Biochimica et Bioph)'sica Acta. 1118 {19921277-287 ~:, 1992 El.,,cvicr Science Publishers B.V. All rights reserved 0167-4838/92/$05.01b

277

BBAPRO 34079

Phosphorylation of the cardiac isoform of calsequestrin in cultured rat myotubes and rat skeletal muscle Steven E. Cala and Kathryn Miles

*

Laboraton" of Mtrlecular and Celhdar Neuroscience, The Rocktfeller Unicersitv. New York, IVY (U.S.A.)

(Received 18 April 1991) (Revised manuscript received 8 August 19911

Key words: Calsequestrin: Casein kinase II; M~'otube: Development: Sarcoplasmic reticulum: (Muscle)

Calsequestrin is a high-capacity Ca~+-binding protein and a major constituent of the sarcoplasmic reticulum (SR) of both skeletal and cardiac muscle. Two isoforms of calsequestrin, cardiac and skeletal muscle forms, have been described which are products of separate genes. Purified forms of the two prototypical caisequestrin isoforms, dog cardiac and rabbit fast.twitch skeletal muscle calsequestrins, serve as excellent substrates for casein kinase II and are phosphorylated on distinct sites (Cala, S.E. and Jones, L.R. (1991) J. Biol. Chem 266, 391-398). Dog cardiac calsequestrin is phosphorylated at a SO to 100-fold greater rate than is rabbit skeletal muscle calsequestrin, and only the dog cardiac isoform contains endogenous Pi on casein kinase 11 phosphorylation sites. In this study, we identified and examined both calsequestrin isoforms in rat muscle cultures and homogenates to demonstrate that the cardiac isoform of calsequestrin in rat skeletal muscle was phusphorylated in vivo on sites which are phosphorylated by casein kinase !! in vitro. Phosphorylatiou of rat skeletal muscle calsequestrin was not detected. in tissue homogenates, cardiac and skeletal muscle calsequestrin isoforms were both found to be prominent substrates for endogenous casein kinase il activity with cardiac calsequestrin the preferred substrate. In addition, these studies revealed that the cardiac isoform of calsequestrin was the predominant form expressed in skeletal muscle of fetal rats and cultured myotubes.

Introduction

Calsequestrin is a high-capacity CaZ+-binding protein that is abundant in striated muscle SR [1-11]. Calsequestrin is found in junctional SR of both skeletal and cardiac muscle as part of an electron-dense matrix that juxtaposes the sarcolemma [3-6]. Though it is widely believed that calsequestrin serves as the primary storage for calcium in the SR, its function has not been established. Certain biochemical characteristics of calsequestrin are not adequately explained by a simple paradigm in which calsequestrin functions as a passive

* Present address: SUNY/HSC at Brooklyn, Department of Anatomy and Cell Biology. Box 5. 450 Clarkson Ave., Brooklyn. NY 11203-2098. Abbreviations: SR, sarcoplasmic reticulum: SAC. Staphylococcus au. reus cells; SAP, 8taphylococclts aureus proteinase; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis. Correspondence: S.E. Cala. Krannert Institute of Cardiology, !!il West 10th Street, Indianapolis, IN 462024800, U.S.A.

Ca-"+ store. First, calsequestrin undergoes a change in conformation upon binding of Ca z+ that results in the loss of a hydrophobic site [9,12-15]. This site imparts to calsequestrin a property of Ca2+-dependent binding to hydrophobic surfaces [9-14]. A 26 kDa protein has been identified in SR vesicles that binds to calsequestrin in vitro in this same manner [14]. Other studies have also suggested that interactions occur between SR proteins and calsequestrin [16,17]. Determination of the function of calsequestrin is further complicated by the presence of several other prominent Ca" +-binding proteins of unknown function in both the free and junctional SR [11,18-20]. Many of these intralumenal SR proteins have recently been cloned and sequenced

