in the literature varies greatly. When examined at ph 6.8 or 8.2, the effect of calmodulin has been found to vary from none to a 7-fold activation (4, 20, 21). It has been postulated that the variation in calmodulin effect may be due to proteolysis or variation in the degree of phosphorylation of phosphorylase kinase (4). In support of the former suggestion, it has been shown that limited proteolysis, resulting in the loss of the integrity of the a and p subunits of phosphorylase kinase, destroyed the ability of the enzyme to interact with calmodulin (20, 21). Furthermore, Sharma et al. (33) have recently demonstrated that the red muscle isozyme of phosphorylase kinase, which contains modified a subunits, is not sensitive to activation by calmodulin. These results imply that calmodulin may interact with phosphorylase kinase at both the Q and /3 subunits. The second potential explanation of the variation in calmodulin activation of phosphorylase kinase involves variations in the state of phosphorylation of the enzyme. Both Walsh et al. (21) and Cohen (34) have reported that phosphorylase kinase that has been phosphorylated by CAMP-dependent protein kinase is relatively insensitive to activation by added calmodulin. We hypothesized that the reverse situation may also be true. The presence of bound calmodulin may interfere with

5 12732 Calmodulin Inhibition the interaction of the catalytic subunit of CAMP-dependent protein kinasewith phosphorylase kinase. This possibility might be useful to help identify the location of calmodulin binding to phosphorylase kinase. If protein kinase and calmodulin interact with phosphorylase kinase at widely separated sites, then the combination of their actions would be no less than the action of either agent alone. However, if these proteins interact at the same site or at closely spaced sites then the prior addition of calmodulin to phosphorylase kinase might interfere with the binding of protein kinase and the phosphorylation reaction. An effect of calmodulin on the interaction of CAMP-dependent protein kinase and phosphorylase kinase has been shown to occur. When phosphorylase kinase is incubated in the presence of calcium and calmodulin there is a clear inhibition of the enzyme-catalyzed activation and phosphorylation of phosphorylase kinase. This inhibition is significant at calmodulin:phosphorylase kinase molar ratios of as low as 1:1. Evidence that this inhibitory reaction is due to calmodulin rather than contaminating protein kinase inhibitors is 3-fold. First, this reaction is clearly calcium-dependent. Inhibition of phosphorylation occurred only when both calcium and calmodulin were present (see Table 11). Walsh et al. (35) first described a heat-stable protein inhibitor of CAMP-dependent protein kinase. This protein has been purified from skeletal muscle and characterized by D le et al. (36) and shown to possess some characteristics similar to those of calmodulin. This inhibitor is a small, heat-stable, acidic protein (Mr = 11,300). Szmigielski et al. (37) have describedtwo distinct protein kinase inhibitors in rat brain that also are heat-stable and acidic in nature. The Type I inhibitor is believed to be analagous to the skeletal muscle protein. The Type I1 inhibitor was found to inhibit cyclic nucleotide-dependent as well as cyclic nucleotide-independent protein kinases. It was specifically tested for calmodulin activity with phosphodiesterase and found to be ineffective. Moreover, the inhibitory activity of this protein is calcium-independent. The skeletal muscle inhibitor (36) is also independent of calcium.' The second line of evidence that this is a calmodulin effect is the demonstrated reversibility by trifluoperazine (see Table 11). Since the discovery of Levin and Weiss (38) that the antipsychotic drug, trifluoperazine, binds specifically to calmodulin thereby inhibiting its activity, that property of this compound has been frequently used to establish a requirement for calmodulin in putatively calmodulin-dependent processes. The data presented here clearly show that low levels of trifluoperazine completely abolish the effect of added calmodulin. Finally, calmodulin samples prepared by two quite different methods were equally effective in the inhibitory reaction (data not shown). All of these findings support a role of calmodulin in this reaction rather than contamination with other protein kinase inhibitors. We believe that the inhibitory effect of calmodulin on the interaction between CAMP-dependent protein kinase and phosphorylase kinase is best explained by an interference with the binding