TWE JOURNAL OF BIOLOGICAL CHEMI8'rRP Vol. 246, No.23.Isaueof Dec,f%nber 10,~~.6317-6323,1970

Printedin U.S.A.

Inactivation of Glycogen Synthetase and Activation of Phosphorylase Kinase by Muscle Adenosine 3’, S-Monophosphate-dependent Protein Kinases*

(Received for publication, August 3, 1970)

THOMAS R. SODERLING,~ JOHN P. J~.ICXENBOTTOM,S$ ERWIN M. REIMANN,~ FELIX L. HUNKELER,[[ D. A. WALSH, AND EDWIN G. KREBS

From the Department of Biological Chemistry, School of Medicine, University of California, Davis, California 95616 and the Department of Biochemistry, University of Washington, Seattle, Washington 98105

SUMMARY

Rabbit skeletal muscle glycogen synthetase I has been purified and obtained essentially free of phosphor-y&e, phos- phorylase khmse, and glycogen synthetase kinase. Using the purified glycogen synthetase as substrate, it was deter- mined that two separable adenosine 3’,5’-monophosphate (cyclic AMP)-dependent protein kinase fractions from skele- tal muscle each catalyze the conversion of glycogen synthetase I to glycogen synthetase D; activity changes during the reac- tion correlate closely with the uptake of phosphate by glyco- gen synthetase. These same protein kinase fractions also catalyze the cyclic AMP-dependent phosphorylation and ac- tivation of phosphorylase kinase. The specific activities of the two protein kinase fractions towards phosphorylation of glycogen synthetase and phosphorylase kinase were enriched equally during puriCcation. Further evidence that phos- phor&se kmase phosphorylation and glycogen synthetase phosphorylation are common activities of the same enzyme was obtained by studying heat inactivation of the protein kinase, destabilization by cyclic AMP, inhibition by a protein inhibitor, and the cyclic nucleotide specificity. Evidence is presented that the cyclic AMP-dependent protein kinase acts directly on glycogen synthetase I and not through a second kinase as occurs in the phosphorylase activation system.

Rabbit skeletal muscle glycogen synthetase I sediments as a single peak with an szo ,W of 14.3. Preliminary studies

* Supported by Grant AM12842 from the National Institute of Arthritis and Metabolic Diseases, United States Public Health Service, and by a grant from the Muscular Dystrophy Association of America, Inc.

$ The material presented is taken from theses submitted to the Graduate Faculty of the University of Washington in partial ful- fillment of the requirements for the Ph.D. degree.

§ Present address, Department of Pharmacology, School of Pharmacy, University of Mississippi, University, Mississippi 38677.

7 Postdoctoral Fellow of the National Institutes of Health. Present address, Department of Biochemistry, Medical College of Ohio, Toledo, Ohio 43614.

11 Present address, Laboratoire de Chimie Biologique Speciale, University of Geneve, 1211 Geneve 4, Switzerland.

indicate that the enzyme probably exists as a tetramer com- posed of subunits having a molecular weight between 90,000 and 100,000. On conversion of the I form to the D form, the enzyme incorporates 1 mole of phosphate per 91,000 g of protein.

Certain enzymes are known to exist in aivo in two forms possessing different activities. For example, three key enzymes of glycogen metabolism, phosphorylase (cr-1,4-glucan:ortho- phosphate glucosyltransferase, EC 2.4.1. l), phosphorylase kinase (ATP : phosphorylase phosphotransferse, EC 2.7.1.37), and glycogen synthetase (UDP-glucose : glycogen cr-4-glucosyl- transferase, EC 2.4.1.11) all exist in two forms which are in- terconverted by phosphorylation-dephosphorylation reactions. Both phosphorylase and phosphorylase kinase are converted to more active forms by phosphorylation (1, 2), but glycogen synthetase is converted from the active I form to the less active D form through phosphorylation (3). Control of the interconver- sion reactions of these enzymes provides one of the most thor- oughly studied examples of hormonal regulation via adenosine 3’,5’-monophosphate. For example, it is known that epineph- rine increases tissue concentrations of cyclic AMP1 (4,5), thereby activating phosphorylase kinase which in turn activates phos- phorylase (6, 7). Epinephrine also causes an inactivation of glycogen synthetase by conversion of this enzyme to the D form (8, 9). Glycogen levels are thus lowered by epinephrine because of both stimulation of glycogen degradation and diminu- tion of glycogen synthesis.

1 The abbreviations used are: cyclic AMP,, adenosine 3’,5’- monophosphate; dibutyryl cyclic AMP, N6,02 -dibutyryl adeno- sine 3’,5’-monophosphate; cyclic IMP, inosine 3’,5’-monosphos- phate; cyclic CMP, cytidine 3’,5’-monophosphate; cyclic GMP, guanosine 3’,5’-monophosphate; cyclic UMP, uridine 3’,5’- monophosphate; cyclic dAMP, deoxyadenosine 3’,5’-monophos- phate; cyclic TMP, thymidine 3’,5’-monophosphate; EGTA, ethylene glycol his@-aminoethyl ether)-N,N’-tetraacetic acid.

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The mechanism by which cyclic AMP stimulated phosphoryl- ase kinase activation was clarified by the recent finding of a cyclic AMP-dependent protein kinase which catalyzed the activation of phosphorylase kinase (10). This enzyme was also referred to as phosphorylase kinase kinase (11). The existence of a cyclic AMP-stimulated glycogen synthetase kinase has been described by Huijing and Larner (12). Thus, the role of cyclic AMP in the stimulation in vitro of phosphorylase kinase phosphorylation and activation (1, 10) and of glycogen synthetase phosphoryla- tion and inactivation (3, 12) has now been well documented.

Because of the similarities between phosphorylase kinase activation and glycogen synthetase inactivation, it seemed that the same cyclic AMP-dependent protein kinase might catalyze both reactions, i.e. phosphorylase kinase kinase and glycogen synthetase kinase might be the same enzyme. The observation that in an impure skeletal muscle fraction both reactions were inhibited by the same heat-stable protein from muscle (13) was suggestive of a single cyclic AMP-dependent kinase catalyzing both reactions. Recently, Schlender, Wei, and Villar-Palasi (14) have suggested that glycogen synthetase kinase and phos- phorylase kinase kinase might be the same enzyme (14).

In a preliminary report some of the evidence that a single enzyme catalyzes both phosphorylase kinase activation and glycogen synthetase inactivation was summarized (15). The present paper describes this evidence in more detail. Most of the experiments were carried out utilizing the method originally described for preparing the cyclic AMP-dependent protein kinase (lo), but during the course of this study it became established, using a modified purification technique, that at least two protein kinases are present in skeletal muscle (16). The possibility existed that one of these cyclic AMP-dependent protein kinases was phosphorylase kinase kinase while the other was glycogen synthetase kinase. Evidence that this is not the case, however, is presented here. Villar-Palasi and Schlender (17) have also indicated that they have obtained two cyclic AMP-dependent protein kinase fractions from muscle and were unable to achieve separation of glycogen synthetase kinase from phosphorylase kinase kinase.

MATERIALS AND METHODS

Preparation of Ademsine 3’,5’-Monophosphate-dependent Protein Kinase

Jfethod I-The cyclic AMP-dependent protein kinase used for most of the experiments was prepared by the method of Walsh, Perkins, and Krebs (10) except that the Sephadex G-200 gel filtration step was omitted. The fraction obtained from the next to the last step, chromatography on Whatman No. DE-52 cellulose, was precipitated with ammonium sulfate, dissolved in 5 IRM glycerol-P, 2 mM EDTA, pH 7.0, and desalted by chroma- tography on a Sephadex G-25 column (1.25 X 25 cm) equilibrated in the same buffer.

