Phosphorylation and Activation of Rabbit Skeletal ... - Semantic Scholar

THEJOURNALOF BIOLOGICAL CHEMISTRY Val. 257, No. 22, Issue of November 25, pp. 13379-133&, 1982 Printed in U.S.A

Phosphorylation and Activationof Rabbit Skeletal Muscle Phosphorylase Kinase by a Cyclic Nucleotide- and Ca2+-independent Protein Kinase* (Received for publication, July 11, 1982)

Toolsee J. Singh, Akira Akatsuka, and Kuo-Ping Huang From the Endocrinology and Reproduction Research Branch,National Instituteof Child Health and Human Development, National Institutesof Health, Bethesda, M a u l a n d 20205

Phosphorylase kinase fromrabbit skeletal muscle can be phosphorylated and activated by a cyclic nucleotide- and Ca2‘-independent protein kinase previously identified as a glycogen synthase kinase (Itarte, E., and Huang, K.-P. (1979) J. Biol. Chem. 254, 4052-4057). This independent kinase phosphorylates the j3 subunit of phosphorylase kinase approximately 15 times faster than it does the a subunit. The CAMPdependent and -independent kinases separately catalyze the incorporationof 1 mol of phosphate into thej3 subunit. Analyses of the tryptic peptides from the j3 subunit phosphorylated with eitherkinase by isoelectric focusing and peptide mapping indicate that both kinases phosphorylate the same site on the /3 subunit. Activation of phosphorylase kinase catalyzed by the independent kinase is only 60% of that observed with CAMP-dependent kinase. If phosphorylase kinase is first incubated with the independent kinase to phosphorylate the,8 subunit, subsequent addition of CAMPdependent kinase results in a predominant phosphorylation of the cy subunit. This additional phosphorylation of the a subunit is accompanied bya further activation of phosphorylase kinase to the same extent as that achieved by CAMP-dependent kinase alone. Hence, the phosphorylationof the a subunit is clearly required for full activation of phosphorylase kinase, even at low

of (a&& has been proposed for the enzyme (14). Previously, phosphorylase kinase has been shown to be activated afterphosphorylation by the CAMP-dependent protein kinase (3,4),itself (6, 7), cGMP-dependent proteinkinase (15, X), and Ca2’- and phospholipid-dependent kinase (17, 18). The most extensive studies have been carried out on autophosphorylation and phosphorylation catalyzed by A-kinase.’ It hasbeen shown that when assayed in the presence of 1-2 mM Mg2+A-kinase catalyzes the incorporation of 1mol of phosphate into each of the (Y and ,l3 subunits (3). Phosphorylase kinase activation was found to correlate best with the rapid phosphorylation of the @ subunit (3, 4). The involvement of the a subunit phosphorylation in the activation process under these assay conditions appears tobe minimal. In a subsequent analysis (5) it was found that in the presence of 10 mMMg2’, A-kinase catalyzes the incorporation of 1 mol of phosphate into the p subunit and greater than 5 mol of phosphate into the a subunit. Similar stoichiometries of phosphate incorporation into thea and ,B subunits were also observed under the conditions favoring autophosphorylation (7). Phosphorylase kinase phosphorylated under these two conditions exhibits a 3-fold greater activitythan thatcontaining 1mol of phosphate in each a and ,8 subunit. Whether the higher degree of activation is partially a result of the phosphorylation of a different site in the ,8 subunit has not been resolved. The phosphorylCMg2+1. ation of the a subunit by A-kinase in the presence of 10 mM Mg2‘ or by autophosphorylation does increase the extent of activation (5-7). In the present study we report the activation of phosphoRabbit skeletal muscle phosphorylase kinase exists in nonactivated and activated forms. Conversion of the nonactivated rylase kinase by a cyclic nucleotide- and Ca*’-independent to theactivated form occurs by protein phosphorylation (1-7). protein kinase which was named CAMP-independent glycogen synthase (casein) kinase-1 (19). A-kinase and CK-1 are differThe reverse reaction is catalyzed by protein phosphatase(s) (8-10). Although nonactivated phosphorylase kinase can be ent in their substrate specificities, sensitivities to activation activated by limited proteolysis (3,4), thisprocess is irrevers- by CAMP and inhibition by the heat-stable inhibitor of Akinase, and thesites of phosphorylation in glycogen synthase ible and thoughtnot to be of physiological importance. Phosphorylase kinase has been purified to apparent homo- (20,Zl). Both A-kinase and CK-1 phosphorylate serineresidue geneity from rabbit skeletal muscle (3, 11).The enzyme was in the ,8 subunit much more rapidly than in the a subunit. initially shown to be composed of three types of subunits Activation of phosphorylase kinase by CK-1 is only 60% of termed a,,8, and y (3) or alternatively, A, B, and C (11) in that observed with A-kinase. Addition of A-kinase after the order of decreasing molecular weights. Recently,a fourth initial incubation of phosphorylase kinase with CK-1 results phosphorylation of the a subunit. This subunit, termed 6,was discovered (12). The 6 subunit was inapredominant shown to be similar (13) to the multifunctional calcium-bind- additional phosphorylation is accompanied by a further actiing protein, calmodulin, and is thought to mediate the Ca2+ vation of phosphorylase kinase to the same level as that responsiveness of phosphorylase kinase. A molecular formula achieved by A-kinase alone. These results indicate that phosphorylation of the a subunit is clearly required for full acti* This work was supported in part by a grant from the American vation of phosphorylase kinase even at low [Mg”]. Diabetes Association. 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.

