Phosphorylation of sites 3 and 2 in rabbit skeletal muscle glycogen

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc.

Val. 260, No. 22, Issue of October 5, pp. 12287-12292,1985 Printed in U.S.A.

Phosphorylation of Sites 3 and 2 in Rabbit Skeletal Muscle Glycogen Synthase by a Multifunctional Protein Kinase (ATP-Citrate Lyase Kinase)*

(Received for publication, March 6, 1985)

Virender S. SheorainS, Seethala Ramakrishnag, William B. Benjamin$, and Thomas R. SoderlingS From the $Howard Hughes Medical Institute and Department of Physiology, Vanderbilt Medical School, Nashville. Tennessee 37232 and the 6Deaartment of Phvsiology and Biophysics, School of Medicine, State University of New York, Stony Brook, New York 11794-

, ”

A multifunctional protein kinase, purified from rat liver as ATP-citrate lyase kinase, has been identified as a glycogen synthase kinase. This kinase catalyzed incorporation of up to 1.5 mol of 32P04/mol of synthase subunit associated with a decrease in the glycogen synthase activity ratio from 0.85 to a value of 0.15. Approximately 65-70% of the 32P04 was incorporated into site 3 and 30-35% into site 2 as determined by reverse phase high performance liquid chromatography. Release of 32P04 from the phosphopeptides during automated Edman degradation confirmed the site 3 and 2 assignment. Thermal stability studies established that the phosphorylations of sites 3 and 2 were catalyzed by the same kinase. This multifunctional kinase was distinguished from glycogen synthase kinase-3 on the basis of nucleotide (ATP versus GTP) and protein substrate (glycogen synthase, ATP-citrate lyase, and acetyl-coA carboxylase) specificities. Since the phosphate contents in glycogen synthase of sites 3 and 2 are altered in diabetes and by insulin administration, the possible involvement of the multifunctional kinase was explored. Glycogen synthase purified from diabetic rabbits was phosphorylated in vitro by this multifunctional kinase at. only 10% of the rate compared to synthase purified from control rabbits. Treatment of the diabetics with insulin restored the synthase to a form that was readily phosphorylated in vitro.

Several of the metabolic effects of insulin are mediated by alterations in the phosphorylation state of serine or threonine residues in regulatory enzymes. Insulin treatment of isolated tissues and cells has been shown to both increase and decrease 32P contents of different proteins (1-5). Identification of the kinases and/or phosphatases whose activities are altered by insulin has been difficult. Recently it has been demonstrated that the insulin receptor is a protein kinase that is activated and catalyzes autophosphorylation upon binding of insulin (6). However, the insulin receptor protein kinase specifically phosphorylates tyrosine residues. The relationship, if any, between this tyrosine kinase activity and most of the cellular phosphorylations on serine/threonine residues affected by insulin action is not clear.

In addition to the insulin receptor, other proteins whose net phosphorylations are elevated include ribosomal protein S6 (7, 8), acetyl-coA carboxylase (9), and ATP-citrate lyase

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “uduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(1-5). ATP-citrate lyase i n vivo contains several sites of phosphorylation in two tryptic peptides, peptides a and b (10). When rat epididymal fat pads, prelabeled for 90 min with [32P]phosphate, are incubated with insulin, the 32P-content of peptide a increases whereas the total labeling of peptide b is unaltered (11). However, if the fat pads were “chased” for 6 h with nonlabeled phosphate between the 32P-labeling period and the exposure to insulin, then the calculated specific radioactivity of peptide a increased &fold but that of peptide b decreased 3-fold (11). Peptide A is phosphorylated i n vitro by the CAMP-dependent protein kinase whereas peptide B is phosphorylated by an independent kinase (ATP-citrate lyase kinase) (12). Elution profiles for peptides a and b (derived from i n vivo phosphorylations) were the same as those of peptides A and B (obtained from in vitro phosphorylations). Phosphorylation of peptide B is dependent on prior phosphorylation of peptide A (12). The ATP-citrate lyase kinase that specifically phosphorylates peptide B has been highly purified and characterized (13, 14). Because this kinase also phosphorylates other proteins including acetyl-coA carboxylase (E), it will be referred to as multifunctional protein kinase. To date, no specific regulatory effectors of this kinase have been identified.

