Eur. J. Biochem. 188, 367-376 (1990) 0 FEBS 1990

Localization of phosphoserine residues in the a subunit of rabbit skeletal muscle phosphorylase kinase Helmut E. MEYER’, Gerhard F. MEYER’, Herbert DIRKS’ and Ludwig M. G. HEILMEYER Jr’ Institut fur Physiologische Chemie, Abteilung fur Biochemie Supramolekularer Systeme, Ruhr-Universitat Bochum Gesellschaft fur Biotechnologische Forschung mbH, Abteilung Instrumentelle Analytik, Braunschweig, Federal Republic of Germany (Received September l/November 11, 1989) - EJB 89 1072

The CI subunit of skeletal muscle phosphorylase kinase, as isolated, carries phosphate at the serine residues 1018, 1020 and 1023. Employing the S-ethyl-cysteine method, these residues are found to be phosphorylated partially, i.e. differently phosphorylated species exist in muscle. Serine 1018 is a site which can be phosphorylated by the cyclic-AMP-dependent protein kinase. The serine residues 972, 985 and 1007 are phosphorylated by phosphorylase kinase itself when its activity is stimulated by micromolar concentrations of Ca2 . These phosphorylation sites are not identical to those found to be phosphorylated already in the enzyme as prepared from freshly excised muscle. A ‘multiphosphorylation loop’ uniquely present in this but not in the homologous p subunit contains all the phosphoserine residues so far identified in the a subunit. +

Variable amounts of covalently bound phosphate have been found in freshly isolated phosphorylase kinase and have been determined to be present in the subunits, CI and p (reviewed by Pickett-Gies and D. Walsh [l]). Enzyme preparations containing between 2 - 5 mol phosphate/mol (ajy6) protein have been obtained; 31P-NMR studies indicate that all of these phosphates are covalently linked to serine [2]. Separation of the subunits by HPLC has shown that the CI subunit contains 2.7 f:0.4 mol phosphate/mol protein 131. In the test tube, this subunit can be phosphorylated additionally by a variety of protein kinases. Best studied are phosphorylations catalyzed by the cyclic-AMP-dependent protein kinase as well as by phosphorylase kinase itself, termed selfphosphorylation. The surrounding sequence with only one serine in the c1 subunit, phosphorylated by the cyclic-AMPdependent protein kinase, is known [4]; the sites phosphorylated by self-phosphorylation, by cyclic-GMP-dependent protein kinase, by Ca2+-calmodulin-dependent protein kinase as well as by casein protein kinase are unidentified as yet [5 - 81. By protein and cDNA sequencing, we have determined recently the complete primary structure of the CI subunit comprising 1237 amino acids [9]. This knowledge is the basis by which to localize phosphorylation sites in the primary structure of this high-molecular-mass protein. If self-phosphorylation is a mechanism occurring in the intact cell, we would expect to find identical sites labelled Correspondence to L. M. G. Heilmeyer Jr, Institut fur Physiologische Chemie, Abteilung fur Biochemie Supramolekularer Systeme, Ruhr-Universitat Bochum, D-4630 Bochum 1, Federal Republic of Germany Nomenclature of peptides. Peptides are labelled as follows: the first letter refcrs to peptides derived by cleavage at lysine, arginine, glutamate or methionine residues with endoproteinase Lys-C (K), trypsin (T), Glu-specific endoproteinase V8 (E) or CN-Br (M), respcctively ; the numbering follows the known primary structure starting with the first peptide at the N-terminus. Enzymes. Phosphorylase kinase, ATP:phosphorylase-b phosphotransferase) (EC 2.7.1.38); cyclic-AMP-dependent protein kinase (EC 2.7.1.37); endoproteinase Lys-C (EC 3.4.21.SO); trypsin (EC 3.4.21.4); Glu-specific endoproteinase V8 (EC 3.4.21.19).

during self-phosphorylation in the test tube and phosphorylated in the intact organ. Therefore, an attempt was made to localize phosphoserines endogenously present in the isolated a subunit by employing a new method developed in our laboratory. Thereby phosphoserine is converted into S-ethylcysteine [lo]. The present publication will show that all of the sites phosphorylated either during self-phosphorylation or in intact muscle, as determined here, are clustered in a unique domain of the CI subunit including the site previously shown to be phosphorylated by cyclic-AMP-dependent protein kinase [I 11. MATERIALS AND METHODS Enzymes and reagents