[21-241. Two genetically distinct isoforms of calsequestrin, a skeletal muscle and a cardiac form, have been cloned and sequenced from rabbit fast-twitch skeletal muscle [25] and dog heart [15], respectively. The amino acid sequences for these two isoforms are only 65% identical and cardiac calsequestrin contains a highly acidic 31-amino acid extension [15]. Recently, both purified

278 calsequestrin isoforms were shown to be excellent substrates for casein kinase II and the sites of phosphoD'lation were localized to distinct COOH-terminal residues in the two ca|sequestrin isoforms [2~. Rabbit skeletal muscle calsequestrin is phosphorylated on Thr353 while dog cardiac calsequestrin is phosphorylated at a much greater rate on a cluster of three serine residues 1Ser-378382.386) contained within the cardiac-specific 31-amino acid tail [26]. Moreover, purified dog cardiac calsequestrin, but not rabbit skeletal calsequestrin, ~as shown to contain endogenous P, (!.0-1.5 mol P, per tool calsequestrin) localized to the same cluster of serines that are labeled in vitro by casein kinase ii [26]. These studies on purified calsequestrin isoforms suggest that the cardiac isoform of calsequestrin undergoes a phosphorylation by casein kinase I! in vi,,'o that does not occur for the skeletal muscle form. To further examine the phosphoD'lation of calsequestrin isoforms by casein kinasc ii. we examined rat skeletal muscle and cultured rat myotubes which contain both protein isoforms. Despite the fact that both calsequestrin isoforms were shown to be major substrates for an endogenous casein kinase ll-like act(rib' in muscle homogenates, in vivo we could only detect phosphoD'lation of cardiac calsequestrin. Levels of skeletal muscle cr.i~sequestrin in myotubes, as well as in fetal muscle. ~verc greatly reduced compared to adult muscle, and cardiac calsequestrin appeared to be the predominant isoform in fetal preparations. Materials and Methods

Materials Adult rats and pregnant female rats were obtained from Charles River Laboratories. [~-'P]orthophosphoric acid, [-~:P]ATP and [IZ~l]protein A were from New England Nuclear. Formalin-ftxed Staphylococcus attreus cells (SAC, Pansorbin) were from Chemicon (Los Angeles). Protein A-Sepharose was from Pharmacia. Staphylococcus aureus proteinase (SAP) was from ICN lmmunoBiologicals. Aprotinin (Trasylol) was from Mobay Chemical (New York). Nitrocellulose membranes were from Schleicher and Schuell. Acrylamide was from Serva. Phosphocellulose paper (P81) was from Whatman. A synthetic peptide (RKREEETEEE) substrate for case~.a kinase II [27] was from Peninsula Laboratorie':. Poly-t-!ysine (MW 30000-70000) and heparin were from Sigma. Other chemicals were from Sigma and Fisher. Casein kinase 11 from rabbit skeletal muscle was kindly provided by Drs. Ken Mackic and Angus Nairn (The Rockefeller University., New York, NY). A peptide (5-24 amide) inhibitor of cyclic AMPdependent protein kinase [28] was synthesized and purified by the Yale Protein and Nuclcotide Chemistry Facility.

Preparation of mttscle and myombe homogenates Hind limb music was removed from 20-21-day rat embD'os and frozen at - 70 °C. Frozen tissue was added to I0 vols. of 20 mM Hepes (pH 7.8), 2 mM EDTA. 0.2 mM dithiothreitol, 0.002% aprotinin, 20 # g / m l leupeptin. 4 ttg/ml pepstatin A and 20 a g / m l ch~mostatin (added as a 1/100{! dilution of stock dissolved in DMSO). and homogenized using a Polytron tissue homogenizer (Brinkmann instruments) at 4O% of full speed. Homogenates were immediately frozen in small aliquots and stored at - 7 0 ° C for subsequent use. For myotube cultures, media was removed and dishes were washed twice with cold saline. Cells were suspended by trituration in homogenization buffer then homogenized and frozen as described above. Protein concentrations were determined following the procedure of Lo,,vr." etai. [29].

