the of biological vol. 259. no. 6, lsaue march 25, pp. 3420 … · 2001-09-01 · the journal of...

THE JOURNAL OF BIOLOGICAL CHEMISTRV 0 1984 hy The American Society of Biological Chemists, Inc.

Vol. 259. No. 6, lsaue of March 25, pp. 3420-3423,1984 Printed in U.S.A.

Glycogen Synthase Kinases CLASSIFICATION OF A RABBIT LIVER CASEIN AND GLYCOGEN SYNTHASE KINASE (CASEIN KINASE-1) AS A DISTINCT ENZYME*

(Received for publication, March 21, 1983)

Zafeer Ahmad, Marcella CamiciS, Anna A. DePaoli-Roach, and Peter J. Roach8 From the Department of Biochemistry, Indiana University School of Medicine, Indianapolis, Indinm 46223

A protein kinase, able to phosphorylate casein, phosvitin, and glycogen synthase, was purified approximately 9000-fold from rabbit liver, and appeared analogous to an enzyme studied by Itarte and Huang (Itarte, E., and Huang, K.-P. (1979) J. Biol. Chem. 254,4052- 4057). This enzyme, designated here casein kinase-1, was shown to be a distinct glycogen synthase kinase and in particular to be different from the protein kinase GSK-3 (Hemmings, B.A., Yellowlees, D., Kerno- han, J. C., and Cohen, P. (1981) Eur. J. Biochem. 119, 443-451). Casein kinase-1 had native molecular weight of 30,000 as judged by gel filtration. The enzyme phosphorylated &casein A or B better than K-

casein or aS1-casein, and modified only serine residues in @-casein B and phosvitin. The apparent K , for ATP was 11 pM, and GTP was ineffective as a phosphoryl donor. The phosphorylation of glycogen synthase by casein kinase-1 was inhibited by glycogen, half-maximally at 2 mg/ml, and by heparin, half-maximally at 0.5-1.0 gg/ml, but was unaffected by Ca2’ and/or calmodulin, or by cyclic AMP. Phosphorylation of muscle glycogen synthase proceeded to a stoichiometry of at least 6 phosphates/subunit with reduction in the T glucose-6-P activity ratio to less than 0.4. Phosphate was introduced into both a COOH-terminal CNBr fragment (CB-2) as well as a NH2-terminal fragment (CB-1). At a phosphorylation stoichiometry of 6 phosphates/subunit, 84% of the phosphate was associated with CB-2 and 6.5% with CB-1. The remainder of the phosphate was introduced into another CNBr fragment of apparent molecular weight 16,500. Phosphorylation by casein kinase- 1 correlated with reduced electrophoretic mobilities, as analyzed on polyacrylamide gels in the presence of sodium dodecyl sulfate, of the intact glycogen synthase subunit, as well as the CNBr fragments CB-1 and CB-2.

The subunit of glycogen synthase (EC 2.4.1.11), the enzyme that regulates glycogen synthesis, is known to undergo phosphorylation at multiple sites through the actions of several different protein kinases (see Refs. 1-4 for a review). Several groups have contributed toward establishing the multiplicity

*This work was supported in part by Grants AM27221 and AM27240 from the National Institutes of Health and by the Sho- Walter Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Permanent address, Istituto di Biochimica, Biofisica e Genetica, Facolta di Scienze, Universita di Pisa, Italy.

$ Recipient of Research Career Development Award AM01089 from the National Institutes of Health.

of glycogen synthase kinases, especially the enzymes present in muscle (Refs. 3, 5-9 are selected citations). Cyclic AMP- dependent protein kinase, the first enzyme shown to phosphorylate glycogen synthase (lo), is considered an important glycogen synthase kinase. A second category of glycogen synthase kinases would include Ca2+-activated enzymes of which two are currently known. One is phosphorylase kinase (11- 16) and the second is a calmodulin-dependent glycogen synthase kinase. The latter enzyme, observed first in liver (17, 18), has recently been identified in muscle (19). A third category of glycogen synthase kinases would include those enzymes whose activities are not modified by cyclic nucleo- tides, Ca2+, or calmodulin. Most workers would agree that the enzymes of this group have presented most difficulty in cor- relating the results of different laboratories. Over the last couple of years, however, significant progress has been made in classifying these enzymes.

One glycogen synthase kinase of this group, called PC0.7 in our work (7) , is now relatively well characterized and has been purified to near homogeneity from rabbit muscle (20) and liver (21). This enzyme, which is also an effective casein and phosvitin kinase, has the characteristics of using GTP as phosphoryl donor, of modifying threonine residues in some substrates, and of being inhibited by very low heparin concentrations. As a glycogen synthase kinase, the enzyme has been studied by Huang et al. (8), Itarte et al. (22), and more recently by Cohen et al. (5). From characterization of this glycogen synthase kinase (23), it became apparent that the enzyme is identifiable as a widely distributed casein and/or phosvitin kinase that has been studied in a variety of contexts besides glycogen metabolism, the casein kinase I1 class of enzyme as reviewed by Hathaway and Traugh (24) (see this review for individual citations).

In our original studies of PC0.7 (71, we also distinguished another glycogen synthase kinase, designated that could not utilize GTP and which appeared to have a similar site specificity on glycogen synthase as phosphorylase kinase. Further purification of the enzyme (see under “Results”) has established that it is almost completely devoid of casein and phosvitin kinase activity. This enzyme appears to correspond to the enzyme denoted glycogen synthase kinase-4 by Cohen et al. (5) .

Another clearly distinct protein kinase is the enzyme called glycogen synthase kinase-3 as purified by Hemmings et al. (25) from rabbit skeletal muscle. This enzyme has the distin- guishing characteristic of activating (FA activity) the ATP- Mg+-dependent phosphatase of Merlevede and colleagues (26, 27), and we will refer to it as FJGSK-3. We have identified this enzyme also in rabbit liver (28) (see below). In the work of Cohen and colleagues (5, 291, FA/GSK-~ was shown to phosphorylate three serine residues (sites 3a, 3b,

3420

by guest on March 27, 2020

http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Glycogen Synthase Kinases 3421

and 3c) close to the site phosphorylated by PCo.,. Recently, we found that phosphorylation by PCo., potentiated the action of FA/GSK-3 through site-site interactions (28). Furthermore, phosphorylation by FdGSK-3 caused a significant alteration in the mobility of the glycogen synthase subunit when analyzed by polyacrylamide gel electrophoresis in the presence of SDS,' altering the Map,, from 85,000 to 90,000.

A protein kinase not embraced in the above classification is a casein and phosvitin kinase first studied as a glycogen synthase kinase by Itarte and Huang (30). These workers initially purified the enzyme extensively from rabbit muscle, but have also identified it in rat liver (22). One characteristic of the enzyme was the ability to phosphorylate glycogen synthase to a very high stoichiometry, namely 4 phosphates/ subunit (30). None of the glycogen synthase kinases purified in our Iaboratory appeared to conform, at least superficially, to the properties expected of the enzyme of Huang and Itarte, especially with regard ta the stoichiometry of glycogen synthase phosphorylation, but we had no direct experimental evidence on this point. Furthermore, Cohen et al. (5) suggested that the enzyme studied by Huang and Itarte could be ex- plained as a mixture of FA/GSK-~ and glycogen synthase kinase 4 (PC0.J. Since we had available all the other glycogen synthase kinases discussed above, we decided to establish whether or not this casein and glycogen synthase kinase, which we designate "casein kinase-1," was a distinct enzyme, particularly with respect to FdGSK-3. We can report that casein kinase-1 is unquestionably a separate glycogen synthase kinase. In addition, we present some novel properties of this protein kinase, and, in particular, its inhibition by glycogen and by heparin.

