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Phosphorylase Kinase (Protein Phosphorylation/Glycogen Metabolism/Calcium) THOMAS R

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Phosphorylase Kinase (Protein Phosphorylation/Glycogen Metabolism/Calcium) THOMAS R Proc. Natl. Acad. Sci. USA Vol. 76, No. 6, pp. 2536-2540, June 1979 Biochemistry Phosphorylation and inactivation of glycogen synthase by phosphorylase kinase (protein phosphorylation/glycogen metabolism/calcium) THOMAS R. SODERLING, ASHOK K. SRIVASTAVA, MARTHA A. BASS, AND BALWANT S. KHATRA Howard Hughes Medical Institute Laboratories, Department of Physiology, Vanderbilt University Medical School, Nashville, Tennessee 37232 Communicated by Sidney P. Colowick, February 26, 1979 ABSTRACT Skeletal muscle glycogen a4-synthase (EC cause the observations by Roach et al. (7) could have been due 2.4.1.11) has been purified free of all synthase kinase and phosphatase activities by chromatography on a Glc-N-6P- to stimulation of the kinase, which contaminated their synthase, Sepharose affinity column and then on a phosphocellulose by the CDR present in the added phosphorylase kinase. This column. This preparation of glycogen synthase was tested as a possibility is strengthened by the observation by Cohen et al. substrate for purified skeletal muscle phosphorylase kinase (9) that the CDR in purified phosphorylase kinase can activate (ATP:phosphorylase-b phosphotransferase, EC 2.7.1.38). Phos- phosphodiesterase and myosin light chain kinase. The study phorylase kinase (1-10 .ug/ml or 0.03-0.3 MM) catalyzes rapid reported in this paper utilized a preparation of glycogen syn- phosphorylation of glycogen synthase (4.5 AM) associated with conversion of the active a form to the less active b form. In the thase that did not contain any detectable synthase kinase ac- reaction, >95% of the 32P incorporation from [oy-32PJATP goes tivity. into the synthase subunit almost exclusively in the trypsin-in- sensitive region which is responsible for synthase a-to-b con- METHODS version. Synthase phosphorylation or inactivation catalyzed by phosphorylase kinase is blocked by ethylene glycol bis(P-ami- Glycogen a4-synthase was purified from rabbit skeletal muscle noethyl ether)N,N,N',N'-tetraacetic acid, is ATP dependent, through the Glc-N-6-P-Sepharose affinity column step as de- is 10-fold more rapid at pH 8.6 than at pH 6.8, and is increased scribed (5). An additional purification step, to remove con- 10-fold by prior activation of the phosphorylase kinase with taminating synthase kinases and phosphatase, was chroma- MgATP and cyclic AMP. With activated phosphorylase kinase tography on phosphocellulose. The a4-synthase from the at pH 8.2 the apparent Km and Vmax are approximately 70 AM affinity and 4 Mmol/min per mg with glycogen synthase as substrate and column in 25 mM f.-glycerophosphate, pH 6.8/1 mM 70 MM and 9 Mmol/min per mg with phosphorylase as substrate. EDTA/30 mM 2-mercaptoethanol, 10% (wt/vol) sucrose was It is concluded that glycogen synthase is a substrate in vitro for applied to a phosphocellulose column (25-50 ml) equilibrated phosphorylase kinase, a Ca2+-dependent enzyme. The possible with the same buffer. The glycogen synthase passes through the physiological significance of this reaction is discussed. column whereas the phosphatase and kinase activities are re- tained and can be eluted with 1 M NaCl. The glycogen synthase Phosphorylation and conversion of the active form of skeletal was placed in dialysis tubing, concentrated with solid sucrose muscle glycogen synthase (UDP glucose:glycogen 4-a-glucosyl to 7-10 mg/ml, and stored frozen at -70°C in aliquots. These transferase, EC 2.4.1.11), a4-synthase, to the inactive form, synthase preparations do not contain phosphatase activity as b4-synthase, is a complex reaction involving phosphorylation assayed with either 32P-labeled phosphorylase or 32P-labeled of several sites on the synthase subunit (1, 2). Phosphorylation synthase in the absence or presence of 0.1-1 mM MnCl2 (10). can be catalyzed by several protein kinases including cyclic Additionally, they do not contain synthase kinase activity as- AMP (cAMP)-dependent protein kinase and cAMP-indepen- sayed at pH 6.8 or 8.6 in the absence or presence of CDR dent synthase kinases (3-6). These kinases have relative speci- (8). ficities for phosphorylation of different sites or regions on the Phosphorylase kinase was purified in the nonactivated form synthase molecule as determined by analysis of 32P-labeled from rabbit skeletal muscle by the procedure of Hayakawa et CNBr peptides and tryptic peptides (5). al. (11). Phosphorylase b was purified as described by Fischer Recently there has been interest in possible regulation of and Krebs (12). synthase kinase(s) by Ca2+. Roach et al. (7) have reported that Phosphorylation and a-to-b conversion of glycogen synthase their synthase preparation contains a synthase kinase that is were measured in 50-;tl reaction mixtures containing 50 mM stimulated by Ca2+. Addition of phosphorylase kinase (ATP: Tris, 50 