Tosylphenylalaninechloromethane-treated trypsin was obtained from Worthington; endoproteinase Lys-C and Gluspecific endoproteinase V8 was from Boehringer, Mannheim, FRG. Ethanethiol, triethylamine and trifluoroacetic acid were obtained from Pierce, Rodgau, FRG. Hexafluoroacetone (pro synthesis) and glycerol 2-phosphate were from Merck, Darmstadt, FRG. HPLC columns and packings for protein and peptide separations were from Vydac, Hesperia, CA, USA. Chemicals and solvents for automated protein sequence analysis were from Applied Biosystems, Weiterstadt, FRG. All other chemicals and solvents were of the highest purity available. [Y-~’P]ATP was prepared according to Walseth and Johnson [12]. Rabbit skeletal muscle phosphorylase kinase was purified according to Cohen [l 11 with the modifications introduced by Jennissen and Heilmeyer [13] and by Hessova et al. [14]. Phosphorylase kinase self-phosphorylation

Phosphorylase kinase (3 mg/ml) was dialyzed against 2 x 250 ml SO mM Hepes pH 6.8 containing 1.5 mM glycerol 2-phosphate and 1 mM dithioerythreitol at 4°C for 12 h. Self-phosphorylation was carried out in a mixture of 2.1 ml dialysate, 150 p1 22.45 mM CaCl,, 150 pl 20 mM EGTA,

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150 p160 mM ATP (containing 1.5 - 3.0 mCi [Y-~~PIATP) and 200 p1 buffer (0.5 M Hepes, 15 mM glycerol 2-phosphate, 10 mM dithioerythreitol, pH 6.8). The reaction was started with 150 p1180 mM MgC12dissolved in 10 mM Tris/HCl pH 6.8. The resulting free-Ca2+ concentration was calculated to be 100 pM [15]. After 1 h at 24"C, the reaction was stopped by cooling on ice. Aliquots of the mixture were taken for determination of protein-bound radioactivity and of protein by phenylthiocarbamoyl analysis as described previously [161. Separation of phosphorylase kinase subunits The phosphorylase kinase subunits were separated by reversed-phase HPLC on Vydac 10-pm C4 wide-pore material (214 TPB 10) on a 20 x 50-mm column according to Crabb and Heilmeyer [3, 171. Up to 1.5 mg phosphorylase kinase ( 5 nmol) was applied. Subunits were eluted with a gradient of 0.1YOtrifluoroacetic acid to 84% acetonitrile/0.08% trifluoroacetic acid. A flow rate of 7.8 ml/min was used and the fractions containing the separated subunits were collected. Combined fractions from several separations were provided with SDS (7.5 p1 of a 10% solution for 60 ml total volume) prior to volume reduction to 3 ml, yielding a final SDS concentration of 0.025%. Precipitated protein was solubilized completely by addition of a concentrated Tris/EDTA solution giving a final concentration of 25 mM Tris/O.l mM EDTA. As soon as the precipitate was dissolved completely the pH was adjusted to 8.6 with diluted HCl. Prior to proteolytic digestion, the amount of protein and of protein-bound radioactivity were determined. Protease cleavage of the u subunit The isolated solubilized M subunit (about 5 nmol in 3 ml) was digested with two portions of 2.5% (by mass) Lys-C at 3 7 T , pH 8.6, each digestion lasting 2 h. For tryptic digestion,

the SDS supplement to the combined pool was omitted and the protein was cleaved by addition of two portions of 10% (by mass) tosylphenylalaninechloromethane-treatedtrypsin. Incubation was for two 2-h periods at pH 7.8 and 37°C. Peptide purijication Tryptic peptides of the 32P-labelled M subunit were separated by reversed-phase HPLC using a 5-pm Vydac CI8 column (TP 218; 4 . 6 150 ~ mM) applying a gradient of 0.1% trifluoroacetic acid to 84% acetonitrile/0.08% trifluoroacetic acid at a flow rate of 1.0 ml/min. Fractions containing radioactivity were purified further employing the same column with a changed gradient profile. The mixtures of 32P-labelled or unlabelled peptides generated by Lys-C digestion were separated by HPLC on 10-pm Vydac (228TP104) pH-stable reversed-phase material (4.6 x 250 mm). A solvent system consisting of 0.2% hexafluoroacetone/ammonia pH 8.6 (solvent A) and 84% acetonitrile/0.03% hexafluoroacetone/ammonia pH 8.6 as solvent B was employed at a flow rate of 1 ml/min. Fractions containing radioactive peptides were purified to homogeneity with the same column and solvent system but the pH adjusted to 7.2. Fractions containing phosphoserine (see below) were further purified on the reversed-phase material employing the solvent system described for purification of tryptic peptides. Determination of phosphoserine as S-ethyl-cysteine Conversion of phosphoserine to S-ethyl-cysteine was carried out according to Meyer et al. [lo, IS]. The dried peptide (0.05 - 1 nmol) was incubated for 1 h at 50°C in a reaction mixture containing 0.5 M sodium hydroxide, 33% dimethylsulfoxide, 13% ethanol and 10% ethanethiol. After cooling to room temperature, the reaction mixture was acidified with 10 pl acetic acid. The material was applied directly

