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. The preincubation reaction 0.6 24 E mixture was diluted 1:500 before synthase phosphorylation was conducted, which accounts for the lack of synthase phos- phorylation by cAMP-dependent kinase present in the phos- phorylase kinase. Polyacrylamide Gel Electrophoresis of 32P-Labeled Syn- thase. In all of the experiments, control reactions containing 0 5 10 15 20 phosphorylase kinase but no glycogen synthase were run in Time, min order to correct for 32P incorporation into the phosphorylase FIG. 1. Phosphorylation (solid symbols) and a-to-b conversion kinase. At the low concentrations of phosphorylase kinase used, of glycogen synthase (open symbols) catalyzed by phosphorylase ki- 32p incorporation in these control reactions was always less than nase. Reaction mixtures (50 Al) contained 50 mM Tris/50 mM 5% relative to 32p incorporation into the glycogen synthase. It >glycerophosphate (pH 8.6), 10 mM magnesium acetate, 386 (ig of glycogen synthase per ml, 11.4 ,ug of phosphorylase kinase per ml, and is possible, although unlikely in view of the associated a-to-b 0.5mM ATP or [y-32P]ATP. Phosphorylation was determined in the conversion, that addition of glycogen synthase stimulated the presence of phosphorylase kinase and glycogen synthase (A), absence autocatalytic phosphorylation of phosphorylase kinase. of phosphorylase kinase (-),and absence of glycogen synthase (-). Synthase was phosphorylated by using phosphorylase kinase Phosphorylation is expressed as pmol Of 32p incorporation per 10so and then subjected to polyacrylamide gel electrophoresis in the of reaction mixture (- ) and as mol Of 32p incorporated per mol of of sodium sulfate. Essentially all of the 32p 90,000-dalton synthase subunit ( ). Synthkinaa-to-b conversion, presence dodecyl expressed in terms of the synthase activity ratio (without Glc-6-P/ was located in the 90,000-dalton subunit of glycogen synthase with Glc-6-P), was determined in the presence of phosphorylase ki- (Fig. 3). The a- and 3-subunits of phosphorylase kinase mi- nase and glycogen synthase with ATP (C) and without ATP (X) or grated in the 1.0 to 1.5-cm range and contained <3% of the in the absence of phosphorylase kinase (0). 32p. Downloaded by guest on September 26, 2021 2538 Biochemistry: Soderling et al. Proc. Natl. Acad. Sci. USA 76 (1979)

Table 1. Effects of EGTA and pH on synthase phosphorylation and activity ratio 32p incorporation Synthase activity ratio Reaction conditions 5 min 10 min 20 min 5 min 10 min 20 min Exp. I No phosphorylase kinase 0.00 0.01 0.00 ND 0.76 0.74 With kinase; no EGTA 0.14 0.23 0.42 ND 0.54 0.42 With kinase and EGTA 0.03 0.05 0.07 ND 0.77 0.71 Exp. II No phosphorylase kinase pH 6.8 0.00 0.00 0.01* 0.69 0.64 0.64* pH 8.6 0.00 0.01 0.00* 0.67 0.69 0.68* With kinase pH 6.8 0.01 0.03 0.08* 0.65 0.62 0.60* pH 8.6 0.17 0.34 0.55* 0.33 0.25 0.26* Synthase phosphorylation (mol 32p incorporated per mol of 90,000-dalton subunit) and inactivation were measured as described in Fig. 1 except for the following. Experiment I was at pH 6.8 and used 11.4 (phosphorylation) or 3.8 (activity ratio) jig of phosphorylase kinase per ml and 1 mM EGTA as indicated. Experiment II was at the indicated pH with 1.1 (phos- phorylation) or 4.4 (activity ratio) ,ug of phosphorylase kinase per ml. ND, not done. * At 30 min. Kinetic Constants. The experiments reported above establish Trypsinization of Synthase Phosphorylated by Phos- that phosphorylase kinase can catalyze the phosphorylation and phorylase Kinase. By analysis of 32P-labeled CNBr and tryptic inactivation of glycogen synthase. In order to determine the peptides, we have defined two phosphorylation regions per relative activity of phosphorylase kinase toward these two synthase subunit (5). Low concentrations of trypsin degrade the substrates, the Michaelis constants were determined (Table 2). synthase subunit from 90,000 daltons to about 73,000 daltons With activated phosphorylase kinase at pH 8.2, the Km values by removing peptides from the carboxyl terminus (14, 18). The for glycogen synthase and phosphorylase were about equal. trypsin-insensitive region is within the 73,000-dalton peptide, However, the Vmax for phosphorylase was twice that for gly- and the trypsin-sensitive region corresponds to the small tri- cogen synthase. chloroacetic acid-soluble peptide(s) removed by limited trypsinization (5). Table 3 shows that phosphorylase kinase catalyzes incorporation of 32P almost exclusively (92%) into the trichloroacetic acid-insoluble or trypsin-insensitive region of synthase. Recent results show that the phosphorylation site is 1V serine residue 7 from the amino terminus (unpublished data). For comparison, 32P-labeled synthase phosphorylated by cAMP-dependent kinase catalytic subunit was also trypsinized under identical conditions.