Skeletal muscle glycogen synthase, that is dephosphoryl- ated and activated in response to insulin administration, has been extensively investigated. I n vitro studies have identified eight sites of phosphorylation (sites la, lb, 2,3a, 3b, 3c, 4 and 5) that can be catalyzed by CAMP-dependent, calmodulin- dependent, and independent protein kinases (see Table I for summary of site specificities of kinases). None of the protein kinases tested to date phosphorylated both sites 3 and 2. This phosphorylation specificity is of particular interest relative to the effects of diabetes and subsequent insulin treatment. Skeletal muscle glycogen synthase purified from alloxan- diabetic rabbits has an increased phosphate content in sites 3 and 2 (16, 17). Administration of insulin to the diabetics decreased the phosphate contents of sites 3 and 2 to control values (16, 17). We are, therefore, interested in identifying kinases or phosphatases which specifically act upon these two sites as potential mediators of the insulin-dependent activation of glycogen synthase. This paper describes the phosphorylation site specificity towards glycogen synthase of the multifunctional protein kinase that also phosphorylates ATP- citrate lyase and acetyl-coA carboxylase.

EXPERIMENTAL PROCEDURES

Purification of Enzymes-Glycogen synthase a was purified from rabbit skeletal muscle as described earlier (18) with the exception that synthase was precipitated by polyethylene glycol instead of by (NH,),SO, after DEAE-cellulose (19). Glycogen synthase b was pu-

12287

12288 Phosphorylation of Sites 3 and 2 by a Multifunctional Protein Kinase

TABLE I Phosphorylation specificities of various protein kinases on rabbit

skeletal muscle glycogen synthase phosphorylation sites Phosphorylation specificities were established and described pre-

viously for some of these kinases (28). Phosphorylation of sites 3 and 4 in addition to sites 2, IA, and 1B by CAMP-dependent protein kinase were established recently (29). Sites 3 and 2 for multifunctional protein kinase were determined and have been described in this manuscript.

Kinase Phosphorylation sites

2 3ABC 4 5 1A, 1B

CAMP-dependent ki- + t + + + Casein kinase I1 t Independent kinase 3 t Independent kinase 4 + Phosphorylase kinase + Calmodulin-dependent + + Multifunctional protein + +

nase

kinase I

kinase (ATP-citrate lyase kinase)

rified from normal, diabetic and insulin-treated diabetic rabbits as reported earlier (20). ATP-citrate lyase kinase was purified from rat liver (13, 14). ATP-citrate lyase and acetyl-coA carboxylase used as substrates were also purified from rat liver by the methods of Linn and Srere (21) as modified (22) and Song and Kim (23) , respectively. The heat-stable inhibitor of CAMP-dependent protein kinase was purified from rabbit skeletal muscle (24). Glycogen synthase kinase- 3 was purified from rabbit skeletal muscle as described elsewhere (25).

Phosphorylation Reaction.-Standard phosphorylations were carried out by incubating0.1-0.5 mg/ml synthase with 10 mM magnesium acetate, 5 mM MESlbuffer (pH 6.7) 0.05 mM EDTA, 0.03 mM EGTA, 1.5 mM 2-mercaptoethanol, 10-100 PM [Y-~’P]ATP, heparin (2 units/ ml), and the heat-stable inhibitor of CAMP-dependent protein kinase (0.2 units/ml). Reactions were initiated by addition of kinase(s). Glycogen synthase phosphorylation stoichiometries were determined by spotting an aliquot on 1-cm2 Phosphocellulose papers and washing with 75 mM H3P0, (26). Aliquots were taken from parallel reactions containing nonradioactive ATP for glycogen synthase activity ratio assays (27).

