Unique Phosphorylation Site on the Cardiac Ryanodine Receptor ...

T H EJOURNAL OF BIOLOGICAL CHEMISTRY 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266, No. 17, Issue of June 15, pp. 11144-11152, 1991 Printed in U.S.A.

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Unique PhosphorylationSite on the Cardiac Ryanodine Receptor Regulates Calcium Channel Activity* (Received for publication, November 26, 1990)

Der’rickR. Witcher, Richard J. KovacsS, HowardSchulmans, Dominic C. Cefali, and Larry R. Jones7 From the Krannert Institute of Cardiology and the Departmentsof Medicine and Pharmacology, Indiana University Schoolof Medicine, Indianapolis, Indiana 46202, $Department of Medical Research, Methodist Hospital of Indiana, Indianapolis, Indiana 46202, and §Department of Pharmacology, Stanford University School of Medicine, Stanford, California 94305

Ryanodine receptors have recently been shown to be the Ca2+release channels of sarcoplasmic reticulum in both cardiac muscle and skeletal muscle. Several regulatory sites arepostulated to exist on these receptors, but to date, none have been definitively identified. In the work described here, we localize one of these sites by showing that the cardiac isoform of the ryanodine receptor is a preferred substrate for multifunctional Ca2+/calmodulin-dependentprotein kinase (CaM kinase). Phosphorylation by CaM kinase occurs at a single site encompassing serine 2809. Antibodies generated to this site react only with the cardiac isoform of the ryanodine receptor, and immunoprecipitate only cardiac r3H]ryanodine-binding sites. When cardiac junctional sarcoplasmic reticulum vesicles or partially purified ryanodine receptors are fused with planar bilayers, phosphorylationat this site activates the Ca2+ channel. In tissues expressing the cardiac isoform of the ryanodine receptor, such as heart and brain, phosphorylation of the Ca2+release channel by CaM kinase may provide a unique mechanismfor regulating intracellular Ca2+release.

Ca2+ conductances (2-7), protease sensitivities (11, E ) , calmodulin-binding capabilities ( l l ) ,and modulation by allosteric regulators such as Ca2+, Mg2+, ATP, and calmodulin (13-15), they also exhibitseveral differences in protein structure and function. Quantitative differences have been noted on the effects of modulators on ryanodine binding to thetwo proteins (16-18), as well as on Ca2+ channel kinetics (13, 14). In addition, the cardiac ryanodine receptor exhibits a slightly smaller apparent molecular weight than the skeletal muscle receptor on SDS-PAGE ( l l ) ,and monoclonal antibodies can be made which react with the cardiac receptor but not the skeletal receptor (16). Recent work oncharacterization of the two ryanodine receptors has culminated in elucidation of the primary structures of the proteins by sequencing of their cDNAs (19-21). Consistent with thedifferences between the two protein isoforms noted above, the cardiac and skeletalmuscle receptors have been found to be the products of different genes, with overall amino acid identities of 66% (21). Both protein isoforms are very large, containing approximately 5,000 amino acids and exhibiting predicted molecular weights of 564,711 for the cardiac protein (21) and 565,223 (19) or 563,584 (20) for the skeletalmuscle protein. In the native state, ryanodine receptors are arranged as tetramers(1-7). In an earlier study (22), we demonstrated that the canine Ca2+release from the SR’ causes an increasemyoplasmic in Caz+concentration which leads to muscle contraction (1). cardiac high molecular weightprotein (or ryanodine receptor; Recently, the sites of Ca2+ release have been identified and Ref. 3) was an excellent substrate for the multifunctional purified from both cardiac (2-4) and skeletal muscle SR (5- CaM kinase(23,24) endogenous to junctional SR membranes. In the work described here, we show that phosphorylation of 7) and shown to be the same as the ryanodine receptors or highmolecularweightfeet proteins,thestructures which the cardiac receptor by CaM kinase occurs a t a single site, which isnotsubstantiallyphosphorylatedintheskeletal attach the transverse tubules to the junctional SR both in muscle receptor,andthatphosphorylation of thecardiac intact tissues and isolated membrane fractions(1, 8-10). Although the Ca2+release channels from cardiac and skel- ryanodine receptorat this site activates the Ca2+ channel. Our data are the first to support the theoretical model of Otsu et etal muscle show many similarities such as nearly identical al. (21), that the modulator-binding sites of the cardiac ry* This work was supported by Grants HL28556 and HL06308 (to anodine receptor are contained withinresidues 2619-3016. L. R. J.) from the National Institutes of Health, an American Heart (Indiana Affiliate) Predoctoral Fellowship award (toD. R. W.), Grant 880970 from the American Heart Association (to H. S.), and by the Herman C. Krannert Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must thereforebe hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact. ll To whom reprint requests should be sent: Krannert Institute of Cardiology, 1111 W. 10th St., Indianapolis, IN 46202-4800. The abbreviations usedare: SR, sarcoplasmic reticulum; SDSPAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; CaM kinase, multifunctional Ca’+/calmodulin-dependent protein kinase;cAMPkinase,CAMP-dependentproteinkinase;CHAPS, 3[ (3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid;HEPES,N-2-hydroxyethylpiperazine-N’-2-ethanesulfonicacid;MOPS,3-(N-morpho1ino)propanesulfonic acid.