8 DISCUSSION The results presented in this paper indicate that skeletal muscle glycogen synthase can be phosphorylated and inactivated by phosphorylase kinase. These results cannot be accounted for by the stimulation of a kinase present in the synthase prepara- 6 x tion by the CDR in the phosphorylase kinase. Whereas our E previous preparation of glycogen synthase contained a synthase kinase that was stimulated 10-fold by CDR (8), the preparations 02 of synthase used in this investigation contained no detectable synthase kinase activity as assayed at pH 6.8 or 8.6 in the ab- 4 sence or presence of CDR. Analysis of the reaction mixture showed that 97% of the 32p was in the 90,000-dalton synthase subunit. The properties of the synthase phosphorylation reaction are 2 characteristic of phosphorylase kinase. For example, the reac- Table 2. Michaelis constants for glycogen synthase and phosphorylase with activated phosphorylase kinase Vmaxq Substrate MM gmol/min/mg 0 1 2 3 4 5 6 Km, Distance, cm Glycogen synthase 59; 50; 100; 63 4.3; 3.6; 4.3; 3.3 71 8.9 FIG. 3. Sodium dodecyl sulfate/polyacrylamide gel electropho- Phosphorylase 54; 55; 100; 8.9; 9.5; 9.1; resis of glycogen synthase phosphorylated by phosphorylase kinase. Phosphorylations of glycogen synthase (6-50 MM) and phos- Glycogen synthase was phosphorylated as in Fig. 1, and an aliquot phorylase (30-200MM) were determined with activated phosphorylase containing 8 Mg of synthase was subjected to disc gel electrophoresis kinase (see legend to Fig. 2) at pH 8.2. Apparent Km (calculated using on 7.5% polyacrylamide gels in the presence of sodium dodecyl sulfate. subunit molecular weights) and Vmax values from four experiments The gel was photographed and then sliced and assayed for 32p. were estimated from double-reciprocal plots. Downloaded by guest on September 26, 2021 Biochemistry: Soderling et al. Proc. Natl. Acad. Sci. USA 76 (1979) 2539 Table 3. Trypsin-sensitivity of 32P-labeled synthase Table 4. Properties of protein kinases that catalyze phosphorylated by phosphorylase kinase and phosphorylation and inactivation of glycogen synthase cAMP-dependent kinase Phosphor- 32p, mol/mol synthase Activity ylation Trypsinization Phosphorylase cAMP-dependent Kinase ratio EGTA specificity Ref. time, min kinase protein kinase Phosphorylase 0 0.52 1.63 kinase 10 Inhibits 90 This 2 0.48 0.61 paper 5 0.48 0.64 cAMP-dependent 10 0.47 0.62 protein kinase 1 No effect 30 (5) Glycogen synthase was phosphorylated for 30 min as in Fig cAMP-independent phosphorylase kinase (4.6 ,g/ml) or cAMP-dependent protein synthase kinase 1.5 No effect 90 (5) catalytic subunit (5 X 104 units/ml) (2). The phosphorylation r CDR-dependent was terminated by addition of EDTA to 20 mM. Trypsinizati synthase kinase 10 Inhibits 60-70 (8) performed as described (5), with 25 ,ug of trypsin per ml. Activity ratio refers to the ratio of rates ofsynthase phosphorylation at pH 8.6 and 6.8. Phosphorylation specificity refers to the percentage of the first mole of 32P per 90,000-dalton synthase subunit incorpo- tion rate was 10-fold more rapid at pH 8.6 than at pH 6 rated into the trypsin-insensitive region. was inhibited by EGTA. Additionally, the rate of syi phosphorylation was 10-fold more rapid when the act form of phosphorylase kinase was used than when the r derling et al. (5) are distinct from phosphorylase kinase. It is tivated form was used. In light of the characteristics of t also apparent that the CDR-dependent synthase kinase (8) and action, it can be concluded that the synthase phosphory phosphorylase kinase, which may also be CDR-dependent (9), is catalyzed by phosphorylase kinase and not some hypotl are similar with respect to synthase phosphorylation. Recent contaminating kinase. These results do not agree with th studies (unpublished data) have led us to conclude that the Friedman and Larner (19) who concluded that phospho CDR-dependent synthase kinase we previously observed (8) kinase had little or no activity with glycogen synthase a is the same as phosphorylase kinase. strate but do agree with the report of Roach et al. (7). T1 Although phosphorylase kinase can catalyze the synthase incorporation into synthase was almost exclusively inl a-to-b reaction in vitro, the physiological relevance of this re- trypsin-insensitive region of the synthase subunit. In e: action is unknown. At pH 8.2 the Km values for synthase and ments performed to date the degree of synthase