ATP-citrate lyase kinase and cyclic AMP-dependent protein kinase ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Communication

Vol. 256, No. 20, Issue of October 25, pp. 10213-10216, 1981 Pnnted m U.S.A.

ATP-citrate Lyase Kinase and Cyclic AMP-dependent Protein Kinase Phosphorylate Different Sites on ATP-citrate Lyase* (Received for publication, June 2, 1981, and in revised form, August 3, 1981)

Seethala Ramakrishna, Dominick L. Pucci, and William B. Benjamin From the Diabetes Research Laboratory, Departmentof Physiology, Health Sciences Center, State University of New York a t Stony Brook, Stony Brook, New York 11794

ATP-citrate lyase was phosphorylated by highly purified cyclic AMP-independent protein kinase (ATP-citrate lyase kinase) o r the catalytic subunit of cyclic AMP-dependent protein kinase. Each kinase phosphorylated ATP-citrate lyase equally but the combination of both kinases increased ATP-citrate lyase phosphorylation additively. When ATP-citrate lyase was phosphorylated with each kinase and partially digested with either L-1-tosylamido-2-phenylmethylchloromethyl ketone-treated trypsin o r Staphylococcus aureus protease followed by electrophoresisof the proteolytic products on sodium dodecyl sulfate-polyacrylamide gels o r when the phosphorylated lyase was completely digested by these proteases followed by chromatography and electrophoresis, the results showed that the site phosphorylated by ATP-citrate lyase kinase was different from that phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase. Only phosphoserine was found in lyase phosphorylateia by the catalytic subunit of cyclic AMP-dependent protein kinase whereas phosphoserine and phosphothreonine were found in ATP-citrate lyase phosphorylated by lyasekinase.

The binding of insulin to adipocyte plasma membranespecific receptors elicits profound changes in cell metabolism (1). However, molecular events that couple binding to the biological response are unknown (2). Recently it has been found that ATP-citrate lyase in mammalian tissues and cells can be phosphorylated in response to the action of insulin, glucagon, and P-agonist (3-7). These results suggested that there are at least two pathways leading to ATP-citrate lyase phosphorylation in viuo, one pathway responding to insulin action and the otherresponding to agents that raise intracellular cyclic AMP levels (6). Since insulin action stimulates in vivo ATP-citrate lyase phosphorylation (3-7), we suggested (8) that anearly event in the mechanism of insulin action was the stimulation of an insulin-sensitive cyclic AMP-independ* This investigation was supported by Grant AM 18905 from the National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health. A preliminary report of a portion of these findings was presented at the 72nd Annual Meeting of the Federation of American Society of Biological Chemists, Fed. Proc. (1981) 42, 1720 (Abstract No. 1046). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “uduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

ent protein kinase which phosphorylates ATP-citrate lyase. Subsequently we described (9) in liver a cyclic AMP-independent protein kinase (ATP-citrate lyase kinase) which phosphorylates ATP-citrate lyase in vitro to 0.6 mol/subunit. Guy et al. (10) also found in agreement with in vivo results (4,8 ) that the catalytic subunit of cyclic AMP-dependent protein kinase phosphorylates ATP-citrate lyase. Therefore, it was important to determine if ATP-citrate lyase phosphorylates the lyase independently of cyclic AMP-dependent protein kinase and whether the site phosphorylated is separate from that phosphorylated by cyclic AMP-dependent protein kinase. Should the sites be different and the effects of the kinases additive as would bepredicted by in vivo experiments ( 4 7 ) these findings would support our hypothesis that insulin action in vivo increases the phosphorylation of ATP-citrate lyase by activating an insulin-sensitive cyclic AMP-independent protein kinase. Here we present evidence that the lyase kinase-responsive phosphorylation of ATP-citrate lyase is different from the cyclic AMP-dependent protein kinase-responsive system by showing that different sites of the ATP-citrate lyase molecule are phosphorylated in reponse to the two different kinases. We also demonstrated that serine is the only amino acid phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase while interestingly both serine and threonine residues are phosphorylated in response to lyase kinase. EXPERIMENTALPROCEDURES