Jfethod Z-The second method of purifying the cyclic AMP- dependent protein kinase was similar to Method 1 except that a calcium phosphate gel fractionation step was substituted for one of the column chromatography steps and certain other minor alterations were made. A preliminary report of this method has appeared (16) and a detailed description will be given in a forth- coming publication.2 Briefly, the fractionation scheme is one

2 E. M. Reimnnn, 11. A. Walsh, and E. G. Krebs; manuscript in preparation.

involving a series of steps that follow the precipitation of phos- phorylase kinase from rabbit skeletal muscle extract at pH 6.1 (2). Taking the pH 6.1 supernatant as Fraction 1, the other fractions can be described as follows: Fraction 2, obtained after precipitation of additional protein impurities at pH 5.5, ammo- nium sulfate precipitation, dialysis, and centrifugation at 78,000 X g; Fraction 3, the enzyme after adsorption and elution from calcium phosphate gel; and Fractions 4a and 4b, two peaks of cyclic AMP-dependent protein kinase activity (Peaks 1 and 2) obtained using ion exchange chromatography on DE-52. The enzyme obtained by Method 1 chromatographs on DE-52 in the position of Peak 1 and is presumably identical to Peak 1.

Preparation of Phosphorylase b and Phosphorylase Kinase

Phosphorylase b and phosphorylase kinase were prepared as described previously (2) with the following minor modifications in the purification of the kmase. Centrifugation at 36,000 rpm for 3 hours in the 42 rotor of the Spinco L2-65B ultracentrifuge was carried out in tubes containing 3 ml of 20% sucrose layered in the bottom; this prevented aggregation and denaturation of the kinase. The Sephadex G-200 column was replaced by chromatography on a column of Sepharose 4B equilibrated with 50 mu glycerol-P, 2 mu EDTA, and 10% sucrose, pH 6.8. The purified phosphorylase kinase contained no detectable glycogen synthetase activity.

Inhibitor of Adenosine 3’) 5’-Monophosphate-dependent Protein Kinases

This protein inhibitor was prepared from the supernatant of the pH 6.1 acid precipitation in the phosphorylase kinase purifica- tion method (2) by a modification of the procedure of Appleman, Birnbaumer, and Torres (13). Major steps in the purification involve boiling the pH 6.1 supernatant, precipitating with tri- chloracetic acid, chromatographing on DEAE-cellulose at pH 5, and chromatographing on Sephadex G-75. Details of the purifi- cation and properties of this inhibitor are soon to be published.3

Other Materials

T-~~P-ATP was prepared by the procedure of Glynn and Chappell (18) as modified by Dr. John Perkins in this laboratory. The reaction mixture, containing 20 mCi of 32P (Tracerlab), was the same as that given by Glynn and Chappell except for the addition of 10 ~1 of 15 mM NAD+ and 0.2 ml of 10 mM KH*POr K2HP0, buffer, pH 7.4. The IO-ml reaction mixture was incubated at 30” for 1 hour, and then 1 ml of 1 N KC1 followed by 250 mg of Norit (Pfansteill Laboratories, Waukegan, Illinois) were added. Since the adsorption properties of the Norit vary from lot to lot, the amount required was determined with non- radioactive ATP for each lot. After mixing and standing for 10 min, the mixture was filtered through two Millipore filter pads (0.45 p). The Norit was washed with 20 ml of Hz0 and the T-~~P-ATP was eluted with two 15.ml washes of a 50% ethanol solution containing 0.15 N NHkOH. The eluent was evaporated to dryness on a rotary evaporator, the residue was dissolved in 50 ml of HzO, and the solution was again evaporated to dryness. The T-~~P-ATP was dissolved in 10 ml of HSO and stored frozen. The procedure yielded a solution of approxi- mately 4.5 MM ATP and 3 X log cpm per ml. Contamination

3 D. A. Walsh, C. D. Ashby, C. G. Gonzales, D. Calkins, E. H. Fischer, and E. G. Krebs, manuscript in preparation.

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by ADP was less than 1 %, and less than 1% of the radioactivity was present as 32Pi*

Human salivary a+amylase was prepared according to the method of Shainkin and Birk (19) carried through the Sephadex G-25 step. The enzyme was lyophilized and stored at -15”. Cyclic 3’,5’-nucleotides were a gift of Boehringer Mannheim. Concentrations were determined from published molar extinction coefficients (20, 21) except that a value of 16.0 x lo3 at 272 rnp and pH 7.0 was used for dibutyryl cyclic AMP and the extinction coefficient of 5’-IMP was used for cyclic IMP. 14C-UDP-glucose (uniform label in the glucose moiety) was obtained from New England Nuclear; UDP-glucose and “slightly lysine-rich” histone from calf thymus were from Sigma; and bacteriological dextrin was from Mann. Casein, from Nutritional Biochemicals, was adjusted to pH 9.5 and maintained at this pH during boiling for 10 min. The suspension was cooled, adjusted to pH 6, and clarified by centrifugation before use. Shellfish glycogen, from Krishell Laboratories, Inc., Portland, Oregon, was precipitated twice in 60% ethanol before use. Other chemicals and materials were obtained in the purest available grades from commercial sources.

Glycogen Synthetase Assay-Glycogen synthetase was assayed by a modification of the method of Villar-Palasi et al. (22) by following the incorporation of 14C-glucose into glycogen utilizing i4C-UDP-glucose as substrate. The synthetase was diluted to the desired activity in a solution at pH 7.5 containing 50 mu Tris, 90 mM 2-mercaptoethanol, 1 ml1 EDTA, bovine serum albumin, 0.25 mg per ml, and 0.5% glycogen. The reaction was initiated by adding 0.1 ml of the diluted enzyme to 0.15 ml of an assay mixture at pH 7.5 containing 2.5 mM 14C-UDP-glucose, glycogen, 1.33 mg per ml, 83.3 mM Tris, 1.7 mM EDTA, 20.8 mM MgC12, and 8.33 mM glucose-6-P. When assaying for glycogen synthetase I, the glucose-6-P was omitted. Following an incu- bation at 30” for 15 min, the reaction was terminated by adding 0.25 ml of 60% KOH and heating for 15 min in boiling water. The glycogen was precipitated by addition of 0.1 ml of 1% NaCl plus 0.9 ml of 95% ethanol. After 15 min at 0” the assay tubes were centrifuged, decanted, and allowed to drain for 15 min. The glycogen pellets were dissolved in 0.1 ml of 1% NaCl plus 0.5 ml of H20, reprecipitated with 0.9 ml of 95% ethanol, and centrifuged as before. The pellets were then dissolved in 1 ml of Hz0 and counted in 15 ml of scintillation fluid in a liquid scintillation spectrometer. The scintillation fluid contained 125 g of naphthalene, 7.5 g of 2,5-diphenyloxazole, 0.375 g of p-bis[2-(5-phenyloxazole)]benzene dissolved in 1 liter dioxane (23). The synthetase reaction was linear up to an incorporation of 30% of the UDP-glucose into glycogen. One unit of activity is defined as the amount of enzyme which catalyzes incorporation of 1 pmole of glucose from UDP-glucose into glycogen per min under the standard assay conditions.

Methods of Following Aderwsine 3’,5’-Morwphosphate- dependent Protein Kinase-catalyzed Reactions

Xethod A: Conversion of Glycogen Synthetase I to Glycogen Synthetase D-The conversion of glycogen synthetase from its I to its D form (24) was measured at 30” and pH 7.0 in reaction mixtures of 100 ~1 (unless otherwise noted) containing 26 mu glycerol-P, 1.4 mM EDTA, 5% sucrose, 20 mM 2-mercaptoetha- nol, 2 InM magnesium acetate, 0.4 mu ATP, 1 X 10d5 M cyclic AMP (when added), 40 to 150 pg of purified glycogen synthetase, and cyclic AMP-dependent protein kinase. Dilutions of the

cyclic AMP-dependent protein kinase were made in 5 mM glyc- erol-P, and 2 mM EDTA, pH 7.0. The reaction was initiated by addition of ATP and magnesium acetate. At several time points 20-/d aliquots were removed and diluted 1: 20 or more to stop the conversion reaction in a solution at pH 7.5 and 0” containing 0.5% glycogen, bovine serum albumin, 0.25 mg per ml, 90 InM

2-mercaptoethanol, 50 mM Tris, and 1 mM EDTA. These were assayed for glycogen synthetase I activity as described above. If the reaction was being used as an assay system to determine cyclic AMP-dependent protein kinase activity, initial velocities (-Aunits of glycogen synthetase I per min) were calculated from the initial linear portion of the time course. When the phos- phorylation of glycogen synthetase was being followed, y-“P- ATP was used, the volume of the reaction mixture was increased, and lOO+l samples were removed for determination of protein- bound 3zP. These samples were prepared as described for Method B.