mM f3-glycerophosphate buffer (pH 6.8 or 8.6), 10 mM phosphorylase-b phosphotransferase, EC 2.7.1.38) to this syn- magnesium acetate, 0.5 mM ATP or ["t-32P]ATP, glycogen thase preparation increased synthase phosphorylation. In our a4-synthase, and phosphorylase kinase. Aliquots were analyzed laboratory, the existence of a calcium-dependent regulator for 32P-labeled protein and the synthase activity ratio as de- (CDR)-dependent kinase that catalyzes synthase phosphoryl- scribed (2). ation and a-to-b conversion has recently been noted (8). This [y-32P]ATP was synthesized according to the procedure of CDR stimulation of synthase phosphorylation is not mediated Walseth and Johnson (13). by myosin light chain kinase because this enzyme has little or no activity with glycogen synthase as substrate (8). We therefore RESULTS decided to investigate whether phosphorylase kinase, a CDR-containing enzyme (9), would phosphorylate and inac- Effect of Phosphorylase Kinase on Glycogen Synthase tivate glycogen synthase. This was particularly important be- Activity and Phosphorylation. Skeletal muscle glycogen a4- synthase purified on the phosphocellulose column did not The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- Abbreviations: cAMP, adenosine 3',5'-cyclic monophosphate; CDR, vertisement" in accordance with 18 U. S. C. §1734 solely to indicate calcium-dependent regulator; EGTA, ethylene glycol bis(3-aminoethyl this fact. ether)-N,N,N',N'-tetraacetic acid. 2536 Downloaded by guest on September 26, 2021 Biochemistry: Soderling et al. Proc. Natl. Acad. Sc. USA 76 (1979) 2537 contain detectable synthase kinase (see below). This preparation of synthase therefore was an excellent subsrate for of synthase kinases and phosphatases. t,r V Addition of phosphorylase kinase to this preparation of gly- cogen synthase resulted in phosphorylation and a-to-b con- version of the synthase (Fig. 1). Of the total 32p incorporated in the reaction, >95% went into the synthase (380Wg/ml) and <5% went into the phosphorylase kinase (1.1 sg/ml). In the absence of added phosphorylase kinase there was no incorpo- ration of 32p into the synthase and no a-to-b conversion [even in the presence of added CDR (results not shown)], thereby establishing the absence of contaminating kinase activity in the synthase preparation. The synthase a-to-b conversion by phosphorylase kinase was ATP-dependent, thus excluding the possibility of a-to-b conversion by limited proteolysis (14). The decrease in synthase activity ratio resulted from a decrease in synthase activity assayed without Glc-6-P with no change in activity assayed with Glc-6-P. Preincubation time, min Calcium and pH Dependence of Synthase Phosphoryl- ation and Inactivation. Phosphorylase kinase has an absolute FIG. 2. Synthase phosphorylation catalyzed by nonactivated and dependence on Ca2+ for activity and can be inhibited by the activated phosphorylase kinase. Nonactivated phosphorylase kinase (1.14 mg/ml) was preincubated at 300 for the indicated times in the divalent cation chelator ethylene glycol bis(f3-aminoethyl presence of 50 mM Tris/50 mM f3-glycerophosphate (pH 8.6), 10 AM ether)-N,N,N',N'-tetraacetic acid (EGTA) (15). Synthase cAMP, and 10 mM magnesium acetate in the presence (triangles) or phosphorylation and a-to-b conversion catalyzed by phos- absence (squares) of 0.5 mM ATP. Aliquots from the preincubation phorylase kinase were blocked by 1 mM EGTA as expected reaction mixture were diluted 1:500 in 10 mM Tris/10 mM f3-glycer- (Table 1). Another property of the nonactivated form of ophosphate, pH 6.8/1 mM EDTA and assayed (10 min) for synthase phosphorylase kinase is its much higher activity at pH 8.6 than phosphorylation as in Fig. 1 except that the pH was 6.8. Synthase at pH 6.8 (16). Table 1 shows that the rates of synthase phos- phosphorylation was measured in the absence (solid symbols) and phorylation and a-to-b conversion were about 10-fold more presence (open symbols) of 1 mM EGTA. rapid at pH 8.6 than at pH 6.8. In the absence of phosphorylase kinase there was no phosphorylation or a-to-b conversion of the Nonactivated versus Activated Phosphorylase Kinase. The synthase. experiments described above utilized nonactivated phos- phorylase kinase as catalyst. The activated form can be obtained by preincubation with MgATP plus cAMP. The cAMP stimu- lates the trace contaminant of cAMP-dependent protein kinase 0.8 32 present in preparations of phosphorylase kinase (17), thereby increasing the rate of the activation reaction. Fig. 2 shows that when phosphorylase kinase was preincubated with MgATP plus 06~~~~~~~~~~ cAMP there was a time-dependent 8-fold increase in the E no . phosphorylation of glycogen synthase. When ATP was omitted iE'xfSz in- from the preincubation, there was no such increase. The 0 crease in synthase phosphorylation cannot be accounted for by 00 cAMP-dependent kinase catalytic subunit because the synthase phosphorylation was blocked by EGTA and because both 0~~~~~~~~~~~~~ preincubations contained cAMP.
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