Purified proteins Casein kinase Ii was purified from rat heart as previously described [26]. Skeletal muscle and cardiac calsequestrins were purified using phenyl-agarose as previously described [9]. In cases where phenyl-agarose chromatography led to co-purification of both skeletal muscle and cardiac isoforms of calsequestrin, further purification was achieved by chromatography on Mono O (Pharmacia) using a fast-protein liquid chromatography (FPLC) unit. Using an NaCi concentration gradient, skeletal muscle calsequestrin eluted after cardiac calscquestrin.

Production of antisera Skeletal muscle calsequestrin was purified from adult rat hind limb muscle using phenyl-agarose and Mcno Q. The purest fractions of skeletal muscle calsequestrin were used to immunize rabbits (anti-skeletal CSQ). Antiserum raised in rabbits against the canine cardiac calsequestrin (anti-cardiac CSQ), purified using phenyl-agarose as described [9], was provided by Larry Jones (Indiana University School of Medicine, Indianapolis, IN).

lnmumolabeling of electrophorelic transfers ]?om SDSPAGE Samples of homogenates (20 ag) were added to one-fifth volume of a solution containing 15'~, glycerol, 150 mM Tris-HCi (oH 6.8), 10% SDS, 5 mM dithiothreitol and 0.01% bromophenol blue (dissociation buffer) and electrophoresed on SDS-polyacrylamide gels (7% act3,1amide) using the Laemmli buffer system [30]. EIectrophoretic transfers were done as described by Towbin et al. [31] using Tris-glycine in 20% methanol as transfer buffer. Transfers were made onto 0.2 a m nitrocellulose using 200 mA for 12-16 h. Membranes were stored in cold H , O until use. immunolabeling of transfers was done essentially as described by Burnette

279 [32]. All steps of immunolabeling were carried out at ambient temperature in 20 mM sodium phosphate (pH 7.4), 150 mM NaCi, 0.05% Tween 20 (buffer A). Membranes were washed once in buffer A for 30 min, then treated for 2 h with fresh buffer A containing antiskeletal CSQ, anti-cardiac CSQ or a mixture of both antisera, each at I/I000 dilution. Membranes were washed three times for I0 rain, then incubated with 1 /~1 [l~l]protein A/ml buffer A for I h. Following extensive washing, membranes were dried and exposed to Kodak XAR-5 film without an intensifying screen.

In ritro pltosplzorylation of tissue homogenates Phosphorylation assays were done with 20 ~g of protein in 10O ill of a buffer consisting of 20 mM Mops (pH 7.4), 0.5 mM EGTA, 10 mM MgCI_,, 150 mM NaCI, 0.1% Triton X-10O and 10 #M [3'-P]ATP (approx. 5~Ci/tube), with other additions indicated in figure legends. A 2 rain preincubation (prior to ATP addition) and 10 rain reaction were carried out at 30 "C. Reactions were stopped by addition of 10/zl of 10% SDS, followed by a 1 rain incubation at 60 °C. Two 50 p.I aliquots were then removed. One aliquot was combined with 10 ~ti of dissociation buffer, and a 50 #1 aliquot of this was analyzed by SDS-PAGE. The second aliquot was used for immv:~oprecipitation.

hnmunoprecipitation o[ #J ritro-phosphorylated calsequestrin To prepare SAC, 10 ml of the stock was boiled in 100 ml 3% SDS and 2% O-mercaptoethanol for 10 rain, pelleted, then washed three times with immunoprecipitation buffer (20 mM Tris-HCI, pH 7.6, 125 mM NaCL 50 mM NaF, 10 mM EDTA) and finally resuspended in the same buffer containing 10% bovine serum albumin and 0.5 mM phenylmethylsulfonyl fluoride. 50/~1 of phospho~lated homogenate were added to 20 #1 of immunoprecipitation buffer and 30 ~1 of 15% Triton X-100. To "preclear" the samples, 25 pi of 10% SAC (w/v) were added and the tubes were incubated for 15 rain and then centrifuged at 10000 × g for 10 rain. 5 ttl of anti-skeletal CSQ plus 5 ttl of anticardiac CSQ were added to the samples and incubzted at room temperature for 2 h, at which time 100 #1 of SAC was added. After a 30 rain incubation, the immune complexes bound to SAC were centrifuged at 10000 ×g for 10 rain. The pellets were sonicated in 1 ml immunoprecipitation buffer containing I% Triton X-100, then re-pelleted. A second wash was done using the same buffer without Triton X-100. The final pellet was resuspended in 60 ~1 of a solution made up of 50 tL! H.,O and 10 #1 dissociation buffer. Following centrifugation, a 50 ttl aliquot of the supernatant was analyzed by SDS-PAGE.