EXPERIMENTAL PROCEDURES

Glycogen Synthase-The enzyme was purified from rabbit skeletal muscle by the method consistently used in our studies (31) to give enzyme of similar specific activity, purity, and endogenous phosphate content. The preparation used for most of the work contained approximately 65% GS, and 35% GSB, the two electrophoretically distinct forms of the subunit described in a recent study (28; also see later discussions).

FA/GSK-3-This enzyme was identified, on the basis of its FA activity, as the Ca2'- and calmodulin-insensitive protein kinase fraction separated by gel filtration during the purification from rabbit liver of the calmodulin-dependent glycogen synthase kinase (Fig. 1 of Ref. 18; see also Ref. 28). This protein kinase fraction was then applied to a column (1.5 X 10.3 cm) of DEAE-cellulose (Whatman DE52) equilibrated with Buffer A plus 0.04 M NaCl. Buffer A contained 20 mM 1,4-piperazinediethanesulfonic acid, 1 mM EGTA, 1 mM dithiothreitol, 0.5 mM PMSF, 0.1 mM TLCK, 250 rglliter of leupeptin, and 10% (v/v) glycerol, adjusted to pH 6.8. The FJGSK- 3 activity did not bind to the DEAE-column and was then applied (120 ml) to a column (4 X 10.5 cm) of carboxymethyl Sephadex (Pharmacia) equilibrated with Buffer A plus 0.04 M NaCI. The column was eluted with a gradient formed of 400 ml each of Buffer A plus 0.04 M NaCl and Buffer A plus 0.4 M NaC1. FJGSK-3 activity, which eluted at 0.2 M NaCI, was taken to 60% saturation of ammonium sulfate. The precipitate was dissolved in 20 mM Tris-C1, 0.5 rnM dithiothreitol, and 5% (v/v) glycerol, pH 7.0, and dialyzed against 3

then applied to a column (10 ml) of Affi-Gel Blue (Bio-Rad) equili- liters of the same buffer, with one change, for 3 h. The enzyme was

brated with Buffer A. The FdGSK-3 was either eluted with a gradient formed of 50 ml each of Buffer A plus 0.15 M NaCl and Buffer A plus 1.0 M NaCI, or else eluted with Buffer A plus 0.5 M NaCl. This enzyme was stored at -70 'C and was used in the studies described later. In

The abbreviations used are: SDS, sodium dodecyl sulfate; EGTA, ethylene glycol bis(8-aminoethyl ether)-N,N,W,N"tetraacetic acid; TLCK, N"-p-tosyl-rAysine chloromethyl ketone HCI; PMSF, phe- nylmethylsulfonyl fluoride; IdIuIp, apparent molecular weight determined from electrophoretic mobility on polyacrylamide gels run in the presence of SDS (see Ref. 28).

TABLE I Purification of casein k ime-1

Total activity activity

Specific Purifi- cation

Crude extract High speed centrifu-

gation Ammonium sulfate

precipitation First phosphocellu-

lose column Phosvitin-Sepharose Bio-Gel A-1.5m p-Casein A-Sepha-

Second phosphocellu- rose

lose column

nmffmin mg nnwl/min/ mg

620 17,160 0.036 445 9,300 0.048

502 6,970 0.072

152 18.5 8.2

34.2 2.2 15.6 31.0 0.13 238.5 20.6 0.076 271.0

17.7 0.053 334.0

95 -fold

100 1.0 72 1.3

81 2.0

24.5 227

5.5 433 5.0 6625 3.3 7528

2.8 9277

a previous report, we have described how such enzyme contained undetectable contamination by cyclic AMP-dependent protein kinase, PCo.?, phosphorylase kinase, and the calmodulin-dependent glycogen synthase kinase (28).

PC,,The PC,, fraction from rabbit skeletal muscle (see Fig. 1 of Ref. 7) was diluted with an equal volume of 50 mM Tris-C1, 2 mM EDTA, 0.25 mM PMSF, 0.1 mM TLCK, 5% (v/v) glycerol, pH 7.5, and solid ammonium sulfate added to give 60% saturation. After centrifugation at 10,000 X g for 15 min, the pellet was dissolved in the above buffer and dialyzed for 3 h against the same buffer plus 0.2 M KC1. After centrifugation for 10 min at 10,000 X g to remove insoluble material, the enzyme was applied to a column of Bio-Gel A-1.5m, and the active fractions were collected and stored at -70 "C. Analysis of the enzyme by gel filtration on a Sephadex G-200 column indicated that the PCo., had apparent molecular weight of 100,000.

Casein Kinuse-1-In the initial stages, the purification was based on that of Itarte et al. (22) in which rat liver was used. In the present procedure, rabbit liver (270 g) was removed from three male New Zealand rabbits killed by administration of pentobarbital (3 ml/ rabbit) via the marginal vein of the ear. The liver was homogenized, in two batches, in a 1-liter Waring Blendor a t high speed for 20 s with 200 m1/100 g of liver of a solution containing 4 mM EDTA, 250 mM sucrose, 1 mM ammonium sulfate, 0.5 mM PMSF, 0.5 mM p - aminobenzamidine, 0.1 mM TLCK, 250 pglliter of leupeptin, and 1 mM dithiothreitol, adjusted to pH 7.0. The homogenate was centrifuged for 45 min at 13,000 X g, and the resulting supernatant then centrifuged for 90 min at 38,000 rpm in a Beckman 45-Ti rotor. The supernatant was adjusted to 60% saturation with ammonium sulfate and, after 30 min at 4 "C, the precipitate was collected by centrifugation at 13,000 X g for 30 min. The precipitate was dissolved in Buffer B to give a final volume of 140 ml. Buffer B contained 50 mM Tris-C1,1 mM dithiothreitol, 0.1 mM TLCK, 250 pglliter of leupeptin, and 5% (v/v) glycerol, pH 7.5. After dialysis for 3 h against 4 liters of Buffer B, with one change, the enzyme was applied to a column (4 X 20 cm) of phosphocellulose (Whatman P-11) washed as described by Kish and Kleinsmith (32) and equilibrated with Buffer B. The column was washed successively with 500 ml of Buffer B and 1000 ml of Buffer B plus 0.35 M NaCI. Approximately 90% of the starting FA activity was recovered in the above washes, most being found in 0.35 M NaCI wash. The column was then eluted with a gradient formed of 400 ml each of Buffer B plus 0.35 M NaCl and Buffer B plus 1.2 M NaCI. The phosvitin kinase activity eluting at 0.5-0.6 M NaCP was pooled, dialyzed against 4 liters of Buffer B for 3 h with one change, and applied to a column (1.6 X 9.5 cm) of phosvitin- Sepharose equilibrated with Buffer B. After washing with Buffer B plus 0.2 M NaCI, the phosvitin-Sepharose column was eluted with Buffer B plus 0.6 M NaCl and the peak of phosvitin kinase activity (9 ml) was applied to a column (2.6 X 82 cm) of Bio-Gel A-1.5m and eluted with Buffer B plus 0.04 M NaCl (Fig. 1). The peak of phosvitin kinase activity was applied to a column of @-casein A-agarose, to which 100% of the enzyme bound, and was eluted with Buffer B plus 0.6 M NaCI. After dilution to 0.2 M NaCI, the enzyme was applied to

~ ~~~~

This fraction eluted before and was most often clearly resolved from, the PC0.7 fraction.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

3422 Glycogen Synthase Kinuses a small (4 ml) phosphocellulose column to permit concentration. The protein kinase was eluted with 50 mM Tris-C1, 1 mM dithiothreitol, 5% (v/v) glycerol, 0.6 M NaCl, pH 7.5, and stored in 100-pl aliquots at -70 "C. We also experimented with the use of a glycogen synthase- Affi-Gel 15 column (prepared as in Ref. 25 with 10 mg of glycogen synthase). Although the protein kinase bound to the column and could be eluted with Buffer B plus 0.6 M NaC1, little purification was achieved. The purification table (Table I) indicates that over 9000- fold purification with respect to the initial phosvitin kinase activity was achieved. We elected to measure phosvitin kinase activity because we had used this substrate to monitor previous protein kinase puri- fications (7, 20, 21).