Muteriuls-ATP-citrate lyase (specific activity, 9-9.5 units/mg of protein) was purified from rat adipose tissue (to be described elsewhere). TPCK-trypsin’ was purchased from Worthington. Staphylococcus aureus protease was obtained from Miles Laboratories. [y-”PI ATP (3000 Ci/mmol) was purchased from Amersham. All other reagents were of highest purity obtainable. Purification of Protein Kinases-ATP-citrate lyase was purified from rat liver as described elsewhere (9).This kinase was substantially purified (1500 times) and was not associated with casein, histone, and phosvitin kinase activities.’ Catalytic subunit was prepared from rabbit skeletal muscle as described by Bechtel et ul. (11) with an additional second gel fdtration step. When ATP-citrate lyase was phosphorylated with either protein kinase, the 115,000-daltonsubunit of the lyase was phosphorylated with only minimal phosphorylation of lower molecular weight proteolytic fragments of the parent molecule (Fig. 1, lane A , without trypsin). Catalytic subunit as previously shown by Guy et al. (10) also phosphorylated ATP-citrate lyase (Fig. 1, lane B, without trypsin). When catalytic subunit was incubated with all components of the kinase assay except for ATP-citrate lyase, the catalytic subunit was minimally autophosphorylated compared to the phosphorylation of ATP-citrate lyase. This autophosphorylation did not render the results confusing (data not shown). Limited Proteolysisand Peptide Mapping-ATP-citrate lyase was phosphorylated as described previously (9) but the reaction was terminated with the addition of sample buffer without 2-mercaptoethanol (final concentrations: 50 mM Tris-HC1, pH 6.8, 10%glycerol, 0.5% SDS, 0.001% bromphenol blue) and heated at 100 “C*for3 min. Proteolysis and peptide mapping on SDS gels was by a modification The abbreviations used are: TPCK-trypsin,~-1-tosylamido-2phenylethyl chloromethyl ketone-treated trypsin; SDS, sodium dodecyl sulfate. Catalytic subunitis the catalytic subunit of cyclic AMPdependent protein kinase. * ATP-citrate lyase kinase appears to be a 47 kilodalton protein by gel filtration, glycerol density gradient centrifugation, and SDS gel electrophoresis and is not autophosphorylated when incubated with ATP.

10213

10214

Different Phosphorylation Sites of ATP-citrate Lyase

"" -. . a m . . . 500

200

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C B A C B A C B A

20

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0

C B A C B A C B A C B A

" "

"

0 . 0

" " "

