Proteolytic Cleavage of Phospholamban Purified

THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc.

Vol. 261, No. 11, Issue of April 15, pp. 5154-5159, 1986 Printed in U.S.A.

Proteolytic Cleavageof Phospholamban Purified from Canine Cardiac Sarcoplasmic Reticulum Vesicles GENERATION OF A LOW RESOLUTION MODEL OF PHOSPHOLAMBAN STRUCTURE* (Received for publication, September 9,1985)

Adam D. Wegener, Heather K. B. Simmerman, Juris Liepnieks, and LarryR. Jones$ From the Krannert Instituteof Cardiology and the Departmentof Medicine, Indiana UniuersitySchool of Medicine, Indianapolis, Indiana 46202

Purified phospholamban isolated from canine carpholamban was shown to be the principal membrane protein diac sarcoplasmic reticulum vesicles was subjected to phosphorylated in intact heart in response to P-adrenergic proteolysis and peptide mapping tolocalize the differ- stimulation (3). As such, phospholamban is of interest as a ent sites of phosphorylation on the protein and to gainpotential regulatory protein in myocardium, which responds further information on its subunit structure. Five dif- to changing levels of intracellular CAMP (4). Recently, it has ferent proteases (trypsin, papain, chymotrypsin, elas- been shown that phospholamban is also a substrate for a tase, and Pronase) degraded the oligomeric 27-kDa Ca’+/calmodulin-dependent protein kinase endogenous to phosphoprotein into a major 21-22-kDa protease-re- cardiac sarcoplasmic reticulum (5, 6) and for soluble protein sistant fragment. No 32Pwas retainedby this protease- kinase C prepared from rat brain (7, 8). Biochemical (9-12) resistant fragment, regardless of whether phospholamban hadbeen phosphorylated by CAMP-dependent pro- and immunohistochemical (13) methods have localized phospholambin to sarcoplasmic reticulum in cardiac tissue, where teinkinase, Ca’+/calmodulin-dependent proteinkinase, or protein kinaseC. Phosphoamino acid analysis it may regulate the Caz+pump (3). Phospholamban has also twitch fibers) and thin-layer electrophoresis of liberated phospho- been identified in slow twitch fibers (but not fast peptides revealed that 1 threonine and 2 serine resi- of canine skeletal muscle with use of immunohistochemical dues were phosphorylated in phospholamban and that methods and specific phospholamban antibodies (14). Phosphorylation of phospholamban in cardiac sarcoplasmic 1 of these serine residues and the threonine residue were in close proximity. Only serine wasphosphoryl- reticulum vesicles has been correlated with stimulation of Ca2+transport and Ca2+-dependent ATPase activity in nuated byCAMP-dependent proteinkinase,whereas Ca2+-calmodulin-dependentproteinkinase phospho- merous studies (1, 2, 15-18). In cardiac membrane vesicles rylated exclusively threonine. incubated under conditions resulting in phospholamban phosThe results demonstrate that phospholamban has a phorylation, the sarcoplasmic reticulum Ca’+ pump shows an large protease-resistant domain and a smaller proincreased apparent affinity for Ca’+, but no change in stoitease-sensitive domain, the latterof which contains all chiometric coupling of Ca2+transport toATP hydrolysis (19of the sites of phosphorylation. The 21-22-kDa pro- 22). A physical association between phospholamban and the tease-resistant domain, although devoid of incorpo- Ca2* pump has been difficult to prove with use of isolated rated 32P,was completely dissociated into identical cardiac sarcoplasmic reticulum vesicles, however,because the lower molecular weight subunitsby boiling in sodium protein composition of these membranes is complex (23), dodecyl sulfate, suggestingthat thisregion of the mol- making potential interactions between phospholamban and ecule promotes the relatively strong interactions that the Ca2+ pump difficult to discern. Definitive proof that hold the subunits together. The data presented lend phospholamban is capable of forming a regulatory complex further support for a model of phospholamban strucwith the Ca2+pump will require purification of both proteins ture in which several identical low molecular weight subunits are noncovalently bound to one another, each and their successful reconstitution into a system that retains containing one site of phosphorylation for CAMP-de- the ability to hydrolyze ATP andtransport Ca2+ and is pendent protein kinase and another site of phospho- modulated by phosphorylation. Wehave recently purified phospholamban from cardiac Ca2+/calmodulin-dependentproteinkirylationfor tissue and gained some insight into its structure(10, 24). The nase. protein appears to be composed of five identical phosphorylatable subunits, which are indistinguishable by electrophoretic mobility in SDS1-polyacrylamide gels(10,24). Each subunit contains a unique phosphorylation site for CAMPPhospholamban is an integralprotein of cardiac sarcodependent protein kinase and Ca’+/calmodulin-dependent plasmic reticulum which was first identified in isolated memprotein kinase (24). Phosphorylation may modify the conforbrane vesicles asthe major substrate phosphorylated by mation of the protein in that itmarkedly alters its isoelectric CAMP-dependent protein kinase (1, 2). Subsequently, phospoint (10) and also decreases its electrophoretic mobility in * This work was supported by Grants HL28556 and HL06308 from SDS gels (24). It is obvious from the results of these experithe National Institutes of Health and by the Herman C. Krannert ments (10, 24) and others (11)that thestructure of phosphoFund. The costs of publication of this article were defrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Established Investigator of the American Heart Association.

The abbreviations used are: SDS, sodium dodecyl sulfate; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraaceticacid; MOPS, 3-(N-morpho1ino)propanesulfonic acid; TES, 2-([2-hydroxy-l,l-bis(hydroxymethy1)ethyllaminojethanesulfonicacid).

5154

Mapping Peptide

Phospholamban of Cardiac

lamban is more complex than originally postulated (4). To gain further insight into thesubunit structureof phospholamban and the nature of its different sites of phosphorylation, we have analyzed the protein by proteolytic cleavage and peptide mapping. The results described here allow us to make some conclusions regarding phospholamban structure, particularly to identify two principal domains of the molecule, one of which is phosphorylated and theother of which appears to promote association of the multiple subunits. The methods and cbnclusions derived from this work should aid future studies addressing phosphorylation of phospholamban in intact cardiac tissue, where the activities phosphorylating phospholamban have not yet been identified with certainty (25). EXPERIMENTAL PROCEDURES AND RESULTS’

5155

A M 0

0

M R M

0 E

P-P-

6

.