Other Protein Kinases-Phosphorylase kinase was purified from rabbit skeletal muscle by a slight modification of the method of Cohen (35). Rabbit liver PC,, and calmodulin-dependent glycogen synthase kinase were purified as described previously (18, 21). Homogeneous catalytic subunit of heart cyclic AMP-dependent protein kinase was the generous gift of Dr. David Brautigan, Brown University.

Other Protein Substrates-Phosphorylase b was purified from rabbit skeletal muscle by a modification of the method of Fischer and Krebs (33). Phosvitin (Sigma Lot No. 60F-9555) was used without any treatment to remove endogenous covalently attached phosphate. The individual casein components, a,l-casein, &casein A, @-casein B, and K-casein were generously provided by Dr. Elizabeth W. Bingham, Eastern Regional Research Center, United States Department of Agriculture, Philadelphia.

Assay of Glycogen Synthase-Glycogen synthase activity was measured using the method of Thomas et al. (34) either in the presence or absence of 7.2 mM glucose-6-P. The glycogen synthase activity ratio is defined as the ratio of activity observed in the absence of glucose-6-P to that observed in its presence.

Assay of Protein Kinase Activity and of Protein Phosphoryhtwn- The standard phosphorylation reaction contained, at pH 7.5, 75 mM Tris-C1, 1 mM EDTA, 0.4 mM EGTA, 6 mM magnesium acetate, 0.1 mM [Y-~*P]ATP (approximately 1000 cpm/pmol) and the indicated protein substrates and protein kinases. Phosvitin and glycogen synthase were routinely present a t 2 and 0.2 mg/ml, respectively. For the assay of protein kinase activity, the following protein kinase concentrations were used casein kinase-1, 1.4 pg/ml (purified enzyme); FA/ GSK-3, 0.38 unit/ml; catalytic subunit of cyclic AMP-dependent protein kinase, 9.5 pg/ml; PCar, 0.04 unit/ml; phosphorylase kinase, 2.6 rg/ml; PCO.?, 1.8 pglml; calmodulin-dependent glycogen synthase kinase, 0.3 pg/ml. When included, CaC12 was present a t 0.4 mM in excess of EGTA, and calmodulin was present a t 10 pg/ml. When included, [T-~'P]GTP had specific activity of 1000 cpm/pmol. Assays for activity were of 20 min duration at 30 "C. Protein phosphorylation was determined either by the chromatographic method (Assay 1 of Ref. 18) or by the filter paper technique (Assay 2 of Ref. 18). A unit of protein kinase activity is defined as the amount of enzyme that catalyzes the incorporation of 1 nmol/min of phosphate into glycogen synthase.

ATP-Mg2+-dependent Protein Phosphatase (Fd and Its Actiuating Factor (FJ-The Fc phosphatase was partially purified from rabbit muscle through the gel filtration step by the method of Yang et al. (26). This fraction was then used to monitor the activity of the protein activating factor, FA, in different fractions. FC was incubated in 20 mM Tris-C1, 0.5 mM dithiothreitol, 0.2 mM ATP, 2 mg/ml of bovine serum albumin, 0.4 mM magnesium acetate, pH 7.0, with the protein fraction to be tested at 30 'C for 10 min. The phosphorylase phosphatase activity of Fc was then determined by incubating an aliquot (40 pl) in a reaction (70 pl) containing 20 mM Tris-Cl, 0.5 mM dithiothreitol, 5 mM caffeine, 1 mg/ml of bovine serum albumin, and 1 mg/ml of [32P]phosphorylase a (3 X 10' cpm/mg), pH 7.0. The incubation was for 10 min at 30 "C, a t which time 50 pl of ice-cold 30% (w/v) trichloroacetic acid and 30 bl of 10 mg/ml bovine serum albumin were added. After 30 min at 0 "C, the sample was centrifuged and duplicate 5O-wl aliquots of the supernatant were removed to quantitate the 32P release from phosphorylase a. A unit of phosphatase activity is defined as the amount of enzyme that releases 1 nmol of Pi/min. A unit of F A activity is defined as the amount of protein that produces 1 unit of activated Fc after a 10-min incubation, as described above.

Polyacrylamide Gel Electrophoresis-Polyacrylamide gel electrophoresis in the presence of SDS followed a slight modification of the method of Laemmli (36) as described previously (28) with 0.75-mm thick slab gels. Either 7 or 6-20s acrylamide was used. The term Mm is used to describe the apparent molecular weight of a polypep-

tide obtained by reference to the electrophoretic mobilities of standard proteins. Use of Mm is preferable in certain cases where polypeptide mobility is significantly dependent on factors besides molecular weight, for the present discussion, phosphorylation state. For further discussion and other details of the calibration; see Ref. 28. Gels were stained either with Coomassie blue or the sensitive silver technique (37). Where required, autoradiograms were made by placing dried gels in contact with Cronex 4 x-ray film (DuPont). Quantitation of stained gels or autoradiograms was effected by scanning at 565 nm with a Beckman DU-8 spectrophotometer equipped to integrate selected absorbance peaks.

Analysis of Phosphoamino Acids-Phosvitin or &casein B, phosphorylated under the conditions described above using [-y-32P]ATP, was precipitated by the addition of trichloroacetic acid to a concentration of 25% (w/v) and left on ice for 30 min. After centrifugation, the precipitate was washed twice with cold 25% (w/v) trichloroacetic acid and finally extracted with ice-cold diethyl ether. The precipitate was dissolved in 6 N HCl and hydrolyzed for 4 h at 110°C. After evaporation of the HCl, the protein hydrolysates were analyzed by thin layer electrophoresis at pH 1.9 (38). Autoradiograms were made and the location of the 32P-labeled material was compared with the migration of standards of phosphoserine and phosphothreonine.

Other Methods-CNBr fragmentation of glycogen synthase was performed as described previously (28). For glycogen synthase or phosphorylase kinase, protein concentration was determined by the method of Lowry et al. (39). For other protein kinases, the Coomassie blue binding assay was used (40). Bovine serum albumin was the standard. The concentrations of individual caseins were determined spectrophotometrically as described previously (41), as were the concentrations of 13*P]ATP and [32P)GTP.

Other Materials-Calmodulin from rabbit skeletal muscle was prepared by a modification of the method of Dedman et al. (42). Rabbit liver glycogen (Sigma Type 111) was treated before use to remove ions as described previously (43). ["PIATP and 13'P]GTP were from Amersham-Searle and ICN, respectively. Chemicals for electrophoresis were from Bio-Rad. CNBr and p-aminobenzamidine were from Aldrich. TLCK, PMSF, heparin (Grade I), and leupeptin were from Sigma.

RESULTS

Purification of Casein Kinase-1-The purification scheme, described under "Experimental Procedures," was based on that of Itarte et al. (22) through the phosphocellulose chromatography step, although the starting material was rabbit not rat liver. An important feature of the purification scheme was that FA activity was separated at the stage of the first phosphocellulose column. Of the starting FA activity, approximately 20% was recovered in the initial washing of the phosphocellulose column, and 70% was eluted with buffer containing 0.35 M NaC1. Analysis of the purified casein kinase-1 indicated very low F A activity as compared with FA/ GSK-3 (Table 11).