’ The abbreviations used are: A-kinase, CAMP-dependent protein kinase; CK-1, CAMP-independent glycogen synthase (casein) kinase1.

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EXPERIMENTAL PROCEDURES

for 25 h at 25 "C. The gel slurry was then placed in test tubes (10 X 75 m m ) and centrifuged a t 8000 X g for 10 min. The supernatantwas carefully withdrawn. Two additional extractions were carried out for 12 h each. The recoveries of radioactivity from the gels ranged between 50 and 80%. To the pooled supernatants 100 pgof bovine serum albumin was added and the proteins were precipitated with 10% trichloroacetic acid as previously described (20). The protein pellets dissolved in 75p1of 50 mM Tris (pH 8.5) containing 35 mM dithiothreitol were subjected to tryptic digestion (trypsin:protein ratio = 1:5) a t 25 "C for 24 h. Analysis of Tryptic Peptides-Isoelectric focusing of the 32P-labeled tryptic peptides was carried out as previously described (20) at 5 "C for 90 min using 1 ml of Ampholine pH 3.5-10 and 0.5 ml of Ampholine pH 2.5-4 per 25 ml of gel solution. Peptide mapping on cellulose-coated thin layer plates was carried out by electrophoresis (40watts) for 3 hat pH 6.5 (pyridine:glacialacetic acidwater, 25:1:225) in the fust dimension and ascending chromatography (I-butanol: pyridine:glacial acetic acidwater, 15103:12) inthe second dimension. Protein Concentration-Phosphorylase and phosphorylase kinase concentrations were determined spectrophotometrically at 280 nm using absorbance indices of 13.1 and 12.7 (3), respectively, for 1% solutions. The concentrations of other proteins were determined by the dye binding method of Bradford (26).