Muscle cell culture and metabolic labeling Primary muscle cell cultures were established from hind limb muscles of 20-21-day rat embryos. Myoblasts were enzymatieally dissociated with 0.5% trypsin and cultured in Dulbecco's modified Eagle's medium (GIBCO) supplemented with 20% (v/v) fetal calf serum and 33 mM glucose. After 2 days this medium was replaced with medium containing 10% (v/v) horse serum and 2% (v/v) chicken embryo extract in order to promote muscle cell differentiation. Experiments were performed on 7-12-day-old myotube cultures. For metabolic labeling with radioactive phosphate, the culture medium was replaced with phosphate-free Eagle's minimal essential medium (Flow Laboratories, McLean, VA). [-~zP]Orthophosphate (2 mCi/ml)was added and the cells were incubated for 5 h, a procedure found to be sufficient to achieve isotopic equilibrium of ATP.

hmmmoprecipitation of metabolically labeled calsequestrin Cuhured myotubes were rinsed with isotonic phosphate-buffered saline and calsequestrin was solubilized from each culture dish by using 500 pl of a lysis solution containing 0.5% Triton X-100, 25 mM "Iris (pH 7.4), 75 mM NaCI, 5 mM EDTA, 5 mM sodium pyrophosphate, 50 mM NaF, i mM NaVO 3 (ortho), 50 mM PMSF, 5 mM N-ethylmaleimide, 5 /~g/ml leupeptin, 5 /.tg/ml pepstatin, 5 /~g/ml chymostatin, 5 #g/ml antipain, 5 U/ml Trasylol. Insoluble material from each culture extract was removed by centrifugation for 5 rain at 10000 x g, and the supernatants containing calsequestrin were incubated with 5 #l anti-cardiac CSQ plus 5 ~1 anti-skeletal CSQ for I h at 4"C. The immune complexes were isolated by incubating for 1 h with protein A-Sepharose followed by sedimentation at 10000×g for 1 rain. The protein A-Sepharose pellets (50 ~l) were washed with 3 × I ml i]/sis solution before 200 ~! of 1% (w/v) SDS was added to each pellet. The suspensions were vortexed and pelleted. The resulting supernatants were removed, combined with 200 txl Tris-buffered saline (pH 7.4) and 100 #1 25% Triton X-100 and subjected to a second cycle of immunoprecipitation [33], The immune complexes were eluted from the final protein A-Sepharose pellets with 150 #! of a buffer made by a one-sixth dilution of dissociation buffer in HzO, and analyzed by SDS-PAGE and autoradiography.

Limited proteolytic phosphopeptide maps ProteoIysis of proteins contained in gel pieces using SAP was performed as described by Cleveland et al. [34]. Gel pieces containing phosphorylated calsequestrin were excised from a dried gel, then reswollen in 1 ml of a solution made by a one-sixth dilution of dissociation buffer in H20 for 10 rain at 30°C with

280 occasional shaking. The pieces were pressed into wells of a 0.75 mm thick SDS-polyacrylamide gel, made with a 15% acrylamide separating gel and a 6 cm, 3cA separating gel. 10 ~tl of 0.5 mg/ml SAP solution was overlaid on the gel pieces, and the gel was run at 5 mA. The gel was dried without fixation, then placed against Kodak XAR-5 film. R~.sults

