autophosphorylation of the catalytic subunit of camp-dependent

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 267, No. 35, Issue of December 15, pp. 25174-25180,1992 Printed in U.S.A.

Autophosphorylation of the Catalytic Subunit of CAMP-dependent Protein Kinase*

(Received for publication, May 4, 1992)

Jean Toner-Webb$$, Scott M. van PattenT, Dona1 A. Walshq, and Susan S. TaylorSII From the $Department of Chemistry, University of California, San Diego, La Jolh, California 92093-0654 and the llDepartment of Biological Chemistry, School of Medicine, University of California, Davis, California 95616

The catalytic subunit of CAMP-dependent protein kinase contains two stable phosphorylation sites, Thr- 197 and Ser-338 (Shoji, S., Titani, K., Demaille, J. G., and Fischer, E. H. (1979) J. Biol. Chern. 254, 6211- 6214). Thr-197 is very resistant to dephosphorylation and thus cannot typically be autophosphorylated in vitro once the stable subunit is formed. Ser-338 is slowly dephosphorylated and can be rephosphorylated autocatalytically. In addition to these two stable phosphorylation sites, a new site of autophosphorylation, Ser-10, was identified. Phosphorylation at Ser-10 does not have a major effect on activity, and phosphates from Ser-10 or Ser-338 are not transferred to physiological substrates such as the type I1 regulatory subunit. Autophosphorylation at Ser-10 is associated with one of the two major isoelectric variants of the catalytic subunit. The form having the more acidic PI can be autophosphorylated at Ser-10 while the more basic form of the catalytic subunit cannot. Phosphorylation at Ser-10 does not account for the two isoenzyme forms. Since the reason for two isoelectric variants of the catalytic subunit is still unknown, it is not possible to provide a structural basis for the difference in accessibility of Ser- 10 to phosphorylation. Either Ser- 10 is not accessible in the more basic form of the catalytic subunit or some other type of post- or cotranslational modification causes Ser-10 to be a poor substrate. Whether the myristoyl group at the amino-terminal Gly is important for Ser-10 autophosphorylation re- mains to be established. The isoenzyme forms of the catalytic subunit do not correspond to the gene products coded for by the C, and CB genes.

Many protein kinases are phosphoproteins, and CAMP- dependent protein kinase is no exception. The major, rapid and reversible phosphorylation associated with CAMP-dependent protein kinase is the autophosphorylation of the type I1 regulatory subunit (R"-subunit)' at Ser-95 (1, 2). Phos-

* This research was supported in part by National Institutes of Health Research Grant GM-19301 (to S. S. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertise- ment" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

CA 92714. § Current address: Chiron Opthalmics, 9342 Jeronimo Rd., Imine,

I( To whom correspondence and reprint requests should be ad- dressed Department of Chemistry 0654, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0654. Tel.: 619-534-3677; Fax: 619-534-8193.

The abbreviations used are: R-, R1-, R"-subunits, regulatory, type I, and type I1 subunits; C-subunit, catalytic subunit; HPLC, high performance liquid chromatography; CL-CM, cross-linked carboxymethyl; PAGE, polyacrylamide gel electrophoresis; TPCK, L-l-tosylamido-2-phenylethyl chloromethyl ketone; MES, 4-morpholineetha- nesulfonic acid.

phorylation of the R"-subunit can occur as an intramolecular event that does not require holoenzyme dissociation (3). Thus, this segment of the regulatory subunit occupies the peptide binding site of the catalytic subunit (C-subunit) when the regulatory and catalytic subunits are associated in the holoenzyme complex. The only known consequence of this phosphorylation is a 10-fold decrease in affinity of the R"-subunit for the C-subunit (4, 5), a difference which could be quite significant in vivo (6). The type I regulatory subunit (RI- subunit) is not autophosphorylated readily. The homologous segment of the R'-subunit contains a pseudophosphorylation site, Ala-97 (7). The R'-subunit can in fact be phosphorylated at Ser-99, but the time course of this phosphorylation is very slow relative to the autophosphorylation of the R"-subunit.' This phosphorylation at Ser-99 is catalyzed more readily by cGMP-dependent protein kinase (8, 9). The R"-subunit also can be phosphorylated in vitro at Ser-74 and Ser-76 by casein kinase I1 (10, 11) and at Ser-44 and Ser-47 by glycogen synthase kinase 3 (11). The physiological consequences of these phosphorylations are not known, although recent evidence showing that this region of the R-subunit is important for recognizing other proteins, such as MAP2 and p150 in brain, makes it likely that these phosphorylations may have physiological relevance (12, 13).