Materials All proteins used in these studieswere prepared from rabbit skeletal muscle. Phosphorylase (22), nonactivated phosphorylase kinase (3), A-kinase (21). CK-1 (19), and the heat-stable inhibitor (23) of Akinase were prepared as described previously. [y3*P]ATPwas purchased from New England Nuclear. Ampholine solutions were from LKB Instruments and cellulose-coated thin layer plates were from Eastman. Methods Activation of Phosphorylase Kinase-Phosphorylase kinase was activated either by A-kinase or CK-1 in a reaction mixture (100 pl) containing phosphorylase kinase, 0.40 mg/ml; 2-mercaptoethanol, 20 m ~ EDTA, ; 0.23 mM; [y3'P]ATP, 0.20mM; ethylene glycol bis(Paminoethyl ether)-N,N,N',N"tetraacetic acid 0.50mM; KF, 5 mM; magnesium acetate, 1 mM; sodium P-glycerophosphate buffer (pH 6.8),50 mM; and either 0.3 unit of A-kinase or 0.6 unit of CK-1. Reaction mixture for activation by A-kinase also contained 5 p~ CAMP.The reactions were initiated by the addition of ATP. Aliquots were removed at timed intervals for the determination of 32Pincorporation into protein (24) or for the assay of enzyme activity (11). The rate of phosphorylation of phosphorylase kinase by either Akinase or CK-1 is proportional to the amounts of enzyme added. A unit of phosphorylase kinase activity is defined as the amount of enzyme catalyzing the incorporation of 1 pmol of phosphate into phosphorylase b/min at 30 "C. A unit of A-kinase or CK-1 is defined as the amount of kinase catalyzing the incorporation of 1 nmol of phosphate into 4 mg/ml of histone-IIA (for A-kinase) or casein (for CK-1) per min at 30 "C. Polyacrylamide Gel Electrophoresis and the Quantitation of "P Zncorporation into the a and B , Subunits-Electrophoresis of 32Plabeled samples was carried out in 7.5% polyacrylamide gels in the presence of sodium dodecyl sulfate as described (25). The gels were stained for 30 min in a solution of 50% trichloroacetic acid containing 0.25% Coomassie brilliant blue. Destaining was in a solution of7% acetic acid containing 40% ethanol followed by 7% acetic acid alone. The destained gelswerecovered with cellophane and exposed to Kodak X-Omatic XAR-5 f i i with the aid of an intensifying screen (DuPont Cronex) for autoradiography. The extent of phosphorylation in the a and /3 subunits was determined by multiplying the total protein-bound radioactivity with the percentage of total intensity in each subunit after the autoradiogram was scanned in a densitometer (Zeineh Soft Laser Scanner, LKB Instruments) equipped with an integrator. Preparation of Tryptic Peptides from a and /3 Subunits-Phosphorylase kinase was phosphorylated either by CK-1 or A-kinase as outlined above using [y3'P]ATP with a specific activity of approximately 2000 cpm/pmol. Samples were subjected to electrophoresis in the presence of sodium dodecyl sulfate, and the gel was dried on a gel dryer without prior fiiing or staining. The dried gel was exposed to Kodak X-Omatic XAR-5 f h for autoradiography. The a and /3 subunits (25 pg each) were excised from the gel and extracted with 2 ml of50mM NH4HC03 (pH 8)containing 10 mM 2-mercaptoethanol

A. 2.5

RESULTS

Phosphorylation and Activation of Phosphorylase Kinase by A-kinase and CK-1-Incubation of phosphorylase kinase with A-kinase or CK-1 results in the phosphorylation of phosphorylase kinase (Fig. lA). The reaction catalyzed by M or80 pg of CK-1 is unaffected by the addition of 10 ~ L CAMP partially purified heat-stable inhibitor(sufficient to inhibit 0.3 unit of A-kinase by more than 90%).In the absence of added kinase, phosphorylase kinase undergoes slight autophosphorylation. Phosphorylation of phosphorylase kinase by A-kinase is accompanied by a rapid activationwhen the 1st mol of phosphate is incorporated (Fig. 1B). Additional phosphorylation by A-kinase results in a small (about 20%) increase in the activity. These phenomena have been observed previously and the activation of phosphorylase kinase was reported to correlate primarily with the rapid phosphorylationof a sitein the /3 subunit (3). The activation of phosphorylase kinase catalyzed by CK-1 appears to follow the same kinetics as the incorporation of phosphate into the enzyme. The incorporation of approximately 1 mol of phosphate/phosphorylase kinase monomer results in a16-foldactivation. Incorporationof greater than 1.2 mol of phosphate catalyzed by A-kinase results in a 25-fold activation. Activation by autophosphorylation is less than 2-fold when approximately 0.2 mol of phosphate is incorporated. We have considered possible ways by which the extent of

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w -Kinase + Independent Kinase bd

+ A-Kinase

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M

1.0-

. E

FIG. 1. Phosphorylation (A) a n d activation (I?) of phosphorylase kinase by A-kinase and CK-1. Nonactivated phosphorylase kinase was phosor Aphorylated by itself (El), CK-1 (O), kinase (A). Aliquots were removed at different times for the determination of 32Pincorporation into protein (A) or for assaying enzyme activity ( B ) .