The protein kinase was purified approximately 9,000-fold with respect to phosvitin kinase activity which we elected to use for routine measurement. Since the crude extract contains more than a single phosvitin kinase activity, this figure is probably an underestimate. The specific activities towards

TABLE II F A actiuity of casein kinase-1 and F4IGSK-3

Protein kinase FA activitp Protein kinaseb (FdpJprotein kinase) Ratio

units,assay rnilliunitsl assay

Casein kinase-1 1.9 4.6 0.41 R/GSK-3 98.6 3.8 25.9

FA activity was determined as described under "Experimental Procedures" using partially purified Fc. Unstimulated FC gave 13.9 units of phosphorylase phosphatase activity/assay.

The amount of protein kinase, expressed as milliunits of glycogen synthase kinase activity, corresponding to the amount of enzyme used to measure FA activity.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


3t 1 2 3 4 5 I

fnetlon number

FIG. 1. Gel filtration of casein kinase-1. The enzyme fraction eluted from phosvitin-agarose was applied to a column (2.6 X 82 crn) of Bio-Gel A-1.5m equilibrated with Buffer B plus 0.04 M NaCl and eluted with the same buffer a t 30 ml/h. Fractions of 5 ml were collected and assayed for phosvitin kinase activity ( 0 Assay 2). The broken line corresponds to the absorbance a t 280 nm. The numbers correspond to the elution of standard proteins used to calibrate the column: 1 , thyroglobulin (600,000); 2, aldolase (158,000); 3, bovine serum albumin (67,000); 4, ovalbumin (43,000); 5, ribonuclease (13,700). The numbers in parentheses are the values for molecular weight used for the calibration.

-67

FIG. 2. Gel electrophoresis of casein kinase-1. Casein kinase- 1 (0.13 pg) was analyzed by 10% polyacrylamide gel electrophoresis in the presence of SDS, and stained ( A ) by the silver technique. The two dominant species of Mapp = 67,000 and 34,000 are clearly visible (right two tracks of A) . Scarcely visible are trace amounts of species running with Ma, = 36,000 and Ma, = 18,000-19,000. The origin and the dye front are marked by dashes. The left track of A shows standard proteins, in order of decreasing mobility: phosphorylase, bovine serum albumin, catalase, ovalbumin, lactate dehydrogenase, carbonic anhydrase, trypsinogen, soybean trypsin inhibitor. B shows an autoradiogram of a similar electrophoretic analysis of casein kinase-I that had first been incubated with [-y-"PIATP. The numbers beside the gels indicate values of Mspp X

phosvitin (2 mg/ml) and glycogen synthase (0.2 mg/ml) were 300 and 50-60 nmol/min/mg, respectively. From the gel filtration step (Fig. l ) , a value of 30,000 was obtained for the apparent molecular weight of the enzyme. Polyacrylamide gel electrophoresis in the presence of SDS of the purified enzyme (Fig. 2) indicated two dominant polypeptide species of Mapp = 67,000 and 34,000. On some gels, the species of Mapp = 34,000 appeared as two scarcely resolved polypeptides. Less abundant species, difficult to see in Fig. 2, were observed with Mapp = 36,000 and 18,000-19,000. Incubation of the enzyme with [y -"P]ATP and Mg2+ in the standard phosphorylation assay led to the introduction of phosphate into the species of Mapp = 34,000 and 36,000 (Fig. 2). This phosphorylation did not correlate with any alteration in activity as judged by the standard assay (not shown). Quantitation of relative mass

from scanning silver stained gels spectrophotometrically is probably not definitive. However, such analysis indicated that the species of Mapp = 34,000 represented approximately 50% of the stained material.

Specificity of Glycogen Synthase Kinases-The substrate specificity of casein kinase-1 is compared with that of other glycogen synthase kinases in Table 111. In particular, protein kinase activities toward defined casein components (ael-casein, B-casein A and B, and K-casein) were tested. With glycogen synthase phosphorylation as the reference, casein kinase-1 and PCo., were clearly the most effective casein and phosvitin kinases. Only phosphorylase kinase was effective toward phosphorylase. PCo.4 displayed singularly low relative activity towards any of the substrates tested besides glycogen synthase. From the relative phosphorylation of individual casein components, the protein kinases fell into three cate- gories. The two most effective casein kinases, casein kinase- 1 and PCo.?, showed the preference &casein A or B > K-casein and aSI-casein. Cyclic AMP-dependent protein kinase phosphorylated p-casein B better than p-casein A, asl-casein, and K-casein. This relative specificity for the genetic variant p- casein B over &casein A was first observed by Kemp et al. (44). None of the other glycogen synthase kinases discrimi- nated significantly between 0-casein A and p-casein B. A third category is formed of FA/GSK-~, the calmodulin-dependent synthase kinase, and phosphorylase kinase, for which the relatively low casein kinase activities showed the order of preference K-casein > &casein A or B and aSl-casein. This preference was most marked for phosphorylase kinase.

Nucleotide Specificities for Casein Kinase-1 and FJGSK- 3-Casein kinase-1 exhibited close to hyperbolic dependence of protein kinase activity on the ATP concentration, with either phosvitin or glycogen synthase as substrate. The apparent K, for ATP was 11 p~ with either substrate. F,/GSK- 3, using glycogen synthase as substrate, had apparent K , for ATP of 27 p~ and also displayed hyperbolic kinetics. Neither protein kinase was highly effective using GTP as the phosphoryl donor. Casein kinase-1 activity was essentially proportional to the GTP concentration up to 600 PM, formally defining a K , in excess of 300 PM but probably indicative of an even higher value. The glycogen synthase kinase activity at 10 p~ GTP was 1.5% of the corresponding rate with 10 PM ATP. FA/GSK-3 activity increased with the GTP concentration and was not saturated at 600 PM, the highest concentration used. An approximate value of 500 PM for the apparent K,,, was obtained from a double reciprocal plot, with a V,. 40% of that observed with ATP. These cannot be considered highly reliable estimates, however. At 10 p~ nucleotide, the rate with GTP was 2.1% of that with ATP. These results are summarized in Table IV. The main conclusion is that GTP is a relatively poor substitute for ATP with both protein kinases.

Phosphorylation of Glycogen Synthase by Casein Kinase-1- Casein kinase-1 phosphorylated rabbit skeletal muscle glycogen synthase to a higher stoichiometry than any of the other protein kinases that we have studied. In Fig. 3 is shown the introduction of more than 4 phosphates/subunit, with a con- comitant reduction in the glycogen synthase activity ratio from 0.97 to 0.35. In fact, phosphorylation to as many as 6 phosphates/subunit has been observed (see Fig. 6). For com- parison, phosphorylation of glycogen synthase to 1.6 phosphates/subunit by FA/GSK-3 is shown, in this case causing a reduction in glycogen synthase activity ratio to 0.2 (Fig. 3). It is evident that, simply in terms of total phosphate introduced, phosphorylation by FA/GSK-3 was significantly more effective than casein kinase-1 in inactivating glycogen synthase.

As discussed briefly in the Introduction, recent studies have


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

3424 Glycogen Synthase Kinases TABLE 111

Substrate specificity of glycogen synthase kinases Protein kinase activities were determined as described under "Experimental Procedures" using Assay 2 and

referred to glycogen synthase phosphorylation as 100%.

Substrate dependent Phosphorylase dependent (mglml) protein kinase" protein

kinase kinase

Cyclic AMP- Calmcdulin-

FdGSK-3 PCor PCo.7 kinase-l Casein

Glycogen synthase (0.2) 100 100 100 100 100 100 100 Phosphorylase (2) - b 907 5 <2 <2 c 2 <2 Phosvitin (2) 15 40 <2 1009 565 Casein (2) - b 2 34 12 8 813 778

20 @-Casein A (0.64) 2 17 11 <2 916 637 191 @-Casein B (0.64) 2 20 12 C2 1234 591

a.,-Casein (0.64) 5 2 18 5 4 65 74 52 K-Casein (0.64) 31 34 20 3 71 240

b b - -

"Data taken from DePaoli-Roach et al. (41) and combined with unpublished data. For phosphorylase kinase only, the caseins and phosphorylase were present at 1 mg/ml.