The catalytic subunit of CAMP-dependent protein kinase also is phosphorylated. Peters et al. (14) and Bechtel et al. (15) first reported acid-stable protein-bound phosphates in the catalytic subunit, and these sites were identified subsequently as Thr-197 and Ser-338 (16). These sites were termed "silent" phosphorylation sites because neither could be removed readily by phosphatases. The functional consequences of these phosphorylations has never been ascertained. A mu- tation at the residue corresponding to Thr-197 in the yeast C-subunit (TPK1) leads to little loss of activity but an im- paired ability to reassociate with the R-subunit (17).

Kochetkov et al. (18,19) reported that the catalytic subunit purified from type I1 CAMP-dependent protein kinase in porcine heart could be autophosphorylated to a molar ratio of nearly 1.0 mol of P04/mol of C-subunit. The phosphate- enzyme linkage was identified as an N-phosphohistidine, and the phosphorylated C-subunit was predicted to be an intermediate in the phosphotransferase reaction, since the enzyme- bound phosphate could be transferred to histone. Ho et al. (20), however, subsequently demonstrated that the reaction proceeds by a direct in-line transfer of the yPO, of ATP to the peptide with an inversion of configuration. Chiu and Tao (21) reported a low level autophosphorylation (3-10%) for the rabbit skeletal muscle C-subunit in which the enzyme-linked phosphate appeared to be on serine or threonine. In this case the phosphate did not appear to be involved in catalysis.

J. A. Toner-Webb and S. S. Taylor, unpublished results.

25174

CAMP-dependent Protein Kinase: Autophosphorylation of C-subunit 25175

The autophosphorylation of the porcine heart catalytic subunit is described here. The following questions are ad- dressed 1) What is the relationship of autophosphorylation to activity and to the reassociation of the catalytic subunit with the R"-subunit? 2 ) Is the phosphoamino acid an intermediate in catalysis? 3) What specific residues are autophosphorylated in vitro? In addition, differences between the two isoelectric variant forms of the C-subunit are described.

EXPERIMENTAL PROCEDURES

Reagents-Reagents were purchased form the following sources: histone (IIA), Kemptide, and cross-linked carboxymethyl-Sepharose (CM Sepharose CL-GB), Sigma; [Y-~*P]ATP (15-30 Ci/mmol), Amer- sham Corp.; HPLC grade trifluoroacetic acid, Pierce Chemical Co.; acetonitrile, Fisher; L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (TPCK-trypsin), U.S. Biochemical Corp.; What- man 3 and P-81 paper, Whatman; Cytoscint and Betaphase, West Chem; and synthetic peptide was synthesized by the UCSD Peptide and Oligonucleotide Synthesis Facility.

Protein Purification-The R"- and C-subunits of type I1 CAMP- dependent protein kinase were purified from frozen porcine cardiac tissue as described previously (22-24) with minor modifications. The final step in the purification of the catalytic subunit is elution from CL-CM Sepharose (5 ml/kg starting tissue). When purified isoelectric variants of C-subunit were required, the final elution from CL-CM Sepharose was modified by using a more shallow gradient from 20 to 180 potassium phosphate (15 ml of total gradient/ml of CL-CM Sepharose). The purified catalytic subunit showed a single band on SDS-PAGE following polyacrylamide gel electrophoresis.The R'-subunit was purified as described previously (25), and holoenzyme was prepared according to Bubis and Taylor (26). The heat stable protein kinase inhibitor was prepared according to Van Patten et al. (27).

Assays-Three methods were used to assay enzymatic activity. 1) [y-32P]ATP-phosphorylated histones were precipitated with perchlo- ric acid onto Whatman 3 filter discs as described previously (28). 2) Phosphorylated histones were directly spotted onto P81 paper and washed with phosphoric acid according to the method of Witt and Roskoski (29). In both cases the washed and dried discs were counted in Cytoscint. 3) The integrity of holoenzyme was determined by assaying for activation in the presence and absence of CAMP according to the coupled spectrophotometric method of Cook et al. (30) using the synthetic peptide substrate, L-R-R-W-S-L-G (31) or Kemp- tide, L-R-R-A-S-L-G. Protein concentration was determined as described by Bradford (31).