Quantitation of Phosphorylation Stoichiometries of Each Site-The details of this method have been described earlier (28, 29). In brief, 32P-synthase was separated from [Y-~’P]ATP by precipitation with trichloroacetic acid. The 32P-synthase was then digested by trypsin (1 mg/ml) for either 5 or 16 h. The complete tryptic digest was chromatographed by reverse phase HPLC to separate the 32P-peptides. The 16-h digestion did not change the distribution of 32P04 into various peptides except site 2 peptide was further degraded to a second peak which eluted at about 110-112 min (see Fig. 1 under “Results”). The percentage of 32P04 in each peptide was multiplied by the total phosphorylation stoichiometry, determined by Phospho- cellulose paper assay, to estimate the stoichiometry for each site. The 32P-peptides separated by reverse phase HPLC were analyzed for 32P release during automated Edman degradation in a Beckman model 890 C sequenator.

Other Meth~ds-[y-~’P]ATP and [Y-~’P]GTP was prepared by the method of Walseth and Johnson (30). GDP used for GTP synthesis was repurified on reverse phase HPLC in order to remove the contaminating ADP. Protein determinations were done by the method of Lowry et al. (31). Sodium dodecyl sulfate slab gel electrophoresis was run using Tris/glycine buffer (32).

Materials-Trypsin (L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated, protein 88%) was obtained from Cooper Biomedical. ADP, ATP, GDP, GTP, and bovine serum albumin were obtained from Sigma. Trifluoroacetic acid (HPLC) was purchased from Pierce Chemical Co., and CH3CN was from Burdick and Jackson Labora-

The abbreviations used are: MES, 4-morpholineethanesulfonic acid; HPLC, high performance liquid chromatography; EGTA, eth- ylene glycol bis(P-aminoethyl ether)-N,N,N‘,N’-tetraacetic acid; SDS, sodium dodecyl sulfate.

tories, Inc. Gel electrophoresis reagents were obtained from Bio-Rad. All other reagents were of analytical grade.

RESULTS

Phosphorylation and Identification of Sites 3 and 2-A tryptic digest of 32P-glycogen synthase that was phosphorylated by the multifunctional protein kinase was chromatographed on reverse phase HPLC (Fig. 1). Major 32P-peptides were eluted that corresponded to sites 3 (40-60 min) and 2 (112 and 125 min). The peak at 112 min is derived from the site 2 peptide (125 min) upon prolonged trypsin digestion (see “Experimental Procedures”). A minor 32P-peptide eluting at 30 min was consistently observed, but, because it constituted less than 5% of the total cpm, it was not further analyzed. It may contain sites 4 and or 5 (29). Because the peptide containing site 2 contains other serine residues that could poten- tially be phosphorylated, it was important to determine the actual site(s) of phosphorylation. When this tryptic 32P-peptide was subjected to automated Edman degradation, the 32P was released at the third cycle (not shown). When the phosphopeptides containing sites 3a, 3b, and 3c were likewise analyzed, there was release of 32P at cycles 3, 7, and 11 as expected for site 3 (not shown).

Electrophoretic Mobility-Synthase phosphorylated by multifunctional protein kinase was resolved on SDS gel (Fig. 2). The main synthase band appeared to be split into two bands. Autoradiography revealed that although 32P04 was detected in both bands most of it was associated with the slower migrating species.

Distribution of 32P04 in Sites 3 and 2-Fig. 3 shows a time course of the phosphorylation and 32P04 distribution between sites 3 and 2. Phosphorylation reached a plateau in about 60- 90 min with 1-1.2 mol of 32P04 incorporated per mol of synthase subunit (90,000 9). The distribution of 32P04 between sites 3 and 2 was 65-70 and 35-30%, respectively (see also Fig. 6).

Effect of Phosphorylation on Glycogen Synthase Activity

RETENTION TIME (min) FIG. 1. HPLC separation of tryptic phosphopeptides de-

rived from glycogen synthase. Glycogen synthase a (0.5 mg/ml) was phosphorylated by multifunctional protein kinase in vitro up to 0.72 mol of 32P04/90,000 g (see “Experimental Procedures” for phosphorylation conditions). 32P-synthase was isolated hy trichloroacetic acid precipitation method and subjected to tryptic digestion (1 mg/ ml for 16 h). The mixture of tryptic 32P-peptides was chromatographed on reverse phase (C,) HPLC. The column was developed using CH&N gradient (0-5% for 10 min, 5-38% for 100 min, and 38-50% for 10 min) in 0.1% trifluoroacetic acid.

