Eur. J. Biochem. 142, 51 1-520 (1984) (’ FEBS 1984

Control of glycogen synthase phosphorylation in isolated rat hepatocytes by epinephrine, vasopressin and glucagon

Carlos CIUDAD, Marcella CAMICI, Zafeer AHMAD, Yuhuan WANG, Anna A. DePAOLI-ROACH, and Peter J. ROACH Department of Biochemistry, Indiana University School of Medicine, Indianapolis, Indiana

(Received February 20/April 25, 1984) - EJB 84 0195

Isolated rat hepatocytes were incubated in a medium containing 0.1 mM [32P]phosphate (0.1 mCi/ml) before exposure to epinephrine. glucagon or vasopressin. 32P-labeled glycogen synthase was purified from extracts of control or hormone-treated cells by the use of specific antibodies raised to rabbit skeletal muscle glycogen synthase. Analysis of the immunoprecipitates by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that a single 321’-labeled polypeptide, apparent M, 88 000, was removed specifically by the antibodies and corresponded to glycogen synthase. Similar electrophoretic analysis of CNBr fragments prepared from the immunoprecipitate revealed that 32P was distributed between two fragments, of apparent M, 14000 (CB-1) and 28000 (CB-2). Epinephrine, vasopressin or glucagon increased the 32P content of the glycogen synthase subunit. CB- 2 phosphorylation was increased by all three hormones while CB-lwas most affected by epinephrine and vasopressin. These effects correlated with a decrease in glycogen synthase activity. From studies using rat liver glycogen synthase, purified by conventional methods and phosphorylated in vitvo by individual protein kinases, it was found that electrophoretically simi1a.r CNBr fragments could be obtained. However, neither cyclic-AMP-dependent protein kinase nor three different Ca2 +-dependent enzymes (phosphorylase kinase, calmodulin-dependent protein kinase, and protein kinase C) were effective in phosphorylating CB-2. The protein kinases most effective towards CB-2 were the Ca2+ and cyclic-nucleotide-independent enzymes casein kinase 11 (PC,,,) and F,/GSK-3. The results demonstrate that rat liver glycogen synthase undergoes multiple phosphorylation in whole cells and that stimulation of cells by glycogenolytic hormones can modify the phosphorylation of at least two distinct sites in the enzyme. The specificity of the hormones, however, cannot be explained simply by the direct action of any known protein kinase dependent on cyclic nucleotide or Ca2+. Therefore, either control of other protein kinases, such as F,/GSK-3, is involved or phosphatase activity is regulated, or both.

The activity of glycogen synthase, the rate-limiting enzyme of glycogen metabolism, is controlled by several hormones and, in terms of mechanistic details, most is known of the control of the skeletal muscle enzyme (see [I -41 for a review). It is of considerable importance, however, to expand knowledge also of the control of liver glycogen synthase. In liver, glucagon, epinephrine, vasopressin and angiotensin I1 have been shown to cause a decrease in glycogen synthase activity 14-91 and to alter enzyme kinetic parameters [lo]. From such results, it is inferred that the above agents promote phosphorylation of liver glycogen synthase. However. attempts at direct measure- ment of hormone-induced phosphorylation of the enzyme have been limited to the studies of Garrison and colleagues [II , 121. The hormones that inactivate glycogen synthase may be placed in two broad categories. One would comprise those agents that increase cyclic AMP levels and would include glucagon and epinephrine acting as a P-adrenergic agonist. The second category would be formed of the so-called ‘Ca’ +-dependent’ hormones and would include vasopressin, angiotensin I1 and epinephrine acting as an a-adrenergic agonist. (More specifi-

Abbreviations. SDS, sodium dodecyl sulfate; glucose-6-P, glucose 6-phosphate.

Enzymes. Phosphorylase kinase (EC 2.7.1.38); cyclic-AMP- dependent protein kinase (EC 2.7.1.37); glycogen synthase (EC2.4.1.11); protein phosphdtase (EC3.1.3. -).

cally, epinephrine action on glycogen synthase is probably mediated by a,-adrenergic receptors on the basis that prazosin is a more effective antagonist than yohimbine; C. Ciudad, M. Goldinan & P. J. Roach, unpublished data.) The action of the latter group ofhormones is thought to involve alterations of cellular Ca2 + distribution and stimulation of membrane phos- pholipid metabolism (see [I 3 - 161 for a review).

Although significant progress has been made in under- standing the actions of the above-mentioned hormones, the detailed mechanisms linking them to the control of liver glycogen synthase activity are not completely elucidated. Prevailing opinion certainly holds that covalent phosphory- lation of glycogen synthase is likely to be an important determinant of glycogen synthase activity in the liver, thus implying control of protein kinases andlor phosphatases. However, even though enzymological characterization of liver glycogen synthase is not yet complete, it is apparent that this enzyme, like its muscle counterpart [I -41, is subject to a complex multiple phosphorylation of its subunit by several different protein kinases [I 71. These may be conveniently placed in three categories. The first is simply formed by cyclic- AMP-dependent protein kinase. A second important group is of the Ca2 +-sensitive enzymes, and includes phosphorylase kinase [IS] and a calmodulin-dependent glycogen synthase kinase [19]. Recent indications are that another enzyme must be included [20], namely the Ca2 +- and phospholipid-dependent

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enzyme (protein kinase C) studied first by Nishizuka and his colleagues [21]. This enzyme is also activated by diacylglycerols or tumor-promoting phorbol esters [22]. The final class of glycogen synthase kinases is of the cyclic-nucleotide- and Ca2+-independent enzymes [I -41. Included are the two en- zymes generally known as casein kinase 1 and casein I1 (PC,,,) (reviewed in [23]). Another entry is the enzyme first purified by Cohen and co-workers [24] and which has the property of activating certain phosphatases [25]. We designate this enzyme