Phosphorylation of the Catalytic Subunit with [-p3'P/ATP-The reaction buffer was 25 mM MES, pH 6.3, 5% glycerol, 3 mM mercaptoethanol. The catalytic subunit (1.2 p ~ ; 50 pg/ml) was incubated with [T-~*P]ATP (100 /IM) and 500 /IM M F at 37 "C for the indicated times. Proteins were immediately denatured after the reaction with either SDS-gel buffer and subjected to SDS-PAGE or with 6 M guanidine-HCI followed by exhaustive dialysis and treatment with TPCK-trypsin. Autophosphorylation of the C-subunit is inhibited by greater than 90% when the reaction is carried out in the presence of 6 p M protein kinase inhibitor.

Proteolysis-Proteins were incubated at 37 "C with 1: lOO w/w TPCK-trypsin in 25 mM ammonium bicarbonate, pH 8.3, for 4 h. Incubation was continued overnight following the addition of another equal aliquot of trypsin. Tryptic peptides were resolved by HPLC. Samples were lyophilized to 2 ml when volume reduction was necessary.

HPLC-Reverse phase HPLC was performed using an Altex 3200 system with a Vydac C18-column (4.6 mm inner diameter X 25 cm, 5 pm, 300 A pore size). The gradients employed were (a ) 10 mM sodium phosphate, pH 6.8-6.9, to CH3CN (Fisher, HPLC grade), or (b) from 0.1% trifluoroacetic acid, pH 2.1 (Pierce), to CH,CN. All buffers were filtered and degassed. The majority of peptides were eluted with a 180-min linear gradient from 0 to 60% CH3CN a t a flow rate of 1 ml/ min. Absorbance was monitored at 219 nm using a Hitachi spectrophotometer equipped with a flow-through cell or at 280 nm using a Kratos spectrophotometer with a flow-through cell. Recovery of peptides was typically approximately 80%.

Peptide Sequencing-Amino-terminal residues were identified by manual sequencing using the Dansyl-Edman procedure according to the method of Hartley (32). Solid phase sequencing was carried out using a sequencer designed according to Doolittle et al. (33). Peptides were coupled to derivatized glass beads as described earlier (26).

Amino acids were converted to phenylthiohydantoin-derivatives by incubation with rnethanolic HCl (1.5 N) for 10 min at 80 "C, evapo- rated to dryness, dissolved in acetonitrile containing a norvaline phenylthiohydantoin standard, and identified by HPLC. Gas phase sequencing was carried out using an Applied Biosystems model 470 A protein sequenator. Phenylthiohydantoin amino acids were identified by HPLC, as described by Hunkapillar and Hood (34), on an IBM cyano column.

SDS-PAGE-SDS-PAGE was carried out according to Laemmli (35) utilizing 10% or 12.5% acrylamide in the lower gels. Proteins were stained overnight with 0.25% Coomassie Blue R-250, 25% iso- propyl alcohol, 10% acetic acid, and destained in the same solution without Coomassie dye. When autoradiography was necessary, the dried gels were exposed on Kodak X-Omat film.

Separation and Identification of C, and CB by Discontinuow Non- denaturing Gel Electrophoresis-C-subunit mixtures were incubated with protein kinase inhibitor in the presence of MgATP and were separated by non-denaturing gel electrophoresis according to Van Patten et al. (27). In some cases a 1-mm slab gel was run instead of 5-mm tube gels. Electrophoresis was performed at constant voltage (70 V) until the dye front reached the end of the gel. Gels were stained overnight and destained as mentioned above.

Separation of C, and CB by Isoelectric Focusing Column-Isoelectric focusing was performed with a linear glycerol gradient from pH 3 to 10 with 1% Bio-Rad ampholytes, a 110-ml LKB 8101 isoelectric focusing column, using the procedure of Russell et al. (36). Samples were pooled to obtain homogeneous CA and CB, and these samples were used for comparative autophosphorylation studies.

Amino Acid Analysis-Lyophilized peptides were hydrolyzed in Vacuo at 100 "C in 6 N HCl for 20 h. Analyses were performed on a LKB 4400 automatic acid analyzer using a single column system.