EXPERIMENTALPROCEDURES

Materials-CaM kinase was isolated from rat brain as described previously(25). cAMPkinase(thecatalyticsubunit frombovine heart) was purchased from Sigma or prepared according to Beavo et al. (26). Identicalresults were obtainedwitheithercAMPkinase preparation. [y-”PJATP and ”‘I-protein A were obtained from Du Pont-New England Nuclear.Bovine brain calmodulin, protein Aagarose, iminodiacetic acid-agarose, and Sephacryl S-500 were purchased from Sigma. MembranePreparation-Canine cardiacjunctionalSR vesicles (vesicles sedimenting at the1.0/1.5 M sucrose interface)were isolated as described previously (3) and extracted with 50 mM Tris, 300 mM KC], and 25 mM sodiumpyrophosphate(pH 7.0). Salt-extracted skeletal muscle SR membranes were isolated (27) from predominately fast (tensor fasciae latae) and slow (vastusintermedius) muscles.

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Cardiac Ryanodine Receptor Phosphorylation Autoradiogram

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p g of hrain CaM kinase and 2 p g o f ralmodulin. Phosphorylation hv CAMP kinase ( 1 . 4 p g o f the ratalytir srrhunit) PK was conducted under identical conditions with the omission o f ('a'' + + + + CaM and calmodulin from the incuhation huffer. lieartions wrre terminated hy adding 20 pl of SDS tlissoriation medium. andsamples were suhjected to SIIS-PAGE in 6"; 1.5-mm thickness gels ( 2 % ) .followed hv autoradiography ( 11). To achieve the greatest resolutiono f ryanodine receptors. gels were electrophoresed at :{5 mt!, and elrrtrophoresiswascontinuedfor 20-30 min alter the dye front rxitrd the -200 hottom of t h e gels. Purification ~f f ' h o s p h o ~ l n t c d('nrdinr Rynnrdinr. Hccrptnr 50 mg of cardiac junctional Sf< vesicles were phosphonlatetl for 5 min at 30 " C in 50 ml of huffer containing 50 mM hLIOI'S ( p H 7 . 4 ) . 1 0 mM " ~ -" '" ' 6 97 MgCI,, 4.8 mM EGTA, mM CaCl,, 0.5 mM S a F . 0.4 mg of ralmotlulin, 25 pg of CaM kinase, and 40 p M [-,- I']ATl'. The mrmhranes - ".si.. were then rapidly pelleted at 4 "(' and resuspended t o 4 ml in 0.25 M "66 sucrose, 90 mM histidine (pH 7.4). SDS and Sd'l were added t o linnl concentrations of 4"; and 0.5 M. respertively, antl the sample was \ heated to 50 "C and sedimented. The supernatant. rontaining solu1 2 3 4 5 h l H 9 1 2 3 4 5 6 7 8 9 hilized ryanodine receptors, was loaded onto a 90 X 'L.I)-rmSephnrryl S - 5 0 0 column equilihrated with 20 mM Tris. 0.5 M NaC'l. 0 . 2 ' ; SDS. FIG. 1. Phosphorylation of canine cardiac and canine fast and slow skeletal muscle ryanodine receptors in S R mem- a n d 1 mM dithiothreitol(pH 8).T h e column was run at r o o m temperature at a llow rate of 12 ml/h.'Three-mlfrartions were branes by CaM kinase. J'hosphorylat ion reactions were conducted collectedwhilemonitoringahsorhance at 2x0 nm. XIO-400 p g of for 5 min and stopped with S I N 46 p g of memhrane protein were ryanodine receptor protein were routinely recovered from :io m g of electrophoresed per gel lane. 'The left panel shows the Coomassie memhrane protein. Protein concentrations wrre dc.terminrd hy the 13lue-stained gel, and the right panel is the corresponding autoradimethod of Schaffner and Weissman ( 2 9 ) . ogram. The arrou~hcnds and small orrows indicate the cardiac and T n p t i r I'rotco/ysis a n d Isolntion of I'h~~splrr~~ptidr--liOO-HOO p g nf skeletal muscle ryanodine receptors, respectively. The nstrrisk d e s ignates a phosphorylated protein visible in the slow skeletal fractions purified phosphorylated ryanodine receptors frnm two rnlumn runs not related to the ryanodine receptor. Purified CaM kinase ( I ' K ) and weredialyzed against 4 liters of50 mM NH,HCO,. 1 nlM dithiothreitol for I6 h at room temperature. Tr.ypsin was ad(letl at a 1/50 weight as indicated.Mobilitystandards calmodulin(C'nM)wereadded f o r 24 h ;It :{7 '('. T h e digestwas ratio. and proteolysis contlucted (M. X 10") are displayed hetween the two panels. then lyophilized, soluhilized in 0 . 1 M acetic arid. and applied t o a 0 . 4 ml column of iminodiacetic arid-agarose snturatrd wrth Fr'*as (le. scrihed (30). The column was washed with two rrrlumn vnllrnres of 0.1 M acetic acid (load), then three rolumn volumes o f the following huffers: 0.1 M sodium acetate (pH 5.2): 0.1 %f ammonium arrtate (pH 5.9); 0.1 M ammonium acetate (pH H . 6 ) : 0.1 Sf ammonium arrtate ( p H 10);and 0 . 2 M I