An essential consideration is whether the actions of the various hormones that inactivate liver glycogen synthase can be related to the control of specific protein kinases and hence particular phosphorylation sites of the enzyme. For cyclic- AMP-mediated hormones, the simplest expectation is that cyclic-AMP-dependent protein kinase would be involved. For the Ca2 +-dependent agents, all three Ca2 +-sensitive protein kinases can be posed as possible candidates. Our recent demonstration that tumor-promoting phorbol esters can in- activate glycogen synthase in rat hepatocytes [26] places special interest in protein kinase C. The present study is an attempt to analyze the phosphorylation of glycogen synthase in whole cells by using specific antibodies to isolate enzyme labeled with 32P in intact rat hepatocytes. Included is an analysis of the phosphorylation of purified rat liver glycogen synthase by several different protein kinases. We conclude that vasopressin, glucagon and epinephrine affect the phosphorylation of at least two sites on glycogen synthase. The specificity of the hormones, however, cannot be explained simply by the direct action of any of the known cyclic-nucleotide- or Ca2 +-dependent protein kinases. This result has important implications for the mech- anism of action of epinephrine, vasopressin and glucagon in controlling glycogen synthase phosphorylation.

F,/GSK-3.

EXPERIMENTAL PROCEDURE

Piepa ra t io n of cells

Hepatocytes were prepared from the livers of overnight- fasted, male, Wistar rats, 150 - 190 g (Harlan Industries, Indianapolis). The preparation followed a modification of the collagenase perfusion technique [27] as described by Geelen et al. [28]. In addition, 5 mM glucose was present in all perfusion buffers. The cells were suspended finally (1 8 ml/g of cell pellet) in Krebs-Henseleit bicarbonate buffer, pH 7.4, modified to contain 5 mM glucose and 0.1 mM phosphate. More than 95 % of the cells in the preparations used excluded trypan blue. The ATP content of the cells after incubation was 2.61 kO.27 pmo1,'g wet weight, as defined below.

Ivicubntioii of cells with hormones

Aliquots (5ml) of suspended cells were placed in 25-ml conical flasks and maintained with shaking at 37 "C under an atmosphere of 95 %: 5 o/, O,/CO,. After initial incubation for 15 min ["Plphosphate (New England Nuclear) was added when desired to give a final concentration of 0.1 mCi/ml, and at the same time glucose was added to give a final concentration of 20 mM. After a 60-min exposure to 32P, hormones were added as required for a further period of 8 min. The incubation was terminated by centrifuging 4ml of the cells for 30s with a bench-top centrifuge. A separate I-ml sample was taken for analysis of ATP, as described later. The supernatant of the 4-ml cell sample was carefully removed and the cell pellet im-

mediately homogenized on ice, using a Polytron at setting 7 for 25s after addition of 0.5ml of a homogenization buffer containing 50mM Tris/HCl, 10mM EDTA, 2mM EGTA, 100mM NaF, 0.1 mM N"-p-tosyl-i2-lysine chloromethane, 10 mM p-aminobenzamidine, 1 mM phenylmethylsulfonyl fluoride, and 10 pg/ml leupeptin, at pH 7.2. The protease inhibitors were added fresh just before use except for leupeptin which was diluted from a concentrated stock solution. The homogenates were kept on ice no more than 32min until they were centrifuged at 12000 x g for 30 min. Extracts were then processed immediately for enzyme activity or glycogen syn- thase isolation using the antibody technique.

Establishrneizt o j incubation conditions

The time of exposure to [32P]phosphate and other in- cubation conditions were dictated by several factors. Conditions were required in which [32P]phosphate could be provided at high specific radioactivity with minimal total radioactivity present. To achieve this, the phosphate concen- tration in the Krebs-Henseleit bicarbonate buffer was reduced to 0.1 mM, as had been used previously [Il l . The time of exposure to [32P]phosphate had to be sufficient to allow significant labeling of the glycogen synthase, while maintaining the viability and the responsiveness of the cells to the hormones to be tested. A period of 60 min was taken as a best compro- mise. By 60 min, the y-P of ATP had reached a constant specific activity (164 & 14cpm/pmol) and sufficient 32P was incorpo- rated into glycogen synthase for our analytical purposes. From preliminary experiments, in which the labeling of glycogen synthase was determined, using the methods described below, as a function of time of incubation with ["Plphosphate, we found that the 32P incorporation into the glycogen synthase subunit had not reached a plateau level after 60min (not shown). Though attainment of steady-state labeling would be the ideal situation, recent studies of rat diaphragm, using a similar experimental approach, have shown that significant information on hormone action can be obtained even without reaching a constant level of 32P associated with glycogen synthase [29]. The 60-min incubation period gave cells that responded to hormones (see Results) and which had good viability as judged from trypan blue exclusion and ATP content (see above).

Purijication o j glycogen syizthase ,from hepatocyte extracts by the use o jspec f ic antibodies

Antibodies, raised in guinea pigs to rabbit skeletal muscle glycogen synthase (see [29]) were found also to recognize rat liver glycogen synthase. In the work described, we used IgG fractions purified from immune or non-immune sera. The following procedure was developed for the purification of glycogen synthase from extracts of 32P-treated hepatocytes and is a variant of the techniques successfully applied to rat diaphragm muscle by Lawrence et al. [29]. The main difference is a pretreatment of the extract with control IgG which we found to reduce non-specific binding.

To 0.4ml of cell extract, control IgG was added to a final concentration of 0.2mg/ml and Triton X-100 to a final concentration of 0.1 (v/v). After 15 min at room temperature, 50 p1 of a 10 % suspension of inactivated Staphylococcus aureus of Cowan strain I (Pansorbin, Calbiochem-Behring) were added. Following 15min on ice with occasional shaking, the bacteria were removed by centrifugation at 6300 x g for 10 min. From the supernatant, two aliquots of 0.22 ml were removed

51 3

for further processing. The remainder served for measurements of glycogen synthase activity if required, as described later. To the two aliquots were added either control IgG or immune IgG to a final concentration of 0.2mg/ml and the tubes incubated for 30 min at room temperature. S. aurws were added, 30 p1 of a 10% suspension, for 30min on ice. A I-ml volume of NaCl/P,,'EDTA plus 1 % (w/v) bovine serum albumin, 0.1 % (v/v) Triton X-100 and 0.3 M NaCl was added and the bacteria harvested by centrifugation at 6300 x g for 10min. NaCl/P,/EDTA contains 0.1 M NaCl, 10mM sodium phos- phate, and 1 mM EDTA, pH 7.5. Where needed to estimate glycogen synthase removal from the extract, the supernatant was assayed for glycogen synthase activity. The bacterial pellet was washed twice with 2 ml of NaCl/P,/EDTA plus 0.1 % (v/v) Triton X-100 and 0.3M NaC1. Nomally, for subsequent electrophoretic analysis, the glycogen synthase was released from the bacteria with 70"/, formic acid. In some cases, the bacterial pellet was resuspended to assay for associated glyco- gen synthase activity.