Radioactiuity-Radioactivity was measured by counting aliquots in Cytoscint (up to 0.2-ml sample) or in Betaphase (up to 0.5-ml sample). Radioactive bands from gels were excised and counted both dry (37) and after adding Cytoscint.

RESULTS

Autophosphotylation of CAMP-dependent Protein Kinase Catalytic Subunit-The time course for autophosphorylation of C-subunit in the presence of [y3'P]ATP is shown in Fig. 1. In order to determine stoichiometry, samples were subjected to SDS-PAGE and gel bands were excised and counted as described under "Experimental Procedures." On this basis, the stoichiometry of covalent 32P incorporation was determined to be 1.1 mol of 32P/mol of C-subunit. Stoichiometry also could be established by comparing the autophosphorylation of the R"-subunit with that of the catalytic subunit (Fig. 1, last lane). The stoichiometry for R" autophosphorylation is 1.0 mol of 32P/mol of R" monomer, and phosphorylation is exclusively at Ser-95 (9). The incorporation of 32P into the C- subunit, relative to the R"-subunit was calculated to be 1.2 mol/mol of C-subunit. A final method for quantitating covalent 32P incorporation involved denaturing the autophosphorylated C-subunit with guanidine-HC1, followed by extensive dialysis. This method also showed 1.2 mol of 32P incorporated per mol of C-subunit.

Localization of the Autophosphorylation Site-In order to localize the autophosphorylation site(s), the C-subunit was autophosphorylated with [ Y - ~ ~ P I M ~ A T P , digested with TPCK-trypsin, and the resultant tryptic peptides resolved by HPLC (pH 2.1). As indicated in Fig. 2, one major peptide, as well as several smaller peaks, were radiolabeled. The major radiolabeled fraction was rechromatographed on the same HPLC column using an alternate solvent system at pH 7.0 as shown in the insert of Fig. 2. This yielded a pure peptide which had an NH2-terminal lysine and had the amino acid composition summarized in Table I. When the peptide was immobilized on glass beads and sequenced, the radioactivity was associated with step 3. The sequence of the major autophosphorylated peptide is shown in Table I along with a summary of the supporting data discussed above. The auto-

25176 CAMP-dependent Protein Kinase: Autophosphotylation of C-subunit

phosphorylation site associated with the major peak was thus unequivocally identified as Ser-10 in the linear sequence of the C-subunit by analogy with the known sequence of bovine C-subunit (38). The identification of the minor radioactive peaks will be discussed later.

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

TIME (rnln)

B

PROTEIN

"R

"c

"R RADIOACTIVIT I --- -C

5 IO 15 2025 3035 4060 INCUBATION TIME (min)

R added at 50' _t FIG. 1. Time course and s toichiometry of autophosphory-

lation of the C-subunit. C-subunit phosphorylation was carried out at 37 "C as described under "Experimental Procedures." Aliquots were removed at the times indicated and subjected to SDS-PAGE. A, the stoichiometry of "'P incorporation was determined by excising and counting the gel bands. R, staining (upper panel) and autoradiography (lower panel) were performed as indicated under "Experi- mental Procedures."

Potential Phosphoryl Transfer from Phosphoenzyme to the Regulatory Subunit and Histone-Kochetkov et al. (18) reported the presence of a phosphoenzyme intermediate in porcine brain CAMP-dependent protein kinase-catalyzed re- actions, while the results of Chiu and Tao (21) implied that the rabbit skeletal muscle protein kinase did not exhibit such an intermediate. Consequently, it was important to establish: 1) whether the autophosphorylated C-subunit was catalyti- cally active; 2) whether it could serve as an intermediate in the phosphotransferase reaction; and 3) whether the 32P- labeled C-subunit was capable of transferring this particular phosphate group on Ser-10 to an appropriate substrate mol- ecule.