Not measured.

TABLE IV Nucleotide smcificitv of casein kinuse-1 and FJGSK-3

A- - K, Activity ratio

GTPIATP at substrate ATP G" 10 p~ nucleotide

B a

6 a

P M

Casein kinase-1 Phosvitin 11" -b b -

Glycogen synthase 11" >3W 0.015 FJGSK-3 Glvcogen synthase 27" -5ood 0.021

a Values derived from double reciprocal plots with ATP varied in the concentration range 2.5 to 300 pM.

Not determined. The rate was proportional to GTP concentration in the range 10

to 600 p~ GTP, formally defining a K,,, greater than 300 pM. GTP was varied in the range 10 to 600 p ~ , and so the extrapo-

lation of double reciprocal plots clearly involves significant error for an apparent K , of -500 pM.

FIG. 3. Phosphorylation and inactivation of glycogen synthase by FJGSK-3 and casein kinase-1. Rabbit muscle glycogen synthase was incubated with either 0.4 unit/ml of FJGSK-3 (A ) or 1 pg/ml of casein kinase-I ( E ) under the conditions of the standard assay. At different times, aliquots were removed either for the deter- mination of 32P incorporation (circles), or, after dilution 1 in 35 with 50 mM Tris-HC1, 5 mM EDTA, 2 mM EGTA, 1 mg/ml of glycogen, pH 7.8, for measurement of the glycogen synthase activity ratio (triangles). The loss of activity measured in the presence of glucose- 6-P after 120 min of incubation was approximately 30% of the zero time value.

shown that, as purified, muscle glycogen synthase contains variable amounts of two electrophoretically distinct forms of the glycogen synthase subunit, as is seen also in Fig. 4 (Track I, for example). These species were designated GS, ( M , = 85,000) and GSB (Mam = 86,000). It was further found that appropriate phosphorylation of the enzyme could result in the sequential generation of two other species of reduced electrophoretic mobility, GS, (Mapp = 88,000) and GSa (Mapp = 90,000). The formation of GS, and GSa was linked directly to phosphorylation by FA/GSK-3 but not by any of the other

1 2 3 4 5 6 7 8 910 FIG. 4. Gel electrophoretic analyses of phosphorylated gly-

cogen synthase. Glycogen synthase (2 pg) was analyzed with 7% polyacrylamide gels in the presence of SDS. Coomassie blue-stained gels (A ) or corresponding autoradiograms ( B ) are shown. Tracks 1-7, respectively, glycogen synthase phosphorylated by casein kinase-1 for 0 (O), I/z (2.0). 1 (3.4), 2 (4.5), 3 (5.0), 4 (5.4), and 5 h (6.0); Track 8, phosphorylated by FJGSK-3 (2.2); Track 9, phosphorylated by PCo., plus FJGSK-3 (4.0); Track 10, phosphorylated by FJGSK-3 plus casein kinase-1 (5.7). Tracks 8-10 are directly comparable with Track 4. The numbers in parentheses indicate the corresponding phosphorylation states, as phosphates/subunit.

protein kinases tested? The action of FA/GSK-3 alone converted GSB to GSa so that the resulting enzyme contained GS, and GSa (Track 8, Fig. 4). Phosphorylation by PCo., plus FA/ GSK-3 also converted GS, to GSa so that the resulting enzyme contained predominantly GSa (Fig. 4, Track 9). The subunit forms just discussed could be visualized, after electrophoretic analysis, as relatively discrete species (Fig. 4 and Ref. 28). Examination 6f glycogen synthase phosphorylated by casein kinase-1 indicated that reduction of the electrophoretic mobility of the subunit did occur (Fig. 4) but was of a qualitatively different nature than that associated with FA/GSK-~ action. After phosphorylation by casein kinase-1, the electrophoretically retarded material ran diffusely. I t was difficult to assign values of Mapp to the retarded subunit, but the lowest mobility species ran with Map,, = 90,000-93,000. As a function of increasing phosphorylation, a greater proportion of protein was associated with the higher Mapp species (Fig. 4). From autoradiograms of polyacrylamide gels (Fig. 4), it was also

In the early study (28), the casein kinase-1 was not available.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


apparent that a disproportionate amount of phosphate was associated with the higher Mapp species, suggesting that they corresponded to higher phosphorylation states. That the mobility shift associated with casein kinase-l action was in some way different from that due to FA/GSK-3 was substantiated by allowing both enzymes to phosphorylate glycogen synthase simultaneously. In this case, the resulting electrophoretic pattern (Fig. 4, Truck 10) was distinctive. As expected from our knowledge of FA/GSK-3 action, species analogous to GS, and GSa were visible, but both appeared to run with diffuse trailing edges characteristic of the effects of casein kinase-1 action. It is as though the alterations in electrophoretic mobility due to the individual protein kinases were to some degree superimposable.

CNBr Fragments of Glycogen Synthase Phosphorylated by Casein Kinuse-1-The seven defined phosphorylation sites of rabbit skeletal muscle glycogen synthase are all contained in two CNBr fragments of the subunit (29): a smaller, NH2- terminal fragment, CB-1, of Mapp = 13,100 in our studies (28), and a larger, COOH-terminal fragment, CB-2, whose electrophoretic mobility is very sensitive to its phosphorylation state (28, 29). In our previous work, the Mapp ranged from 22,400 to 27,300, depending on its phosphorylation (28). Electropho- retic analysis of 32P-labeled CNBr fragments of glycogen synthase phosphorylated by casein kinase-1 is shown in Fig. 5. Quantitatively, most phosphate was introduced into polypeptides in the mobility range of CB-2. Although three phos- phopeptides, of Mapp = 22,400, 24,000, and 28,800, were fairly clearly resolved, the autoradiogram indicated a diffuse region of exposure, reminiscent of the behavior of the intact subunit (see Fig. 4). Limited tryptic proteolysis of muscle glycogen synthase is known to cleave in the COOH-terminal region of the subunit that corresponds to CB-2 (29). Exposure of glycogen synthase phosphorylated by casein kinase-1 to 0.5 pg/ ml of trypsin for 10 min a t 30 "C (beforeCNBr fragmentation resulted in almost total loss of radioactivity in the mobility range of CB-2 (not shown). This result suggests that all of the phosphorylated species discussed above correspond to CB- 2 and that the observed electrophoretic heterogeneity is correlated with a diversity of phosphorylation states. This sug- gestion is supported by another observation. FA/GSK-3 action leads to the generation of a dominant 32P-labeled CB-2 of Mapp = 26,200, and a much smaller amount of phosphate running at Mapp = 22,400 (Ref. 28; see also Fig. 5, Track 8). Analysis after the combined action of casein kinase-1 and FA/ GSK-3 indicated that the species of Mapp = 26,200 was no longer present, most phosphate being associated with lower mobility species of Mapp = 29,500 and 27,500 (Fig. 5, Track

16.5 13.7 .13.7

.13.1)cB-1

1 2 3 4 5 6 7 8910

FIG. 5. Gel electrophoretic analyses of CNBr fragments of phosphorylated glycogen synthase. CNBr fragments of glycogen synthase were separated on 6-20% gradient polyacrylamide gels and an autoradiogram is shown. The samples correspond to the experi- ment shown in Fig. 4. Tracks 1-6 correspond to %-, 1-, 2-, 3-, 4-, and 5-h incubations of glycogen synthase with casein kinase-1; Track 7, phosphorylated with cyclic AMP-dependent protein kinase to 1.9 phosphates/subunit; Tracks 8-10 correspond to Tracks 8-10 of Fig. 4. The numbers alongside indicate Maw X Not all species are labeled so as to simplify the figure.