Preparation of CNBr fkagments and gel electrophoresis

The formic acid extract of the S. aureus pellet was normally divided into two samples, one of which received CNBr. After 60 min at room temperature, the formic acid was removed from both by the use of a centrifugal vacuum evaporator (Speedvac, Savant). The samples were analyzed by polyacrylamide gel electrophoresis in the presence of SDS using a variant of the method of Laemmli [30] as described in detail by DePaoli- Roach et al. [31]. Slab gels, 0.75-mm thick, were used. The uncleaved subunit was run on 7 % acrylamide gels and CNBr fragments on 6 - 20 % gradient gels. After running, 7 % gels were stained with Coomassie blue and dried between cel- lophane sheets. The gradient gels were dried directly onto filter paper without staining. Autoradiograms were prepared using Cronex 4 X-ray film (Du Pont). Autoradiograms were quan- tified by scanning in a spectrophotometer. For a given gel, values are referred to the intensity of the enzyme from control cells.

Glycogen syizthase assays

Glycogen synthase activity in cell extracts was measured by a modification of the method of Thomas et al. [32] as described by Guinovart et al. [33]. The UDP-['4C]glucose concentration was 0.2mM (specific activity 2.5 x 10- cpm/mol) and the assay was performed at low (0.25 mM) or high (10mM) glucose-6-P. The ratio of activities low glucose-6-P/high glucose-6-P has been shown to reflect activity changes caused by hormones and other agents. When activity was measured in extracts of 32P- labeled cells, the final dilution in the assay was 1 : 7 or 1 : 14, and the reaction was for 15 or 30 min at 30 'C. The counting window was adjusted to contain 86 'x of I4C with less than 0.03 spillover from 32P. Controls were always run in which the 32P-labeled extract was incubated in a glycogen synthase assay that lacked UDP-[14C]glucose to calculate a 32P blank.

Determination of' ATP levels and specific radioactivity of ATP Samples (1 ml) of the cell incubation were centrifuged and

0.5 ml or 1 ml of 5 % perchloric acid added to the cell pellet. After immediate mixing with a vortex mixer, the samples were kept on ice until centrifuged at 6300 x g for 10min. The supernatants were neutralized with a solution of 3 M KOH plus 0.1 M Tris. ATP was measured in the extract [34] and related to

cell mass as follows. Aliquots of the original cell suspension were dried to determine a dry weight which was converted to a value of wet weight using the factor 3.7 [35]. The specific radioactivity of the y-P of ATP was determined by the method of England and Walsh [36].

Purification of vat liver glycogen synthase

The enzyme was purified from the livers of starved and refed male Wistar rats, 150-200 g (Harlan Industries, Indianapolis). For a preparation, 10-13 rats were killed by guillotines, and their livers removed and homogenized within 3 min by a team effort. Purification then followed exactly the procedures that we have applied successfully to the isolation of rabbit liver glycogen synthase in a dephosphorylated form [37]. Briefly, the low-speed supernatant was centrifuged at 35 000 rpm in a Beckman 45Ti rotor to harvest glycogen pellets. These were resuspended, incubated to allow phosphatase action and applied to a column of DEAE-cellulose. The enzyme eluted from the column was applied to a second column of concanavalin-A - Sepharose. The enzyme bound and was eluted with buffer containing 0.2 M glucose. The procedure gave a yield of 15 o/, with 8000-fold purification. The resulting enzyme had a specific activity of 15 ~ 16 pmol of UDP-glucose converted min- ' (mg of enzyme)-' using the standard assay of Thomas et al. [32] in the presence of glucose-6-P. The activity ratio, - glucose-6-P/ + glucose-6-P, in the standard assay was 0.83 - 0.96.

Other enzymes

The purification of rabbit skeletal muscle glycogen synthase and phosphorylase as well as several protein kinases used in the study are referenced in a recent publication [38]. The catalytic subunit of cyclic-AMP-dependent protein kinase was the generous gift of David Brautigan, Brown University. Protein kinase C was partially purified as described by Kikkawa et al. [21]. The phosphorylase kinase, cyclic-AMP-dependent protein kinase, casein kinases I and 11, and the calmodulin-dependent protein kinases contained minimal cross-contaminations. The protein kinase C, though not pure, had very low activity as a glycogen synthase kinase in the absence of Ca2+ and phospho- lipids, suggesting that contaminating activities were low. Our F,/GSK-3 preparation contained undetectable contamination by casein kinase I or casein kinase 11, cyclic-AMP-dependent protein kinase, calmodulin-dependent protein kinase or phos- phorylase kinase [31,38]. The enzyme was not affected by Ca2+ or phospholipids. However, our enzyme preparation does introduce a lesser amount of phosphate into the CB-1 region of muscle glycogen synthase, in contrast to the highly purified preparation of Hemmings et al. [24]. This might be due to a contaminating activity.

Other methods

Protein phosphorylation was determined as described in [I 71. The enzymatic synthesis of UDP-['4C]glucose was essen- tially as described by Tan [39]. The concentration of ATP used in protein kinase reactions was determined spectrophotometri- cally.