The effect of autophosphorylation on catalytic activity was first characterized using histones as a substrate. When the C- subunit was preincubated with either unlabeled MgATP or [r-"P]MgATP and then assayed using histone as a substrate, autophosphorylation appeared to have little effect on enzymatic activity (data not shown). Similar results were obtained using peptide substrate and the spectrophotometric assay. The phosphorylated C-subunit also formed holoenzyme that was fully dependent on CAMP for activation. In order to establish definitely whether the autophosphorylated C-subunit is capable of transferring its phosphate group to R- subunit, the C-subunit was autophosphorylated with [-y-"P] MgATP, and the reaction was terminated by adding EDTA prior to the addition of the R"-subunit. Under these conditions, the 32P-labeled C-subunit is incapable of transferring its phosphate to R" (Fig. 3). To eliminate the possibility that M e might be required for phosphate transfer, a mixture of hexokinase and &D-glUCOSe was added following autophosphorylation of the C-subunit in order to remove any residual ATP prior to addition of R". The gels shown in Fig. 4 demonstrate that, even in the presence of M$+, the autophosphorylated C-subunit cannot transfer its phosphate to the R"-subunit. Fig. 4, lane 2, indicates extensive autophosphorylation of the C-subunit as determined qualitatively by comparison to phosphorylation of the R"-subunit. Lane 3 demonstrates clearly that the R"-subunit cannot be phosphorylated under these conditions when ATP is removed by hexokinase. The same R"-subunit can be phosphorylated if

FRACTION NUMBER

FIG. 2. H P L C separation of the tryptic peptides f r o m the autophosphorylated C-subunit. The [y-32P]MgATP-modified C-subunit was denatured with 6 M guanidine-HC1 and extensively dialyzed. Following digestion with TPCK-trypsin at pH 8.3, the resulting peptide were separated by HPLC using a gradient of 0-60% solvent B in 180 min where solvent A was 0.1% trifluoroacetic acid (pH 2.11) and solvent B was CH3CN. Radioactivity was determined by counting 100-pl aliquots of fractions containing 1 ml total. Solid line, absorbance a t 210 nm; dashed line, radioactivity (32P cpm). Arrow indicates the major peak of radioactivity. The inset shows the purification of the major autophosphorylated tryptic peptide of C-subunit by HPLC in a second solvent system. The major 32P-labeled peptide from the HPLC was rechromatographed using HPLC with a gradient from 0.01 N sodium phosphate (pH 6.9) to 30% CH&N in 90 min. Top, radioactivity (32P cpm); bottom, absorbance a t 219 nm.


TABLE I Sequence analysis of the major autophosphorylated peptide of C-subunit

1.8 (21, Glv 3.3 (4). Glv 1.1 (1). Ala 0.7 (1). Val 0.8 (1). Leu 0.9 (1). Phe 1.0 (1). Lys 3.0 (3). The composition of the peptide, based on duplicate analyses, was as follows with the theoretical composition shown in parentheses: Ser

Residual No.

1 2 3 4 5 6 I a 9 10 11 12 13 14

Gas phase sequencing" Lys Gly X Glu Gln Glu Ser Val Lys Glu Phe Leu Ala Lys Solid phase sequencing Gly X Glu Gln Glu X Val 'lP radioactivity (cpm)' 27 26 225 50 24 20 18 18 15 13 12 12 10 12 NH, terminal' Lvs ' Determined from triplicate experiments.

Determined from duplicate experiments.

Protein Autorad.

R-

C-

Preincubation with MgATP

FIG. 3. Inability of the autophosphorylated C-subunit to transfer its phosphate to R" in the presence of EDTA. C- subunit was incubated in the presence and absence of [-y-3'P]MgATP for 30 min a t 37 "C according to the conditions described in Fig. 1. EDTA was added to the incubation a t 20 min to remove M e . After 30-min samples were brought to 0 "C and a molar equivalent (1.2 PM) of R"-subunit added for 5 min. Samples (50 PI) were then boiled in SDS-gel buffer and subjected to SDS-PAGE.

1 2 3 1 2 3 - Protein Radioactivity

7 p""" TI

4

Preincubation - + + - + + with ATP

Hexokinase - - + - - + FIG. 4. Inability of the C-subunit to transfer its phosphate

to R". C-subunit (3.5 PM) was incubated a t 37 "C for 30 min in 25 mM MES, pH 6.3, 10 pM CAMP, 1 mM MgC12, 5% glycerol, 5 mM 2- mercaptoethanol with or without [32P]ATP (150 PM). A hexokinase, fi-D-ghICOSe mixture was added where indicated to remove excess ATP. R" (1.7 PM) was then added to incubations for 2 min. In the case of lane 1, ["'PIATP (150 PM) was added at the same time as the R"-subunit, since this sample had not been preincubated with ATP. Left panels are protein gels; right panels are autoradiograms.