P E

-

T

M

DISCUSSION

Fig. 7 shows a working model of phospholamban structure, which incorporates several of the features suggested by the data presented here and previously (10, 24). Central to the model are two major molecular domains: a relatively short, protease-accessible domain extending from the sarcoplasmic reticulum membrane and accessible to phosphorylation by different protein kinase activities and a larger protease-resistant domain, which allows the identical subunits to interact, perhaps anchoring them the in membrane. Subunit interactions occurring in theprotease-resistant domain are apparently quite strong; theywere only disrupted after the protein was boiled in SDS (Figs. 1 and 2). Preliminary sequence data demonstrate that thephosphorylated portion of the molecule is located at the protein’s amino terminus and is directly connected to the protease-resistant region, which is highly enriched in hydrophobic amino acid residue^.^ The model of phospholamban as a pentamer (Fig. 7 B ) of identical monomers (Fig. 7 A ) results from several indirect observations and remains tentativeat present. The protein is phosphorylated by the catalytic subunit of CAMP-dependent protein kinase to a level of approximately 200 nmol of PJmg ~ minimum of protein (lo), a t a single serine r e ~ i d u e .By stoichiometry, this suggests a subunit molecular weight of approximately 5,000 for the phospholamban monomers comprising the multimer. Five such monomers wouldyield a molecular weight of approximately 25,000 for the holoprotein, which is the observation made when molecular weight is estimated by SDS-polyacrylamide gel electrophoresis. Moreover, five distinct sets of mobility forms of phospholamban (pentamer through monomer) can be identified in SDS gels by either autoradiography (24) or direct staining (10) when the protein is partially dissociated by heating in SDS prior to electrophoresis. It should be emphasized that this estimate of subunit stoichiometry pertains only to theprotein solubilized in SDS; there is presently no information available on the quaternary structure of the protein in native sarcoplasmic reticulum membranes. There seems little doubt, however, that the purified phospholamban multimer is composed of identical phosphorylatable monomers, even based on our indirect Portions of this paper (including “Experimental Procedures,” “Results,” Figs. 1-6, and Table I) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 85M-3030, cite the authors, and include a check or money order for $5.60 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press. H. K. B. Simmerman, J. H. Collins, J. L. Theibert, A. D. Wegener, and L. R. Jones, manuscript submitted for publication.

P”

R

KINASE

M

0 E

C

P-

CAMP-PKII

PROTEASE

PIJCa/CaM-PK

1

FIG. 7. Hypothetical model of phospholamban structure depicting the protease-resistant domain promoting subunit interactions and the protease-sensitive domain containing the sites of phosphorylation. A , monomeric phospholamban showing the protease-resistant domain inserted in the lipid bilayer and the positively charged, protease-sensitive domain protruding from the membrane. Each monomer contains one site of phosphorylation for CAMP-dependentprotein kinase and one site of phosphorylation for Ca’+/calmodulin-dependent protein kinase. Phosphorylated monomeric phospholamban retains a conformational change, causing a decreased mobility in SDS-polyacrylamide gels and a decrease in its isoelectric point. B, pentameric model of phospholamban showing hypothetical subunit interactions and 10 potential phosphorylation sites for relevant protein kinase activities. The pentamer is composed of five identical monomers, each containing one phosphorylation site for CAMP-dependentprotein kinase and one phosphorylation site for Caz+/calmodulin-dependentprotein kinase. Phosphorylation of the pentamer also causes a conformational change, giving the protein a slower mobility in SDS-polyacrylamide gels. C, protease effects on phospholamban. The protease-sensitive domain contains the phosphorylated residues: serine for CAMP-dependent protein kinase (CAMP-PK) and threonine for Ca2+/calmodulin-dependent protein kinase (Ca/CaM-PK). These two phosphorylated residues are localized to thesame small peptide fragment.

observations. The monomers exhibit identical mobilities in SDS gels when dephosphorylated and identical mobility shifts after phosphorylation by the catalytic subunit of CAMPdependent protein kinase (24). Proteolysis of phosphorylated phospholamban by trypsin or papain yields clipped monomers with identical apparent molecular weights (6,000 for trypsin and 7,000 for papain), and the monomers all lose their radioactivity by thistreatment. Phospholamban migrates asa single protein bandin isoelectric focusing gels in the presence o f Triton X-100 and urea, whether focused as the intact molecule (PI 10) or after proteolysis by trypsin (PI 6.0) or papain(PI 8.0). Taken together, all of these observations strongly suggest that phospholamban is composed of identical monomers that can each be phosphorylated.

5156

Mapping Peptide

Phospholamban of Cardiac

All proteases tested on phospholamban yielded large protease-resistant fragmentswith very similar mobilities in SDS gels. This suggests that there is a small accessible region of phospholamban that is especially prone to proteolytic cleavage. Previous studies on protease treatment of phospholamban in intact cardiac sarcoplasmic reticulum vesicles have yielded apparently conflicting results. The phosphorylated protein has been reported to be either protease-resistant (2, 34) or readily degraded by proteases (1, 8, 35). No difference in the rate of proteolytic hydrolysis of purified phospholamban, whether phosphorylated or dephosphorylated, was detected in this study. We did observe that hydrolysis of phospholamban phosphorylated in sarcoplasmic reticulum vesicles was very slow unless membranes were first solubilized with any of a number of detergents (data not shown). The resistance of phospholamban to proteolysis in intact membranes reported previously by others (2, 34) may depend upon some type of interaction of phospholamban with the lipid bilayer or with other membrane components. Thin-layer electrophoresis of radioactive phospholamban peptides along with partial acid hydrolysis suggested that each phospholamban monomer contained 3 residues that could be phosphorylated a threonine residue phosphorylated by Ca2+/calmodulin-dependentprotein kinase or protein kinase C, a serine residue phosphorylated by CAMP-dependent protein kinase or proteinkinase C, and asecond serine residue phosphorylated only by protein kinase C. Protein kinase C thus seems less selective than the otherprotein kinases with respect to phospholamban phosphorylation. The physiological significance of phospholamban phosphorylation by protein kinase C,however, has recently been questioned (36, 37).4 Consistent with this, we observed phospholamban phosphorylation by protein kinase C to be much more rapid in the reconstituted particulate fraction than in native sarcoplasmic reticulum vesicles. Another protein kinase, glycogen synthase kinase FA/GSK-3 (38), was virtually inactive toward phospholamban in intact sarcoplasmic reticulum vesicles, but rapidly phosphorylated purifiedphospholamban primarily at the serine residue phosphorylated by CAMP-dependent protein kinase (data not shown).Phosphorylation of phospholamban by Ca2+/calmodulin-dependentprotein kinase, by contrast, was very rapid in sarcoplasmic reticulum vesicles. This protein kinase activity appears to copurify with phospholamban (5, 11, 24), suggesting, perhaps, a tight association between Ca2+/calmodulin-dependent protein kinase and phospholamban in the sarcoplasmic reticulum membrane. The catalytic subunit of CAMP-dependentprotein kinase also rapidly phosphorylated phospholamban at all stages of purification. Previous studies suggesting different phosphorylation sites on phospholamban for CAMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase relied on indirect assessments of site specificities: measuring additivity of phosphorylation catalyzed by different protein kinases (5, 24, 35), mobility changes in the phosphorylated protein in SDS gels (24), or analyzing peptide maps of the phosphorylated protein (8). Demonstration that CAMP-dependent protein kinase phosphorylates exclusively serine on phospholamban and that Ca2+/calmodulin-dependent protein kinase phosphorylates exclusively threonine shows unambiguously that indeed different residues are phosphorylated. This information should be useful for determining more precisely which protein kinase activities phosphorylate phospholamban in intactcardiac tissue (25).4 Early observations with cardiac sarcoplasmic reticulum vesA. D. Wegener, J. P. Lindemann, and L. R. Jones, manuscript in preparation.