Other materials

Collagenase (type I) was from Worthington. (-)Epine- phrine (+)-bitartrate, vasopressin (arginine), glucagon, bovine

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I I I I I 0 0.1 0.2

IgG, w / m l

Fig. 1 . Rrinoval of glycogrri synihase uctivity from hcytocy tc~ extructs bj sprcifi'c untihndies. Hepatocytes were prepared as described under Experimental Procedure except that no radioactive label was in- cluded. The cells were finally exposcd to 1 pM epinephrine or 0.01 pM glucagon for 8 min. Control cells received only the vehicle. Using the procedure described, glycogen synthase was removed from cell extracts with different amounts of immune IgG, and the activity remaining in the extract determined, either at low (A) of high (B) glucose-6-P concentration. The amount of S. uuwus cells was always in excess in terms of IgG binding capacity. In the figure, the activity measured is referred to that of extracts manipulated identically but exposed only to control IgC. Control (0); epinephrine-treated (0); glucagon-treated (MI

serum albumin (fraction V), and protease inhibitors (except p-aminobenzamidine) were from Sigma. Chemicals for gel electrophoresis were from Bio-Rad. CNBr and p-amino- beuamidine were from Aldrich.

RESULTS

Purification of glycogen synthase from rat hepatocytes using specific antibodies

Antibodies raised t o rabbit skeletal muscle were able to remove glycogen synthase from extracts of rat hepatocytes. Shown in Fig. 1 is the glycogen synthase activity remaining in the extract after exposure to different concentrations of IgG. Some 70 - 80 % of the activity determined in the presence of high glucose-6-P was removed by the antibodies. Similar curves were obtained for activity measured a t low glucose-6-P con- centration. Also, the results were not significantly different if extracts from epinephrine-treated or glucagon-treated cells were used (Fig. I), indicating that the antibodies did not discriminate between the glycogen synthase present in control and hormone-treated cells. Glycogen synthase activity could be measured in resuspensions of the Staphylococcus aureus pellet. In the experiment of Fig. 1, for example, some 53 - 55 of the enzyme activity was detected in association with the bacterial pellet.

Polyacrylamide gel electrophoresis of the immunopreci- pitates from cells incubated with [32P]phosphate indicated that a single main 32P-labeled polypeptide, of apparent M , 88000, was recognized specifically by the antibodies (Fig. 2). Some non-specific "P-labeled material was sometimes present and was seen also in association with control IgG. To prove that the XX-kDa polypeptide was indeed glycogen synthase, com- petition experiments were performed in which unlabeled, purified rabbit muscle glycogen synthase was added t o extracts

Fig. 2. Electrophoretic unalysis of inimunnl)recipitateugl~cogen s.yizthase. Hepatocytes were labeled with 32P and the cell extracts processed with antibodies as described under Experimental Procedure. The material associated with the final S. aureus pellet was eluted and analyzed by polyacrylamide gel electrophoresis in the presence of SDS. Autoradiograms are shown in the figure. (A) From an early experiment in which the putative degraded species are clearly visible. Tracks 1 and 3, control IgG ; tracks 2 and 4, immune IgG. (B) Extract processed by the final protocol, with a single, dominant 3ZP-labeled species. Tracks 1 and 3, control IgG; tracks 2 and 4, immune IgC. (C) Competition experiment in which a lower immune-IgG concentration (0.028 mgiml) was used and different amounts of unlabeled, purified rabbit muscle glycogen synthase were added to the extract before the immune IgC. Traces of the putative degraded species are present and were also competed. Tracks I - 4 correspond to the addition of 0, 0.5, 2 and 5 pg of muscle glycogen synthase, respectively. The two-digit numbers alongside the gels represent apparent M, x 10-3

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Fig. 3. 3 2 P-labeled CNBr pagments of inimunoprecipitated hepatoc,vte glycogen synthase. Material, such as that analyzed in Fig. 2B, was first cleaved with CNBr before electrophoresis on a 6-20% gradient polyacrylamide gel in the presence of SDS. An autoradiogram is shown. Tracks 3 and 5 correspond to the immunoprecipitate; tracks 4 and 6 are corresponding controls in which only control IgG were used. The ratio of radioactivity CB-I :CB-2 was 0.89 in track 3 and 0.69 in track 5. As standards and for comparison, 32P-labeled CNBr frag- ments of rabbit skeletal muscle glycogen synthase phosphorylated by F,/GSK-3 plus casein kinase 11 (track 1) or cyclic-AMP-dependent protein kindse (track 2) were run. Note that the mobility of the larger muscle fragment is very sensitive to its phosphorylation state (see [31]). The numbers alongside the gels are apparent M , x

from 32P-labeled cells before addition of the immune IgG. As shown in Fig. 2, the muscle enzyme competed with the 32P- labeled enzyme in the extract, reducing the amount of the 88-kDa species bound to the antibodies.

We should note that in our first attempts to isolate glycogen synthase from rat hepatocytes by the use of antibodies, one or two additional 32P-labeled polypeptides were often observed (Fig. 2A). These species had apparent M, of 80000 and 76000 and, in the worst case appeared with similar intensities to the 88-kDa polypeptide. They were also competed by muscle glycogen synthase, suggesting that they are immunologically similar to the 88-kDa species. Since we suspected that these were proteolytic degradation products of the native subunit (see Discussion), we effected numerous trials before arriving at the protocol described under Experimental Procedure, with which the proportion of the higher mobility species could be consistently reduced to a trace (Fig. 2B). The features of the method that we believe to be most important are the inclusion of several freshly dissolved protease inhibitors in the homogeni- zation buffer and the processing of the cells immediately after incubation (in the first experiments the cell pellet was frozen).