0 1 2 3 4 5 time ( h )

FIG. 6. Prolonged time course of glycogen synthase phosphorylation by casein kinase-1. Glycogen synthase was phosphorylated by incubation with casein kinase-1 (3 pg/ml) for the indicated times. Samples were analyzed for 32P incorporation by Assay 1 of Ref. 18 (0). From analyses of 32P-labeled CNBr fragments, as in Fig. 5, the distribution of the phosphate in CB-1 (A), CB-2 (A), and the species of Man = 16,500 (0) were calculated.

10). We conclude that the two protein kinases both phosphorylated CB-2 and that the effects of phosphorylation to reduce mobility were additive.

Casein kinase-1 action also resulted in the appearance of a 32P-CNBr fragment of Mapp = 13,700 (Fig. 5). This species was not lost after limited tryptic proteolysis as described above (not shown) and had slightly lower mobility than CB- 1 phosphorylated by other protein kinases. Either the species of Mapp = 13,700 was a distinct CNBr fragment or CB-1 with reduced mobility. Under our conditions, FI\/GSK-~ action leads to the phosphorylation of CB-1 which runs with Mapp = 13,100 (Ref. 28 and Fig. 5, Track 8). When glycogen synthase phosphorylated by both casein kinase-1 and FA/GSK-~ was analyzed, no species of Mapp = 13,100 was found, only the species of Mapp = 13,700 (Fig. 5, Track 10). Similar results were obtained by analyzing glycogen synthase phosphorylated by casein kinase-1 and phosphorylase kinase, the latter enzyme being specific for the phosphorylation of CB-1 at site 2 (not shown). We conclude, therefore, that the two species, of Mapp = 13,100 and 13,700, both corresponded to CB-1. Phos- phate was also observed, after casein kinase-1 action, in a fragment of Mspp = 16,500.4 The level of phosphorylation was comparable to that of CB-1 (Figs. 5 and 6).

From quantitation of the phosphate distribution in the different CNBr fragments (Fig. 6). it was apparent that phosphorylation in CB-1 and the species of M., = 16,500 proceeded to constant levels of approximately 0.5 phosphate/ subunit in each case. CB-2 phosphorylation, in terms of stoichiometry, occurred more rapidly but was still increasing slowly even after prolonged incubation. At least 5 phosphates/ subunit were introduced into CB-2 and so it is impossible to know whether individual sites were sequentially modified or whether all were being affected simultaneously.

Effectors of Casein Kinase-I Actiuity-Casein kinase-1 was not affected by Ca2+ and/or calmodulin or by the presence of cyclic AMP. The enzyme was inhibited by NaCl, with Zo.6 (the concentration required for half-maximal inhibition) of 0.15- 0.18 M. The effects of glycogen on glycogen synthase phosphorylation by the different protein kinases are shown in Table V. Casein kinase-1 and PC0.7 were significantly inhibited and phosphorylase kinase was activated. The other protein kinases were slightly activated. The concentration dependence of glycogen inhibition of casein kinase-l and PCo,

' A minor species of MW = 15,700 is also visible in Fig. 5.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

3426 Glycogen Synthase Kinases

TABLE V Effect ofglycogen on the actiuity of glycogen synthase kinases

Protein kinase Relative activitf ?6 control

Cyclic AMP-dependent protein kinase 115 Phosphorylase kinase 270 Calmodulin-dependent protein kinase 126 FA/GSK-~ 127 PCo.4 113

Casein kinase-1 21 pc0.7 35

a Protein kinase activity was measured, using glycogen synthase as substrate, as described under "Experimental Procedures" using Assay 2. The relative activity denotes the activity in the presence of 10 mg/ ml of glycogen expressed as a percentage of the corresponding activity in the absence of glycogen.

0 5 10 15 20 g l p g e n (mr/ml)

FIG. 7. Inhibition of Pc0.7 and casein kinase-1 by glycogen. The glycogen synthase kinase activity of PC0.7 (A) or casein kinase-1 (0) was determined as a function of glycogen concentration. Also shown is the effect of glycogen concentration on casein kinase-1 activity toward phosvitin (0).

Ii

O L 0 8 i2 Ib zb 1 4 50 heparin pg/ml

FIG. 8. Inhibition of casein kinase-1 by heparin. Using glycogen synthase (0) or phosvitin (A) as substrate, casein kinase-1 activity was determined with the indicated concentration of heparin.

(Fig. 7) indicated lo.s values of 2 and 4 mg/ml, respectively. In the case of casein kinase-1, the analysis was repeated using phosvitin as a substrate (Fig. 7) and no more than 20% inhibition was seen even at 20 mg/ml of glycogen.

Heparin, a known inhibitor of Pc0.7 and related enzymes (24,45,46), was also tested for effects on the activities of the different glycogen synthase kinases. Inhibition of Pc0.7 and also of casein kinase-1 was observed. Phosphorylase kinase activity was stimulated, as has been reported (47,48), and the activities of P&, FA/GSK-3, calmodulin-dependent protein kinase, and cyclic AMP-dependent protein kinase were un- changed (not shown). The concentration dependence of the heparin inhibition of casein kinase-1 (Fig. 8) indicated an lo.s value of 0.5-1 pg/ml, whether phosvitin or glycogen synthase was the substrate. From analysis of CNBr fragments of gly-

'" ~ r .Y"

P- ser

P-thr * 0

1 2 3 4 FIG. 9. Amino acid specificity of casein kinase-1 and Pc0.7.

Casein kinase-1 was used to phosphorylate phosvitin (Track 4 ) to 21.7 nmol/mg and 8-casein B (Track 2 ) to 17.5 nmol/mg. Pc0.7 was used to phosphorylate phosvitin (Track 3 ) to 5.4 nmol/mg and 8- casein B (Track I ) to 21.7 nmol/mg. After hydrolysis of the substrate proteins, samples corresponding to 4 pg of &casein B or to 10 gg of phosvitin were analyzed by thin layer electrophoresis at pH 1.9 for 3 h a t 500 V (see "Experimental Procedures"). The figure shows an autoradiogram of the thin layer plate. The migration of phosphoserine (P-ser) and phosphothreonine (P-thr) is indicated. The origin is indicated by 0. The anode would correspond to the top of the figure.

cogen synthase, it was established that the phosphorylation of CB-1, CB-2, and the species of Mapp = 16,500 was inhibited (not shown). Since until now only protein kinases of the casein kinase I1 (PcO.7) class had been shown to be inhibited by heparin, it was necessary to prove that the casein kinase- 1 was not contaminated by Pc0.7. The approach we adopted was to analyze the amino acid residues in phosvitin and p- casein B modified by casein kinase-1. As seen in Fig. 9, phosphoserine, but not phosphothreonine, was detected in hydrolysates of p-casein B or phosvitin phosphorylated by casein kinase-1. In contrast, PC0.7 action gave rise only to phosphothreonine in hydrolysates of p-casein B, and phosphothreonine and phosphoserine in hydrolysates of phosvitin. From this result, it is impossible to explain the inhibition of casein kinase-1 by contamination wtih Pc0.7.

DISCUSSION The present investigation was undertaken to improve the

classification of the various glycogen synthase kinases and specifically to assess whether the enzyme, designated here casein kinase-1, was distinct from other protein kinases that we have studied, in particular the enzyme FA/GSK-3. The conclusion is quite clear that casein kinase-1 is not related in any straightforward way to the other glycogen synthase kinases discussed in the Introduction (see also Table VI).