ides suggested that the level of 32Pincorporated into the phospholamban polymer (expressed per milligram of total membrane protein) was approximately the same as the density of Ca2+pumps in sarcoplasmic reticulum vesicles, detected by formation of their acyl phosphoprotein intermediates (4). Such observations led to theearly suggestion of a one-to-one stoichiometry of Ca2+-dependentATPase to phospholamban in the sarcoplasmic reticulum membrane (4), but no firm evidence of a direct interaction between phospholamban and the Ca2+pump protein has been presented to suppoft this idea. We recently proposed that the high molecular weight form of phospholamban is composed of five phosphorylatable subunits and that the multimeric complex can incorporate 5 mol of Pi/mol through CAMP-dependent protein kinase and 10 mol of Pi/mol through thisprotein kinase and Ca2+/ calmodulin-dependent protein kinase together. Thus, phospholamban is a minor component of the total membrane protein in sarcoplasmic reticulum vesicles (1-2%, Ref. 10) but is phosphorylated to a high specific activity due to a high molar phosphorylation stoichiometry. The argument for similar levels of 32Plabeling of phospholamban and the Ca2+ pump in sarcoplasmic reticulum vesicles (4), therefore, does not support aone-to-one stoichiometry between phospholamban and the Ca2+pump, assuming phospholamban exists in its polymeric form in the native sarcoplasmic reticulum membrane. Definitive demonstration that phospholamban directly interacts with and regulates the activity of the cardiac Ca2+ pump will ultimately require in uitro reconstitution of both purified proteins in an intact, functional system. It is hoped that conclusions about phospholamban structure, based on the observations shown here and elsewhere (10, 11, 24), will be useful in designing and interpreting more sophisticated experiments working toward a molecular model of phospholamban function in cardiac sarcoplasmic reticulum membranes. '

Acknowledgments-The technical assistance of David Patterson is gratefully acknowledged. We thank Anna Wells forher secretarial help.

REFERENCES 1. LaRaia, P. J., and Morkin, E. (1974) Circ. Res. 3 5 , 298-306 2. Tada, M., Kirchberger, M. A., and Katz, A. M. (1975) J. Biol. Chem. 250,2640-2647 3. Lindemann, J. P., Jones, L. R., Hathaway, D. R., Henry, B. G., and Watanabe, A. M. (1983) J. Biol. Chern. 2 5 8 , 464-471 4. Tada, M., and Inui, M. (1983) J. Mol. Cell. Cardiol. 1 5 , 565-575 5. LePeuch, C. J., Haiech, J., and Demaille, J. G. (1979) Biochemkt? 19,5150-5156 6. Jones, L. R., Maddock, S. W., and Hathaway, D. R. (1981) Bwchim. Biophys. Acta6 4 1 , 242-253 7. Iwasa, Y., and Hosey, M. M. (1984) J . Biol. Chem. 259,534-540 8. Movsesian, M. A., Nishikawa, M., and Adelstein, R. S. (1984) J. Biol. Chem. 259,8029-8032 9. Manalan, A. S., and Jones, L.R. (1982) J. Biol. Chem. 2 5 7 , 10052-10062 10. Jones, L. R., Simmerman, H. K. B., Wilson, W. W., Gurd, F. R. N., and Wegener, A. D. (1985) J. Biol. Chem. 2 6 0 , 7721-7730 11. Inui, M., Kadoma, M., and Tada, M. (1985) J. Biol. Chem. 260, 3708-3715 12. Presti, C. F., Jones, L. R., and Lindemann, J. P. (1985) J. Biol. Chem. 260,3860-3867 13. Jorgensen, A. O., and Jones, L. R. (1985) Biophys. J. 4 7 , 5 9 a 14. Jorgensen, A. O., and Jones, L. R. (1985) J. Biol. Chem. 2 6 1 , 3775-3781 15. Tada, M., Ohmori, F., Kinoshita, N., and Abe, H. (1978) Adu. Cyclic Nucleotide Res. 9, 355-369 16. Kirchberger, M. A., and Antonetz, T. (1982) J. Biol. Chem. 257, 5685-5691 17. Plank, B., Wyskovsky, W., Hellmann, G., and Suko, J. (1983) Biochim. Biophys. Acta7 3 2 , 99-109

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Peptide Mapping of Cardiac Phospholamban 18. Plank, B., Pifl, C., Hellmann, G., Wyskovsky, W., Hoffmann, R., and Suko, J. (1983) Eur. J. Biochem. 136,215-221 19. Hicks, M., Shigekawa, M., and Katz, A. M. (1979) Circ. Res. 44, 384-391 20. Kranias, E. G., Mandel, F., Wang, T., and Schwartz, A. (1980) Biochemistry 19,5434-5439 21. Tada, M., Inui. M.. Yamada, M., Kadoma, M., Kuzuya, T., Abe, H.,'and Kakiuchi, S . (1983) J. Mol. Cell. Cardiol. 15, 335-346 22. Kranias, E. G. (1985) Biochirn. Biophys. Acta 844,193-199 23. Jones, L. R., Besch, H. R., Jr., Fleming, J. W., McConnaughey, M. M., and Watanabe, A. M. (1979) J. Biol. C h m . 254, 530539 24. Wegener, A. D., and Jones, L. R. (1984) J . Biol. Chem. 259, 1834-1841 25. Lindemann, J. P., and Watanabe, A. M. (1985) J. Biol. Chem. 260, 4516-4525 26. Schaffner, W., and Weissman, C. (1973) Anal. Biochem. 56,502514 27. Jones, L. R., Maddock, S. W., and Besch, H. R., Jr. (1980) J. Bid. Chem. 255,9971-9980

PROTEOLYTIC CLEAVAGE OF PHOSPHOLAMEAN PURIFIED FROH CANINE ULBDIAC SARCOPLASMIC RETICVLlM VESICLES: GENERATION OF A LOW RESOLUTION MODE1 OF PHOSPHOLAMBAN STROCTUBE Adam D. Wegener, Heeather K.B. Simmerman, Juris Liepnieks and Larry R . Jones E X P E R I ~ N T A L PROCEDURES