If the glycogen synthase purified from extracts of 32P- labeled cells was subjected to CNBr cleavage and then analyzed

Fig. 4. Electrophoretic analysis of conventionally purified rat liver gl.ycogen synfhase. Shown is a 7 % polyacrylamide gel run, in the presence of SDS and stained with Coomassie blue. In region A is shown the analysis of the rat liver enzyme in the active fractions eluted from the final step in its purification (i.e. from a concanava1in-A- Sepharose column). In region B, purified rabbit liver glycogen synthase was analyzed; left track, enzyme purified to maintain its phosphory- lation state; right track, enzyme purified to be dephosphorylated. Numbers alongside gels are apparent M , x

by polyacrylamide gel electrophoresis in the presence of SDS, two main 32P-labeled species were resolved (Fig. 3). The larger fragment contained 50- 60 ”/, of the radioactivity. The ap- parent M , of these species were 28000 and 14000, close to values obtained from analogous studies of rat [29] or mouse [40] diaphragm muscle. However, the values are quite different from those of the main CNBr fragments of purified rabbit liver glycogen synthase phosphorylated in vitro with individual protein kinases [17]. Since all our previous enzymological work was directed at the rabbit liver enzyme, this species difference necessitated that we investigate the phosphorylation in vitro of purified rat liver glycogen synthase.

Phosphorylation qf rat liver glycogen synthase by individual protein kinases

Glycogen synthase was purified from rat livers by the method successfully applied to rabbit liver glycogen synthase. The enzyme activity eluting from the final concanavalin-A - Sepharose column coincided with the presence of two polypep- tides visualized after polyacryldmide gel electrophoresis (Fig.4). These species, which represented almost all of the protein present, had apparent M , of 85000 and 76000. Approximately 70 ”/, of the protein was present as the 85-kDa species. A trace species of 80-kDa was also present. In view of the recent report by Huang et al. [41], the lower-molecular-mass species might be proteolytically degraded forms of the subunit,

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Table 1. Phosphoryla~ion ofrat liver glycogen synthase by protein kinase Rat liver glycogen synthase (0.05 mg/ml) was phosphorylated for 135 min by the indicated protein kinase. The reaction conditions were essentially as described in [I71

Protein ki nase Glycogen synthase phosphorylation

Cyclic-A M P-dependent protein kinase Calmodulin-dependent protein kinase Phosphorylase kinase Casein kinase- 1 Casein kinase-I1 (PC,,,) F,/GSK-3 F,/GSK-3 + casein kinase I1 Protein kinase C

mol Plmol subunit 0.5 - 0.9 0.6-1.0 0.6- 1.0 0.2-0.6 0.6-0.7 1.3-1.5 2.2 - 2.8 0.5

Fig. 6. Comparison of 3 2 P-labeled CNBrji.upwnt,s of rat liver glycogen synthase,pho.sphorylatedin vitro OY in w h o l ~ cells. An autoradiogram of a gradient gel is shown. The left four tracks correspond to samples prepared as in Fig. 3: tracks 1 and 3, immune IgG; tracks 2 and 4, control IgG. The ratio of radioactivity CB-1: CB-2 was 0.86 in track 1 and 0.73 in track 3. The right four tracks are from enzymes phosphorylated in the test tube, as described in Fig.5: track 5 , phosphorylated by cyclic-AMP-dependent protein kinase; track 6, by F,/GSK-3 plus casein kinase 11; track 7, by F,/GSK-3; track 8, by casein kinase 11

Fig. 5 . CNBr ,fragments derived j>om rat liver glycogen synthase phosphorylated in vitro. Autoradiograms are shown of 6- 20 ”/, polyacrylamide gels run in the presence of SDS. Track 1 comes from a separate experimcnt. CNBr-cleavage products were prepared from rat liver glycogen synthase phosphorylated by individual protein kinases as in Table 1. Enzyme phosphorylated by: track 1, protein kinase C (0.5); track 2, casein kinase 11 (0.7); track 3, F,/GSK-3 (2.5); track 4, casein kinase I1 plus F,/GSK-3 (2.8); track 5, cyclic-AMP-dependent protein kinase (0.9); track 6 , phosphorylase kinase (1.0); track 7, calmodulin-dependent protein kinase (0.8). The numbers in paren- theses denote the phosphorylation level, as phosphate residues/ subunit, of the glycogen synthase. The ratio of ”P in CB-1 :CB-2: species ‘X’ was as follows: track 1, 100: -0: -0; track 2, .=0:70:20; track 3, 24:50:19; track 4, 16:54:22; track 5 , 91:9:-0: track 6, 85:15: ~ 0 ; track7,lOO: -0: - 0 . h track8isshownthesameanalysis for rabbit liver glycogen synthase phosphorylated by F,/GSK-3 plus casein kinase I1

but, like Huang et al. [41], we have been unable, by con- ventional purification, to obtain enzyme devoid of these smaller polypeptide species.

Thc purified rat liver glycogen synthase was tested as a substrate for several enzymes defined as glycogen synthase kinases on the basis of their phosphorylation of the rabbit liver or muscle enzyme (Table 1). All of the enzymes phosphorylated rat liver glycogen synthase. Phosphate was always introduced

into both the 85-kDa and 76-kDa species, although not always in the same proportion. In terms of phosphorylation stoi- chiometry, FA/GSK-3 was the most effective. There was some synergism in the combined action of casein kinase 11 plus F,/GSK-3, to the extent of about 0.5 phosphate residue/ subunit over purely additive phosphorylation. Phosphoryla- tion by F,/GSK-3 also correlated with a reduction in the electrophoretic mobility of the enzyme subunit, corresponding to an apparent M, of approximately 87000 (not shown). Similar results have been found for rabbit liver and muscle glycogen synthase [17, 31, 371. None of the other protein kinases affected the apparent subunit molecular mass.