w + Independent Kinase M+

A-Kinase

c 3 0.8"

I

0 Time (mid

I

m

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40 Time (mini

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Phosphorylation and Activationof Phosphorylase Kinase phosphorylation and activation of phosphorylase kinase catalyzed by CK-1 can be increased. It was previously shown that the rate and extent of phosphorylation of phosphorylase kinase catalyzed by A-kinase can be increased by raising the concentration of Mg’+ in the reaction mixture (5). We found no difference in the rate or extent of phosphorylation when the CK-1-catalyzed reaction was studied a t 1 and 15 mM Mg”. Addition of a 10-fold molar excess of calmodulin over phosphorylase kinase to the phosphorylation mixturealso did not affect the rate of phosphorylation catalyzed by CK-1. However, a slight stimulation of phosphorylase kinase autophosphorylation was observed (data not shown). Subunit Phosphorylation of Phosphorylase Kinase Catalyzed by CK-1”It has previously been shown that A-kinase catalyzes the incorporationof 1 mol of phosphate rapidly into the p subunit and a 2nd mol of phosphate into thea subunit at a slower initial rate (3, 4). The phosphorylation of the a subunit becomes greatly facilitated after approximately 50% of the p subunit has been phosphorylated (3, 4). We have verified these findingsin thepresentstudy.Therate of phosphorylation of the p subunit by CK-1 is approximately 15 times faster than that of the a subunit (Fig. 2). The rateof phosphorylation of the a subunit is not significantly affected by the extent of phosphorylation of the /3 subunit. After 60 min of incubation phosphate incorporation reaches approximately 1 mol/p subunit whereas the incorporation into the a subunit is less than 0.2 mol. It should be noted that the a subunit phosphorylation is the sumof a and a’.Hence, like Akinase, CK-1 phosphorylates sites on the B subunit at a faster rate than on thea subunit. Comparison of the Sites in the /3 Subunit Phosphorylated by A-kinaseand CK-1-The p subunit of phosphorylase kinase, which had been phosphorylated by A-kinase or CK-1, was isolated from the gel after electrophoresisin the presence of sodium dodecyl sulfate. Isoelectric focusing analysis of the “’P-labeled tryptic peptidesderived from the p subunit shows that a major (PI4.7) and a minor (PI5.5) peptide is labeled by each kinase (Fig.3). Further analysis of the ,“’P-labeled tryptic peptides by peptide mapping (see“Methods”) reveals the

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5.5

-.A

”- 0 4.7

A

B

FIG.3. Autoradiogram of 32P-labeledtryptic peptides from the B subunit after isoelectric focusing. Phosphorylase kinase was phosphorylated either by A-kinase ( A ) or CK-1 ( B ) . Tryptic peptides prepared from the ‘”P-labeled p subunit were analyzed by isoelectric focusing as described under “Methods.”

.,

L

a

i,

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Electrophoresis

6 3

FIG. 4. Autoradiogram of 32P-labeledtryptic peptides from the /3 subunit after peptide mapping. Tryptic peptides prepared from the p subunit of phosphorylase kinase phosphorylated by CK-1 were analyzed by peptide mapping as described under “Methods.” 0, origin.

FIG. 2. Phosphorylation of phosphorylase kinase subunits by CK-1. Nonactivated phosphorylase kinase was phosphorylated by CK-1 as described under “Methods.” Aliquots of the reaction mixture were withdrawn at the different times for analysis by sodium dodecyl sulfate-polyacrylamide gels. The phosphorylations of the a subunit (A) and p subunit (0) are shown.

presence of two major ‘”P-labeled peptides. These peptides have equivalent amounts of radioactivity after counting the scraped samples (Fig. 4). This same pattern of separation is observed when the tryptic peptides of A-kinase- or CK-1phosphorylated B subunit are analyzed alone or after mixing. We have also identified phosphoserine by thin layer electrophoresis(27) as the only “’P-labeled amino acid in these peptides. The resolution of tryptic peptides derived from the p subunit phosphorylated by A-kinase into two spots after thin layer chromatography confiims the previous finding by Yeaman et al. (28) who identified the amino acid sequence surroundingthe phosphorylation siteas Lys-Arg-Ser-GlyIle Ser(P)-Val-Tyr-Glu-Pro-Leu-Lys. The existence of two peptides having the same isoelectric point is due to thepresence of either valine or isoleucine adjacent tophosphoserine. This is because of the presence of two alleles for the p subunit of