Method B: Assay for Comparing Glycogen Synthetase I to D Reaction and Phosphorylase Kinuse Activation Reaction by Meas- uring 32P Incorporation-The protein kinase can be assayed with phosphorylase kinase as the substrate by following the activity change of the latter enzyme (II), but such an assay involves multiple dilution steps and is difficult to quantitate. Since the rates of phosphorylation of glycogen synthetase and phosphoryl- ase kinase parallel their activity changes (see “Results” and References 2 and ll), an assay method based on phosphorylation of the protein substrates was developed, thereby allowing these two activities of the protein kinase to be measured under identical reaction conditions. EGTA was included and low concentra- tions of rJ2P-ATP and magnesium acetate were used to prevent autocatalysis of the phosphorylase kinase phosphorylation reac- tion (11). Fluoride was present to inhibit phosphatases and theophylhne to inhibit cyclic AMP phosphodiesterase present when crude fractions were being analyzed.

Reaction mixtures at pH 7.0 contained 0.3 mM EGTA, 0.7 mM

EDTA, 6.3 mM glycerol-P, 0.8% sucrose, 8 mM NaF, 0.8 mM

theophylline, 100 to 150 pg of purified glycogen synthetase or phosphorylase kinase, 1 X low6 M cyclic AMP, 0.12 mM yJ2P- ATP, 1 mM magnesium acetate, and the cyclic AMP-dependent protein kinase in a total volume of 0.5 ml. The yJ2P-ATP (together with the magnesium acetate) was used to initiate the reaction. The time course of phosphorylation at 30” was followed by adding O.l-ml aliquots of the reaction mixture to 0.7 ml of a solution containing 10% trichloracetic acid and 1.25 mg per ml of bovine serum albumin. After all the samples were taken, an additional 1 ml of 5% trichloracetic acid was added to each tube. After standing at 0” for 15 min the tubes were cen- trifuged and decanted, the pellets were dissolved in 1 ml of 0.1 N NaOH, and the proteins were reprecipitated with 1 ml of 10% trichloracetic acid. After an incubation at 0” for 15 mm, the tubes were again centrifuged and the entire procedure was repeated. The final protein pellets were dissolved in 1 ml of 98% formic acid and counted in 20 ml of the same scintillation fluid used in the glycogen synthetase assay. The initial rates of phosphorylation (picomoles of a2P incorporated per min) were calculated from the linear portion of the time course.

Other MethodsPhosphorylase kinase was assayed at pH 6.8 or 8.2 by the previously published method (25). Protein con- centrations were determined by the method of Lowry et al. (26) using bovine serum albumin as the standard. Sedimentation velocity patterns were obtained with a Spinco model E analytical

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ultracentrifuge using schlieren optics and a standard 12-mm double sector cell. Sucrose density gradient centrifugation by the method of Martin and Ames (27) was performed in the SW-40 rotor of a Beckman L2-65B ultracentrifuge.

Disc gel polyacrylamide electrophoresis was performed at 7” using an asparagine buffer system (28) at pH 7.3 containing 1 mu glucose-6-P in the upper reservoir. Prior to electrophoresis of the glycogen synthetase, a preliminary electrophoresis of the gels was executed at 0.5 ma per tube for 2 hours to remove ammo- nium persulfate. Then a 20+1 sample (about 10 pg) of glycogen synthetase in 20% sucrose and 0.01% bromphenol blue was layered on top of the 6% gels, and electrophoresis was accom- plished using 2 ma per tube until the tracking dye approached the end of the gel. The gels were stained for 1 hour in a solution

FRACTION NUMBER (22 id EACH)

FIG. 1. Elution profile of enzymes from Sepharose 4B column. Glycogen synthetase was purified through the DE-52.cellulose columnstep and concentrated with ammoniumsulfate as described in the text. The concentrated enzyme (11.5 ml, 16 mg per ml) in 50 mM glycerol-P (pH 7.0), 2 mrvr, EDTA, 40 mM mercaptoethanol, and 10% sucrose was applied to a Sepharose 4B column (2.5 X 65 cm) equilibrated with the same buffer. Fractions of 2.2 ml were collected every 20 min and assayed undiluted for glycogen syn- thetase (plus glucose-6-P) and phosphorylase kinase (pH 8.2). In the glycogen synthetase and phosphorylase kinase assays, values greater than 12.5 and 300 units per ml (represented by A) exceeded the quantitative limits of the two respective assays for the undiluted enzymes. Absorbance at 280 rnp was measured against a buffer blank.

of 0.1% Coomassie brilliant blue in methanol-acetic acid-water (5: 1:5) and destained overnight by immersion in 7.5% acetic acid, 5% methanol. When glycogen synthetase activity was to be determined, two identical disc gels were run simultaneously. One gel was stained for protein while the duplicate was placed in a test tube (0.8 x 7.8 cm) and surrounded by the standard glycogen synthetase assay mixture containing glucose-6-P (see above) to which 5% bacteriological dextrin had been added. This low molecular weight dextrin can diffuse into the gels (29) and served as primer for the glycogen synthetase assay. The gels were left overnight at room temperature, washed extensively with Hz0 for 3 hours, and then sliced into l-mm sections. Each section was placed in a glass scintillation vial, dehydrated by heating at loo”, and dissolved in 200 ~1 of Hz01 at 60” (30). This solution was then counted in 20 ml of liquid scintillation fluid (see above). In correlating enzyme activity with protein stain- ing, corrections were made for swelling of the gels during staining or assaying. Disc gel electrophoresis in sodium dodecyl sulfate was performed by the method of Shapiro, Viriuela, and Maize1 (31). Nonradioactive protein-bound phosphate in glycogen synthetase was determined in duplicate on 1.6-mg samples by a previously published method (32).

RESULTS

Purijication of Glycogen Synthetase-In order to study the specificity of enzymes catalyzing the phosphorylation of glycogen synthetase it is essential to separate the latter enzyme from other proteins which incorporate phosphate, such as phosphorylase kinase and phosphorylase. In addition, phosphorylase kinase, if present in a high enough concentration, would cause inter- ference, since it is capable of catalyzing a slow conversion of glycogen synthetase from the I to the D form (24). This latter reaction is not stimulated, however, by cyclic AMP.

The starting material for the purification of rabbit skeletal muscle glycogen synthetase was the pellet containing glycogen and proteins obtained after the centrifugation step at 30,000 rpm in the phosphorylase kinase preparation (2). This fraction contained 50 to 707, of the glycogen synthetase of the crude muscle extract as well as some phosphorylase kinase and large amounts of phosphorylase. The pellets from a preparation utilizing six rabbits (4.2 kg of muscle) were suspended in a Teflon- pestle, glass-tube homogenizer in 240 ml of 50 mM Tris, 1 mM EDTA, pH 7.5, and, for convenience, were stored frozen at -15” for up to 4 months before further purification. The suspension

TABLE I

Purification of glycogen synthetase from phosphorylase kinase and phosphorylase Glycogen synthetase was prepared as described in the text from ence of 5’-AMP, and phosphorylase kinase at pH 8.2. Protein

4.2 kg of fresh skeletal muscle from six rabbits. At each major step in the purification, aliquots were assayed for glycogen sgn-

was determined by the method of Lowry et al. (26) after pre- liminary preciuitation wit.h 5% trichloracetic acid.

thetase in the presence of glucose-6-P, phosphorylase in the pres- -- - ,.