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Fig. 2. Separation of endoproteinase Lys-Cpeptides ofphosphorylase kinase CI subunit. The Lys-C digest from 20 nmol isolated CI subunit was applied onto a pH-stable reversed phase HPLC column (4.6 x 250 mm, Vydac 228TP104). Separation was performed using a hexafluoroacetone/ ammonia/acetonitrile linear gradient at pH 8.6 as indicated. Peptides were detected by their absorbance at 214 nm (solid line). Aliquots of 100 pl were taken from each fraction to determine the S-ethyl-cysteine content after conversion of the phosphoserine residues (see Methods). The open bars represent the amount of S-ethyl-cysteine found. The different species of the phosphoserine-containing peptide K,55 are numbered I - IV according to their elution position Table 1. Amido acid composition of phosphopeptides Amino acid analyses were carried out as described in Methods. Values were not corrected for destruction or slow liberation of special amino acids. Cysteine and tryptophan were not determined. Peptides K,55 1 -1V contained endogenous phosphate peptides K,52 and T,103/104 contained phosphate following self-phosphorylation S-Et-Cys, Sethyl-cysteine Amino acid

" 1

Amount in peptide K,55 I

K,55 I1

K,55 I11

K,55 IV

K,52

T,103/104

mol/mol peptide ASP Glu Ser GlY Thr Ala His Pro Arg TYr Val Met S-Et-Cys Ile Leu Phe LYS =PO,

0.9 2.6 8.4 1.6 2.4 1.8 2.3 4.2 2.0

1.1 3.4 8.9 1.7 2.8 1.8 2.3 5.0 1.9

0.9 1.8 9.3 6.6 3.4 1.2 2.7 5.1 2.2

0.7 (2) 2.1 (4) 8.4 (12) 7.1 (6) 3.1 (4) 1.3 (1) 2.6 (3) 5.1 (5) 2.2 (2)

3.1 (2) 3.1 (3) 3.8 (4) 3.3 (3) 1.7 (2) 3.6 (3) 1.1 (1) 2.9 (2) 1.5 (2)

1.9 0.9 2.0 1.0 4.8 2.3 0.4 -

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0.9 (1) 3.2 (4) (0) n.d. 3.0 (3) (0) 2.5 (1) 0.8 (I) 1.9

This peptide contains one phosphate group according to Fastatom-bombardment mass spectrometry. This peptide contains two isoleucine residues which were determined during sequence analysis. Determination of isoleucine during phenylthiocarbamoyl analysis in this case was hampered by a coeluting byproduct of the coupling reaction.

to the glass-fiber filter disk for Edman degradation in the gas-

phase sequencer. Samples designated for phenylthiocarbamoyl analysis were modified without dimethylsulfoxide. Prior

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to hydrolysis, the acidified mixture was dried at room temperature in as short a time as possible by using a high-vacuum system (0.1 Pa) equipped with a small-volume trap (800 ml) cooled to - 80°C. Best results were obtained if not more than ten samples were processed at the same time.