Phosphorylation Activation and

13382

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k

Q 5

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of Phosphorylase Kinase

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IndepdenIKinere A-Kmare at 4omm

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n 0

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0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 Time lminl

0 1 0 2 0 3 3 4 0 5 0 6 0 7 0 Time (minl

FIG. 5. Phosphorylation and activation of phosphorylase ki- the same conditions as shown in A . Aliquots were taken at timed nase by the Combination of A-kinase and CK-1. A, phosphate intervals for the analyses of phosphate incorporation into a (A, A) incorporation into phosphorylase kinase in the presence of CK-1 (0)and p ( 0 , O ) subunits by CK-1 (A, 0) and the combination of CK-1 or the combination of A-kinase and CK-1 (A).The reactions were and A-kinase (A,0).C, activation of phosphorylase kinase catalyzed carried out under standard assay conditions. After 40 min of incubation with CK-1, aliquots were removed and supplemented with 5 PM CAMP and A-kinase. B , phosphate incorporation into the a and p subunits of phosphorylase kinase. Reactions were carried out under

phosphorylase kinase in New Zealand White rabbit populations (29). There are two potential serine resudues that can be phosphorylatedby CK-I; however, the phosphorylation of the serine residue on the carboxyl-terminal side of arginine wiU partially protect thisbond from hydrolysis by trypsin (30) and thereby produce tryptic peptides having different isoelectric points from that phosphorylated by A-kinase. Based on this reasoning and the results presented here, we tentatively conclude that A-kinase and CK-1 phosphorylatethe samesite on the /3 subunit. Role of the a Subunit Phosphorylationin the Activation of Phosphorylase Kinase-Under optimal conditions, autophosphorylation (6, 7) or phosphorylation catalyzed by A-kinase in the presence of10 mM Mgz+ (5) results in greater than 5 mol of phosphate being incorporated into the a subunit. In these studies it was clearly established that maximal activation of phosphorylase kinase is achieved only when sites on the a subunit arephosphorylated, in addition to a site on the p subunit (5-7). When A-kinase-catalyzed phosphorylation of phosphorylase kinase is studied in the presence of 1-2 mM Mg2+,the (Y and p subunits each become phosphorylated to the extent of 1 mol (3). In these circumstances the role of a subunit phosphorylation in the activation of phosphorylase kinase is unclear. However, the suboptimal activation achieved by the CK-I-catalyzed reaction (Fig. 1B) and the very low level of a subunit phosphorylation observed (Fig. 2) suggest that complete phosphorylation of this subunit to the extent of 1mol may be required for full activation (compared to the A-kinase-catalyzed reaction, see Fig. 1B) of phosphorylase kinase. Addition of A-kinase after an initial incubation of phosphorylase kinase with CK-1 results in afurther phosphorylation (Fig. 5A). During the initial 40 min of incubation 0.8 and 0.1 mol of phosphate are incorporated into the/3 and a subunits, respectively (Fig. 5B). The subsequent addition of A-kinase results in a slight increase in the/3 subunit phosphorylation. By contrast, the 01 subunit is greatly phosphorylated. This additional phosphorylation of the a subunit appears to correlate better with a further increase in phosphorylase kinase activity (Fig. 5C). The extent of activation resulting from the combination of both kinases is comparable to that by Akinase alone. Addition of CK-1 after theinitial phosphorylation of phosphorylase kinase with A-kinase does not result in a further phosphorylation or activation (data notshown). The involvement of the a and /3 subunit phosphorylations

by CK-1 and A-kinase. Phosphorylase kinase activities were deterand mined after incubation with A-kinase alone (0).CK-1 alone (O), the combination of both kinases (A).