._ _

Fraction Vdllllle

- 1

ml

Muscleextract............................ 10,000 Washed glycogen-protein pellet.. . 254 Supernatant after amylase digestion.. 225 After DE-52 column (concentrated). 6.4 Aft,er Sephadex G-25 column 11

?rotein Glycogen synthetase Phosphorylase kinase

w/ml total units

14.0 1,760 21.0 1,219 11.0 1,080 22.7 352

3.2 165

units/ % YC- total units unitsj mg coocry x 10-s m&T

0.01 100 72 514 0.23 70 5.6 1,060 0.44 60 5.2 2,110 2.4 20 0.3 2,150 4.7 9 0.008 250

% 5Y‘OW”y

100 7.8 7.2 0.4

<O.l

- I

-

Phosphorylase

to&d unils x 10-8 units/mg

18.3 130 4.2 790 3.6 1,409 0.001 8.8 0.0002 7.1

% rccove*y

100 23 20

<O.l <O.l

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was thawed and the pellets were further dispersed by use of a Teflon-pestle, glass-tube homogenizer. The preceding and following steps were all done at 5” unless otherwise noted. The turbid suspension was centrifuged in the No. 30 rotor of a Spinco model L ultracentrifuge for 90 min at 30,000 rpm, and the super- natant, containing substantial amounts of phosphorylase kinase and some of the contaminating phosphorylase, was discarded. Very little or no glycogen synthetase was lost by this procedure. Since further washings by the same procedure failed to solubilize any additional phosphorylase kinase or phosphorylase, only one such step was used. The washed glycogen-protein pellets were suspended as above in 100 ml of 50 InM Tris, 1 InM EDTA, 40 mu 2-mercaptoethanol, 5% sucrose, 10 my magnesium acetate, pH 7.5. Three milligrams of lyophilized human salivary (Y- amylase were added, and the mixture was incubated at 30” for 1 hour with occasional stirring. During this incubation, glyco- gen synthetase was solubilized and the enzyme was converted from the D to the I form. Centrifugation at 18,000 rpm yielded a slightly yellowish supernatant and a large pellet of insoluble material. The amylase supernatant was applied to a Whatman No. DE-52 cellulose column (2.5 X 40 cm) equilibrated with 150 KIM Tris, 1 mM EDTA, 5% sucrose, 40 InM .%mercapto- ethanol, pH 7.5. The column was washed with the same buffer until the absorption at 280 rnp decreased to the base line value. The synthetase was then eluted with pH 7.5 buffer of the same composition except that 500 mM Tris was substituted for the 150 InM Tris. The proteins in the 500 mu Tris wash were pre- cipitated by adding solid ammonium sulfate to a concentration of 1.5 M and stirring for 30 min. The precipitated protein was collected by centrifugation at 15,000 g for 20 min. The pellets were dissolved in 8 ml of 50 MM glycerol-P, 2 mM EDTA, 40 mM 2-mercaptoethanol, 10% sucrose, pH 7.0, and this solution was applied to a Sepharose 4B column (2.5 x 65 cm) equilibrated with the same buffer. Elution of the column with the same buffer yielded two main protein peaks (Fig. 1). Phosphorylase kinase was present in the first, turbid peak while glycogen syn- thetase eluted in the second peak. The second peak was pooled, poured into dialysis tubing, and concentrated to 4 to 6 mg per ml by surrounding the dialysis tubing with Aquacide II (Calbio- them). The Aquacide contained some material which inhibited the synthetase I to D conversion, but this substance was removed

FIG. 2. Sedimentation velocity pattern of glycogen synthetase. The sedimentation velocity of purified glycogen synthetase (3.3 mg per ml) in 50 mM glycerol-P, 2 mM EDTA, and 40 mM mercapto- ethanol was determined in a Spinco model E analytical ultra- centrifuge. Sedimentation, from left to right, was at 56,000 rpm and a temperature of 5”. Pictures shown here were taken at 20, 28, and 36 min after maximum speed and with a bar angle of 56”. The major peak has an SZO,~ of 14.3 and the minor component an s2o,u, of 21.7.

by passing the concentrated enzyme through a Sephadex G-25 column (1.25 X 25 cm) equilibrated with 50 mu glycerol-P, 2 mM EDTA, 10% sucrose, 40 mM 2-mercaptoethanol, pH 7.0. Ali- quots of the purified enzyme were frozen in a Dry Ice-ethanol bath and could be stored frozen at -15” for at least 4 months with little loss of activity. When the enzyme was stored in the refrigerator (without freezing) for longer than a week, activity was lost and protein precipitated.

By the above procedure glycogen synthetase was purified about 500-fold from muscle extract with a 9% recovery (Table I). Assaying the enzyme in the presence and absence of glucose-6-P gave a ratio (-glucose-6-P to +glucose-6-P) of 0.5 to 0.6. Determination of alkaline-labile phosphate in the purified glycogen synthetase I gave values between 0.1 to 0.5 mole of phosphate per 105 g of protein. The final enzyme preparation contained, on a weight basis, only 0.3% phosphorylase kinase and 0.4% phosphorylase based on specific activities, determined in this laboratory, of 100,000 units per mg and 1,600 units per mg for pure phosphorylase kinase and pure phosphorylase, respectively. The DE-52 cellulose column removed the phos-

TOP FRACTION NUMBER BOTTOM

FIG. 3. Sucrose density gradient centrifugation of glycogen synthetase, phosphorylase b, and phosphorylase kinase. Fifty- microliter samples (about 150 pg of protein) of each of the three purified enzymes were combined and layered on 13 ml of a 5 to 20% sucrose density gradient in 50 mM glycerol-P, 2 mM EDTA, pH 7.0. Centrifugation, at 30,000 rpm and 5” for 164 hours, was per- formed in the SW-40 rotor of a Beckman L2-65B ultracentrifuge. Equal-volume fractions (0.44 ml) were collected and assayed un- diluted for total glycogen synthetase, total phosphorylase, and phosphorylase kinase at pH 8.2.

FIG. 4. Dodecyl sulfate-polyacrylamide gel electrophoresis of glycogen synthetase and phosphorylase b. Proteins (0.5 mg per ml) were denatured and reduced by incubation in 10 mM Pi (pH 7.1), 0.1% sodium dodecyl sulfate, 0.1% mercaptoethanol, and 20% sucrose at 60” for 30 min. Fifty-microliter samples (approxi- mately 25 pg of protein) were applied to 7.5% gels containing 0.1% sodium dodecyl sulfate and subjected to electrophoresis at room temperature at 8 ma per tube using bromophenol blue as the tracking dye. Purified glycogen synthetase (gel A) and phos- phorylase b (pel B) have essentially the same relative migration rates.

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ui t

- : 800- I 3 F 2 600-

% 2 2 400- 5 ci

FIG. 5. Disc gel electrophoresis of glycogen synthetase in the presence and absence of glucose-6-P. Twenty-microliter samples (8 pg of protein) of purified glycogen synthetase were subjected to electrophoresis at 2 ma per tube on duplicate 6% polyacrylamide gels in a Tris-asparagine buffer system at pH 7.3 (28) either con- taining (gel A) or without (gel B) 1 mM glucose-6-P in the upper buffer reservoir. In each case one gel was stained for protein while the duplicate was assayed (see “Materials and Methods”) qualitatively for glycogen synthetase activity. Synthetase activity for gel A is represented by -; for gel B by -.

A-

* C

lBI D z E p0 I I I I I J

10 2 4 6 8 IO 12 ”

TIME OF REACTION (MINUTES)

FIG. 6. Effect of cyclic AMP and the cyclic AMP-dependent protein kinase on the conversion of glycogen synthetase I to glycogen synthetase D. Reaction mixtures were made up accord- ing to Method A (see “Materials and Methods”). Aliquots were removed at the indicated times for determination of glycogen syn- thetase activity in the absence of glucose-6-P. The amount of glycogen synthetase used was 40 pg in a 100-J reaction mixture. The cyclic AMP-dependent protein kinase (7 pg) used was pre- pared by Method 1. Curve D, complete system; Curve A, no cyclic AMP or protein kinase; Curve B, no protein kinase; Curve C, no cyclic AMP. Glycogen synthetase activity given on the ordinate is calculat.ed on the basis of units per ml of the original synthetase preparation containing 1.9 mg per ml.

phorylase in the first wash, and the Sepharose 4B column sepa- rated the phosphorylase kinase from the glycogen synthetase.