_Isolatioo _ _ of Dephosphorylated Phoephorylatedpurified Phosphalamban From Canine Cardiac from canine cardiae membranes Sarcoplasmic Retievlum Veeicles - Phospholamban was

enriched in sarcoplasmic retieulvm exactly as described previously ( 1 0 ) . The dephoaphorylared pratein vas purified t o apparent homogeneity from sarcoplasmicreticulum veaiclea by selectivl extraction of membranes with sodium cholate, followed by adsorption of the protein t o Ca oxalate and dialysia at 4' C t o yield the "reconstituted partienlate fraction" (IO). Phaspholwban was solvbilieed from the reconstituted parlicvlatian fraction with w e of the detergent Zwittergent 3 - 1 4 , and subsequently by svlfhydryl gravp affinity chromatography employing p-hydroxymercuriben.oafe purified agarose (10). Protein eoncenrrariooa were decemined by the method of Scbaffnn and Weisaman (261. Purified phoaphalambsn was phosphorylated by the Catalytic wbunit of eANP-dependent protein kinase prior to treatment with different protease acrivities. Io B typical phosphorylafioo experiment. 1.0 pg of purifiedphoepholamban was phosphorylated by 5.0 units of the catalytic subunitOf CAMP-dependent protein kinase for 10 minutes at 3OoC, in 50 pl of reaction oediur containing ZOaH TES (pH 7.5) and 10 mM &$I2: Phoapharylatiqq reactions were started by addition of 0.2 mM of Na ATP conrainlng a trace amovnl of [y- PIATP. Beactions were terminated by brief immerhioi of sample tubes in boiling R 0 prior to ptotealysia, or by adding 10 p l of eleetrophoresie s t o p Solution containin; SDS (2-42 final concentrstion) (9). +

In some experiments, a partially pvrified phospholamban preparation ( t h e reconstitvted particulate fraction) was phoaphorylared by differentprotein kinase aetivitiea, prior to further pvrificatinn of phospholamban for use in proteolysis experiments. This reconetitoted psrticulate fraction, obtained during thenormal purificnfian of phasphaIarjpn, ~ 8 enriched 6 5-fold in phosphal-ban (101, and contained its own endogeoovr Ca 1csl.odulin-depeodenr protein kinyp. of which phasphalamban vas the principal eubsfrafe ( 2 4 ) . In the presence of Ca and enlmodulin, phoapholamban was phosphorylated in this fraction to greater than 904 of the level obtained using the added catalytic subunit of e m d e p e n d e n t protein kinase. Harimal phosphorylation of phosphalsmban by aolvble prorein kinase C prepared f q y rat brian was achieved in the lipid environment endogenom LO chis fraction, when CB was included in the assay (data not shovn). Io a typical phosphorylation a ~ 6 a yvith this fraction, 10 pg of reconstituted particulate fraction vas incubated at 30' C io SO p l of buffer mgraining 20 mm TES (pB 7 . 5 ) and 10 mp( MgC12. Othlf additions r e r e 0.10 mM CaCl and 10 H calmoddin (to activate the endogenous Ca iealmodulin-dependenr proteii kinase), 0.1 mn EGTA and 20 units of the catalytic subunit of cAMP-depen$p protein kinase. or 0.15 mn CSCl and 10 p l of protein kinase C suspension. I XM IT- ]ATP vas added Lo start phosphorylation reactions, and incubations were temioated after 20 minutes by addition of 10 p l of SDS-stop eolvfion, or by brief heating prior to proteolysis. For certain experiments (Figs. 3 and 4), phosphorylated phospholamban was further purified from the reconstituted parficvlare fraction by svlfhydryl group affinity chromatography (10). Approximately 400 pg of the reconstituted parrievlntefraction vas phosphorylated as described above, with the inenbation volume increased t o 1.0 ml. After phosphorylation, protein vas sedimented sf 30,000 i g for 30 minutes and the pellet resuspended in 0.2 ml of 10 mM HOPS (pB 7.0). Phoapholamban v a s eolvbilized by addition of 1% Zvittergenr 3-14 and the suspension sedimenfed aC 30,000 x g for 30 minutes. The resulting ~ n p e r n ~ t a n containing r phosphorylated phoapholambaoUBI applied to a 0.2 m l bed volume of p-hydrorymercuriben=oare agarose in a tuberculin syringe. preequilibrated with 10 mM HOPS (pH 7.0) p l u s 0.1% Zvittergent 3-14. The column with loaded prorein vas then washed with 1.0 ml (five columo volumes) of 10 mM MOPS (pH 7.0). 0.1% Zwiftergent 3-14 and 0.5M NaC1, then with 1.0 m l of the 88me buffer lacking Nacl. Phoaphalamban va8 subsequently eluted from the column with three consecutive 1.0 ml buffer washes of the following composition: Wash 1 . 10 mM HOPS (pH 7.0). 20 mM dirhialhreifol, and 0.11 Zwittergent 3-14; Wash 2 , 10 mU MOPS (pH 7.0). 20 d l dithiothreitol, and 0.2% Zvittergent 3 - 1 4 ; Wash 3 , 10 mm MOPS (pH 7.0), 20 mM dithiothreitol, 0.22 Zwitrergenf 3-14, and SO mN NaC1. Column eluates enriched in phospholamban were concentrated in an Amicon Centricon.

(pronase) (behriqer-khnnheim 165511). Protease activities lire given as weight of protein added per weight of pure phoapholambanor weight of t o t a l membrane protein for less purified fractions. Aliquata of control phoapholsmban or of the phosphorylation l TES, mixtures described above were diluted into an equal volume of buffer conraiaing20 d 1.5 mM CaCl 2.0 mM dithiafhreitol. and 0 . 5 % Zwittergent 3-14 (PA 7.5) containing appropriafe2~~oncenrrsriaos of freshly prepared protease. Proteolysis was condncted f r w 2 t o 16 hours a t room temperature. Additional details are given in figure legends.

28. Porzio, M. A., and Pearson, A. M. (1977) Biochirn. Biophys. Acta 490,27-34 29. Merril, C.R., Goldman, D., Sedman, S. A., and Ebert, M. H. (1980) Science 211, 1437-1439 30. Jones, L. R., and Cala, S. E. (1981) J. Biol. Chern. 256, 1180911818 31. Hartzell, H. C., and Glass, D.B. (1984) J. Bid. Chem. 259, 15587-15596 32. Walton. G. M.. SDiess. , J.., and Gill. G.M. (1982) . . J. Biol. Chem. 257,'4661-466i) 33. Hashimoto. E.. Takeda. M.. Nishizuka. Y., Hamana,. K.,. and Iwai, K. (1976) J. B i d . Chem. 251, 628716293 34. Bidlack, J. M., and Shamoo, A. E. (1980) Biochim. Biophys. Acta 632,310-325 35. LePeuch, C. J., LePeuch, D.A.M., and Demaille, J . G. (1983) Methods Emymol. 102, 261-278 36. Presti, C. F., Scott, B. T., and Jones, L. R. (1985) J . Biol. Chem. 260.13879-13889 37. Movsesian, M. A., Thomas, A. P., Selak, M., and Williamson, J. R. (1985) FEBS Lett. 185, 328-332 38. DePaoli-Roach. A. A,. Ahmad. Z.. Camici. M.. Lawrence. J. C.. Jr., and Roach, P. J: (1983) 2. Bid. Cheh. 258, 10702-10709 '