=

0

0.2 04 06 08 10 Phosphate lncorporatlon (mol/mol B -subunit)

Kinase Activlty (units/rng)

FIG. 6. Relationship between activation of phosphorylase kinase and the phosphorylation of the a and /3 subunits. A , phosphorylase kinase was phosphorylated either by A-kinase (A)or by CK-I (0)and the extent of activation measured. Incorporation of phosphate into the a and p subunits were determined as described under “Methods.” Activity is expressed only as a function of p subunit phosphorylation. B , phosphorylase kinase was phosphorylated by Akinase and the extent of activation and phosphorylation of the subunits measured as in A . The phosphorylations of the a (A)and /3 (0) subunits are plotted as functions of the kinase activity.

in phosphorylase kinase activation is shown in Fig. 6. There is a linear increase of enzyme activity with p subunit phosphorylation up to 0.6 mol of phosphate for both the CK-1and A-kinase-catalyzed reactions. Further phosphorylation of the /3 subunit by A-kinase leads to agreater increase in enzyme activity as compared to the CK-1-catalyzed reaction (Fig. 6A).This second phase increase in enzyme activity with increasing phosphorylation in the A-kinase-catalyzed reaction is a resultof both a and p subunit phosphorylations(Fig. 6B). In the fistphase the enzyme is activated approximately 60% of maximum mainly as a resultof the incorporation of 0.6 mol of phosphate into the /3 subunit. In the second phase enzyme activity increases with both a and p subunit phosphorylation. After the incorporation of 1 and 0.3 mol of phosphate (up to 0.4mol in other experiments) into p and a subunits, respectively, enzyme activity becomes insensitive to further phosphorylation of the a subunit. Comparison of the Sites in the a Subunit Phosphorylated by A-kinase and CK-1 Alone or in Combination-Under certain conditions many sites on the a subunit can be phos-

Phosphorylation Activation and

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o f Phosphorylase Kinase

13383

that both A-kinase and CK-1 phosphorylate the same site on the p subunit (Figs. 3, 4 and 5B). The substrate specificityof A-kinasehasbeenextensivelycharacterizedbyusingsynthetic peptides (31-33). The serine residues phosphorylated by A-kinase usually have one or two basic amino acids placed close bythe NH2-terminal side. Ry comparison, our knowledge on the substrate specificity of t h e cyclic nucleotide-independent protein kinases has been derived from the studies using various casein variants (see Ref.34 for review). These kinases recognize sites proximal to acidic amino acid residues in the protein. The aminoacid sequencein thep subunit surrounding the