Physical Properties of Glycogen Synthetase I-The glycogen synthetase preparation (3.3 mg per ml) in the analytical ultra- centrifuge showed a major, somewhat asymmetric peak with an

pg ADDED CYCLIC AMP-DEPENDENT PROTEIN KINASE

FIG. 7. Dependence of glycogen synthetase I activity change on added cyclic AMP-dependent protein kinase. Each reaction contained 49 fig of glycogen synthetase, 6.7 X 10m6 M cyclic AMP, the purified cyclic AMP-dependent protein kinase (Method l), and other components as described in Method A. The ordinate gives the initial rates of activity change (see Method A) of the original synthetase preparation containing 1.9 mg per ml.

s20,W of 14.3 and a small amount of heavier material (Fig. 2). The sedimentation coefficient at a much lower protein concen- tration (0.02 mg per ml), determined by density gradient cen- trifugation (27) using phosphorylase b and phosphorylase kina& as markers, was approximately 14.0 (Fig. 3). Additional knowl- edge of the structure of glycogen synthetase was obtained by disc gel electrophoresis in the presence of sodium dodecyl sulfate QI- 34). By this technique the enzyme was found to migrate as a single major component plus a minor band (Fig. 4, Gel A). Estimation of the molecular weight of the major component, using phosphorylase b monomer, ovalbumin, and the two y- globulin chains as standards (33), gave a value between 90,000 to 100,000. This would suggest that native glycogen synthetase may exist as a tetramer, since, based on an ~20,~ of 14, the native enzyme would probably have a molecular weight around 400,000, assuming that it is a typical globular protein. In disc gel poly- acrylamide electrophoresis without sodium dodecyl sulfate at pH 7.3 in a Tris-asparagine buffer (28) containing 1 mM glucose- 6-P, glycogen synthetase gave a single protein band which con- tained synthetase activity (Fig. 5, Gel A). If glucose-6-P was omitted from the running buffer, two protein components were present, neither of which exhibited significant glycogen synthe- tase activity (Fig. 5, Gel B). The effects of glucose-6-P on electrophoresis of glycogen synthetase were not duplicated by glucose-l-P. When the polyacrylamide gel concentration was varied between 4% and 6%, only one protein band was observed in the presence of glucose-6-P, further indicating the homo- geneity (29) of the glycogen synthetase.

Cbnversion of Glycogen Synthetuse I to Glycogen Synthetase D- Glycogen synthetase I, purified by the method described above, is essentially free of the enzyme which catalyzes the ATP- dependent conversion of the synthetase from its I to its D form (12). It was determined, however, that the cyclic AMP-depend- ent protein kinase originally purified from skeletal muscle by utilizing phosphorylase kinase as a substrate (10) would catalyze the synthetase conversion. A typical experiment illustrating

*The molecular weight of phosphorylase kinase has recently been determined to be 1.3 X lo6 daltons by Dr. Taro Hayakawa in this laboratory.

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0.0 I I 0 2 4 6 8 IO 12 14 I6

MINUTES

” L 4 b 8 IO 12 14 MINUTES

FIG. 8. Correlation of activity change and phosphorylation of glycogen synthetase. The 2-ml reaction mixture (see Method A) contained 0.85 mg of purified glycogen synthetase I, 0.07 mg of cyclic AMP-dependent protein kinase (Method l), 1 X 10V6 M

cyclic AMP, 0.6 mM r-@P-ATP, and 1.8 mM magnesium acetate. At each time point 204 aliquots were diluted 1: 100 and assayed for glycogen synthetase in the presence (total activity) or absence (I activity) of glucose-6-P. Phosphorylation was followed by determining the protein-bound 32P in NO-~1 aliquots. Glycogen synthetase activity given on the ordinate is calculated on the basis of units per ml of the original synthetase preparation con- taining I.7 mg per ml. Curve A, total glycogen synthetase activ- ity; Curve B, glycogen synthetase I activity; Curve C, picomoles of protein-bound phosphate in the loo-p1 sample.

these points is given in Fig. 6. In the presence of the protein kinase and cyclic AMP (Curve D) there was a rapid conversion of the synthetase to a form that has a low level of activity in the absence of glucose-6-P. In the absence of either cyclic AMP (Curve C) or of the protein kinase (Cuwe B), the rate of the I to D conversion was drastically reduced. Omission of both cyclic AMP and the protein kinase (Curve A) completely prevented the reaction. Total glycogen synthetase activity, as determined by assays carried out in the presence of glucose-6-P, showed no change during the I to D reaction. This is not illustrated in Fig. 6 but was shown in other experiments (see Figs. 8 and 13). The initial rates for the conversion of glycogen synthetase I to glycogen synthetase D were proportional to the amount of cyclic AMP-dependent protein kinase added (Fig. 7), thus making this reaction suitable as the basis for assaying the latter enzyme. Friedman and Larner have described a similar assay system for glycogen synthetase kinase (24).

Relationship between Phosphorylation and Change in Enzyme Activity in Glycogen Synthetase I to Glycogen Synthetase D Reac- tion-The conversion of glycogen synthetase from the I to the D form was studied using rJ2P-ATP in order to determine the relationship between phosphorylation of the enzyme and change in its activity. As is demonstrated in Fig. 8, there was a good correlation between the loss of glucose-g-p-independent synthe- tase activity (Ccrve B) and 32P uptake by the protein (Curve C). Total glycogen synthetase activity (Curve A) was constant during the reaction. Arbitrarily using the 8-min values to indicate the point of maximal change, half-maximal phosphorylation was found to occur in * min and half-maximal loss of activity to take place in approximately * min. The maximal extent of phos- phorylation in the experiment of Fig. 8 amounted to 1.3 moles of 32P per lo6 g of protein. To gain further information on total phosphate uptake by the synthetase, data were examined from a

6323

FIG. 9. Phosphorylation of glycogen synthetase during con- version to its D form. The time-dependent incorporation of 32P into glycogen synthetase in the presence of cyclic AMP and the cyclic AMP-dependent protein kinase (prepared by Method 1) was determined (Method B) in 10 separate experiments using 3 different preparations of glycogen synthetase. Protein-bound 32P was determined on NO-~1 aliquots of the reactions mixtures as described under “Materials and Methods.” Mean values and standard deviations are plotted.

pq ADDED CYCLIC AMP- DEPENDENT PROTEIN KINASE

FIG. 10. Dependence of phosphorylation of glycogen synthetase and phosphorylase kinase on added protein kinase. All reactions (Method B) contained +P-ATP, magnesium acetate, cyclic AMP, and the amount of protein kinase (prepared by Method 1) indicated on the abscissa. In A, each reaction contained 100 pg of glycogen synthetase; in B, 135 pg of phosphorylase kinase. Initial rates of phosphorylation are expressed as picomoles of 32P incorporated per min in the loo-p1 aliquot.

number of experiments in which the time course of phosphoryla- tion of several different preparations of glycogen synthetase was followed. These data, expressed in Fig. 9 as mean values with standard deviations, show that phosphorylation leveled off at 1.1 f 0.1 moles of 32P per lo6 g of synthetase.5

Assay of Cyclic AMP-dependent Protein Kinase by Measuring Incorporation of a2P into Glycogen Synthetase or Phosphorylase Kinuse-In order to study the specificity of cyclic AMP-depend- ent protein kinases catalyzing the conversion of glycogen synthetase I to glycogen synthetase D and the activation of phos- phorylase kmase, it was important to develop quantitative assay procedures that would be applicable using either substrate. As

5 The exact amount of phosphate incorporated into glycogen synthetase during its conversion from the I to the D form must remain open until it is known with certainty that one is starting with the enzyme completely in the dephospho form. Although the preparative procedure contains a step in which glycogen syn- thetase phosphatase, present as a contaminant at that point, is allowed to act, analysis of the purified synthetase I still shows traces of alkaline-labile phosphate present as described earlier.