Amino acid analysis Aliquots containing peptides or subunits were dried in vacuum and hydrolyzed in an atmosphere developed from 6 M HCl at 150°C for 1 h. To obtain optimal recoveries of Sethyl-cysteine, extreme caution was taken to remove all oxygen from the hydrolysis vessel by repeated evacuations and flushings with argon. Derivatization of the free amino acids with phenylisothiocyanate was carried out as described pre-

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Fig. 4. Sequence unulysis ofpeptide KJ5. (A) 200 pmol peptide K,55 I was sequenced as described in Methods. Serine 1018 (cycle 8) yields the dithiothreitol adduct of dehydroalanine (D-S) exclusively (DPTU = N , N'-diphenylthiourea, DMPTU = N-dimethyl-N'-phenylthiourea). (B) 200 pmol peptide K,55 I was sequenced after modification of phosphoserine to S-ethyl-cysteine (S-El-Cys) (see Methods). Serine 1018 (cycle 8) yields the phenylthiohydantoin of S-ethyl-cysteine. (D-S = dithiothreitol adduct of dehydroalanine)

viously [18, 191. The phenylthiocarbamoyl derivatives were separated by reversed-phase HPLC on a 3-pm Spherisorb ODs-2 column (4.6 x 125 mm). Alternatively amino acid analyses were carried out using the 420A derivatizer and the 130A separation system of Applied Biosystems. In general, phenylthiocarbamoyl analysis was also used for protein determination.

plied in 30-p1 aliquots or they were dried and then dissolved in 90 p1 50% formic acid and applied onto the polybrenecoated glass-fiber disk. The resulting derivatives were analyzed using an Applied Biosystems model 120A on-line phenylthiohydantoin analyzer. The yield of amino acid in each cycle was calculated by adding the overlap of the following cycle and subtracting the background of the previous cycle.

Sequence analysis

Mass spectrometry

Edman degradations were performed on an Applied Biosystems 470A gas-phase sequencer using the 03RPTH standard program. Underivatized samples were either directly ap-

Fast-atom-bombardment mass spectrometry was performed on a Kratos MS 50 R F with a high-field magnet equipped with a Kratos FAB source, using a 5 - 7-kV xenon

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Fig. 5. Sequence analysis of peptide KJ.5 I and II. 200 pmol of a mixture of peptides K,55 I and I1 was sequenced after modification of phosphoserine to S-ethyl-cysteine (S-Et-Cys) (see Methods). All serine residues (1018,1020, 1023, cycle 8, 10, 13, respectively) yield Sethyl-cysteine, the dithiothreitol adduct of dehydroalanine (D-S) and serine

beam at a source pressure of 1 mPa. Samples were dissolved in a small amount of methanol containing a trace of acetic acid. Glycerol was used as matrix on a copper tip or alternatively in dimethylsulfoxide-3-nitrobenzyl alcohol containing a small amount of oxalic acid as matrix. The spectra were recorded at an accelerating potential of 8 kV with a magnet scan rate of 300 s/decade at a resolution of 4000. RESULTS 5’-Ethyl-cysteine appears as a new peak in a chromatogram of the phenylthiocarbomoyl derivatives of amino

acids if modification of phosphoserine is carried out prior to hydrolysis. From the result demonstrated in Fig. 1, it is calculated that the a subunit as isolated contains 2.6 mol phosphoserine/mol protein. When self-phosphorylation is catalyzed by the high-affinity Ca2+-dependentactivity Al up to 4.7 mol phosphoserine/mol are incorporated additionally (for definition of the partial activities A b , A1 and A2 see [20]). Taken together, these results indicate that minimally seven serine residues can be phosphorylated in the a subunit. The number of sites involved may be even higher if partial phosphorylation of serine residues occurs. Conversion of phosphoserine into S-ethyl-cysteine can also be employed as a screening method to detect phosphopeptides in the separated peptide fractions of a proteolytic digest. Several peptides, generated by Lys-C digestion of the a subunit, can be seen to contain this derivatized amino acid upon S-ethyl-cysteine modification (Fig. 2). The peptides which are obtained in homogeneous form, eluting between 52-64 min, are labelled I-IV (Fig. 2). According to Nterminal analysis (not shown) as well as amino acid composition (Table 1) four fractions contain the same peptide K,55. Their S-ethyl-cysteine (i.e. phosphoserine) content decreases with increasing elution time. Peaks 1 and I1 each contain 2 mol/mol, whereas peaks 111 and IV contain only 1 mol phosphoserine/mol (Table 1). The localization of phosphoserine residues in these peptides is unusually difficult: K,55 contains at position 16 an asparagine-glycine bond which forms a cyclic amide when exposed to acidic conditions during isolation or during Edman degradation. Therefore, aspartic acid, and not asparagine as expected from the cDNA sequence, is identified in this position. Moreover, a drop in repetitive yield occurs from cycle 15 to 36 (Fig. 3) which, together with an increased overlap beginning at this point, hampers identification of phosphoserine residues further downstream. The presence of a cyclic amide bond is also confirmed by fast-atom-bombardment mass spectrometry analysis. For example, K,55 peak 111 yields a molecular mass of 5363 Da. This mass is in accordance with the known composition of the peptide which contains the amide bond and additionally one phosphate group (data not shown). The peptides eluting between 65-80 min (Fig. 2) could not be obtained in homogeneous form. Furthermore, S-ethylcysteine may have been formed from cysteine residues which can also undergo p-elimination and S-ethyl-cysteine formation in special environments [21] (see Discussion). During Edman degradation phosphoserine residues can be identified in two ways. Without modification, phosphoserine yields exclusively the dithiothreitol adduct of dehydroalanine during automatic Edman degradation and ‘on line’ analysis of the phenylthiohydantoin derivatives (for details see [ 10, 161). Following modification, the phenylthiohydantoin of Sethyl-cysteine can be identified in the chromatogram. Fig. 4A and 4B are examples for these two methods. Without modification, the phenylthiohydantoin derivative of the dithiothreitol adduct of dehydroalanine is formed in cycle 8 exclusively, no peak corresponding to that of serine being found (Fig. 4A). The identified phosphoserine represents amino acid 1018 in the whole sequence. The following serine 1020 identified in cycle 10 yields the usual mixture of dithiothreitoladduct of dehydroalanine and serine which is characteristic for a non-phosphorylated serine (Fig. 4A). Following S-ethylcysteine modification the same result is obtained. Indeed, it can clearly be seen that in cycle 8, corresponding to serine 1018, the phenylthiohydantoin derivative of S-ethyl-cysteine arises whereas the normal mixture of serine and the