Electrophoretic analysis of phosphorylated CNBr frag- ments associated with the actions of the different protein kinases is shown in Fig. 5 and is compared with the pattern resulting from phosphorylation in hepatocytes in Fig. 6. The phosphopeptide maps displayed significant similarities (Fig. 6). The larger fragment labeled in cells (apparent M , 28000) was identifiable with the phosphopeptide preferentially phosphory- lated by F,/GSK-3 or casein kinase TI plus F,/GSK-3. The fragment phosphorylated in whole cells, of apparent M , 14000, had similar mobility to species derived from phosphorylation in vitro. Zn vitro, however, two peptides, apparent M , 13 500 and 14000, were resolved in this region. We cannot be sure whether these two peptides came from different regions of the subunit or simply differed as to phosphorylation state, slight proteolysis, or some other chemical modification. Our suspicion ts that they are different forms of the same peptide. Another 32P-labeled CNBr fragment of apparent M , 22000 was seen when glycogen synthase was phosphorylated in vitro. This peptide may derive from the degraded subunit. It was most prominent when the 28-kDa species was heavily phosphorylated (Fig. 5 ) and may

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Table 2. Effects of vasopressin, glucagon and epinephrine on rat hepatocyte glycogen synthase activity and phosphorylation Hepatocytes, incubated with [32P]phosphate, were exposed, for 8 min, to 10 pM epinephrine, 0.1 pM glucagon or 10 pM vasopressin. Glycogen synthase activity ratios and 32P contents were measured as described under Experimental Procedures. For the 32P contents, the values for hormone-treated cells were normalized to the control values of the corresponding experiment. The results represent three identical experiments, with different cell preparations; in each experiment, each condition was run with duplicate incubations. Values are expressed as the mean k SEM. For enzyme from control cells, the average ratio of 3zP content CB-1: CB-2 was 0.81

Treatmen t Activity ratio 32P content of

intact subunit CB-1 CB-2

Control 0.26 2 0.05 1 1 1 Glucagon 0.17 2 0.02 2.21 0.20 1.43 0.58 2.53k0.38 Epinephrine 0.19 0.01 2.35 & 0.47 2.17 0.1 5 2.74k0.75 Vasopressin 0.18 f 0.01 2.03 k 0.34 2.02 0.35 1.89k0.30

The combined and synergistic phosphorylation by casein kinase I1 and F,/GSK-3 was directed also at CB-2. All of these results were similar to those found with rabbit muscle glycogen synthase [31, 421. Note that F,/GSK-3 introduced a lesser amount of phosphate also into CB-1 and, as noted under Experimental Procedures, it is not totally excluded that CB-1 phosphorylation is related to a trace contaminant of our F,/GSK-3. Casein kinase I was somewhat less effective in phosphorylating rat liver glycogen synthase under our con- ditions but most phosphate was detected in CB-2 (not shown). Cyclic-AMP-dependent protein kinase and the three Ca2+- activated enzymes all showed a marked preference for phos- phorylation in CB-1. Cyclic-AMP-dependent protein kinase and protein kinase C exerted some specificity for CB-lb whereas phosphorylase kinase and calmodulin-dependent pro- tein kinase were more specific for CB-la.

Fig. 7. Ej'ect of glycogenolytic hormones on glycogen synthase pho- sphorylation in rat hepatocytes. As described in the text, hepatocytes, after incubation with [32P]phosphate, were exposed for 8 min to 0.1 pM glucagon (section G), to 10 pM epinephrine (section E), 0.1 pM vasopressin (section V) or no hormone (section C). Shown are regions of autoradiograms of gel electrophoretic analyses of the intact subunit (in A) or the corresponding CNBr fragments (in B). For each treatment, the analysis of two cell incubations is shown. I n each section, tracks 2 and 4 correspond to immune IgG while tracks 1 and 3 correspond to control IgG. The ratio of radioactivity CB-1: CB-2 in B was as follows: section C: track 2,0.79; track 4,0.80; section G: track 2, 0.34; track 4, 0.40; section E : track 2, 0.72; track 4, 0.85; section V: track 2, 0.61; track 4, 0.68

contain some of the same phosphorylation sites. Also, this species was prominent in enzymes labeled in whole cells when the extracted subunit was itself degraded (not shown but note its trace presence in Fig. 6). For convenience of discussion, we will refer to the phosphorylated fragment of M, 28 000 as CB-2 andthefragmentsofM, 13500and 14000asCB-laandCB-lb, respectively.

The protein kinases displayed an interesting specificity for phosphorylation in the different CNBr fragments (Fig. 5) . Casein kinase 11 was specific for CB-2. Likewise, this fragment was the preferred target of F,/GSK-3 and phosphorylation caused a small reduction of mobility (to apparent M , 28000).

Efyects of glucagon, epinephrine and vasopressin on the phosphorylation of glycogen synthase in rat hepatocytes

Rat hepatocytes were incubated in the presence of [32P]phosphate and exposed to glucagon (0.1 pM), epinephrine (10pM) or vasopressin (10pM) as described under Ex- perimental Procedure. Glycogen synthase activity and phos- phorylation was analyzed in the cell extracts (Table 2 and Fig. 7). The three hormones had no effect either on the ATP content of the cells or the specific radioactivity of the y-P of the ATP. All three hormones caused reductions in the activity ratio of glycogen synthase (0.26 to 0.17-0.19) with concomitant increases, 2-2.5-fold, in the 32P associated with the glycogen synthase subunit. When the 32P content of the corresponding CNBr fragments was analyzed, it was apparent that all three hormones had comparable effects to increase the 32P- associated with CB-2. Epinephrine and vasopressin also led to clearly elevated 32P in CB-1. Glucagon had a lesser effect to increase the "P associated with CB-1.

DISCUSSION

The results demonstrate the applicability of the use of specific antibodies for the rapid isolation of glycogen synthase from rat hepatocytes incubated with [32P]phosphate. The glycogen synthase obtained in this way had electrophoretic mobility corresponding to an apparent M, of 88000. This finding has bearing on current discussions of the subunit M , of the rat livcr enzyme [41, 43, 441. Enzyme purified by con- ventional methods has been reported to have a subunit M , of

518

either 77000-80000 [45, 461, 85000 [47, 481, or 87000 2000 [49]. Huang et al. [41] recently described the presence of both an 85-kDa species as well as variable amounts of an 80-kDa polypeptide. These latter results are comparable to those of the present investigation except that we found only a trace of the 80-kDa species and more of an even higher mobility polypep- tide. The matter is further complicated by the recent report of Bahnak and Gold [43] who described a translation product obtained in vitro with a relatively low subunit M , of 77000- 80000. The fundamental question is how, and indeed if, these different species are related. One possibility would be the existence of multiple isozymes that were selected to different, perhaps variable, degrees in purification. There is really no strong evidence for this hypothesis and certainly we have shown that a major fraction, up to SO%, of the glycogen synthase in rat liver can be recovered, through the use of antibodies, as a single electrophoretic form.