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FIG. 1. Autoradiogram of phosphorylated ATP-citrate lyase after limited proteolysis. Proteolysis was for 30 min with left, TPCK-trypsin,and right, S. aureus protease as described under "Experimental Procedures." Lanes A, ATP-citrate lyase phosphorylated by lyase kinase (1370 cpm); lanes B, the lyase phosphorylated by catalytic subunit (990 cpm); lanes C, the lyase phosphorylated by both lyase kinase and catalytic subunit (2000 cpm). Only half the of Cleveland et al. (12). TPCK-trypsin or S. aureus protease was added to thesample and proteolytic digestion was carried out a t 37 "C for 30 min. Proteolysis was stopped by the addition of SDS and 2mercaptoethanol to final concentrations of 4 and 2%, respectively, and boiling for 3 min. About 35 pl of each samplecontaining identical amounts of ATP-citrate lyase were electrophoresed through a 15% gel (8).Gels were acid treated and autoradiograms were developed (9). Regions 1, 2, 3, and 4 and bands a-f of Fig. 1 were arbitrarily designated to describe the radioactive band patterns. Essentially similar results were obtained as shown in Fig. 1 in at least 3 independent experiments. Complete Digestion of Phosphorylated ATP-citrateLyase-Purified ATP-citrate lyase from adipose tissue (300 p g ) was phosphorylated by incubation a t 30 "C for 4 hwith 30 pm [y-"*P]ATP (1000cpm/ pmol) and 30 pg of ATP-citrate lyase kinase or 50 pg of catalytic subunit. The reaction mixture was chromatographed on a Sephadex G-75 column (1.5 X 30 cm) equilibrated with 10 mM Tris-HC1, pH 7.5, containing 0.1 M NaCI, 10 mM 2-mercaptoethanol. Fractions (0.3 ml) within the void volume were pooled and lyophilized. The protein was dissolved in 1 ml of 5 M guanidine hydrochloride containing 0.2 M 2mercaptoethanol, reduced, carboxymethylated (13, 14), and dialyzed against 0.05 M NH4HCO:,(IO00 volumes with 3 changes). The dialyzed protein sample was divided into two equal volumes. One portion was treated with S. aureus protease, 25 pg/ml a t 30 "C, and after 6 h an additional 7 g / m l of protease was added and incubated for another 6 h. The otherportion of the sample was digested with TPCK-trypsin for 24 h a t 30 "C by adding 25 pg/ml of TPCK-trypsin followed by 25 were separatedonthin layer cellulose g / m l 6 hlater.Peptides chromatographic plates (E. Merck) using a chloroform/methanol/ ammonium hydroxide (22:l) solvent system in the ascending chromatography for the f i t dimension and electrophoresis (1 kV for 1 h, cathode on the right) in a pH 3.5 buffer system (pyridine/acetic acid/ H20, 2:20:978) for the second dimension (15). Phosphoamino Acid Analysis-ATP-citrate lyase was phosphorylated, reduced, carboxymethylated and digested using TPCK-trypsin or S. aureus protease as described above. The digested lyase sample (100 pl) was made to 6 N HCI, sealed, and hydrolyzed for 3, 6, and 12 h a t 110 OC. The acid hydrolysates were dried under a stream of nitrogen and dissolved in electrophoresis buffer, pH 1.9. Samples (20 p l ) were spottedtogether with nonradioactive phosphoserine, phosphothreonine, and phosphotyrosine (a gift from Dr. Paul Rothberg of the StateUniversity of New York, Stony Brook) on a cellulose thin layer chromatographic plate. The samples were electrophoresed a t pH 1.9 (acetic acid/90% formic acid/HnO (7825897)) for 5 h a t 1 kV with the anode at the top which separated phosphothreonine from phosphotyrosine (16). The plates were dried at 110 "C for 15 min, sprayed with ninhydrin, and heated a t 110 "C to locate the positions of the phosphoamino acids.

amounts of each kinase as added to samples in lanes A and B were added to sample in lunes C. The radioactivity associated with the 115-kilodalton subunit of ATP-citrate lyase is given in parentheses above. The concentrations of TPCK-trypsin (0,200, and 500) and S. aureus protease (0,5, 10,and 20) in micrograms/ml are given at the top of the gel.

TABLE I Additive phosphorylationof ATP-citrate lyase by ATP-citrate lyase kinase a n d catalytic subunit The assays were performed as described under "Experimental Procedures." The reaction was started by the addition of 5 pl of protein kinase (fmtaddition) to a 40-pl incubation medium and after incubation for 15 min an additional 5 pl of the same or different protein kinase or buffer (second addition) as indicated were added and theincubation continued for another 15 min. First addition"

Second addition

Phosphorylation O f lyase pmol/rnoI subunit

PK-A Buffer 0.45 PK-A PK-A 0.56 0.90 PK-A PK-C PK-C Buffer 0.48 PK-C PK-C 0.56 PK-C 0.99 PK-A PK-A is ATP-citrate lyase kinase and PK-C is catalytic subunit of cyclic AMP-dependent protein kinase. RESULTS AND DISCUSSION