Phosphorylation of Sites 3 and 2 by a Multifunctional Protein Kinase 12289

A B C D

94 kDa

64 kDa

42 kDa

30 kDa

22 kDa

14 kDa

FIG. 2. SDS gel electrophoresis on glycogen synthase phosphorylated by ATP-citrate lyase kinase. A 12.5% gel was run for 4 h at a constant current of 15 mA/slab. Gel was fixed, stained, destained, and dried. An autoradiogram was done using Kodak X- Omat RP film. Lane A represents molecular mass standards (94 kDa, phosphorylase b; 64 kDa, bovine serum albumin; 42 kDa, ovalbumin; 30 kDa, carbonic anhydrase, 22 kDa, soybean trypsin inhibitor; and 14 kDa, lyosozyme). Lane B represents glycogen synthase a incubated with [r-32P]ATP-M92+ mixture without kinase. Lane C represents glycogen synthase a phosphorylated by multifunctional protein kinase up to 0.59 mol of 32P04/90,000. Lane D is an autoradiogram of lane C.

Ratio-Fig. 4 shows the linear regression between changes in the activity ratio of glycogen synthase and the total 32P04 incorporation per subunit. With three different preparations of glycogen synthase and using two separate preparations of the multifunctional protein kinase, it was observed that the activity ratio dropped from an initial value of 0.76-0.84 to about 0.15-0.2 with 1-1.2 mol of 32P04/90,000 g. The coeffi- cient of correlation was 0.91.

Thermal Actiuation-The thermal stability of the multifunctional protein kinase was examined, particularly with respect to the phosphorylation site specificity. Kinase activity was inhibited about 85% in 20 min at 50 “C (Fig. 5A). The distribution of 32P04 between sites 3 and 2 was calculated at different degrees of thermal inactivation (Fig. 5B). When moles of 32P04/mole of site were plotted against total 32P04 incorporated, the distribution between sites 3 and 2 appeared

1,2

‘e 0,8 8 N c)

Oa6

v, W J

0.4

0.2

C

e / e

20 40 60 80 100

INCUBATION TIME (Min)

FIG. 3. Time course of glycogen synthase phosphorylation by multifunctional protein kinase and phosphate distribution between site 3 and 2. Glycogen synthase (0.1-0.5 mg/ml) was phosphorylated by multifunctional protein kinase over a period of 90 min (see “Experimental Procedures” for reaction conditions). At each time point, an aliquot was spotted on filter paper for total 32P04 incorporated (moles/90,000 g, M) as well as for HPLC run (conditions for HPLC run were identical to those described under Fig. 1). Mol of 32P04/mol of site 3 (A-A) or site 2 (X-X) were calculated from per cent of total counts in respective peaks multiplied by total phosphate (mol of 32P04/90,000 g) estimated by paper assay.

-I ” 0,2 0.4 0.6 0,8 I ,O 1,2

TOTAL 32P04 INCORPORATED (Moles/90,000g)

FIG. 4. Relationship between glycogen synthase activity ratio and degree of phosphorylation by multifunctional protein kinase. Phosphorylation reaction and activity ratio were carried out as described under “Experimental Procedures.” The number of ob- servations was 21 and the r value was calculated to be -0.91. G6P, glucose 6-phosphate.