372

T i m e (min I Fig. 6. Separation of a Lys-C digest of self-phosphorylated phosphorylase kinase a subunit. 720 pmol of self-phosphorylated a subunit was injected onto a pH-stable Vydac C8 column after endoproteinase Lys-C digestion (see Methods). Peptides were eluted applying a linear hexafluoroacetone/ammonia/acetonitrilegradient at pH 8.6 as indicated; they were detected by their absorbance at 214 nm (-). 32Pradioactivity (----) was determined in aliquots of each fraction (see Methods)

dithiothreitol-adduct of dehydroalanine is found in cycle 10 (Fig. 4B). The second phosphoserine residue present in this peptide according to amino acid composition (Table 1) could not be localized. Most probably, it is present C-terminally to the asparagine-glycine bond mentioned above. Following S-ethyl-cysteine modification, partial phosphorylation of a particular serine residue can also be shown. An example is demonstrated in Fig. 5. In this instance, a mixture of peaks I and I1 was analyzed. In cycles 8, 10 and 13 all three phenylthiohydantoin derivatives were observed, namely those of S-ethyl-cysteine, dithiothreitol-adduct of dehydroalanine and serine. It shows that the serine residues in positions 1018,1020 and 1023 are partially phosphorylated. This mixture was analyzed due to an incomplete separation of peaks I and I1 which can be seen in Fig. 2. Furthermore, this result demonstrates the presence of differently phosphorylated species of a subunit which show up as partially phosphorylated sites in these three positions (Fig. 5). The peptides K,55, peak 111 and peak IV, are not phosphorylated at these three serine positions (not shown); the position of the phosphoserine residues must be located further downstream which could not be identified due to the reasons given above. Following self-phosphorylation, essentially the same strategy is employed to isolate the labelled a subunit which is digested with trypsin or Lys-C specific protease. As an example, Fig. 6 shows the separation of radioactively labelled Lys-C peptides. Two major radioactively labelled peptides are visible. The major Lys-C peptide, K,52, contains 1.9 mol [32P]phosphate/mol radioactively labelled peptide (Table 1) and includes the sequence of the tryptic peptide T,100/101 (not shown). Sequence analysis of this Lys-C peptide was carried out with and without modification of phosphoserine to S-ethyl-cysteine. Following modification, this phenylthiohydantoin derivative was identified in cycles 7 and 20 (Fig. 7). Even though the amount of S-ethyl-cysteine is very low in cycle 20, it clearly indicates the presence of phosphoserine at this position. Calculation of the repetitive