Nevertheless, it is true that 20-3076 of the glycogen synthase activity was not removed by the antibodies. The same antibodies can effect 100 ”/, removal of activity from extracts of rabbit hepatocytes, rat muscle and mouse muscle. We cannot explain this peculiarity of rat liver.

The other major possibility is that the various subunit forms are related chemically. Both limited proteolysis and covalent phosphorylation state are factors that can contribute to the electrophoretic behavior of the glycogen synthase subunit from rabbit liver and muscle [I, 17, 411. Partly by analogy, our current interpretation is that the dephosphorylated subunit of rat liver glycogen synthase has an apparent M , of 85000 and that species smaller than this most likely result from pro- teolysis. When phosphorylated appropriately, however, the subunit displays lower electrophoretic mobility, corresponding to the form recovered from cell extracts by the antibodies. Whether or not the higher mobility species normally are found as such in whole cells is difficult to know but the fact that we could consistently eliminate their presence in immunopreci- pitates makes us suspect that they are artifacts of the processing.

From the study of the phosphorylation in vitro of rat liver glycogen synthase, it was apparent that the enzyme was subject to phosphorylation by the same set of protein kinases able to phosphorylate its rabbit muscle [I - 41 or liver [I 71 counterparts (see model in Fig.8). Mapping of phosphorylated CNBr fragments revealed some further similarities. For example, in all three glycogen synthases, the two protein kinases, casein kinase 11 and F,/GSK-3, preferentially phosphorylate the same large CNBr fragment, and the phosphorylation is synergistic. This is suggestive evidence that the CB-2 region in the different enzymes is analogous and may contain conserved features in the form of phosphorylation sites for F,/GSK-3 and casein kinase 11. Some differences in protein kinase specificity were also found. The most important was the relative lack of phosphorylation of the larger CNBr fragment (CB-2) of rat liver glycogen synthase by any of the three Ca2 +-activated enzymes or by cyclic-AMP-dependent protein kinase. At least one of these four protein kinases effectively phosphorylates CB-2 in rabbit muscle or liver glycogen synthase. Just after submission of this paper, a report appeared from Exton’s laboratory [49] on the phosphorylation of rat liver glycogen synthase by several protein kinases. In terms of protein kinase specificity for CB-1 versus CB-2, our results are very similar except for one point. Exton’s group reported that, although cyclic-AMP-dependent protein kinase phosphorylated more rapidly in CB-1, significant phosphate could be introduced into CB-2. This differs slightly from our results in which we never

epinephrine

vasopressin

I Fig. 8. Model of’ rat liver glycogen synthase pkosphorylation. The subunit of rat liver glycogen synthase is depicted in the center with identification of two phosphorylatable regions, corresponding to CB-I (14K) and CB-2 (28K). The distinction between CB-la and CB-lb is left out for simplicity. To the left, the specificity of individual protein kinase for phosphorylation in CB-1 or CB-2 is indicated, and to the right the corresponding analysis for hormones when acting on whole cells. CK-I and CK-TI, casein kinase I and 11; CAMP, cyclic-AMP- dependent protein kinase; Phos K, phosphorylase kinase; CaM PK, calmodulin protein kinase; PK-C, protein kinase C

observed more than 10 - 15 :< of the total phosphorylation in CB-2. The reason for this difference is not clear but there is agreement that CAMP-dependent protein kinase phosphor- ylates preferentially in CB-1.

Incubation of rat hepatocytes with [32P]phosphate led to the incorporation of 32P into glycogen synthase. An immediate conclusion, therefore, is that phosphate turns over on the enzyme within the time scale of the incubations. The 32P was distributed between two CNBr fragments of the subunit and the 32P-labeled CNBr fragments obtained from phosphory- lation in whole cells or in vitro using protein kinases gave remarkably similar gel electrophoretic patterns. There remains a question as to whether the CB-1 phosphorylated in cells would correspond to CB-la or CB-lb or both. As it happens, this uncertainty does not influence the main conclusions of this work. The fact that 32P was introduced into two distinct CNBr fragments of the enzyme subunit provides direct proof that multiple sites, at least two, are phosphorylated in whole cells. The most important result of the present study concerns the effects of hormones on glycogen synthase phosphorylation.

Glucagon, epinephrine and vasopressin all caused increases in the 32P content of the glycogen synthase subunit. Since these effects were correlated with reductions in the glycogen synthase activity ratio, it is likely that the altered 32P levels were related to an increase in the phosphorylation state of the enzyme. All three hormones affected the phosphorylation of CB-2. Epinephrine and vasopressin caused large changes in the phosphorylation of CB-1 while glucagon exerted a much lesser effect. These findings suggest first that hormonal control affects the phosphorylation of multiple, two or more, phos- phorylation sites of glycogen synthase. Secondly, it appears that, at least in a quantitative sense, glucagon influenced glycogen synthase phosphorylation differently than vaso- pressin or epinephrine. Full substantiation of the latter con- clusion will require much more detailed study. Nonetheless, this concept would be consistent with the work of Bosch et al. [lo] who reported that glucagon and epinephrine caused different alterations in the kinetic properties of glycogen synthase. Also, Garrison et al. [I 1, 121 have reported increased 32P incorporation into rat hepatocyte glycogen synthase after cxposure of cells to either glucagon or vasopressin. From electrophoretic analysis of V8 protease digests, the same

519

authors suggested that the two hormones differentially affected phosphorylation sites on the enzyme, although visually the differences do not appear very strikmg. There is suggestive evidence that different hormones display some degree of site- specific control of glycogen synthase phosphorylation. However, in our opinion, this important question requires more detailed study in order to have a definitive answer.