0.5 I I I I 100

INCUBATION TIME AT 50°C (Min)

1 I I I I

B

1

0.1 On2 0.3 044 0 .5 0,6 TOTAL 32P04 INCORPORATED (Moles/90,000g)

FIG. 5. Effect of temperature on the activity of multifunctional protein kinase. Several aliquots of kinase were incubated at 50 “C for different time periods (up to 20 min). These were then assayed for synthase phosphorylation. Phosphorylation conditions were the same as described under “Experimental Procedures” except reactions were terminated at 15 min in order to be in the linear range of phosphorylation. Panel A shows the total 32P04 incorporation (U) as well as per cent inhibition (X- - -X) of kinase activity. Panel B shows the distribution of 32P04 between site 3 and site 2 of glycogen synthase phosphorylated by control as well as phosphorylated by thermal inactivated kinase. Phosphorylations were carried out with varying degrees of thermal inactivated kinase and these were matched with phosphorylation by control enzyme (by using different concentrations of kinase). Total phosphorylations were determined by filter paper assays, and distribution between sites 3 and 2 were calculated by subjecting various samples to HPLC (as described in the legend for Fig. 3). M indicate site 3 and 0- - -0, site 2 of glycogen synthase phosphorylated by control enzyme whereas A- A indicate site 3 and A- - -A site 2 of glycogen synthase phosphorylated by thermal inactivated kinase.

to be about the same in synthase phosphorylated by thermal inactivated enzyme compared to synthase phosphorylated by control enzyme. Total 32P04 incorporations between the samples were approximated by using lower concentrations of the control enzyme compared to the thermally inactivated enzyme.

Nucleotide Specificity-Glycogen synthase phosphorylation reactions were carried out using either [y3’P]ATP or [y-”P] GTP as phosphoryl donors. The multifunctional protein kinase used [y-32P]ATP about 20 times better than [y3’P]GTP (Fig. 6). When the 60 min [ Y - ~ ~ P I G T P sample was analyzed by peptide mapping, 74% of the 32P04 was in site 3 and 26% was in site 2. In the same experiment, the phosphorylations

INCUBATION TlME(m1n)

FIG. 6. Phosphorylation of glycogen synthase by ATP-citrate lyase and glycogen synthase kinase-3. Phosphorylation reactions were carried out by glycogen synthase kinase-3 (0 and 0) and multifunctional protein kinase (A and A) using either [-p3’Pp1 ATP (-) or [y3’P]GTP (- - -). Sixty-minute samples were analyzed for peptide mapping on HPLC (see “Results”).

catalyzed by glycogen synthase kinase-3 were examined. The activity of this kinase was only 5 times better with [y-”P] ATP when compared to that with [T-~~PIGTP (Fig. 6).

Protein Substrate Specificity-Since the multifunctional protein kinase can catalyze the phosphorylation of several proteins, the relative rates of phosphorylation of these proteins using this protein kinase were compared to the relative rates of phosphorylation by glycogen synthase kinase-3. In all cases, the subunit concentrations of the three protein substrates were about 1.2 p ~ . Under the conditions tested, glycogen synthase was the best substrate for both kinases (Fig. 7). The multifunctional kinase phosphorylated ATP-citrate lyase and acetyl-coA carboxylase, whereas glycogen synthase kinase-3 did not significantly phosphorylate these proteins.

Phosphorylation of Glycogen Synthase b-In vitro phosphorylation of glycogen synthase b purified from normal, diabetic, and insulin-treated diabetic rabbits was carried out using the multifunctional protein kinase. The time course of these phosphorylations is shown in Fig. 8. Glycogen synthase b obtained from diabetic rabbits was a poor substrate when compared to synthase from normal rabbits. Glycogen synthase b purified from insulin-treated diabetics was as good a substrate for this kinase as synthase b from normal rabbits. Distribution of 32P04 into sites 3 and 2 was determined for the several synthase b species (Table 11). In the cases of normal and insulin-treated diabetics, out of 0.80-0.83 mol of 32P04/90,000 g, 66% was in site 3 and 25% was in site 2. The remaining 9% was distributed in other sites, mainly the peptide containing sites 4 plus 5. Using synthase purified from diabetics, the total 32P04 incorporation was 0.13 mo1/90,000 g with 54% in site 3 and 38% in site 2. Therefore, the in vitro phosphorylation of synthase from diabetic rabbits was mark- edly decreased in both sites 3 and 2.