phosphorylation

Lys-Arg-Ser-Gly-Ser(P)-Val-TyrIle site,

Glu-Pro-Leu-Lys (28). consists of basic and acidic residues and seems to satisfy the substrate specificities of both kinases. Phosphorylation of the a subunit under the condition favoring a higher level (greater than 5 mol/subunit) of phosphate incorporation has been shown to augment the activation FIG. 7. Autoradiogram of “P-labeled tryptic peptides from of phosphorylase kinase (5). However. the role of it subunit the a and p subunits after isoelectric focusing. Phosphorylase phosphorylation in the activation was not clear when approxkinase was phosphorylated either by CK-I alone ( 0 ) .A-kinase alone imately 1 mol of phosphate is incorporated into each of the ( b ) ,or A-kinase added after CK-I (c) as described in the legend to a and /3 subunits (3, 4 ) . Addition of A-kinase after phosphoFig. 5A. Trvptic peptides were prepared from the isolated n subunits rylation of phosphorylase kinase with CK-I results in a pre( h and c) or from native phosphorylase kinase (both o and /Isubunits dominant phosphorylation of the a subunit anda concomitant present) ( a ) and isoelectric focusing was carried out as described activation (Fig. 5). The site(s) in the (1 subunit phosphorylated under “Methods.” under this condition is similar to that phosphorylated by Akinase alone (Fig. 7 ) . These results demonstrate that phosphorylated by A-kinase, resulting in enhanced activation of phorylation of the a subunit by A-kinase in the presence of phosphorylase kinase(5). We did consider the possibility that 1-2 mM Mg’+does contribute to phosphorylase kinase actithe additional activation resulting from the sequential phosvation as was observed by Hayakawa et al. (4). However, it phorylation of phosphorylase kinase by CK-1 and A-kinase should be noted that the activation of phosphorylase kinase (see Fig. 5) may be occurringat asite in thea subunit different 1 by A-kinase reaches a constant level after approximately from the one normally phosphorylated by A-kinase alone. To and 0.3 mol of phosphate are incorporatedinto ,l3 and a check this possibility we analyzed the tryptic peptides in the subunits, respectively. Phosphorylase kinase holoenzyme con(Y subunit phosphorylated by A-kinase alone or by the addition 0.3 sists of four (a/3yS) monomers; it is not known whether of A-kinase after phosphorylation with CK-1 as was outlined mol of phosphate/a subunit represents the partial phosphoin Fig. 5. The major““P-labeled tryptic peptides derived from rylation of every, or complete phosphorylationof some, phosthe a subunit phosphorylated under these two conditions have a phorylase kinase molecules. Although CK-1 also catalyzes the same isoelectric point (Fig.7, lanes h and c ) . This peptide low level of phosphorylation in the a subunit (Fig. 2), the is absent in the tryptic digest of phosphorylase kinase phosactivation of phosphorylasekinase is not as great as that phorylated by CK-1 alone (Fig.7, lane a).The major peptide achieved by A-kinase. This may be because the site(s) in the (PI 4.7) observed in this latter pattern is that derived from the a subunit phosphorylated by CK-1 and A-kinase is different /3 subunit as was shown inFig. 3. It can therefore be concluded (Fig. 7 ) . a subunit phosphorylated by Athat the major site in the Skeletal muscle phosphorylase kinase has been shown to be kinase at 1-2 mMMg’+ is not phosphorylated by CK-1 and phosphorylated by A-kinase ( 3 , 4), GMP-depedent protein also that prior phosphorylation of t h e /3 subunit by CK-1 does kinase (15, 161, Ca”-andphospholipid-dependentprotein not subsequently change the specificity of A-kinase for sites kinase (17, 18), phosphorylase kinase itself ( 6 , 7 ) . and CK-1 on the a subunit. (present study). It is interesting to note that all these kinases also phosphorylate glycogen synthase. Therefore, the activaDISCUSSION tions of these kinases should facilitate glycogenolysis through In the present study we have demonstrated that phospho- the concurrent inactivation of glycogen synthase and activrylase kinase can be phosphorylated and activated by CK-1, tion of phosphorylase kinase. The activation of phosphorylase a cyclic nucleotide- and Ca”-independent kinase from skeletal kinase in cGvo by A-kinase in response to adrenalin has been muscle. In addition to the known differences in the properties reported (35). However, the physiological significance of the of A-kinase and CK-1 (19), the phosphorylation of phospho- other kinases in the regulation of phosphorvlase kinase activrylase kinase by these kinases differs in some respects. First, ity has yet to be determined. The regulatory mechanism for although both A-kinase and CK-1 phosphorylate [jthe subunit t h e cyclic nucleotide- and Ca’”-independent protein kinasesis at a faster rate than the a subunit, the phosphorylation of a unknown. This class of protein kinases has been shown to be subunit by CK-1 is not significantly affected by the extent of present in manymammaliantissuesandphosphorylatea phosphorylation on the p subunit. The phosphorylation of a variety of substrates (34) including casein and phosvitin,glysubunit by A-kinase is greatly facilitated after approximately cogen synthase, phosphorylase kinase (present study), initia505%of t h e /3 subunit has been phosphorylated. Second, the tion factors, nonhistone chromatin proteins, and eukaryotic rate and final extent of phosphorylation of phosphorylase RNA polymerase I and 11. The substrates phosphorylatedby kinase catalyzed by A-kinase is greatly affected by increasing these kinases are as diversified as those by A-kinase. T h e Mg” concentrations from 1 to10 mM (5).T h e CK-1-catalyzed questions concerning the possible functional and regulatory reaction is insensitive to increasing Mg”+ concentrations. roles of these kinases for the diverse cellular activities require The results presented in this study support the conclusion further investigation.

a

b

c

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PhosphorylationActivation and REFERENCES

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