Identification of rat calsequestrin isoforms by immunoblm analysis lmmunoblot analyses of rat skele!al muscle and rat heart homogenates were used to determine the distribution of calsequestrin isoforms in rat muscle tissues, and to demonstrate the relative reactivities of the two calsequestrin antisera used in this study. Anti-skeletal CSQ (Fig. 1, left panel) recognized a 65 kDa protein in rat skeletal muscle homogenate (lane 2). This protein

AntiSkeletal

CSQ

Anti-

Cardiac CSQ

11213 11213

Developmental distribution of rat calsequesuin isoforms and characterization of rat calsequestrin immunoreactiv-

i0" Origin

97 S "--4"-

was present in rat heart in only trace amounts (lane 3). The 65 kDa pro!ein co-migrated with rat skeletal muscle calsequestrin that was purified using the phenylagarose method (lane 1), indicating that the 65 kDa protein was skeletal muscle calsequestrin. When these rat muscle preparations were analyzed with anti-cardiac CSQ (Fig. 1, right panel), both the 65 kDa protein and a 55 kDa protein were immunolabeled in rat skeletal muscle homogenate (lane 2, right panel). The 55 kDa calsequestrin co-migrated with calsequestrin in rat heart homogenate (lane 3, right panel) indicating that it was rat cardiac calsequestrin. The phenyl-agarose purified rat calsequestrin from rat skeletal muscle (lane 1. right panel) also contained both isoforms. The set of high molecular-weight immunoreactive protein bands seen in rat heart (lane 3, right panel) have been previously described as calsequestrin-like proteins [9]. Interestingly, the relative immunoreactivities of rat calsequestrins to our two antisera were quite similar to those reported by Damiani et al. [35] for two calsequestrin isoforms in rabbit slow-twitch skeletal muscle using antisera that were not raised in rabbits.

66

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43

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lmmunoblot analysis was used to compare the levels of calsequestrin isoforms in fetal-derived cells with those from tissue preparations. Labeling of immunoblots with anti-skeletal CSQ (Fig. 2, left panel), showed that fetal rat skeletal muscle (lane 2) and myotubes (lane 3) contained significantly lower levels of skeletal muscle calsequestrin than did adult skeletal muscle (lane 1). Furthermore, labeling with anti-cardiac CSQ (middle panel) indicated that, in fetal skeletal muscle and cultured myotubes, cardiac calsequestrin was the predominant isoform. A composite picture was seen for these homogenates when both antisera were used simultaneously (right panel),

29 Phosphoo'lation of rat skeletal and cardiac isofonns of calsequestrin by casein kinase H in vitro

Front Fig. I. tZ~l-autoradiograph of immunoblot identifying calsequestrin i.soforms in rat muscle tissues using antibodies against rabbit skeletal muscle calsequestrin {anti-skeletal CSO) or dog cardiac calsequestrin (anti-cardiac CSQ). Phenyl-agarose purified rat calsequestrins (2/.tg) and rat muscle homogenates (50 pg) were separated by SDS-PAGE and transferred to nitrocellulose paper. Bound antibodies were visualized using tX%labeled protein A and autoradiography. Lane 1.

both panels, phenvl-agarose purified rat calsequestrins from skeletal muscle; lane 2, both panels, rat skeletal muscle homogenate; lane 3, both panels, rat heart homogenate.The exposure time used to obtain the autoradiograph shov,n in the right panel was approx, twice that used for the autoradiographshown in the left panel.