ATP-citrate lyase was phosphorylated with lyase kinase or with catalytic subunit followed by additional increments of either the same or the different kinase. As shown in Table I, the combination of the two kinases increased ATP-citrate lyase phosphorylation additively while the same kinase added twice increased phosphorylation minimally. These resultssuggested that different sites were phosphorylated by the two kinases. To demonstrate that the sites phosphorylated by each kinase were different, ATP-citrate lyase was f i t phosphorylated with either ATP-citrate lyase kinase, catalytic subunit, or both. The phosphorylated lyase was digested with TPCKtrypsin or S. aureus protease and the products of proteolysis wereresolvedby SDS gel electrophoresis as described by Cleveland et al. (12).As shown in Fig. 1, ATP-citrate lyase was phosphorylated (acid-stable phosphorylation) by each kinase to 0.52 mol/mol of subunit with lyase kinase and 0.46 mol/mol of subunit with the catalytic subunit.(Inother experiments, lyase was phosphorylated up to 0.6 mol/mol of subunit by either kinase.) After trypsin or protease digestion, some radioactive fragments derived from ATP-citrate lyase

Different Phosphorylation Sites of ATP-citrate Lyase phosphorylated with lyase kinase were different from fragments generated when catalyticsubunit wasused as the kinase. Only minimal proteolysis was observed in 30 min at concentrations of trypsin up to 50 pg/ml (data not shown) whereas at increasing concentrations, more proteolysis was observed. With 200 pg/ml of trypsin (Fig. 1, left), radioactive band I C was prominent and band l e was absent in lanes A (ATP-citrate lyase kinase-treated sample). In lanes B (catalytic subunit-treated sample), band le was more apparent, band I C was very light, and band l a was absent. When the lyase was phosphorylated with both kinases (lanes C ) the proteolytic fragments generated by trypsin digestion were the sum of those present in lanes A and B. Similar findings were noted in regions 2 and 3 of the gel although the bands in these regions were less intense. Unambiguous examples of differences between the radioactive fragments produced by S. aureus protease treatment of lyase f i t reacted with either ATP-citrate lyase kinase or catalytic subunit are also shown in Fig. 1, right. Radioactive bands le, 36, and 3d were present in the sample phosphorylated by catalytic subunit (lanesB ) whereas bands Id, 3a,and 3c were noted in the lyase kinase-treated sample (lanes A ) . All the radioactive bands present in lanes A and B were noted in lanes C inwhich lyase was phosphorylated with both kinases. In lanes A at a concentration of 20 pg of protease, bands 3e and 3f were present although poorly defined. Band 3e was absent and 3f was more intense and distinct in lanes B. Again, when both the kinases were added together (lanes C ) ,the radioactive profile was the sum of lanesA and B. Note that lower molecular weight fragments were not well resolved on the 15%gel. The less intense bands, 4a (lanes A ) and 4b (lanesB ) seen in the samples treated with 5 pg/ml of protease, were no longer clearly resolved from each other when higher protease concentrations were used. However, the differences between the samples treated with the different kinases were still evident. Band 4c was distinct in lanes A , whereas 4d was more prominent in lanes B, and lanes C contained both 4c and 4d and merged as one large intense band. In general, peptides in B lanes (samples phosphorylated by catalytic subunit) were of slightly lower molecular weight than their counterparts in A lanes (samples phosphorylated by ATP-citrate lyase kinase). Controls (no protease or trypsin) treated similarly did not show any proteolytic peptides other than those present at zero time. The appearance of the complete complement of peptides in lanes C that were inlanes A and B suggests that the differences in the peptides seen in lanes A and B were not dueto protease contamination of any of the added protein kinases. If the observed differences in the peptides in lanes A and B were in fact due to conformational changes in lyase related to the degree of phosphorylation of the same site, one would expect lanes C (in which phosphorylation was 1.5- or 2-fold higher than in lanes A or B, respectively) would show an entirely different pattern and not asimple addition of the fragments seen in lanes A and B. Homogeneous ATP-citrate lyase was phosphorylated with ATP-citrate lyase kinase or with catalytic subunit. Fig. 2 shows the patterns of [”‘PI-phosphopeptides obtained after exhaustive TPCK-trypsin or S. aureus protease digestion of phosphorylated ATP-citrate lyase and two-dimensional separation of peptides by thin layer chromatography, followed by electrophoresis and autoradiography. The autoradiograms of the tryptic peptides produced in response to phosphorylation by lyase kinase (Fig. 2 0 ) were distinctively different from that produced in response to the action of catalytic subunit (Fig. 2 0 . One major spot appeared well above the origin and to the right(cathodal) in the lyase kinase treated samples. Smeared minor spots also appeared in the lane of the major