ATP alone is added back into the reaction mixture, demon- strating that there was sufficient Mg2+ present for the transfer to occur (data not shown). Thus, the fully active autophosphorylated C-subunit is not capable of directly transferring its covalently bound ["PI to the RE'-subunit. Identical results were obtained using histone as substrate (data not shown).

Comparative Autophosphorylation of C, and CB-The extent of autophosphorylation varied between preparations of the C- subunit. In some cases, there was very little phosphate incorporated in contrast to autophosphorylation in amounts in excess of a 1:l stoichiometry as just described. One explanation for this variability could be microheterogeneity within

FREE I -

FIG. 5. Variability in the composition of the C-subunit from two heart preparations. A non-denaturing polyacrylamide gel electrophoresis system (27) was used to distinguish the CA and CB forms of the C-subunit. Heart preparation 46 ( h p 46) contained 30% CA and 70% Ce, whereas heart preparation 58 ( h p 58) contained only CB. Inhibitor protein catalytic subunit complexes, CA:I or CB:I; free inhibitor protein, I.

the C-subunit preparation. Heterogeneity was, in fact, demonstrated in the initial characterization of C-subunit where isoelectric variants were observed (14). The reason for the isoelectric variants of the C-subunit has never been established, but it is likely that the differences are due to post- or cotranslational modifications either in vivo or generated in vitro during purification. Van Patten et al. (27) described functional differences in the capacity of the isoelectric variant forms of the C-subunit to complex with the heat stable protein kinase inhibitor and developed a non-denaturing gel electro- phoretic method for distinguishing the two forms of the C- subunit, CA and C g . This method was used to characterize our preparation of the C-subunit in order determine whether the autophosphorylation was preferentially associated with one form of the C-subunit. Initially two samples of C-subunit from two different preparations were run on this non-denaturing gel system. The sample in the left lane of Fig. 5 represents a preparation of C-subunit which showed autophosphorylation to a stoichiometry of 0.25 mol of 32P incorporated per mol of C-subunit, while the C-subunit in the right lane represents another preparation of C-subunit which showed negligible autophosphorylation. This suggests that autophosphorylation may be preferentially associated with CA, which was enriched in the sample that showed good phosphorylation. CA has the lower isoelectric point and runs faster when it is complexed with protein kinase inhibitor in the non-denaturing gel system described above (27). The potential difference in the autophosphorylation of CA and Cg

25178 CAMP-dependent Protein Kinase: Autophosphorylation of C-subunit

was characterized further. The final step in the purification of C-subunit is elution from CL-CM Sepharose. Our routine procedure utilizes a linear gradient of 17-200 mM potassium phosphate, and fractions containing pure C-subunit based on SDS-PAGE are typically pooled (Fig. 6B) . In many cases a high molecular weight contaminating protein elutes from this column just prior to the C-subunit; therefore, the leading edge of the peak containing C-subunit was frequently discarded. Fractions from various regions of this normal elution profile of C-subunit were characterized for their contents of CA and CB with the results shown in Fig. 6A. It can be seen that homogeneous CB is easily obtained by this gradient, but that C A usually contains some CB as well. Therefore, preparations of pure CA or CB were isolated by elution from the CL-CM Sepharose column with a more shallow gradient: from 20 to 180 mM of potassium phosphate and one and a half times the volume shown in Fig. 6. Fractions of nearly homogeneous CA or CB, based on the non-denaturing gel electrophoresis system described in Fig. 5 were then pooled. CA is the C-subunit eluting first from the CL-CM Sepharose column and represents approximately 30% of the total C-subunit in porcine heart.

The extent of autophosphorylation of CA was always much greater than CB as shown by SDS-gels in Fig. 7. These samples were characterized further by comparing HPLC profiles of tryptic peptides (Fig. 8). The level of phosphorylation of CA was somewhat less than what was shown earlier in Fig. 2, and the HPLC solvent systems used to resolve the tryptic peptides

FREE I -C

FRACTION NUMBER

FIG. 6. A, fractions were examined for their CA or CB content using the non-denaturing polyacrylamide gel electrophoresis system described under “Experimental Procedures.” Fraction numbers are indicated below each gel lane and symbols are as in B. B, isolation of the C-subunit on CL-CM Sepharose. The purification of C-subunit is described under “Experimental Procedures.” The C-subunit was eluted from the CL-CM Sepharose with a linear potassium phosphate gradient (17-200 mM) and absorbance of each fraction was measured at 280 nm.