Our objective was to isolate enzyme analogous to the glycogen synthase kinase studied by Itarte and Huang (30) and Itarte et al. (22), and the purification scheme initially followed that of Itarte et al. (22). The purification achieved was a little less than that obtained for the rabbit muscle enzyme (30) and greater than that for the rat liver enzyme (22). The properties of the purified rabbit liver casein kinase-1 were consistent on several counts with the results of Huang and Itarte (30); included would be the native molecular weight, the K, for ATP, and the high stoichiometry of glycogen synthase phosphorylation located in both CB-1 and CB-2 (49). I t seems probable that the casein kinase-1 described here is analogous to the enzyme of Itarte and Huang (22, 30). There may also be a link with our previous work (7) on the multiplicity of rabbit muscle glycogen synthase kinases. At a stage of relative

In the operational nomenclature used, the subscript referred to the molarity of KC1 at which the fraction eluted from phosphocellulose (see Ref. 7). The PCo.6 fraction of Ref. 7 appeared to have very similar properties to PCo.., and we did not define these fractions as distinct enzymes.


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/


TABLE VI Glycogen synthase kinases

cvelic AMP- Calmodulin- property

dependent Phosphorylase dependent FdGSK-3 pcoA PC07

Casein orotein kinase Drotein kinase-1 kinase kinase

1. Native M, (X10-3) 170 1300 275-500 57 100 150 30 2. Subunit structure R2cz (c.0784 (4% ?a ? ffzsz ?a 3. Autophosphorylation R?I ff ,B a,S a ? B

Heat- 4. Inhibitors ? Trifluo- ? ? Heparin Heparin ff

stable perazine Glycogen Glycogen inhibitor

5. Activators

6. GTPuse 7. Casein/phosvitin

phosphorylation 8. Preferred casein 9. Glycogen synthase

phosphorylation: No. of sites CNBr fragments

Inactivation

protein CAMP

- +b

8-B

3 CB-2> CB-1

+

Caz+ Ca"/ Glycogen Caimodulin Heparin - -

+ + K K

1 3 2 CB- 1 CB-1,

CB-2 + +

? ? ? ?

+ +

- +++ +++ +++

- -

K - p (Aor B) f l (Aor B)

3 3 1 1 CB-2> CB-1 CB-2 CB-1, CB-1 CB-2 +++ i- - ++

36

~-~ ~

The best characterized autophosphorylation is of the Rn-type regulatory subunit. This refers to the very specific phosphorylation of @-casein B.

impurity, two protein kinase fractions: PCo., and PCo.7, were separated by phosphocellulose chromatography. The Pc0.7 fraction was incompletely resolved from a smaller peak of phosvitin and glycogen synthase kinase activity (Fig. 1 in Ref. 7) that we termed PCo,,.5 In later studies we subjected this PCo.6 fraction to chromatography on DEAE-cellulose.' Two peaks of protein kinase activity were resolved, both of which were sensitive to inhibition by heparin, although to different extents. At the time, we thought that both activities were related somehow to PCo.7, but the recognition that casein kinase-1 is inhibited by heparin suggests that the PC0.6 fraction might be identifiable as casein kinase-1.

The casein kinase-1 of this report shares several properties with the casein kinase that has been studied by several groups, casein kinase I as reviewed by Hathaway and Traugh (24). Some properties in common are molecular weight, lack of GTP use as a phosphoryl donor, possibly autophosphorylation of a subunit of approximately 30,000 daltons, and a specificity for serine residues in phosphorylating caseins. One observation interferes with the unequivocal assignment of our rabbit liver enzyme as being casein kinase I, and that is the inhibition by heparin. In a study of rabbit reticulocyte casein kinase I, Hatbaway et al. (46) observed no effect of heparin on protein kinase activity. At present, we have no explanation of this apparent discrepancy. In any event, the observation that two clearly distinct enzymes, casein kinase-1 and PG7, are both inhibited by heparin means that heparin cannot he used as a specific discriminator for the presence of PCo.7-like enzymes.

Several criteria establish that FA/GSK-3 and casein kinase- 1 are distinct enzymes: (i) FA activity was separated from casein kinase-1 during the purification of the latter enzyme, and purified casein kinase-1 had very low FA activity; (ii) casein kinase-1, but not FA/GSK-3, was inhibited by glycogen and by heparin; and (iii) the phosphorylation and inactivation of glycogen synthase by the two enzymes were quite different. These points, together with several others, combine to make

A. A. DePaoli-Roach and P . J. Roach, unpublished observations.

it exceedingly unlikely that casein kinase-1 and FJGSK-3 are the same enzyme. Casein kinase-1 is also distinguishable from all the other glycogen synthase kinases in Table VI. It is interesting that the two most effective casein and phosvitin kinases, PCo., and casein kinase-1, share several properties that distinguish them from the other glycogen synthase kinases. Most notable are the common preference for &casein A or B as substrates over other casein components, inhibition by glycogen and inhibition by heparin. At the same time, casein kinase-1 and PCo.7 are unquestionably different enzymes, by several criteria. For example, GTP use and the ability to modify threonine residues is a characteristic only of Pco.7. Given the precedents of this area of study, we are hesitant to propose any definitive classification of the protein kinases able to phosphorylate glycogen synthase. However, our current assessment, shown in Table VI, is of seven distinct glycogen synthase kinases. In our favor, it should be pointed out that we have studied all of the protein kinases in side by side experiments in the same laboratory.

Our primary interest in casein kinase-1 is its phosphorylation of glycogen synthase. The reaction proceeded to a relatively high phosphorylation stoichiometry with effective inactivation of the enzyme, findings generally consistent with the work of Itarte and Huang (30). Six or more separate phosphorylation sites must be involved although the results do not indicate how many of the sites affect kinetic properties. It can be noted that inactivation continued even after the phosphorylation of CB-1 and the CNBr fragment of Maw = 16,500 had reached constant levels. This argues for a significant influence of CB-2 site(s) on activity. However, CB-2 is phosphorylated to a level of at least 5 phosphates/subunit and a more detailed correlation with activity is not possible at present.

Whether casein kinase-1 acts on any of the sites phosphorylated by other glycogen synthase kinases cannot be an- swered directly by this investigation. However, the evidence does suggest that some sites of casein kinase-1 action are unique to that enzyme. First, the phosphorylated CNBr fragment of MaPp = 16,500 is not generated by any other protein


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

3428 Glycogen Synthase Kinases

kinase and must represent a distinct site. Secondly, phosphorylation of site 2, the only known site in CB-1, does not affect the electrophoretic mobility of the peptide (28). The generation of a phosphorylated form of CB-1 with reduced mobility is therefore an indication of a different phosphorylation site. A similar argument can be applied to CB-2 in the sense that the alterations in mobility caused by casein kinase-1 are qualitatively different from the effects of FA/GSK-~ or cyclic AMP-dependent protein kinase action (28,29). Thus, at least three casein kinase-1 sites must be distinct from those already characterized.

Insufficient information is available to assess rigorously the extent to which casein kinase-1 functions as a physiological glycogen synthase kinase. From the enzymological studies, there is no reason a priori to assign the enzyme less signifi- cance than the other protein kinases in Table VI, and its ability to affect kinetic properties does provide the potential for controlling glycogen synthase activity. The only studies of glycogen synthase phosphorylation in vivo that have at- tempted to analyze the phosphorylation of specific sites were guided in part by knowledge of the phosphorylation sites involved (50, 51). Therefore, it is difficult to judge whether phosphorylation at sites unique to casein kinase-1 would have been detected or not. Further study is required for more definitive evaluation of casein kinase-1 as a glycogen synthase in vivo.

Acknowledgment-We wish to thank Peggy Smith for typing the manuscript.