10215

B

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&

C

D

FIG.2. Phosphopeptides from ATP-citrate lyase. ATP-citrate lyase was phosphorylated by catalytic subunit (A and 0 and lyase kinase ( B and D).ATP-citratelyase was reduced, carboxymethylated, and digested with either S. uureus protease (A and B ) or TPCKtrypsin (C and D).The phosphopeptideswere separated on thin layer cellulose plates by ascending chromatography and electrophoresis in the second dimension as described under “Experimental Procedures.” The originsareindicated by 0. The minorradioactive spot in D enclosed by dushed lines coincided with the major radioactive spot in C. spot while a distinct minor spot appeared cathodally (right) and below the major spot (Fig. 20, enclosed in dashed lines). In the catalytic subunit-treated sample, one major spot appeared above the origin and tothe right. It coincided with the minor spot to the right of the major spot noted in the lyase kinase-treated sample. The autoradiogram of the protease-derived peptides from lyase kinase-treated ATP-citrate lyase was also different from the pattern of peptides derived from the catalytic subunittreated sample (Fig. 2, A and B ) .In thelyase kinase phosphorylated ATP-citrate lyase, one major spot appeared to the right and well above the origin. Material remained at the origin, and smeared radioactive material ran above the origin. A different major spot whichmoved more cathodally was found in the catalytic subunit-treated sample. No radioactive spot corresponding to theregion of the major spotin the lyase kinase-treated sample was found in the catalytic subunit. Analysis of the phosphoamino acids of adipose tissue ATPcitrate lyase which had been phosphorylated with either lyase kinase or catalytic subunit was surprisingly informative. Fig. 3 shows a traceof the separated phosphoamino acid standards and the autoradiograms of the resolved radioactive phosphoamino acids. Phosphoserine was the only detectable radioactive amino acid derived from acid hydrolysis of ATP-citrate

Different Phosphorylation Sites of ATP-citrate Lyase

10216 A

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p-ser P-tY r p-thr

P-PeP

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FIG. 3. Phosphoamino acids from ATP-citrate lyase. Acid hydrolysates of TPCK-trypsin-or S.aureus protease-digested ATPcitrate lyase, phosphorylatedby catalytic subunit( A )or lyase kinase (B), were electrophoresed along with phosphoserine (p-ser),phosphothreonine (p-thr),and phosphotyrosine (p-tyr).Phosphopeptides @-pep)that were present after 3 h of acid hydrolysis (due to incomplete hydrolysis) disappeared after 12 h of acid hydrolysis (data not shown). lyase treated with catalytic subunit. This observation is consistent with results previously reported for rat liver lyase (17). However, in the case of lyase kinase-phosphorylated ATPcitrate lyase hydrolyzed in acid for 3 to 12 h, phosphothreonine and phosphoserine were radiolabeled. Phosphotyrosine was not present in any samples. Furthermore, when trypsin- or protease-generated radioactive peptides (on two-dimensional analysis) were eluted from the plates and hydrolyzed in acid, only phosphoserine was found from lyase samples treated with catalytic subunitof cyclic AMP-dependent protein kinase whereas both phosphothreonine and phosphoserine were found in the acid hydrolysate of the major phosphopeptide from lyase kinase-phosphorylated enzyme. Our results on partial proteolysis of phosphorylated ATPcitrate lyase followed by SDS gel analysis and on complete proteolysis followed bytwo-dimensional peptide mapping and phosphoamino acid analysis of ATP-citrate lyase are consistent with the hypothesis that ATP-citrate lyase possesses at least two phosphorylation sites, serine phosphorylated by cyclic-AMP-dependent protein kinase and serine and threonine phosphorylated by lyase kinase. Whether the serine phosphorylated by lyase kinase is at thesame site phosphorylated by catalytic subunit is not known. Our evidence, which includes the marked differences in the two dimension peptide