DISCUSSION

This paper presents evidence that a rat liver multifunctional protein kinase that is active towards ATP-citrate lyase and acetyl-coA carboxylase also catalyzes the phosphorylation of sites 3 and 2 in skeletal muscle glycogen synthase. This kinase is of potential physiological significance because the phosphorylation states of these three proteins are modu- lated by insulin action.

Phosphorylation of Sites 3 and 2 by a Multifunctional Protein Kinase 12291

W (3 0 V > e J

W v)

I + z v) >

a

FIG. 7. Substrate specificities of multifunctional protein kinase and glycogen synthase kinase-3. All three substrates were tested at 1.2 PM concentration in the reaction mixture. Phosphoryla- tion reactions were carried out as usual except that filter papers were washed with 1 N HCl for 1 h in addition to washings with 75 mM H3P04. HC1 wash was included to get rid of ATP-citrate lyase endogenous phosphorylation of histidine residues. Glycogen synthase was the best substrate for multifunctional protein kinase as well as glycogen synthase kinase-3 with 0.78 and 0.22 mol of 32P04/90,000 g, respectively. This was expressed as 100% activity for both ATP- citrate lyase kinase and glycogen synthase kinase-3. Shaded bars indicate phosphorylation by multifunctional protein kinase; open bars indicate phosphorylation by glycogen synthase kinase-3.

1 00

0” 0.8 0 9 0 al * ‘ 0.6 0 a m N

LL 0.4 0 rn w _J 0 0.2 r

0

I I I I

15 30 45 60 INCUBATION TIME (rnin)

FIG. 8. Phosphorylation of glycogen synthase b by multifunctional protein kinase. Two species of synthase b purified from control, diabetic, and insulin-treated diabetic rabbits were used as substrate. Mean values have been plotted against time of incubation: X-X, control, A-A, diabetic, and 0 4 , insulin-treated diabetics.

The multifunctional protein kinase catalyzed incorporation of up to 1.5 mol of 32P04/mol of synthase subunit. In the linear phase of the reaction, about 65-70% of the phosphorylation is in site 3 and 30-35% in site 2. Determination of 32P04 release during automated Edman degradation confirmed that the phosphorylation occurred at the appropriate serine residues in the tryptic peptides containing site 3 and site 2. The amino acid sequences for the tryptic peptides containing site 2 are Thr-Leu-Ser(P)-Val-Ser-Ser-Leu-Pro-

TABLE rr Distribution of 32P04 into various sites of different species of glycogen

synthase b phosphorylated by multifunctional protein kinase Glycogen synthase b species were purified as described earlier (20).

These were further purified over Phosphocellulose in order to remove trace amounts of contaminating kinases and phosphatases (33). Pro- tein concentrations were 0.019-0.035 mg/ml for different species. [y- 32P]ATP concentration was 107 pM with specific activity of 4098 cpm/pmol. For time course, see Fig. 8. Sixty-minute aliquots from that experiment were processed for HPLC separations of different sites and 32P04 distribution between sites 3 and 2 were calculated as described under “Experimental Procedures.”

Glycogen synthase

b species

Total Moles 3zP04/ mole of site

incorporated 2 3 2 ~ 0 ,

mo1/90,000

Control (2) 0.80 0.52 0.19 Diabetic (2) 0.13 0.07 0.05 Diabetic + insulin (2) 0.83 0.56 0.23

and for sites 3a, 3b, and 3c is Pro-Ala-Sera(P)-Val-Pro-Pro- Serb(P)-Pro-Ser-Leu-Ser”(P)-Arg. This phosphorylation was associated with marked inactivation of glycogen synthase from an initial activity ratio of 0.85 down to 0.15. Phospho- rylation of sites 3 and 2 are known to decrease the activity ratio by increasing the K, for substrate UDP-glucose (34,35).