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6324 Inactivation of Glycogen Xynthetase Vol. 245, No. 23

GLYCOGEN SYNTHETASE PHOSPHORYLATION

250 m PHOSPHORYLASE KINASE PHOSPHORYLATION

PHOSPHORYLATION I

FRACTION: I 2 3 4a 4b

FIG. 11. Copurification of the cyclic AMP-dependent protein kinase activities. The protein kinase was purified by Method 2 which gives two acbive protein kinase peaks (Fractions Qa and 4b). Fractions from the different stages of purification were tested for their abilities to phosphorylate glycogen synthetase and phos- phorylase kinase (Method B). The activity of the endogenous protein kinase in both substrates was subtracted from the activity found for each fraction. Casein phosphorylation was assayed for 20 min at 30” in 100-J reaction mixtures containing 1.8 mM glyc- erol-P (DH 6.8). 4 mM NaF. 0.4 mM theonhvlline. 0.13 mM EGTA. 0.4 mM kDTA,‘2 X KVaa cyclic AMP, casein, 6 mg per ml, 0.2 rni +zP-ATP, 10 mM magnesium acetate, and aliquots of the protein kinase. The three specific activities of the acid supernatant frac- tion, pH 6.1 (Fraction 1), were set equal to 1, and the three relative specific activities of each of the other fractions were then calcu- lated. The fract.ions contained the following percentages of the total protein kinase activity (assayed with casein as the substrate) of the muscle extract; Fraction 1,54%; Fractions, 18%; Fraction 3, 7.8%; Fraction da, 2.7%; Fraction &b, 1.8%. See “Materials and Methods” for details regarding the fractionation steps used.

noted above (Figs. 6 and 7), the glycogen synthetase I to glyco- gen synthetase D reaction followed by measuring the decrease in glucose-6-P-independent activity could serve as a good quantita- tive assay for the protein kinase. Likewise, it would be possible to utilize the change in activity of phosphorylase kinase in assay- ing the protein kinase (11). These methods proved to be cumbersome, however, and were particularly difficult to apply when assaying crude sources of the protein kinase. For this reason, phosphorylation of each of the protein substrates by y-s*P-ATP was used as the basis for assaying the protein kinase. Fig. 10 shows that initial reaction rates of a2P incorporation into either glycogen synthetase (Fig. 1OA) or phosphorylase kinase (Fig. 10B) were proportional to the protein kinase concentration. The data of Fig. 10, A and B, were analyzed by extrapolating the lines to zero initial rates in order to determine the extent of contamination of each of the substrates with the cyclic AMP- dependent protein kinase. This analysis revealed that glycogen synthetase and phosphorylase kinase each appear to contain 0.1% of the protein kinase on a weight basis. Analysis of the data of Fig. 7 by the same method would appear to indicate that glycogen synthetase was contaminated to the extent of 1% with the protein kinase. The discrepancy between these values was probably because different preparations of glycogen synthetase were used in the two experiments. It should be noted that in all of these calculations, the estimates of extent of contamination were maximal since the protein kinase preparation used in Figs. 7 and 10 was only partially pure.

Copuri$cation of Glycogen Synthetase Kinase and, Phospho yluse Kinase Kinase-With the availability of quantitative methods for measuring the activities of the cyclic AMP-dependent protein kinase with glycogen synthetase or phosphorylase kinase as substrates, it was possible to determine whether both specific activities increased in parallel during purification of the protein kinase. While this study was in progress, it was found that the cyclic AMP-dependent protein kinase could be separated into at least two peaks by column chromatography (16). It was par- ticularly important to look at both of these fractions since one fraction might have contained phosphorylase kinase kinase activity and the other glycogen synthetase kinase activity. Fig. 11 gives the results of an experiment in which the phosphoryla- tions of phosphorylase kinase, glycogen synthetase, and casein by the protein kinase at various stages of purification are compared. The acid supernatant fraction at pH 6.1 was used as the starting point since it is essentially free of phosphorylase kinase, which would have interfered if it were present. The three specific activities of this fraction were arbitrarily assigned a relative specific activity of one. As can be seen, the three activities increased equally at each stage of purification. This was so even in the final step that resulted in the separation of the cyclic AMP-dependent protein kinase into two peaks.

E$ect of Heat-stable Protein Inhibitor on Phosphoylation of Glycogen Synthetase and Phosphorylase Kinase-Skeletal muscle contains a heat-stable protein known to inhibit the cyclic AMP- stimulation of both phosphorylase kinase activation and the glycogen synthetase I to D conversion (11, 13, 35, 36). In addition, this protein inhibitor has been shown to inhibit the cyclic AMP-dependent protein kinases from bovine heart (37) and rat adipose tissue (38) but does not inhibit glycogen synthe- tase or cyclic AMP-independent kinases, such as phosphorylase kinase and phosphofructokinase (13). Pertinent to the question of whether the same or different enzymes catalyze the phos- phorylation of phosphorylase kinase and glycogen synthetase, was the ability of the inhibitor to affect these two reactions equally. Fig. 12 shows that, with increasing concentrations of the inhibitor, both reaction rates were decreased in parallel as would be expected if a single enzyme were catalyzing both reac- tions.

The protein inhibitor was also used to probe the question of whether or not the cyclic AMP-dependent protein kinase is involved directly as the catalyst for the conversion of glycogen synthetase I to synthetase D. An alternative role for the protein kinase would be that it might catalyze the phosphorylation and activation of a speci6c glycogen synthetase kinase as illustrated in Reaction 1. The activated glycogen synthetase kinase would then catalyze the conversion of glycogen synthetase from its I to its D form (Reaction 2). The inactive glycogen synthetase

protein kinase Inactive glycogen synthetase kinase >

cyclic AMP, ATP 0)

active glyoogen synthetase kinase

active glycogen synthetase kinase

Glycogen synthetase I > ATP (2)

glycogen synthetase D

kinase could be present as a contaminant either in purified

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pg ADDED INHIBITOR

FIG. 12. Inhibition of glycogen synthetase and phosphorylase kinase phosphorylations by a protein inhibitor from skeletal muscle. Phosphorylation (Method B) of glycogen synthetase (Curve A) and of phosphorylase kinase (Curve B) was determined in the presence of cyclic AMP, 0.95 pg of added cyclic AMP- dependent protein kinase (Method I), and varying amounts of the protein inhibitor. The two protein kinase activities in the ab- sence of any added inhibitor were taken as 100%. The percent- ages of the two protein kinase activities are plotted against the amount of the inhibitor used.

glycogen synthetase or in the protein kinase. Such a scheme would be analogous to the phosphorylase system in which the cyclic AMP-dependent protein kinase catalyzes the phos- phorylation and activation of phosphorylase kinase which in turn catalyzes the phosphorylation of phosphorylase.6 To decide between a direct or indirect role for the protein kinase, the protein inhibitor was added during the course of a glycogen synthetase I to D conversion reaction that had been initi- ated by adding the cyclic AMP-dependent protein kinase. From what was known of the specificity of the protein inhibitor (see above), it was reasoned that if the protein kinase were acting directly on synthetase I, then the reaction would immedi- ately be blocked. On the other hand, if the protein kinase served only to activate a separate cyclic AMP-independent synthetase

kinase, analogous to phosphorylase kinase, then the reaction would not be blocked by the inhibitor. Fig. 13 gives the results

of an experiment of this type. Two identical conversion reac- tions (&rues B and C) were started by addition of magnesium acetate and ATP. At arrow 1 the inhibitor was added to one of

the reactions (B) and, as can be seen, it, completely blocked the reaction. Addition of an excess of the cyclic AMP-dependent. protein kinase to Reaction B at arrow %’ reinitiated the reaction, thus showing that the protein inhibitor was acting on the protein kinase and not on some other component of the reaction, e.g. cyclic AMP or ATP. Total synthetase activity (Curve A) was not affected by addition of the protein inhibitor or excess protein

kinase. Inactivation of Cyclic AMP-dependent Protein Kinase by Heat-

ing in Presence and Absence of Cyclic AMP-Further evidence that the same enzyme catalyzes the conversion of glycogen syn- thetase I to synthetase D and the activation of phosphorylase

6 Preliminary reports describing the role of the cyclic AMP- dependent protein kinase in t.he phosphorylase kinase activation system have already been given (10, ll), and a more complete presentation of the data is in preparation (D. A. Walsh, J. P. Perkins, C. 0. Brostrom, E. S. Ho, and E. G. Krebs)

1 T I 27 *. = . . A

25 - . .