SOS-Palvacrvlsmide Gel Eleetrorhoresis - SDS-polyacrylamide gel electrophoresis was condwted in 0.75 PO thick slab gels "sing a .lightly modified buffer system ( 2 7 ) of Parzio and Pearson (28). 10-15% gradient gels were run with a ratio of acrvlamide to bis-acrylamide Of 20:l. 30-SO p l aliquots of contra1 and PrDfea~e treated phospholwban samples were solubilized in 10 pl Of SDS-slop solution, which eonaisted of 60 mM Trie, 40 mM dithiothreitol, 20% glycerol, 2 to 4% SDS (final concentration) and e treee of bromophenol blve IIS B tracking dye. Some tiampies were boiled for 1 minvte prior t o eleczropharesia t o diasaciate phoapholamban info subunit.. After electrophoresis, g e l s vere fixed in 10% acetic acid, 25% isopropanol, and protein visualized by B sensitive silver staining method (25). Is were then dried between cellophane sheers far autorqdiographie detection of %labeled phasphalamban ( 2 4 ) . The molerular weights (~10- ) of the Protein standards used were B P follws: from Bim-Bad, bovine eerm albumin carbonic anhydrase (30). soybean trypsin inhibitor (21) and (68), ovalbumin (a), lyeolyme (14.3); from Boehringer Hannheim, cytochrome E (12.5). aprotinio (6.!,), and insulin chain B ( 3 . 4 ) .

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T b i n W Eteetmohoresia Proteolytic phosphopepfides deriggdf r w phasphalamban After incubation of P-labeled phorpholamban with proteases, 3 p l aliquot. Of the mixtures containing phosphopeptidea were *potted Onto a 160 vm cellulone adaarbanl sheet (Kodak Chromagram Sheet13255). The sheet was plneed in B Savant chamber, and electrophoresiawas conducted for 2 hours at 700 Poltr in 89% %O 1 LO% acetic acid I 1%pyridine (pH 3.7). After electrophoresis, rrypfie peptides were recovered f r w electrophoresis plates by scraping the e e t l u l a s e adsorbant om the BYPPort sheer and shaking it in a small volume of water for e few hours. "P-labeled peptides could be recovered in good yield by this method and analyzed further by partial acid hydrolysis and phosphaamino acid snalysis. were reaalved by thin layer electrophoresis.

-

Phos hoamino Acid Anal sis 32P-labeled phosphalamban Or tryptic peptides derived from phas~holambeoverehldrolyzedin 6N RCL for 4 hours a t 110' C in evacuated h y d r o W i s tYbeB. After evaporation of theRCL under nitrogen phoaphaamino acids were separated by thin layer eleerropboresia on Kodak Chromatogrsm Sheets (13255) at 500 volt* for 2 hour* in R 0 / 7 . 9 1 acetic acid / 2.5% formic acid (pH 1 . 9 ) . Phosphoserioe and PhosphrrthteOnine vere visualized with ninhydrin. 8ts&ds For phoaphoaoina acid analyais of phospholamban phosphorylatedin ioract sareopla~mie vesicles. 20 pg of purifiedcardiac sarcoplasmic reticulum vesicles (fraction E , ref. 30) were phoephorylated at 30' c in 50 p l oz reaction buffer containing 20 m~ TES (PH 7 . 5 ) , lo mM Mg12 and dther 0.1 mM CaCl and 10 M ' ealmodulio, which activates the endogenous Ca /c.lmodulin-dependenr protein kinase in this fraction (6). or 1.0 mM EGTA and 20 U of the catalytic subunit of AMP-dependent protein kinase. Phosphorylation Y B E starced by addition of 50 pM [:5PlArP and stopped after 30 8econdB by addition of 20 1 of SDS-stop eolurion. Samples were subjected to SDS-polyacrylamide gel electrophoresis Znd phosphorylated phoepholamban was defected by antoradiography of the unfixed gel. The section containing radioactive phoapholnmban was excised from the vet g e l . and phoapholnmban W)IB elvted from the gel pieceby diffusion into 1.0 ml of 0.01% Zvittelgent 3-14 for 24 h. Samples were lyophilized, faken "p in 1.0 ml of 6 N HC1 ilnd then hydrolyzed in evacuated hydrolysis fubee for 4 hours a t 110' C. Thin layer e1e:rrophore.i. was wndvered 8s described above. '

-

Ieoeleetrie roeusinp. of Phosnhalamban and mior Peptide Frazmente I~oelecfrie focusing of phoapholamban or the proteolytic prodnetsof phospholamban vas performed in slab gels containing ampholytes, Z X Triton X-100. and 6 M area, as previously described (10). After eleerropharesia, phospholamben and its major proteolytic fragemnta were visualized by staining with Coomnssie blue. Materials - p-Hydrorymercuribeo.oate a g s r ~ e e ,the catalytic subunit of CAMP-dependent protein kinase, phoBphoserine, and phosphothreonioe were purchased from Sigma. ~~ittergent 3-14 11a= Obtained f r w CalbiochersBehring. Soluble protein kinase C (2-3 l i l m l ) prepared from rat brain andthe glycogen mynthnre kinase F IGSK-3 were generously supplied by 2. Ahmad, A. DePaali-Roach. and P.J. Roach, Indiana bnirersity School of Medicine. RESULTS

a

Protealvsis & Phaspholnmban Phoenhorylafion & cbnP-dependent Protein Kinase - Purified Phospholamban. mexinslly phosphorylatedby the catalytic e u b m u i r __ CAMP-dependent Protein kinase, " 8 6 hydrolyzed L O B limited extent by trypsin shovs the effects of incubating purephoapholmban with increasing Concentra;io:?oflthe protease. The 27-kDe polymeric form of phospholamban was progressively Clewed through a series of intermediate molecular reigbt produets to a limit pcodmct ofapparent Hr = 21,000, which =an a8 a single band in SDS gels (Pig. I, Silver Stain, control). Phoapbolamban *=e fairly resistant t o pmtealyaia, requiring 0.04 "g of trypain t o fully hydrolyze 0 . 2 Pg of the purified protein t o the limit peptide (Fig. 1 Silver Stain control). Higher concentrations of trypsin rested did not further *I& the mobiliiy of the 21-kDa produet (datanot sbovn). Associated with the graded hydrolysis of phospholamban by trypain was B loss of radioactive phosphate ineorpmafed by the catalytic subunit Of CAMP-dependent protein kinase. the 21-kDa limit product retainedno detectable label (Fig. 1, Antoradiogram, Control). The tryptic phosphopeptides liberated from phospholamban did not fix io SDS gels and were therefore not seen by autoradiography.