‘8 ‘A ‘B ‘A

FIG. 7. Autophosphorylation of CA versus CB. C-subunit was separated into CA and CB isoelectric variants as discussed under “Experimental Procedures.” CA or CB (1.0 pM) was incubated at 37 “C with [-p3’P]ATP (100 PM) and Mg(0Ac)z (1.0 mM) in 25 mM MES, pH 6.1, 5% glycerol, 3 mM 2-mercaptoethanol. SDS-gel buffer was added at 60 min, and the samples were boiled and run by SDS-PAGE. The panel on the left is a Coomassie-stained gel: the panel on the

”

rightis an autoradiogram.

FKGPGDTSNFDDYEEEEIRVSINEK

400

a 200 -

0 800- LL I , KGSEOESVKEFLAK

20 40 60 80 100 120

TIME (mln)

FIG. 8. High performance liquid chromatography separation of TPCK-trypsin digest of autophosphorylated Ca and CB. The buffers employed were ( a ) 0.01 N sodium phosphate (pH 6.0) and (b) CH3CN. The tryptic peptide were eluted with a 180-min linear gradient from 0% to 60%. Top panel, absorbance at 219 nm; middle panel, radioactive profile for CB; bottom panel, radioactive profile for CA. 32P-Autophosphorylated residues are indicated by a star in the middle and lower panels. The upper panel also indicates the peptide containing the silent phosphorylation site, Thr-197. All three peptides were sequenced in their entirety on the Applied Biosystems sequenator.

differs in Figs. 2 and 8. However, in each case, for Fig. 2 and for CA in Fig. 8, three main peaks of radioactivity were seen, with the peptide containing Ser-10 having the highest radioactivity in both cases. The other peaks correspond to the tryptic peptides containing Ser-338. Duplicate experiments carried out under the conditions described in Fig. 8 also showed variability in the height of these other peaks (data not shown). The variability in the phosphorylation of this peptide is due presumably to variability in the initial phosphate content in the isolated sample. In each case, the trailing edge of CA and CB, after elution from CM Sepharose, showed greater phosphorylation of Ser-338. The initial breakthrough peak of radioactivity in Fig. 8 (lower panel) was not associated with peptide based on gas phase sequencing or amino acid


analysis. The only major radioactive peak in the central panel corresponding to tryptic peptides from CB was identified as the tryptic peptide containing Ser-338. The sequence of this peptide was confirmed by gas phase sequencing. While vari- able levels of phosphorylation of Ser-338 were observed for different preparations of both CA and CB, no phosphorylation of Ser-10 was ever observed for CB. The other peptide containing a silent phosphorylation site was not autophosphorylated under these conditions; however, this peptide containing Thr-197 was identified by gas phase sequencing, and its location is indicated in the upper panel of Fig. 8. The absence of radioactivity and the absence of even a trace of threonine at step three in this gas phase sequence supports the conclu- sion that this residue was already fully phosphorylated as isolated.

In order to determine whether CA and CB correspond to the two isoforms of the catalytic subunit coded for by the C, and Cp genes, the peptide containing Ser-10 was isolated from CB. The sequence of this unphosphorylated peptide, shown in Table 11, was identical to the phosphopeptide isolated from C A . The deduced sequences from the bovine, human, and mouse C, and Cp genes are also shown and establish that CA and CB do not correspond to the products of these two genes. Some other explanation must account for their differences.

DISCUSSION

The results described here report a major and previously unrecognized site of autophosphorylation, Ser-10, for the catalytic subunit of CAMP-dependent protein kinase. Phos- phorylation at Ser-10 is associated primarily with the CA form of the C-subunit, the form that has the lowest PI. The rate of autophosphorylation of the C-subunit is slow compared to that of R", implying that the C-subunit autophosphorylation may not be physiologically significant. It is also possible that another kinase is responsible for phosphorylating this site in uiuo. Since Ser-10 is preceded by two lysines and a glycine, it fits the substrate requirements for CAMP-dependent protein kinase. However, many other kinases, such as protein kinase C, myosin light chain kinase, and glycogen synthase kinase share these substrate requirements, and some actually prefer Lys rather than Arg residues before the phosphorylation site (39). Protein kinase C, for example, would be a candidate for phosphorylating this site, if phosphorylation of Ser-10 does in fact occur in uiuo. Whether phosphorylation at Ser-10 occurs physiologically or not, the results described here indicate that this region of the protein differs in its accessibility in the two isoelectric variant forms of the C-subunit.