O------J 0 2 4 6 8 IO

MINUTES

6325

FIG. 13. Site of action of the cyclic AMP-dependent protein kinase in the glycogen synthetase I to glycogen synthetase D re- action. Two identical conversion reaction mixtures (Method A) contained 320 pg of glycogen synthetase and 12 pg of the cyclic AMP-dependent protein kinase (Method 1) in a final volume of 0.3 ml. At arrow 1 purified protein inhibitor was added to one of the reaction mixtures (Curve B) to a final concentration of 17 pg per ml. At arrow 8 an excess of the protein kinase (final concen- tration of 26Opg per ml) was added to this same reaction mixture. The control reaction (Curve C) received the appropriate volumes of buffer at both time points. Glycogen synthetase activities in Curves B and C were determined in the absence of glucose-6-P and are given on the ordinate as units per ml based on the original synthetase preparation containing 3.2 mg per ml. Curve A is a control showing the activity (expressed as units per ml of the original synthetase preparation) of glycogen synthetase meas- ured in the presence of glucose-6-P in the reaction mixture tu which the addit,ions were made (Curve B).

kinase was obtained by studying the heat inactivation of the two activities of the purified cyclic AMP-dependent protein kinase. Heat, inactivation was carried out in the presence and absence of cyclic AMP, since the latter compound was known to destabilize the protein kinase (37) and its inclusion added an additional parameter for the possible differentiation of separate kinases. Fig. 14 illustrates an experiment in which aliquots of the protein kinase were incubated for 5 min at the temperatures indicated on the abscissa in the presence (Curves C and D) or absence (Curves A and B) of 5 X 10V6 M cyclic AMP. Activities were then determined in the presence of 1 X 1OV M cyclic AMP in all cases using either glycogen synthetase I (Curves B and C) or phosphorylase kinase (Curves A and D) as substrates. No significant differences in the sensitivities to heat, inactivation were noted regardless of whether phosphorylase kinase kinase or glycogen synthetase kinase activity was being measured. It will be noted that when cyclic AMP was present during the pre- liminary incubation period, both activities were much lower, even at 0”. Destabilization of the protein kinase by cyclic AMP did not occur in the phosphorylation reaction itself as indicated by linear reaction kinetics (not illustrated). This was due to stabilization of the enzyme by the protein substrate in the reaction mixture.

Relative Rates of Phosphorylation of Several Substrates by Cyclic AMP-dependent Protein Kinase-In the original work describing

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6326 Inactivation of Glycogen Synthetase Vol. 245, No. 23

24d - I , TABLE II

\j 30 35 40 45 50 C'

-iFMPERP:URE "C

FIG. 14. Destabilization of the two cyclic AMP-dependent protein kinase activities by heating and cyclic AMP. Samples of the protein kinase (Method 1) were diluted to 12.5 pg per ml in 5 rnM glycerol-P, 2 mM EDTA, pH 7.0, and incubated for 5 min at t,he indicated temperatures in the presence or absence of 5 X 10m6 M cyclic AMP. All samples were then assayed for their abilities to phosphorylate both glycogen synthetase I and phos- phorylase kinase in the presence of 1 X 1W6 M cyclic AMP. Phos- phorylation due to endogenous protein kinase in the substrates was subtracted from all the values. The substrates and pre- liminary incubation conditions were as follows. A, phosphoryl- ase kinase, without cyclic AMP; B, glycogen synthetase, without cyclic AMP; C, glycogen synthetase, with cyclic AMP; D, phos- phorylase kinase, with cyclic AMP.

the cyclic AMP-dependent protein kinase which involved studies on the mechanism of activation of phosphorylase kinase (lo), it was recognized that this enzyme was not a specific phosphorylase kinase kinase, hence the name “protein kinase.” It was shown, for example, that the protein kinase catalyzed the phosphoryla- tion of casein and protamine in addition to phosphorylase kinase. In the present study the relative rates of phosphorylation of several substrates were determined. The two separable protein kinase peaks (Fractions 4a and 4b from Method 2 for preparing the enzyme) were used, and the initial rates of phosphorylation of casein, histone, phosphorylase kinase, and glycogen synthetase were determined in the presence of cyclic AMP (Table II). Specific activities, expressed as picomoles of 32P incorporated per min per pg of protein kinase, are shown in the first two col- umns for both enzymes with each substrate. In this particular preparation of the two protein kinases, the specific activities of Fraction 4b were approximately 0.5 those of Fraction 4a, irrespec- tive of the substrate. The data in the last two columns are expressed as relative specific activities with the specific activity for phosphorylase kinase phosphorylation arbitrarily set equal to 1 for each fraction. It is apparent from these data that the two protein kinases do not differ in substrate specificity at the substrate concentrations used. Phosphorylase kinase and glyco- gen synthetase, even at much lower protein concentrations (about 0.2 mg per ml), were much better substrates than histone and casein at 6 mg per ml. It should be noted, however, that the histone preparation used did not necessarily contain the best substrate among the several known histone fractions (39). A study similar to that in Table II was carried out using the pro- tein kinase prepared by Method 1, except that in this experiment the concentration of all protein substrates was kept constant at 0.27 mg per ml and the reaction conditions were those used in the experiment of Fig. 10, i.e. in the standard assay for the cyclic

Rates of phosphorylation of several substrates by cyclic AMP-de- pendent protein kinase fractions

For the phosphorylation of histone and casein, the reaction mixtures contained the following: 50 mM 2[N-morpholinolethnne sulfonic acid, 1 mM potassium phosphate, 20 mM NaF, 0.3 mM EGTA, 0.2 mM EDTA, 2 mM theophylline, 0.2 mM r-@P-ATP, 10 m&l magnesium acetat.e, 2 PM cyclic AMP, 6 mg per ml of casein (assayed at pH 6.0) or histone (assayed at, pH 6.7), and Fraction 4a, 37 pg per ml, or Fraction 4b, 2Opg per ml, of the protein kinase prepared by Method 2. For the phosphorylation of glycogen synt,hetase and phosphorylase kinase, the reaction mixtures con- tained the following: 6.5 mM glycerol-P (pH 6.8)) 0.8 mM theophyl- line, 8.3 mM NAF, 0.27 mM EGTA, 0.18 mM EDTA, 0.7% sucrose, 5 FM cyclic AMP, 0.14 mM Y-~~P-ATP, 1.25 mM magnesium acetate, phosphorylase kinase, 0.27 mg per ml, or glycogen spnthetase, 0.18 mg per ml, and Fraction 4a, 1.8 pg per ml, or Fraction 4b, 2.0 pg per ml, of the protein kinase prepared by Method 2.

Substrate

Rate of phosphoryl- ation

Fraction Fra;;ion 4a

Rate of phosphoryl- ation relative to that obtained with phos-

phorylase kinase as substrate

Casein....................... 10.3 5.2 0.18 0.16 Histone. . _. _. 19.6 9.6 0.34 0.30 Phosphorylase kinase 58.5 31.9 1.00 1.00 Glycogen synthetase......... 65.5 37.7 1.12 1.18

AMP-dependent protein kinase based on phosphorylation rates (Method U). Under these conditions the initial rates of phos- phorylation (picomoles of phosphate incorporated per min) were as follows: glycogen synthetase, 100; phosphorylase kinase, 76; histone, 14; and casein, 5. Other proteins that were examined as potential substrates for the protein kinase but that were not phosphorylated at 0.27 mg per ml included bovine serum albu- min, rabbit muscle phosphorylase b, rabbit muscle aldolase, phosvitin, egg white lysozyme, and yeast hexokinase.

Cyclic Nuckotide Xpecijkity in Conversion of Glycogen Synthetase

I to Glycogen Xynthetase D and in Activation of Phosphorylase K&use-Several investigators (14, 40) have studied the stimula- tion of the glycogen synthetase I to glycogen synthetase D reaction by various cyclic 3’,5’-nucleotides in liver and muscle. In general, the order of effectiveness in terms of the reciprocal of K, values has been cyclic AMP > cyclic IMP > cyclic CMP N cyclic GMP N cyclic UMP >> cyclic dAMP > cyclic TMP. In the present study the relative effectiveness of the various nucleo- tides was examined with respect to their ability to stimulate either the phosphorylation of glycogen synthetase or phosphoryl- ase kinase using the purified protein kinase (Method 1). This enzyme showed some activity toward each of these substrates in the absence of any cyclic nucleotide, so that a comparison of the effectiveness of these activators had to be based on their ability to augment the basal phosphorylation rate that was present without them. With phosphorylase kinase as substrate the protein kinase exhibited 19% of its maximal activity without cyclic AMP, and with glycogen synthetase as substrate, 8% of its maximal activity. Maximal activity was defined as that which occurred in the presence of 1 X low6 M cyclic AMP. Table III gives the relative effectiveness of the different nucleotides tested

Fraction Fraction 46% 4b

-___

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TABLE III ATP

Relative effectiveness of cyclic nucleotides in stimulation of protein kinase with glycogen synthetase I and phosphorylase kinase as

substrates

Various cyclic 3’,5’-nucleotides were tested at concentrations of 1 X 10-e M and 1 X 1W7 M to determine the relative extent of activation of the protein kinase (prepared by Method 1) with respect to the phosphorylation of glycogen synthetase I and phos- phorylase kinase.