patterns of the lyase kinase- and catalytic subunit-treated samples and the finding that the peptide pattern generated from the addition of both catalytic subunit and lyase kinase to ATP-citrate lyase is the sum of each kinase added separately rather thanthe patterngenerated by lyase kinase alone as would bethe case if the serine sites were the same, suggests that the serine sites are different and indeed might be at different regions of the molecule. However, since the amount of serine phosphorylated in vitro is never greater than 1 mol/ mol with the addition of both kinases, it is difficult to conclude that the serine phosphorylated is different for each kinase. We are currently sequencing the radiolabeled peptides generated by digestion of the lyase phosphorylated by either kinase, which will conclusively show whether different serine residues are phosphorylated by these kinases. The relation of these postulated regions to thephosphohistidine at thecatalytic site isunknown. The relationship of the pathway of lyase kinase-mediated ATP-citrate lyase-specific site phosphorylation demonstrated in vitroto insulin's effects on lyase phosphorylation in vivo are still unknown but our results are consistent with the hypothesis that insulin action activates a specific protein kinase for lyase phosphorylation. Recently (18,19),evidence has been presented on S-6 ribosomal protein phosphorylation in 3T3-Ll cells in vivoand in vitrowhich supports the notion that insulin activates a specific protein kinase. REFERENCES 1. Krahl, M. E. (1961) The Action of Insulin on Cells, Academic

Press, New York 2. Czech, M. (1977) Annu. Rev. Bwchem.46,359-3& 3. Benjamin, W. B., and Singer, I. (1974) Biochim. Biophys. Acta 351.28-41 4. Avruch, J., Leone, G. R., and Martin, D. B. (1976) J.Biol. Chem. 251, 1511-1515 5. Fom, J., and Greengard, P. (1976) Arch. Biochem. Biophys. 176, 721-723 6. Ramakrishna, S.,and Benjamin, W. B.(1979) J.Bwl. Chem. 254, 9232-9236 7. Alexander, M. C., Kowaloff, E. M., Witters, L. A., Dennihy, D. T., and Avruch, J. (1979) J.Bwl. Chem. 254,8052-8056 8. Benjamin, W. B.,andSinger, I. (1975) Biochemistry 14, 33013309 9. Ramakrishna, S.,and Benjamin, W. B. (1981) FEBS Lett. 124, 140-144 10. Guy, P. S., Cohen, P., and Hardie, D. G. (1980) FEBS Lett. 109, 205-208 11. Bechtel, P.J., Beavo, J. A., and Krebs, E.G. (1977) J.Bwl. Chem. 252,2691-2697 12. Cleveland, D. W., Fischer, S. G., Kmhner, M. W., and Laemmli, U.K. (1977) J. Bwl. Chem. 252,1102-1106 13. Renaud, F. L.,Rowe, A. J., and Gibbons, I. R. (1968) J.Cell Biol. 36.79-90 14. Stephens, R. E. (1975) Anal. Biochem. 65,369-379 15. Stephens, R. E. (1978) Anal. Biochem. 84,116-126 16. Eckhart, W., Hutchinson, M. A., and Hunter, T.(1979) Cell 18, 925-933 17. Lmn, T. C., and Srere, P. A. (1979) J.B i d . Chem. 254,1691-1698 18. Lastick, S.M., and McConkey, E. H. (1981) J. Bwl. Chem. 256, 583-585 19. Rosen, 0.M., Rubin, C. S., Cobb, M. H., and Smith, C. J. (1981) J. Biol. Chem. 256,3630-3633