5158

Peptide Mapping of Cardiac Phospholamban in the reconsriruted partienlatefraction by either the added catalytic svbvnir of CAB-dependent protein kinase. by added protein kinaseC. or by the endogenous CB Icslmodulin-dependent protein kinaee. The phoaphorylated protein was then solubilized in Zvittergent 3-14 8nd isolated by sulfhydryl group affinity chromatography (10). Phosphorylated phoapholamban required somewhat more vigorous conditions for e1UtiOn from the xg column than that reported previoualy for dephosphorylated phaspholamban (10). Dephosphorylated phospholanban is eluted mostly in wash 1, which contain* only 0.1% Zviffergenf 3-14 and 20 mU dithiothreifol (10). In contrast, 0.2% Zvittergent 3-14 (Wash 1 , or 0.2% Zvittergent 3-14 plus 50 mU NaCl (Wash 3) both containing 20 diihiorhreitol - were required t o elute pho8pharylated phospholamban from the Colmn completely (autoradiogram depicted in Fig.3). Consecutive rinses of theCOlmn with wash buffers 1 through 3 yielded phoephoproreins with lncresaing apparent molecular weights (decreasing mobilities) in SQS gels (Pig. 3). Notice that the highest apparent mOleCu1s* weight form (29 K, eluted in Wash3 when phospholamban wall phosphorylated by Ce icalmodulin-dependent and cAb-dependent protein kinase activities simultaneously)" 8 8 obtained when phospholambsn vas phosphorylated by either protein kinasealone. We shored previoualy (24) that maximal phosphorylation by either protein kinaae pradveed an identical mobility shift and that simultaneous phosphorylation resulteda further in "additive" shift in mobility. Same of thie 29,000 moleevlar weight form was a l e 0 obtained C (Fig. 3s 29 X). in wash 3 after phospholambsn had been phosphorylated by protein m i s BUggellfll that protein kinase c (unlike CAMP-dependent and Csicalmodulin-dependent kinase) may phosphorylatemore than one site on each phoapholamban eubunit.

-

T r y p s i n

Added

IpgI

A U T O R A D I O G R A M FIG. 1. Tryptic proteolysis of phoaphorylated phospholamban. hrified phospholamban phoephorylated maxilnally by the carslytic subunit CAMP-dependent protein kinase. Aliquots cantainmg 0.2 pg of phosphorylated phospholambsn were then incubated for 16 h at room temperature with 0 , 0.01, 0.02. or 0.04 pg of trypsin. SDS-efop solution wall added to proteolyzed samples to terminate reactions, and SDS-polyacrylamide g e l electrophoresis Y B B then performed. The silver stained gel* and corresponding BUtOrediogrBmsare shown. Results obtained for samples boiled in SOS just prior to electrophoresisare shovn in the t w o panel# on the right. VPB

Boiling pho9pholanhan inSDS prior t o eleetrophoresia caused dissociation of the polymeric protein into its low moleeuler weight svbunits (Fig. 1, Silver Stain, Boil). Incubating the proteinwith increasing concentration* of trypsin progressively comerfed all of the intact 11-kQa phosphorylated subunitt o a product of homogengys mobility of P, as nhovn by apparent molecular weight about 6000. This 6-kQs product contained no Combinations of intact 11-kQa phosphoautoradiography (Pig. I , Autoradiogram, Boil). lamban subunits and clipped 6-kDasubunits rweining associated in "on-boiled samples in SQS gels could prodwe the multiple intermediate mobility forms of molecular veighrs between 27,000 and 21.000 for polymeric phaspholamban observedafter electrophoresis (Fig.1, Silver Stain and Autoradiogram, 0.01 and 0.02 pg trypsin). We emphasize that the lover molecular weight value* reported are only BpprOximBte, a* considerable uncertainty exists in estimatingreliably maleeular weights for the monomeric, dissociated forms Of phoapholamban in SOS palyacrylmide gels (10.24). Phosphorylation of phospholambanby the catalytic subunit of cAHP-dependent protein kinase decreases the mobility of the protein in SQS gels (24). causing it to exhibitan increase in apparent ~ a l e c u l a rweight from 25,000 t o 27,000 (Fig. 2, lanes 1 and 4 ) . This mobility shift " 8 6 abolished by protease treatment of the protein. When the 25-kDa dephosphorylated and the 27-kQa phwphorylated forms of phoapholarnban were incvbafed with trypsin, non-radioactive peptides of identical mobility (2l-kDa) were generated (Fig. 2, lanes 2 and 5 ) . Papain also hydrolyzed only a portion of the molecule, in fhie case giving identical 22-kDa products inSDS g e l s for either dephosphorylated or phosphorylated phospholamban (Fig. 2, lanes 3 and 6). lover concentratione of papainyielded intermediate moblliry forms between 27- and 22-kQa. similar to the results deeeribedabove for fyrpsin. No difference in rates of proteolysis between dephosphorylated and Phoaphorylsted phospholamban were observed for either trypsin or pamin (data not sham).

SDS-PACE ( - ) BOIL I

SDS-PACE (+) BOIL II

I

27K -I 25K

1

2

3

1

2

3

1

2

3

1

2

3

W A SF HR A C T I O N FIG. 3. Avroradiogram depicting interaction of different phosphorylated forms of phospholamban with p-hyd=0xymercuribeo.o.te p a r o o e . Phoephalsmban YBB phosphorylated in the reconstituted particulate fraction by Ca /CBlnOd"lin-dependenr protein kinase (CaiCaM PK), carslytie subunit of CAMP-dependent proteinkinase (CAMP PK), both of these protein kinase activities. or protein kinase C (PK-C). I t was then further purified by sulfhydryl group affinity chromatography as described under EXPERIHBNTAL PROCEDURES, where the compoeition of the different wash fractions(1-3) is indicated. Phosphalambaa eluted from the column in wash fractions 1-3VBB nubjecred t o SQS-polyaerylamlde gel electrophoresis. and the result~nt autoradiogram is shown. Phoapholamban phosphorylated by .the different protein kinase activities vas iaolated hy sulfhydryl gromp affinity chromatography from the reconstituted particvlate fraction 8 8 described above. The purified pho8phOrylafed protein elmredin wash 2 from theHg-column vae then exposed to trypsin or papain and subjected t o SQS-polyacrylamide gel elecrrophoresia and autoradiography (Fig.4). Regardless of which proteinkinase phosphorylated phospholamban cleavage of the phosphorylated proteinby trypsin or papain resnlted in production of homogenous producte of molecular weights 21.000 (trypsin) and 000 (papain) (Fig. 4 Silver Stain). Associated with proteolysisvas lase of all %label in the major kragnents, regardlese of the source of the protein kinase (Fig. 4 , Autoradiogram). Thus, all phoephorylateble residves of phoepholamban were located in protease accessible regions of the proteinwhich together madeup only a small portion of the molecule (Phoapholamban eluted in wash 3 (Fig. 3) couldn o t be used for this experiment, 8s inclusion of NaCl in the wash buffer caused several protein contaminante t o coelvre from thecolumn with phospholamban (10). The electrophoretic mobilities seen in Fig. 4 reflect this aubmalinal phosphorylation).