of protein kinase to phosphorylase kinase by the presence of calmodulin bound to phosphorylase kinase. The strongest evidence for this hypothesis is the specificity of the inhibitory effect. When the a and p subunits were separated by SDS-polyacrylamide gel electrophoresis, it was possible to determine the relative effects of calmodulin on these two phosphate acceptor sites. It is clear from the data presented in Figs. 4 and 5 that the presence of calmodulin results in an inhibition of the rate of phosphorylation of the p subunits with little effect on the a subunit phosphorylation a D. E. Cox and R. D. Edstrom, unpublished observations. of Phosphorylase Kinase Activation rate. If this effect was due to an interaction between calmodulin and protein kinase, an equivalent effect on the rate of phosphorylation of both subunits would be predicted. This is clearly not the case. We have examined the effect of calmodulin on the phosphorylation of alternative substrates for CAMP-dependent protein kinase and have found some inhibition of the phosphorylation of these substrates (see Table 111). The effect was less with histone and casein than with phosphorylase kinase. Therefore, it remains a possibility that calmodulin interacts to some extent with the catalytic subunit. However, an association between calmodulin and histone has been shown (39, 40) and may account for this effect. In a recent report by Wolff et al. (41), calmodulin was shown to influence the dephosphorylation of histones by brain phosphoprotein phosphatase due to an interaction between calmodulin and histone. Srivastava et al. (42) have shown that calmodulin has no effect on the phosphorylation of glycogen synthase by CAMPdependent protein kinase. Thus, the mechanism of the inhibition of phosphorylation of these alternative substrates remains to be determined. The conclusionsfrom these studies are that calmodulin inhibits the phosphorylation and thus the activation of phosphorylase kinase by CAMP-dependent protein kinase. The mechanism of this inhibition appears to be due to a calciumdependent interaction between calmodulin and phosphorylase kinase. Furthermore, this inhibitory effect is specific for the,l3 subunits of phosphorylase kinase with no apparent effect on the CY subunits. This implies either that the site of interaction of exogenous calmodulin with phosphorylase kinase is at the p subunit or that the site of interaction at a different location results in a conformational change that inhibits the interaction of the catalytic subunit of protein kinase with the,b subunit. In support of the former hypothesis, Picton et al. (22) have described a series of cross-linking studies performed in an effort to identify the calmodulin-binding subunits of phosphorylase kinase. They obtained two species with molecular weights corresponding to complexes of calmodulin with the /3 and a subunits. The predominant cross-linked species was the,f3 subunit-calmodulin complex. The present studies confirm the existence of the association with the p subunit. Chan and Graves (43) have clearly shown that exogenous calmodulin activates the ays complex prepared by the dissociation of phosphorylase kinase in 1.8 M LiBr. The isolated ay8 complex contained essentially no p subunit thus excluding the possibility of a calmodulin complex with the,8 subunit. They also isolated the y8 complex and showed that calmodulin was not able to activate that dimer. One must therefore conclude that calmodulin has a direct interaction with the a subunit in the ay6 complex. It is reasonable to assume that it is also the a subunit on the holoenzyme which is responsible for the calmodulin-induced activation of the nonphosphorylated phosphorylase kinase. These results are compatible with the observation of Shanna et al. (33) which indicate that alteration in the a subunit causes loss of the activation of phosphorylase kinase by calmodulin. In order to reconcile the various observations of the interaction of calmodulin with phosphorylase kinase, we propose that calmodulii can interact with both the CY and p subunits. Since phosphorylase kinase can bind just 1 mol of calmoddin/ tetramer, it is possible that the single calmodulin can simultaneously interact with both the a and,8 subunits. In its association with the a subunit, it can stimulate phosphorylase kinase activity independently of activation by protein kinase. Its simultaneous interaction with the,8 subunit causes an inhibition of the activation by protein kinase. We are currently pursuing studies of the mechanism of the

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