Isoelectric variants of the C-subunit were reported when the protein was initially purified and characterized (14, 15, 40); however, no functional differences between these variants were identified for many years. van Patten et al. (27) showed subsequently that the CB-protein kinase inhibitor complex formation was markedly stimulated by MgATP, whereas the formation of CA was only slightly affected by MgATP. These

authors suggested that this difference may imply that CA and CB differentiate in their interaction with some substrates. Kinzel et al. (41) also showed differences between the two isoelectric forms of the C-subunit. Specifically, they demonstrated that the CB isoform was degraded more rapidly by the protease that is highly specific for cleaving the active conformation of the C-subunit and other protein kinases.

The studies presented here have shown that CA can be autophosphorylated in vitro to a major extent at Ser-10, whereas CB cannot. The difference in the autophosphorylation potential of CA uersus CB does not necessarily account for their difference in isoelectric points, as CA is both the more negatively charged isoenzyme and the isoenzyme capable of undergoing additional phosphorylation. Also upon autophosphorylation, CA and CB are not interconverted as one might predict, since enhanced phosphorylation of CA would only serve to lower its PI. This study is consistent with the early report of Peters et al. (14), who showed that CA and CB are not interconvertable by treatment with phosphatase. The difference in CA uersus CB which makes Ser-10 accessible in the former protein, and not in the latter, may be due to structural differences elsewhere in the two molecules or to a post- or cotranslational modification other than phosphorylation. For example, methylation of Lys-7 or Lys-8 could destroy the recognition site for Ser-10. The only other known posttranslational covalent modification of the C-subunit, in addition to phosphorylation, is myristoylation at the amino- terminal Gly (42). Obviously, this myristoyl group must lie in relatively close proximity to Ser-10. C A and CB could conceiv- ably differ at the amino terminus in such a way that CA is more accessible for phosphorylation.

Functions for the autophosphorylation at Ser-10, as well as for the silent phosphorylation sites, Thr-197 and Ser-338, are presently unknown. Thr-197 is the most resistant to dephosphorylation and appears to represent an unorthodox phosphorylation as described by Steinberg (43). This type of phosphorylation would very likely occur cotranslationally and would be inaccessible once the protein was folded into its stable active conformation. The crystal structure of the C- subunit confirms that the phosphate moiety of Thr-197 inter- acts with multiple side chains and this presumably accounts for its resistance to proteases (44, 45). These stable phosphorylated residues may have a structural role in stabilizing conformations of the C-subunit as seems likely for Thr-197, or may contribute to interactions with other proteins in uiuo. The results in this study demonstrate that the phosphorylation of Ser-10 does not dramatically affect the activity of the C-subunit using histone, peptide, or R"-subunit as substrate. Also, this phosphorylation does not prevent reassociation of the C-subunit with the R-subunit. The fact that Ser-10 is not rapidly labeled during the reaction between C-subunit and [ T - ~ ~ P I M ~ A T P argues against its role as a phosphoryl intermediate. Consistent with Ho et al. (20), who demonstrated a direct in-line transfer of phosphate from ATP to peptide, the

TABLE I1 Comparatiue sequences from CA and CB versus the gene products of C, and C,

The peptide corresponding to residues 9 through 26 were isolated from CA and CB, sequenced, and shown to be identical except that SerlO was not phosphorylated in the peptide from CB. The predicted sequences corresponding to C, and C, from bovine, mouse, and human are indicated below. All CBs so far investigated have a Val at position 12 while C, has a Gln.

20 CA: Cg: C,: Cg:

25180 CAMP-dependent Protein Kinase: Autophosphorylation of C-subunit

evidence reported here shows that neither Ser-10 nor Ser-338 are involved in phosphate transfer during catalysis.

Now that the gene encoding the catalytic subunit has been isolated and the protein can be over expressed in Escherichia coli (46), genetic analysis of these residues hopefully will resolve the importance of each of the phosphorylation sites for the function and/or structure of the C-subunit.

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