+

C O N T R O L

21-22K

papain

T r y p s i n

I 15-17K

{

9-11K

5-7K

SILVERSTAIN

CAMP PK

FIG. 2. Trypsin and papain effect. on phoaphorylated and dephosphorylated phospholambsn. 1.0 pg of purified dephosphorylated phospholambanor phoapholambao phoephorylated by the catalytic svbvnit of eANP-dependent proteln kinase Y ~ Bincubated for 16 h with proteolysis buffer alone or buffer plus 0.2 pg of trypsin (T) or 0.2 pg of papain (P). SDS-polyacrylamide g e l electrophoresis and silver staining yere then performed on proreolyred samples. The panel on the right shove resultB obtsined for samplee boiled in SDS j w t prior to electrophoresis. The major protein bands in this panel correspond to the phospholamban monomers. An eutoradiogram of $9' same gel shored t h a t the msjor proteolytic fragments (T andP) were nor labeled virh P. Monomeric phospholsnban aleo exhibits a deereaae in mobility in SQS gels after phosphorylation by the catalytic svbvnif of CAMP-dependent prorein kinase(24). Control phospholamban and phospholamban reacted with papain and trypsin were dissociated t o subunits by boiling in SQS prior to elecfropharesie (Fig. 2, lanes 7-12). The 9-kDa dephosphorylated subunit (lane 7) and the 11-kDa phosphorylated subvnir(lane 10) were each cleaved t o fragment8 of identicalnobilities with apparent molecular veighrs of 6-kQs after trypsin (lanes 8 and 11) snd 7-kQa after papain (lanes 9 and 12). Autoradiographic analysis revealed that the major proteolytic fragment of g$oaphalamban retained in P. Similar res~lrswere SDS-gels after papain cleavage "8s also nor labeled rich obtained with uee of proneae. chymotrypsin. and elmtaae (data not shown). Thus, phospholamban appears to contain I ) relatively small andacceosible phosphorylated region responsible for the phaephorylarion-induced mobility ahifr observed previously inSQS gels (24), 8 8 vel1 811 B large nonphosphorylated. protease-reeistent region. which probably holds the several identicalsubunits together during electrOphoresi8. Proteolysis Phoaoholamban Phomhorvlated Ca2+/Cal.odulihQe~endent Protein Kinase and Protein Kinase C - It was of interest t o determine whether the protease accessible region of purified phospholmban also 5pntained the sites of phoephorylation for soluble proteinkinase C and the intrineic Ca icalmodulin-dependent protein kinase in sarcoplasmic reticulm. However, this latter proteinkinase has not yet been purified, complicating this type of study. Po avercome this problem, we phosphorylated phoepholamban in the reconetitvted psrtieulare fraction, which is obtained during the normal isolation of phoephalambsn. This frac$$on is enriched approximately 5-fold io phospholamban (IO), and contains en active Ce ieal~odulia-dependenrprorein kinase. the predominant wbstrate of which is phospholamban (10,24). Phospholamban v a s phosphorylated

AUTORADIOGRAM

CAMP PK CalCaM PK

-

C

FIG. 4. Remmal of 32P-label from phospholambnn by papain or trypsin, after phqfphorylation by catalytic aubunit ofcm-dependent protein kinase ( C A W PK). CB /calmoduli~-depe~deot protein kinase (Cdcan), or protein kinase C. Phoepholsmban was phosphorylated by the different proteln kinese acfivifles in the reconstituted particulate fraction and pvrified by sulfhydrhyl group affinity chromatogrephy a8 described in the legend t o Fig. 3 . Phosphorlyared phospholanban eampleseluted in vaah 2 , a s well Be dephosphorylated phospholamban, vere then incubated withtrypsin or papain (approximstely 1 ug of phoapholamban plvn 0.2 pg of protease) for 16 h a t room temperature. The samples were then subjected to SQS-polyacrylamide gel electrophoresis followed by silver staining (upper) and ( l o w e r ) . Beeauee phosphorylated the phospholamban samples utilized were those most purified by sulfhydryl affinity ehrmmatography, they were n o t maximally phosphorylated. Therefore, the nobilities shown for phosphorylatedsamples ere n o t directly emparable to those depicted in Fig. 3. The lover apparent mobility of dephosphorylated phospholamban relative to phospholamban phosphorylatedby protein kinase C in nom-proreolyzed esmplen (Control. Silver stain) isartifactual, due t o slower .i8mfiOn Of protein samples in the Outermost lanes of the slabgel in this electrophoresis run.

-

The 21-kDa and Isoelectric Focusing of Haior Proteolvtic Fraxmmente2L PhosDhalsman 22-kQs protease-resistant fragments of phospholamban, producedby frypein and papain, respectively, were examined by isoelectric focusing. The major tryptlc fragment exhibited II PI = 6. and the major papain fragment,a PI = 8 (data not shown). Native dephoaphorylsfed phospholamban is very basic with B PI 10 (10). Thus. the protease accessible region on phospholamban likely containsB large proportion Of poeitively charged residues. which Contrlbute to fhe allraline isoelectric paint of the dephosphorylated protein.

-

Peptide Mapping of Cardiac Phospholamban

5159

-

-a

Thin electrophoresis d Phomhorvlafed Phospholamban P e m i d e s None of the proteases rested liberated phosphopeptides from phospholamban whichwere retained in SDS g e l e , lluggesting that they were relatively small (Figs. 1 and 4). The phoephopeptides were well-resolved end recovered in good yield (65.95%) using thin layer electrophoresis, however. Separation of tryptic phosfppeptidee derived f r w pnosphalamban phosphorylated by eAUP-dependent protein kinase, Ca /celolodulin-dependent protein kinase. or protein kinase C is depicted in Pig. 5. Phonpholambsn yielded a aingle high mobility phosphopeptide if phosphorylated by CAMP-dependent protein insse (peptide 3 ) . and a single low mobility phosphopeptide if phosphorylated by Cs2~/=~lmodulin-dependentprotein kinase (peptide 1 ) . Phosphorylation by protein kinase C produced three well-resolved phoaphopeprides, t w o with mobilities the same all phosphopeptides I and 3. and a third. intermediate mobility phosphopeptide (peptide 2). Phoephopeptide 2 YBB nly observed when phospholambsn Y B B phosphorylated by protein kinase C. and the yield of '*P recovered in this peptide vas fairly Consrant over LI range of tryptic conditione tested. Therefore, phosphopeptide 2 probably contained B vnique residue phosphorylated only by protein kinase C.