a-to-I for phosphorylase are about 70 AM. The cellular concentrations version (i.e., the synthase activity ratio) relative to 32p i of these enzymes are about 4 and 100 MM, respectively (20). poration into the trypsin-insensitive region agrees well wil However, the Vmax for phosphorylase is about 2-fold higher previous observations with a cAMP-independent syntha than for synthase. Although it is clear from these calculations nase (see figure 7 of ref. 5). that phosphorylase is the preferred substrate, it seems reasonable The question arises as to whether phosphorylase kinase that glycogen synthase may also be a substrate. If phosphorylase same as one of the previously reported synthase kinases kinase does catalyze the synthase a-to-b reaction in vivo, this various synthase kinases can be compared on the basis of poses some interesting questions concerning the physiological activities at pH 8.6 and 6.8, effects of EGTA, and specifi roles of the various synthase kinases (Fig. 4). For example, in- of synthase phosphorylation (trypsin-sensitive versus try creased cAMP levels in skeletal muscle will produce an acti- insensitive). From Table 4 it is clear that the cAMP-depex vation of both cAMP-dependent kinase and phosphorylase ki- kinase and the cAMP-independent kinase described b nase. Do both of these kinases catalyze the associated synthase Nonactivated Phosphorylose ts > Kinose < \ /cot

a-PHOSPHORYLASE ( i-SYNTHASE XGLYCOGEN -, (n-I glucose units) FIG. 4. Regulation of glycogen metabolism by Ca2+ and cAMP. Downloaded by guest on September 26, 2021 2540 Biochemistry: Soderling et al. Proc. Natl. Acad. Sci. USA 76 (1979) a-to-b conversion? If only one, which one? More intriguing 6. Brown, J. H., Thompson, B. & Mayer, S. E. (1977)Biochemstry perhaps is the situation that occurs during muscle contraction. 16,5501-5508. The increased Ca2+ concentration stimulates nonactivated 7. Roach, P. J., Roach-DePaoli, A. A. & Larner, J. (1978) J. Cyclic phosphorylase kinase, resulting in phosphorylase b-to-a con- Nucleotide Res. 4, 245-257. version. However, the synthase activity ratio does not decrease 8. Srivastava, A. K., Waisman, D. M., Brostrom, C. 0. & Soderling, from its control value (21). In our laboratory the synthase ac- T. R. (1979) J. Biol. Chem. 254, 583-586. tivity ratio in control rabbit skeletal muscle extracts is about 9. Cohen, P., Burchell, A., Foulkes, J. G., Cohen, P. T. W., Vanaman, 0.2-0.3. It is interesting that we have not been able to decrease T. C. & Nairn, A. C. (1978) FEBS Lett. 92,287-293. 10. Khatra, B. S. & Soderling, T. R. (1978) Biochem. Biophys. Res. the synthase activity ratio below about 0.2 by using phos- Commun. 85, 647-654. phorylase kinase as catalyst. Although the reason for this is not 11. Hayakawa, T., Perkins, J. P., Walsh, D. A. & Krebs, E. G. (1973) presently understood it may explain why the synthase activity Biochemistry 12, 567-573. ratio does not decrease below this value during muscle con- 12. Fischer, E. H. & Krebs, E. G. (1958) J. Biol. Chem. 231, 65- traction. These and other interesting questions concerning the 71. physiological role of phosphorylase kinase in the regulation of 13. Walseth, T. & Johnson, R. (1979) Biochim. Biophys. Acta, in glycogen synthase activity await further investigation. press. 14. Soderling, T. R. (1976) J. Biol. Chem. 251, 4359-4364. 15. Brostrom, C. O., Hunkeler, F. L. & Krebs, E. G. (1971) J. Biol. This work has been supported by National Institutes of Health Grant Chem. 246, 1961-1967. AM 17808. 16. Krebs, E. G., DeLange, R. J., Kemp, R. G. & Riley, W. D. (1966) Pharmacol. Rev. 18, 163. 1. Roach, P. J., Takeda, Y. & Larner, (1976) J. Biol. Chem. 251, 17. Walsh, D. A., Perkins, J. P., Brostrom, C. O., Ho, E. S. & Krebs, 1913-1919. E. G. (1971) J. Biol. Chem. 246, 1968-1976. 2. Soderling, T. R. (1975) J. Biol. Chem. 250,5407-5412. 18. Takeda, Y. & Larner, J. (1975) J. Biol. Chem. 250, 8951- 3. Schlender, K. K. & Reimann, E. R. (1975) Proc. Natl. Acad. Sci. 8956. USA 72, 2197-2201. 19. Friedman, D. L. & Larner, J. (1965) Biochemistry 4, 2261- 4. Nimmo, H. G., Proud, C. G. & Cohen, P. (1976) Eur. J. Biochem. 2264. 68,31-44. 20. Fischer, E. H., Heilmeyer, L. M. G. & Haschke, R. H. (1971) 5. Soderling, T. R., Jett, M. F., Hutson, N. J. & Khatra, B. S. (1977) Curr. Top. Cell. Regul. 4,211-215. J. Biol. Chem. 252,7517-7524. 21. Piras, R. & Staneloni, R. (1969) Biochemistry 8, 2153-2160. Downloaded by guest on September 26, 2021