Purified rat calsequestrin isoforms were readily phosphorylated by casein kinase I1 (Fig. 3, lanes 3 and 4). While both rat calsequestrin isoforms served as effective substrates for casein kinase I1, rat cardiac calsequestrin (lane 4) was much more highly phosphorylated than was rat skeletal muscle calsequestrin (lane 3, upper band) under the reaction conditions used. In fact, the major phosphoprotein in preparations of skeletal muscle caisequestrin was usually cardiac calsequestrin (cf lane 3) though it was a minor protein component of these preparations (cf lane 1). Heparin (2 t~g/ml), an inhibitor of casein kinase II [36], led to decreases in phosphorylation of skeletal muscle and cardiac calsequestrins of 78 and 74%, respectively

281 (lanes 5 and 6). As was previously reported for dog cardiac and rabbit skeletal muscle calsequestrins, stoichiometric amounts of Pi could be incorporated into either rat calsequestrin isoform (approx. 1.0 tool P~ per mol protein) although for skeletal calsequestrin such a level required much higher concentrations of enzyme and longer incubation times (data not shown).

shown). If casein kinase !1 was added to the homogenates, phosphorylation of calsequestrin isoforms was increased and the effects of heparin and polylysine were qualitatively similar (lanes 4-6). A higher concentration of heparin (2 # g / m l ) was needed to inhibit casein kinase II-stimulated phosphorylation of calsequestrin isoforms in these homogenates (data not shown, cf Fig. 5). The right panel of Fig. 4 displays the skeletal muscle homogenates before immunoprecipitation. Calsequestrin isoforms were major substrates for casein kinase II, easily detectable in muscle homogenates whether using the endogenous casein kinose II-like activity or following the addition of casein kinase I1. A notable feature of these data is the stimulation of phosphorylation that occurred for most phosphoproteins following the addition of 0.1 p.g/ml polylysine to the reaction mixture. Similar sets of proteins were phosphorylated when polylysine was added to reactions catalyzed by endogenous kinase or by casein kinase I! (compare lanes 2 and 5, right panel). Phosphorylation of calsequestrin was readily observed in homogenates of rat cardiac and skeletal muscle tissues from both adult and fetal rat (Fig. 5). The identities of rat cardiac and skeletal muscle calsequestrins on SDS-polyacrylamide gels were verified by their co-migration with purified rat ealsequestrins (cf Fig. 1), by their immunoprecipitation using specific

Phosphorylation bz ritro of calsequestrin from rat muscle Using the two caisequestdn antisera described above, we immunoprecipitated calsequestrin from rat muscle homogenates following in vitro phosphorylation of proteins by endogenous protein kinases. Endogenous phosphorylation was compared to that observed when casein kinase II was added to the reaction mixture. As seen in Fig. 4, left panel, skeletal muscle from adult rat contained an endogenous calsequestrin kinase (lane 1). The endogenous kinase was nearly completely inhibited by 0.2 # g / m l hepadn or by 0.1 # g / m l polylysine (lanes 3 and 2, respectively). Polylysine is a known effeetor of casein kinase II-dependent phosphorylation, leading to increased or decrea~d phosphorylation, depending upon the substrate [37-39]. Endogenous phosphorylation was not significantly affected b:.' the addition of the peptide inhibitor of cyclic-AMP-dependent protein kinase or by addtion of 0.75 mM CaCI 2 to the reaction mixture (data not |

|

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Fig. 2. "-~l-auloradiographof immunoblot showing labeling of rat muscle preparations by anti-skeletal CSQ and anti-cardiac CSQ antibodies, Samples of rat skeletal mu~le homogenates (30 pg) from adult (A). fetal (F) and cultured myotubes(M) were separated by SDS-PAGEand transferred to nitrocellulose paper. Bound anti~)dies were visualizedusing ~2~l-proteinA and autoradiography.