9

c P-ser

c P-thr

+Origin i

CAMP PK

Ca ICaM PK

PIG. 6. Phmphosmino acid analyeis of phospholambao phoephorylated in earsoplamic reticulum vesic Phospholsnban was pho6pgPryleted in cardiae sarcoplasmic reticvlum vesicles by [y-%iATP in the presence of Ca and calmoddin tCa/Can PK) or added CAW-dependent protein kinase (cAHP PK). The samples were subjeered t o SDS-polyacrylamide g e l electrophoreaie, and the phosphoprotein wa8 eluted from the unfixed gel. hydrolyzed in HC1. and phosphosmino acids then resolved by thin layer electrophoreeis. Non-radioactive amino acid alsndsrds were visuallied with ninhydrin. P-ser, phosphoserine; P-thr. phoaphorhremine. Phosphomino acid analysis Y ~ B11180 performed OD tryptic phosphopeptides 1-3 made phospholamban eluted from en SDS gel (Table I ) . In the experiment shown, 21): of the P incorporated by CAMP-dependent protein kinase Y B S recovered in phoephopetide I, and 55% io phoaphopeptide2j. Only phosphoserine " 8 6 identified in either peptide. Phosphorylation by Ca /caln~dulin-dependenr protein kinase resultedin detectable "P only in peptide 1. This product was indistinguishable f r w the cAUP-dependent protein kinase peptide 1 by either thin layer electrophoresis or chromatography, but contained exclusively phoaphothreonine rather than phoaphoserine2+ For phospholamban phoaphorylared sirnultaogqusly by cAHP-dependent protein kinase and CB /calmoddin dependent protein kinase. P was recovered only in peptides I and 3 predominantly in peptide I which contained both phosphoserine and phosphofhreonine.

35.m

-

-

TABLE I

properties of ~ r y p t i c Phoephopeptidee Derived from Phospholamban p p h o p e p t i d e s were obtained by tryptic h r p l y s i r of phospholambaa phosphorylated /eal.odulin-dependenr protein kinase (Ca /CsU PK). EAUP-dependent pf9t.i" kinase PK), or protein kinase C (PK-C). Distances migrated and recovery of P label (compared to nowhydrolyzed samples) for peptides 1-3 (defined in Fig. 5 ) are given for a typical experiment. Thin layer electrophoresis was conducted for 2 h at 700 V a t pH 3.7. Resolved phoephopepridee were eluted into H20 and subsequently analyzed for phosphoamino acids. by Ca

FIG. 5. Thin layer eleetcophqfesia of tryptic phosphopeptides derived f r w phospholamban phoaphorylared by Ca /calmodulin-depeodeot protein kioaee tCa/Can PK). the carslytic subunit of CAMP-dependent protein kinase ( C A M P or protein kinase C (PK-C). Phospholsnban was phosphorylated in the presence of [YPlATP and each of the three different protein kinase activities in the reconsrituted particulate fraction. 10 p g of phosphorylated fraction Y A G then incubated with 2 pg of trypsin in 50 p l of proteolysis buffer for 16 h at room temperature. acid Reaction aliquots were spotted on thin layer plates mobilities and resolved by thin-layer electrophoresis, and the resulting autoradiogram isproduced shown. Resolved phosphopeptides are labeled 1 , 2, and 3 in order of increasing mobility. Samples were s m t t e d near ?he anode ( r ) .

Sf),

Peptide

PeptidesProtein kinase

CaZ'/CaM PK

Phosphopeptide 3 , on the other hand, whether originating f r w phoapholamban phosphorylated by CAMP-dependent protein kinase or protein kiosae C. covld be completely converted t o a mobility form identical t o phosphopeptide 1 , when the trypsin concentration used for proteolysis was increased ten-fold (data not shown). Phosphopeptide I. whether geqerated from phospholmban phosphorylated by CAMP-dependent protein kinase or Ca Icelmodulin dependent protein kinase, could not be further resolved in P second dimension of thin layer chromatography in B number of different s 0 1 ~ e n tsystems teated (data nor shown). Thus. the ~ e q ~ e n c eof s phosphopeptidea I and 3 are probably Overlapping.

;:$p

PK

6 .I

1

6 .O 11.9

3

Thr

Phosphosmino

50

21

55

PK-C

-

Partial Acid Hydrolysis Phosphorylated Phospholamban4Peptide8 Phosphosmino acid analysis was performed on phoapholanban phoephorylated in intact cardiae 8srcoplsBmic reticulum ves'clea by added catalytic svbunif of CAW-dependent protein kinaseor by endogenous Ca*t/.~tmodulin-dependenr protein kinase (Fig. 6 ) . The phosphoprotein was eluted f F w an unfixed SDS g e l . avbjected t o partial acid hydrolysis, and phosphoanino resolved by thin layer eleCtrOphoreSi8. Phosphorylation of phospholnmban with /celmodulin-dependent protein kinase occurred only a t threonine residues, and with CAW-dependent protein kinase, only st serine residue? (Fig. 6). These resvlrs clearly demonatrare that CAMP-dependent protein kinase and Ca */calnodulin-dependent protein kinase phosphorylate different sires an phoapholanban. "

CAMP

1

32P-Rec0very

Phosphosaino acid snalyni. of peptides generated f r w h08pholarnban phosphorylated by protein kinase C was more complex. The distribution of "P vas between the three phonphopepfidee 1-3. Both phosphothreonine and phosphoserine were recovered f r w peptide 1 , but only phosphoserine " 8 8 present in peptides 2 and 3 (Table I). Peptide 3 generated f r w phospholamban phoophorylnred by either CAMP-dependent protein kinase or protein kinaae C could be converted to peptide 1 with B high concentration of trypsin (data not shown), but yet coulq be disringuiohed from peptide I originating from phospholamban phosphorylated by Ca +/c.lm~dulin-dependent protein kinase by phosphoanino acid analysis (Table I). Peptide 2, obtained only after phosphorylation by protein kinase C. contained phosphonerine (Table I) and could not be further hydrolyzed by trypsin a t high concentrations. Taken togeeher, our observftions euggest that the t w o residues phosphorylated by the CAMP-dependent and Cs +/calmodulin-dependent protein kinaGea reside within the o m e region on phosphalsmbsn, which may be ieolated as either a limit tryptic peptide (peptide 1 ) or as B larger peptide containing sf least one additional basic residue (peptide 3 ) . Incomplete tryptic hydrolysis a t sites near phosphorylated residues has been reported by others (31-33). These results s 1 m suggest that the serine phosphorylated in pepride 2 by protein kinase C is a second distinct serine residing in B eeparate locua from the serine phosphorylated by CAW-dependent protein kinase. Preliminary sequence data indicate that the phosphorylated serine end threonine residues in peptides and 3 are adjacent t o one anofher, and located near the m i n e terminus of the ~rofein.

4