REFERENCES 1. Roach, P. J. (1981) Curr. Top. Cell. Regul. 20, 45-105 2. Cohen, P. (1982) Nature (Lond.) 296,613-620 3. Soderling, T. R. (1979) Mol. Cell. Endocrinol. 16 , 157-179 4. Preiss, J., and Walsh, D. A. (1981) in Biobgy of Carbohydrates

(Ginsburg, V., ed) Vol. 1, pp. 199-314, Academic Press, New York

5. Cohen, P., Yellowlees, D., Aitken, A., Donella-Deana, A., Hem- mings, B. A., and Parker, P. J. (1982) Eur. J. Biochem. 124 ,

6. Schlender, K. K., Beebe, S. J., Willey, J. C., Lutz, S. A., and

7. DePaoli-Roach, A. A., Roach, P. J., and Larner, J. (1979) J. Biol.

8. Huang, K.-P., Itarte, E., Singh, T. J., and Akatsuka, A. (1982) J.

9. Brown, J. H., Thompson, B., and Mayer, S. E. (1977) Biochem-

21-35

Reimann, E. M. (1980) Biochim. Biophys. Acta 615,324-340

Chem. 254,12062-12068

Biol. Chem. 257,3236-3242

ist&l6,5501-5508 10. Villar-Palasi. C.. and Larner. J. (1971) Curr. TOD. Cell. Reml. 3. . , . . .

195-236 - .

11. Roach, P. J., DePaoli-Roach, A. A., andLarner, J. (1978) J. Cyclic Nucleotide Res. 4,245-257

12. DePaoli-Roach, A. A., Roach, P. J., and Lamer, J. (1979) J. Biol. Chem. 254,4212-4219

13. Embi, N., Rylatt, D. B., and Cohen, P. (1979) Eur. J. Biochem. 100,339-347

14. Walsh, K. X., Millikin, D. M., Schlender, K. K., and Reimann, E. M. (1979) J. Biol. Chem. 254,6611-6616

15. Soderling, T. R., Srivastava, A. K., Bass, M. A., and Khatra, B. S. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 2536-2540

16. Soderling, T. R., Sheorain, V. S., and Ericsson, L. H. (1979) FEBS Lett. 106,181-184

17. Payne, M. E., and Soderling, T. R. (1980) J. Biol. Chem. 255 ,

18. Ahmad, Z., DePaoli-Roach, A. A., and Roach, P. J. (1982) J. Biol.

19. Woodgett, J: R., Tonks, N. K., and Cohen, P. (1982) FEBS Lett.

20. DePaoli-Roach, A. A., Ahmad, Z., and Roach, P. J. (1981) J. Biol. Chem. 256,8955-8962

21. Ahmad, Z., DePaoli-Roach, A. A., and Roach, P. J. (1982) J. Biol. Chem. 257,5873-5876

22. Itarte, E., Mor, M. A., Salavert, A., Pena, J. M., Bertomeu, J. F., and Guinovart, J. J. (1981) Biochim. Biophys. Acta 658,334- 347

23. DePaoli-Roach, A. A., Roach, P. J., Pham, K., Kramer, G., and Hardesty, B. (1981) J. Biol. Chem. 256,8871-8874

24. Hathaway, G. M., and Traugh, J. A. (1982) Curr. Top. Cell. Regul.

25. Hemmings, B. A., Yellowlees, D.. Kernohan. J. C.. and Cohen. P.

8054-8056

Chem. 257,8348-8355

148,5-11

21,101-127

26.

27.

28.

29.

30.

31.

32.

33.

34.

35. 36. 37.

38.

39.

40. 41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

(1981) kur. J. Eiochem. 119, 443-451 '

Yang, S.-D., Vandenheede, J. R., Goris, J., and Merlevede, W. (1980) J. Biol. Chem. 2 5 5 , 11759-11767

Vandenheede, J. R., Yang, S. D., Goris, J., and Merlevede, W. (1980) J. Biol. Chem. 2 5 5 , 11768-11774

DePaoli-Roach, A. A., Ahmad, Z., Camici, M., Lawrence, J. C., Jr., and Roach, P. J. (1983) J. Biol. Chem. 258,10702-10709

Picton, C., Aitken, A., Bilham, T., and Cohen, P. (1982) Eur.J. Biochem. 124,37-45

Itarte, E., and Huang, K.-P. (1979) J. Biol. Chem. 254 , 4052- 4057

Takeda, Y., Brewer, H. J., Jr., and Larner, J. (1975) J . Biol. Chem. 250,8943-8950

Kish, V. M., and Kleinsmith, L. J. (1975) Methods Enzymol. 40, 198-208

Fischer, E. H., and Krebs, E. G. (1958) J. Biol. Chem. 2 3 1 , 65- 71

Thomas, J. A., Schlender, K. K., and Larner, J. (1976) Anal. Biochem. 25,486-499

Cohen, P. (1973) Eur. J. Biochem. 34, 1-14 Laemmli, U. K. (1970) Nature (Lond.) 227 , 680-685 Merril, C. R., Goldman, D., Sedman, S. A., and Ebert, M. H.

Donella-Deana, A., Meggio, F., and Pinna, L. A. (1979) Biochem.

Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.

Bradford, M. M. (1976) Anal. Biochem. 72,248-254 DePaoli-Roach, A. A., Bingham, E. W., and Roach, P. J. (1981)

Dedman, J. R., Potter, J. D., Jackson, R. L., Johnson, J. D., and

(1981) Science (Wash. D. C.) 211 , 1437-1438

J. 179,693-696

(1951) J. Biol. Chem. 193 , 265-275

Arch. Biochem. Biophys. 212,229-236

Means, A. R. (1977) J. Biol. Chem. 252,8415-8422 Roach, P. J., Takeda, Y., and Larner, J. (1976) J. Biol. Chem.

251,1913-1919 Kemp, B. E., Bylund, D. B., Huang, T.-S., andKrebs, E. G. (1975)

DePaoli-Roach, A. A., and Roach, P. J. (1982) Arch. Biochem.

Hathaway, G. M., Lubben, T. H., and Traugh, J. A. (1980) J.

Chrisman, T. D., Jordan, J. E., and Exton, J. E. (1981) J. Biol.

Krebs, E. G., Love, D. S., Bratvold, G. E., Trayser, K. A., Meyer, W. L., and Fischer, E. H. (1964) Biochemistry 3,1022-1033

Vila, J., Salavert, A., Itarte, E., and Guinovart, J. J. (1982) Arch. Biochem. Biophys. 2 1 8 , 1-7

Parker, P. J., Embi, N., Caudwell, B., and Cohen, P. (1982) Eur.

Parker, P. J., Caudwell, F. B., and Cohen, P. (1983) Eur. J.

Proc. Natl. Acad. Sci. U. S. A. 7 2 , 3448-3452

Biophys. 217, 305-311

Biol. Chem. 255,8038-8041

Chem. 256 , 12981-12985

J. BW~hem. 1 2 4 , 4 7 4 5

Biochem. 130,227-234


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

Z Ahmad, M Camici, A A DePaoli-Roach and P J Roachsynthase kinase (casein kinase-1) as a distinct enzyme.

Glycogen synthase kinases. Classification of a rabbit liver casein and glycogen

1984, 259:3420-3428.J. Biol. Chem.

http://www.jbc.org/content/259/6/3420Access the most updated version of this article at

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

http://www.jbc.org/content/259/6/3420.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at


http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/content/259/6/3420

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;259/6/3420&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/259/6/3420

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=259/6/3420&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/259/6/3420

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/259/6/3420.full.html#ref-list-1

http://www.jbc.org/

the of biological vol. 259. no. 6, lsaue march 25, pp. 3420 … · 2001-09-01 · the journal of...

Documents