Even without more detailed peptide mapping, however, the results of the present study raise some fundamental questions about the mechanisms of the hormonal control of glycogen synthase. The principal point is as follows. Glucagon would be expected to function by a cyclic-AMP-mediated mechanism; epinephrine, which will be acting primarily through LX-

adrenergic receptors to inactivate glycogen synthase in our system [50], and vasopressin might be anticipated to act by perturbing cell Ca2 +. Although hormonal stimulation of intact cells provoked phosphorylation in thc larger CNBr fragment (CB-2), neither cyclic-AMP-dependent protein kinase nor any of the Ca2 +-stimulated protein kinases was potent in phospho- rylating this region of the purified rat liver glycogen synthase in our studies. Based on this assessnient of protein kinase specificity, the actions of glucagon, epinephrine, and vasopres- sin are difficult to explain simply by the stimulation of cyclic AMP and/or Ca2 +-activated protein kinases to phosphorylate glycogen synthase directly. For cyclic-AMP-mediated control by glucagon, the results are reminiscent of epinephrine action on muscle. Parker et al. [51] found that epinephrine, which would act through a cyclic-AMP-dependent pathway in skele- tal muscle, increased the phosphorylation of sites for cyclic- AMP-dependent protein kinase as well as sites unique to F,/GSK-3 ; thus, some mechanism beyond the direct phospho- rylation of glycogen synthase by cyclic-AMP-dependent pro- tein kinase had to be invoked. Of the various hypotheses that could be advanced to accommodate the results of the present investigation, three main ones are (a) the assessment of protein kinase specificity in vitro is misleading, (b) control of CB-2- specific protein kinases is involved in the hormonal control, and (c) the hormones regulate protein phosphatase activity. These entries are not necessarily mutually exclusive.

With respect to the first possibility, we are faced with the usual uncertainty in extrapolating from properties observed in vitro to behavior in whole cells. One specific question would be whether interactions among phosphorylation sites could in- fluence the observed site specificity of protein kinases. In other words, could a particular phosphorylation state of the enzyme permit phosphorylation in CB-2 by cyclic-AMP-dependent or Ca2 + -depenent protein kinases ? Examples of site-site in- teractions have been reported for both muscle [31,42] and liver [17,41] glycogen synthase, although these would not appear to explain phosphorylation in CB-2 mediated by cyclic AMP or Ca2.+. A related consideration is whether a much slower dephosphorylation could occur in v i i v at CB-2 as compared with CB-1. Thus, even a relatively slow rate of phosphorylation in CB-2 could nonetheless correspond to a significant absolute level of phosphorylation. We cannot properly judge this suggestion without knowing the relative specificity of liver phosphatases for liver glycogen synthase phosphorylation sites. From studies of muscle enzymes, however, there was no evidence for a great disparity in the rates of dephosphorylating cyclic-AMP-dependent protein kinase sites in CB-2 as com- pared with CB-1 [52]. Nonetheless, it is important to recognize that the effectiveness of any protein kinase in vivo depends also on the activity of opposing phosphatase(s).

However, if the specificity of a protein kinase in vitro is a reliable guide to its dominant action in vivo, then one possibility

is that the hormones control the protein kinase(s) specific for CB-2. The .most effective of these is F,/GSK-3, perhaps in conjunction with casein kinase 11. Casein kinase I has similar specificity. To date, there is no known physiological mech- amism for the control of these three enzymes, either by effectors or by phosphorylation in a cascade system.

The third possibility is that the hormones control phos- phoprotein phosphatase activity. A role for phosphatases in controlling glycogen synthase activity has been discussed for some time, most often in relation to the control of glycogen synthesis (reviewed in [53]). One immediate thought is that cyclic-AMP-mediated control could occur via phosphatase inhibitor-I (see discussion in [51]). The possible role of this protein has been studied mostly in skeletal muscle systems but analogous inhibitors are likely to be present also in liver [54, 551. Phosphatases sensitive to inhibitor-I (i.e. of type 1) are present in liver [56] although the extent of their contribution to dephosphorylating glycogen synthase is not known with cer- tainty. Mechanisms for Ca2 +-mediated inactivation of phosphatase(s) are not clear. Another formal possibility is the control of glycogen synthase dephosphorylation through phos- phorylase, as proposed by Hers and his colleagues [57]. One argument against this last mechanism in our case is that, under the experimental conditions used, the inactivation of rat hepatocyte glycogen synthase could be uncoupled from altera- tions in phosphorylase activity when cells were treated with phorbol esters [26]. Also, it is not immediately obvious how phosphorylase a inhibition of glycogen synthase phosphatase could explain the different effects of glucagon and vasopressin on the labeling of CB-1 and CB-2.

In conclusion, further work is needed to establish the mechanism whereby the three glycogenolytic hormones studied can govern phosphorylation in the CB-2 region of glycogen synthase. Of course, the significance of CB-1 phosphorylation, which can be explained by Ca2+- or cyclic-AMP-activated protein kinases, should not be obscured. The main importance of the work, however, is to provide a direct demonstration of the control of rat liver glycogen synthase phosphorylation by vasopressin, epinephrine and glucagon. The methods de- veloped, especially with further refinement, will provide a valuable tool for this type of study. Already, the results place several interesting constraints on the mechanisms whereby glucagon, vasopressin and epinephrine regulate glycogen syn- thase phosphorylation in liver cells.

This work was supported in part by research grants AM27240 and AM27221 from the National Institutes of Health (Bethesda, MD, USA) and by the Showalter Foundation of Indiana University. Peter J. Roach is recipient of Research Career Development Award AM01089 from the National Institutes of Health. Carlos Ciuddd was recipient of a Fulbright Fellowship. We thank Peggy Smith for typing the manuscript.

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Z. Ahmad, Y. Wang, A. DePaoli-Roach, and P. J. Roach, Department of Biochemistry, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, Indiana, USA 46223 C. Ciudad, Departament de Bioquimica, Facultad de Veterinaria, Universitat Autonomd de Barcelona, Bellaterra, Barcelona, Spain

M. Camici, lstituto di Biochimica, Biofisica e Cenetica, Universita di Pisa, Via Volta 4, 1-56100 Pisa, Italy