Phosphorylation of sites 3 and 2 could be due to the separate actions of two kinases, i.e. synthase kinases 3 and 4 (see Table I), rather than a single kinase. Evidence that a single kinase phosphorylated both sites 3 and 2 is compelling. Although studies in this paper utilized highly purified kinase that on SDS gel electrophoresis gave a single major band on silver staining (14), less pure fractions had the same ratio of site 3 to site 2 phosphorylation. Secondly, if the site 3 phosphorylation were due to synthase kinase 3, then there should have been significant phosphorylation when using GTP rather than ATP. Fig. 6 shows that glycogen synthase kinase 3 does utilize GTP as substrate whereas there is little phosphorylation using GTP and the multifunctional protein kinase. When the 60- min sample using GTP and the multifunctional protein kinase from Fig. 6 was analyzed by peptide mapping, the ratio of site 3 to site 2 was the same as when ATP was used. Thirdly, if two kinases are involved, they may have different thermal stabilities. The experiment of Fig. 5 shows that during thermal inactivation of the kinase at 50 “C, the ratio of site 3 to site 2 phosphorylation did not change compared to the control enzyme.

In addition to differences in nucleotide specificity, the multifunctional protein kinase also differs from synthase kinase-3 in protein substrate specificity. Both kinases phosphorylate site 3 in glycogen synthase. However, the multifunctional protein kinase also phosphorylates ATP-citrate lyase, acetyl-coA carboxylase, and site 2 in glycogen synthase whereas synthase kinase-3 does not catalyze the phosphorylation of these proteins. Thus, synthase kinase-3 and the multifunctional protein kinase appeared to be different kinases.

The specific phosphorylation of sites 3 and 2 in glycogen synthase by the multifunctional protein kinase may be of importance in our understanding the effects of insulin on glycogen synthase. The ability of insulin to increase skeletal muscle glycogen content results from two actions of the hormone: increased glucose transport into the muscle and conversion of glycogen synthase to a more activated form (20). Activation of glycogen synthase by insulin is associated with net dephosphorylation (16, 20, 36). The mechanism involved has not been elucidated and could either involve inhibition of a kinase or activation of a phosphatase. Our


previous studies have shown that diabetes and long-term insulin treatment affect sites 3 and 2 in skeletal muscle glycogen synthase (16,17). Therefore, the kinase or phosphatase involved in mediating these effects of insulin should be specific for sites 3 and 2. This conclusion is reinforced by the data in Fig. 8 and Table 11. Because sites 3 and 2 are more highly phosphorylated i n vivo in diabetic rabbits, synthase purified from these rabbits should be a very poor i n vitro substrate for this multifunctional protein kinase. Fig. 8 shows that synthase purified from diabetic rabbits was phosphorylated in vitro at about 10% the rate compared to synthase purified from control rabbits. Treatment of the diabetics with insulin completely restored the rate of i n vitro phosphorylation. In these experiments, 90% or more of the i n vitro phosphorylation was in sites 3 and 2 as determined by HPLC peptide mapping (Table 11).

The crucial question is whether insulin i n vivo inhibits this multifunctional protein kinase in rabbit skeletal muscle. In general, treatment of isolated cells or tissues with insulin increases the total phosphate content of proteins such as ATP-citrate lyase, acetyl-coA carboxylase, and ribosomal protein S6. All these phosphoproteins have multiple sites of phosphorylation catalyzed i n vitro by several protein kinases. It is possible that with insulin action some sites become more highly phosphorylated while other sites were less phosphorylated. This indeed appears to be the case with ATP-citrate lyase. Insulin treatment of fat pads results in elevated 32P content in peptide a and decreased 32P content of peptide b. Peptide A is phosphorylated in vitro by CAMP-dependent protein kinase and peptide B by the multifunctional protein kinase. Thus, with both ATP-citrate lyase and glycogen synthase, sites specifically phosphorylated by this multifunctional protein kinase contain less phosphate after insulin treatment. Current studies are focused on specifically assaying the activity of this multifunctional protein kinase in extracts of insulin-treated tissues and in identifying allosteric effectors of this kinase that may be regulated by insulin.

Acknowledgments-We would like to thank Martha A. Bass and Elizabeth Shipp for excellent technical assistance and Penelope Stell- ing and Becky Lawson for typing the manuscript.

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Phosphorylation of sites 3 and 2 in rabbit skeletal muscle glycogen