yield (Fig. 7B) reveals that the amount determined in this cycle is exactly within the range expected from the amount determined in the previous and following cycle. The location of these two phosphoserine residues was confirmed by analyzing the unmodified peptide: in cycles 7 and 20 the dithiothreitol adduct of dehydroalanine can be detected exclusively (data not shown). The identification of two phosphoserine residues is in accordance with the stoichiometry of incorporated radioactive phosphate (compare Table 1). Thus, the serine residues 972 and 985 are the two sites phosphorylated during self-phosphorylation. The minor radioactively labelled Lys-C peptide (Fig. 6) was lost during attempts at further purification. However, from the tryptic digest the peptide T,103/104 can be isolated in homogeneous form and is probably part of the minor Lys-C peptide (see above). During Edman degradation the dithiothreitol adduct of dehydroalanine is found exclusively at position 8 (Fig. 8) which identifies the amino acid 1007 as phosphoserine in the whole sequence. Indeed, this phosphoserine location is in accordance with the finding that trypsin does not split the lysine-phosphoserine bond between amino acids 1006 and 1007. In addition, serine 1042 has been found to be phosphorylated after self-phosphorylation and isolation of a chymotryptic peptide starting with amino acid 1036. However, it was not possible to decide whether this site is already phosphorylated endogenously prior to self-phosphorylation. This serine is located in the Lys-C peptide K,55 downstream to the endogenous sites identified (data not shown).

DISCUSSION By employing the S-ethyl-cysteine method a content of 2.6 mol phosphoserine/mol CI subunit has been determined. This shows that all of the intrinsic phosphate is present as phosphoserine, exclusively. This is in accordance with the

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Fig. I.Sequence analysis of thephosphopeptide K J 2 isolatedfollowing self-phosphorylation. (A) Approximately 100 pmol of the phosphopeptide K,52 was modified and applied onto the gas-phase sequencer. Sequence analysis was carried out as described in Methods; the phenylthiohydantoin chromatograms of cycles 6 - 21 are shown. S-ethyl-cysteine is the newly identified amino acid in cycle I and cycle 20. (D-S = dithiothreitol adduct of dehydroalanine, DMPTU = N-dimethyl-N'-phenylthiourea,DPTU = N,N'-diphenylthiourea, S-Et-Cys = S-ethyl-cysteine). (B) Yields of amino acids identified in each cycle are plotted on a logarithmic scale versus cycle number. The peptide contains two serines and two phosphoserines. (S-Et-Cys = S-ethyl-cysteine)

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Fig. 8. Sequence analysis of the 32P-labelled phosphopeptide T,103/ 104. 8 5 pmol of the purified peptide containing about 1.2 mol 32P phosphate/mol peptide was sequenced with the gas-phase sequencer (see Methods). The phenylthiohydantoin chromatograms of degradation steps 6-11 are shown. (D-S = dithiothreitol adduct of dehydroalanine)

31P-NMR data showing that the endogenous phosphate is phosphoserine and not phosphothreonine [2]. Such a good agreement between phosphate analysis, as determined by ashing, and phosphoserine content, determined as S-ethylcysteine, was also obtained with the troponin subunits I and T [22]. Cysteine, which potentially can eliminate hydrogen sulfide to form dehydroalanine also, does not seem to interfere when the holoproteins are used in this method. However, in some isolated peptides we found hydrogen sulfide elimination and therefore S-ethyl-cysteine formation. For example, cysteine 98 of troponin I does not react if holotroponin is employed for determination of phosphoserine. In an identical manner, this cysteine residue does not react when present in