HORMONE l ;yDCELN;SLE

. I CYCLIC AMP

NONACTIVATED PHOSPHORYLASE KINASE 7 g

CYCLIC AMP-DEPENDENT I

ACTIVATED/ PROTEIN KINASE

PHOSPHORYLASE KINASE - I

Cyclic 3’,5’-nucleotides

AMP. . . . . 100 IMP. . . . . . . . . 64 GMP . . . 50 CMP 46 Dibutyryl-AMP. 44 UMP 25 dAMP. 4 TMP 0.5

Concentrations of Concentrations of nucleotides = nucleotides =

1 x 10-s M 1 x lo-’ M

With glycogen

synthetase

- With

glycogen syll-

thetase

I ,

With PhW

,horylase kinase

F

100 78 63 77 36 35 45 10 5 50 5 1 30 7 5 20 6 2

3 3 2 1 2 2

- 0 The increase in protein kinase activity (over the basal rate)

caused by 1 X lO+ M cyclic AMP was assigned a value of 100 for each of the two protein substrates.

at two different concentrations. It will be noted that except for cyclic TMP and cyclic dAMP all of the nucleotides gave signifi- cant activation of the protein kinase when tested at a concen- tration of 10e6 M, but the degree of stimulation was independent of the substrate being used. At a concentration of lo-’ M, only cyclic AMP and cyclic IMP were effective, and again the degree of stimulation was independent of the substrate. The slightly greater effect of 1W7 M cyclic GMP on the phosphorylation of phosphorylase kinase as compared with phosphorylation of glycogen synthetase is of doubtful significance.

DISCUSSION

The purification of rabbit skeletal muscle glycogen synthetase in its I or D form has been described previously by Schlender et al. (14) and Villar-Palasi et al. (22). This paper introduces a new procedure for purification of glycogen synthetase I in an essentially homogeneous form that is free of phosphorylase and phosphorylase kinase and contains very little cyclic AMP- dependent protein kinase activity. il preliminary characteriza- tion of the enzyme indicates that it is an oligomeric protein with a molecular weight of approximately 4 x lo5 daltons. Piras and Staneloni (41) have recently reported a molecula,r weight of 250,000 for rat muscle glycogen synthetase which would appear to indicate that species differences in this property may exist. The subunit size of the rabbit muscle enzyme is similar to that of rabbit muscle glycogen phosphorylase, 92,500 daltons (42), since the two proteins have essentially the same relative mobilities in dodecyl sulfate-polyacrylamide disc gel electrophoresis. A sub- unit molecular weight of 90,000 to 100,000 for glycogen synthe- tase I is consistent with the finding that 1 mole of phosphate is incorporated per 91,000 g of protein when the enzyme is converted from the 1 to the D form. This amount of phospate isconsider-

GLYCOGEN SYNTHETASE 0

PHOSPHORYLASE b PHOSPHORYLASE a_ GLYCOGEN SYNTHETASE I

GLYCOGEN GLYCOGEN (n-1 glucose units) (nt t glucose units1

FIG. 15. Dual role of the cyclic AMP-dependent protein kinase in the regulation of glycogen metabolism in muscle.

ably less than the value of 5 moles of phosphate incorporated into glycogen synthetase per lo5 g of protein reported by Larner, Villar-Palasi, and Brown (43) and Schlender et al. (14). Reasons for this large discrepancy in the stoichiometry of phosphate incorporation during the conversion of glycogen synthetase I to glycogen synthetase D are not readily apparent.

On disc gel electrophoresis (without sodium dodecyl sulfate), glycogen synthetase I migrates as a single band when glucose-6-P is present. In the absence of this compound the synthetase separates into two bands, one of which moves ahead of the band with glucose-6-P and another which moves more slowly. It is possible that the more rapidly migrating band represents a dis- sociated form of the enzyme, whereas the slower band represents undissociated glycogen synthetase. The increased mobility of undissociated enzyme in the presence of glucose-6-P, as compared with this form in the absence of the glucose-6-P, could be due to the increased negative charges contributed by the bound sugar phosphate (29). The single protein band seen with glucose-6-P is associated with glycogen synthetase activity, but no activity was detectable in the gel without glucose-6-P. This result may have been simply due to the known instability of the enzyme in the absence of glucose-6-P (44, 45), but it is also possible that dissociation itself results in a loss of activity. In the absence of glucose-6-P more than half of the enzyme appeared to be present in the rapidly moving band and that remaining in the slow band may have been at too low a level to be detected by the assay system used. The ability of glucose-6-P to promote the forma- tion of or to stabilize a form of glycogen synthetase which does not dissociate readily is consistent with the role of this compound as an allosteric effector of the enzyme (46, 47).

The availability of purified glycogen synthetase and phospho- rylase kinase relatively free of cyclic AMP-dependent protein kinase activity made it possible to determine whether the same or different enzymes catalyze the phosphorylation of these pro- teins. The data obtained support the former view. The rela- tive specific activities of each of two separable protein kinase fractions toward the two substrates increased in parallel during purification. Furthermore, the activity of one of the protein kinase fractions toward these substrates was inhibited equally by the protein inhibitor regardless of which substrate was employed. There was equivalent sensitivity to heat inactivation, similar destabilization of the enzyme by cyclic AMP, and similar degrees

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6328 Inactivation of Glycogen Synthetase Vol. 245, No. 23

of activation by various cyclic nucleotides, all of which were independent of the substrate used. It was also noteworthy that the absolute rates of phosphorylation of glycogen synthetase and phosphorylase kinase by the cyclic AMP-dependent protein kinase were almost identical.

Besides being a valuable tool in helping to establish the dual activity of the cyclic AMP-dependent protein kinase, the skeletal muscle protein inhibitor proved to be useful in showing that the protein kinase acts directly on glycogen synthetase I and not through a second kinase. This means that the glycogen synthe- tase inactivation system has one less potential amplification or control step than is present for phosphorylase activation (Fig. 15). This seems reasonable, since under certain conditions glycogenolysis must be triggered very rapidly whereas it is per- haps not so important that a decreased rate of glycogenesis be brought about as quickly. In skeletal muscle the relative amounts of total phosphorylase and glycogen synthetase are also such that the glycogenolytic response can occur with greater velocity than glycogenesis.

The activation of one enzyme system and inactivation of the opposing enzyme system by the action of a single enzyme pro- vides a unique regulatory mechanism (Fig. 15). This type of control prevents the energetically wasteful use of the newly formed glucose-l-P for resynthesis of glycogen when glucose-l-P is needed to provide energy via glycolysis. Further regulation between these two enzyme systems may be found when the roles of the various phosphoprotein phosphatases are assessed. In particular, it will be of interest to determine whether the same or different phosphatases catalyze the dephosphorylation of phos- phorylase kinase and glycogen synthetase.

Acknowledgments-The technical assistance of Mrs. Peta Hallisey and Mrs. Phyllis Abd-El Al is gratefully acknowledged. We are also grateful for the help of Dr. Taro Hayakawa in carrying out the model E ultracentrifugal analysis.

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Hunkeler, D. A. Walsh and Edwin G. KrebsThomas R. Soderling, John P. Hickenbottom, Erwin M. Reimann, Felix L.Muscle Adenosine 3',5'-Monophosphate-dependent Protein Kinases

Inactivation of Glycogen Synthetase and Activation of Phosphorylase Kinase by

1970, 245:6317-6328.J. Biol. Chem. 

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