a Lys-C peptide of 36 amino acids. However, it undergoes pelimination of hydrogen sulfide if it is present in a V8 peptide consisting of 10 amino acids. In contrast, cysteine 81 of troponin I never reacts either when present in holotroponin or in an isolated peptide (Swiderek and Heilmeyer, unpublished results). Probably, the secondary structure of a peptide can influence the reactivity of a particular cysteine residue. Therefore, the S-ethyl-cysteine content of a particular peptide can not be the only criterion for the presence of phosphoserine. In the Lys-C digest of the tl subunit many peptides were found to form S-ethyl-cysteine, some probably being due also to p-elimination of cysteine. The peptide K,55 does not contain cysteine, thus the amount of S-ethyl-cysteine in this instance represents the amount of phosphoserine. One site clearly containing phosphoserine in the enzyme as isolated is the site known to be phosphorylated by the cyclic-AMP-dependent protein kinase [I I]. Other endogenously present phosphoserine residues are seen to be localized downstream. Two serine residues, 1020 and 1023, assigned as phosphorylation sites, are found to be phosphorylated partially. At least one additional serine must be phosphorylated intrinsically in this polyphosphorylation domain since the peptides K,55 I11 and IV contain 1 mol phosphoserine/mol peptide which has not yet been localized. The elution pattern of the phosphoserine-containing peptides seen in the HPLC chromatogram suggests a distinct arrangement of phosphate groups in K,55 (compare Fig. 2): the peptide eluting as peak I contains serine 1020 and 1023 unphosphorylated but serine 1018 and a serine further downstream in the phosphorylated form. Vice versa, the peptide eluting as peak TI contains serine 1020 and 1023 phosphorylated but serine 1018 and the unknown serine further downstream unphosphorylated. It may be that phosphorylation of one site influences (i.e. promotes or inhibits) the phosphorylation of neighboring sites which could produce such a pattern. Two arginine residues are located two residues upstream from serine 1018 which specifies this serine as a substrate for cyclic-AMP-dependent protein kinase. Serine 1020 has recently been shown to become phosphorylated by 5' AMPactivated protein kinase [23]. Serine 1023 is phosphorylated by a protein kinase of unknown specificity but is situated in a position which resembles sites phosphorylated by myosin light-chain kinase. This protein kinase requires basic amino acids preceding the phosphorylatable serine in upstream positions of 6-8 and 10- 13 amino acids [24]. Self-phosphorylation under a variety of conditions yields variable amounts of phosphate incorporated into the CI subunit [I]. Here, conditions were employed which result in stimulation of the partial activity A l , probably the most relevant activity of phosphorylase kinase during muscle contraction [20]. If self-phosphorylation occurs in vivo, sites phosphorylated upon activation of Al should be identical to endogenous phosphorylation sites. The serine residues 972, 985 and 1007, identified as self-phosphorylation sites in the CI subunit, are all located in the same region as the endogenously phosphorylated serine residues. These sites are unlikely to be phosphorylated by the cyclic-AMP-dependent protein kinase. For all three of these serines, a positively charged residue is found two or three amino acids downstream so that their environment resembles the sites phosphorylated in glycogen phosphorylase h and glycogen synthase by phosphorylase kinase [l]. Therefore, they are most probably phosphorylated by phosphorylase kinase itself, but the function of their phosphorylation remains to be determined. However, they are not identical to those sites found to be phosphorylated endoge-

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Fig. 9. Distribution ofphosphorylation andputative calmodulin-binding sites in the domain structure of the CI and p subunits. The domain structures of the homologous CI and p subunits designated A - F are shown (hatched bars represent the a and p subunit, respectively). The phosphorylation sites in the subunit are located in domain A and at the C-terminal ends of domains B and F [21]. All the phosphorylation sites in CI are located in a ‘multiphosphorykation loop’ (domain E). Phosphalc groups are designated by the letter P, phosphorylation sites which can be phosphorylated by thc cyclic-AMP-dependent protein kinase are marked with , the putative calmodulin-binding sites are marked by filled bars. Peptides describcd in Results and Discussion are marked by a linc above or below the outlined primary structures and labelled according to the footnote on the first page. The first amino acid of each peptide is numbered according to its position in the known primary structures

+

nouslv and therefore, it seems questionable whether they are phosphorylated in vivo by phosphorylase kinase . All phosphorylation sites identified in the a subunit so far, whether phosphorylated by self-phosphorylation or endogenously, are clustered in a region of only 80 amino acids. This stretch contains a large number of glycine and proline residues, a prerequisite for a flexible structure. Therefore, we propose to call this domain a ‘multiphosphorylation loop’. It directly precedes a predicted calmodulin-binding site [9,25] exhibiting a more pronounced basic character than the homologous domain in the p subunit (see Fig. 9). It is tempting to speculate that phosphate incorporation into the ‘multiphosphorylation loop’ may create a pseudo-calmodulin site. The ‘multiphosphorylation loop’ in a may mimic the effect of Ca2+-calmodulin. Upon phosphorylation this domain may bind to a neighboring putative calmodulin-binding site by direct intramolecular interaction. It may cause a further activation of the enzymic activity upon a-subunit phosphorylation which is otherwise correlated with 0-phosphorylation. We are grateful to Dr Magdolna Varsanyi for preparing phosphorylase kinase. We thank Edcltraut Hoffmann-Posorske, Horst Korte and Ullrich Siemcn for their expert technical assistance. This work was supported by grants from Der Minister fur Wissenschaft und Forschung des Landes Nordrhein- Westfalen, Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie.

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