282 antisera (cf Fig. 4). and by the similarity of their phosphopeptide maps and those produced by phosphorylation of purified calsequestrins using casein kinase II (of Fig. 7). In rat cardiac and skeletal muscle homogenates, heparin inhibited phosphorylation of calsequestrins and several other phosphoproteins in a concentration-dependent manner. Heparin appeared to be less effective in neonatal muscle homogenates compared to those from adult rat. For example, in adult tissue homogenates, 10 t~g/ml heparin inhibited caisequestrin phosphorylation nearly completely (Fig. 5, second and fourth panels), while in fetal tissue homogenates, 10 # g / m l heparin led to only a minor decrease in phosphorylation (Fig. 5, first and second panels). This discrepancy was also seen when calsequestrin was immunoprecipitated from adult and fetal skeletal muscle samples (data not shown). Interestingly, cardiac and skeletal muscle isoforms of calsequestrin were the major casein kinase I! substrates observed in cardiac and skeletal muscle homogenates from adult rats (second and fourth panels). Furthermore, consistent with the results obtained by immunoblotting of fetal skeletal muscle and myotube homogenates, in vitro phosphorylation suggested that cardiac calsequestrin was the predominant isoform in fetal skeletal muscle.

Phosphorylation of calsequestrin in cuhured rat myotubes Cultured rat myotubes prepared from fetal rat skeletal muscle were metabolically labeled for 5 h using [3Zp]orthophosphate. Calsequestrin was immunoprecipitated from labeled cell culture homogenates using either anti-skeletal CSQ or anti-cardiac CSQ antisera. The results of a typical immunoprecipitation experiment are shown in Fig. 6. lmmunoprecipitation using anti-skeletal CSQ antiserum did not yield any detectable phosphoprotein of M, = 65 000 (lane 1). This result was consistently obtained in numerous immunoprecipitations from myotube cultures prepared from a number of fetal rat muscle preparations. In contrast, immunoprecipitation from labeled myotubes using anti-cardiac calsequestrin antiserum consistently yielded a phosphoprotein having Mr = 55000 (lane 2). This phosphoprotein exhibited a mobility on SDS-polyacrylamide gels that was indistinguishable from that of purified rat cardiac calsequestrin phosphorylated in vitro (lane 4). Moreover, immunoprecipitation of this 55 kDa phosphoprotein was specifically inhibited by pretreatment of the anti-cardiac CSQ antiserum with purified rat cardiac calsequestrin (lane 3). Immunoprecipitation of high molecular-weight proteins using anti-cardiac CSQ (lanes 2 and 3) was not a consistent observation and in some experiments only a single 55 kDa phosphoprotein was precipitated. The high-molecular-weight phosphoproteins often seen with immuno-

Control

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Auloradiogram Origin

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Skcl Card Skel Card Skel Card .,

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Fig. 3. Protein stain and -~ZP-autoradiographshowingphosphorylalion of purified rat calsequestfinisoformsby casein kinase il and inhibilion bv heparin. Samplesof purified rat skelelal muscle and cadiac calsequestrin(I jzg)were phosphorylatedfor 15 rain using 15 ng of caseinkina.se11and standard reaclionconditions.Lanes ! and ! Coornassieblue stain of purifiedrat skeletalmusclecalsequeslrin {Skel}and rat cardiaccalsequestrin(Card):lanes3 and 4, autoradiograph of phosphofflaledcalscquestrins;lanes5 and 6, same as lanes 3 and 4 buz with 4 gg/ml heparin added to the reaction.Arrows indicate the mobilitiesof skeletalmuscle(S) and cardiac(C) calscquestrin iseforms. A lo~er Coomagsieblue-stainingprotein band appearing in lane ! is probably a proteol~ic fragmentof skeletal musclecalsequestrin.

precipitatk,ns did not exhibit mobilities on SDS-PAGE that were similar to those of the high molecular-weight calsequestvin-like proteins seen by immunoblotting (cf. Fig. 1. lane 6).

Proteolylic phosphopeplide mapping of calsequestrin isoforms phosphorylated b~ L'itro mtd in t'it'o SAP-digestion of 3zP-labeled cardiac calsequestrins generated a unique phosphopeptide pattern that was seen in every one of the cardiac calsequestrin samples examined (Fig. 7, lanes 1-4). A very low apparent molecular weight doublet ( < 1000 Da) of phosphopeptides was seen for cardiac caisequestrin which had been metabolically labeled with 3ZPi in cultured rat myotubes (lane 1) as well as for cardiac calsequestrin phosphorylated in adult or fetal muscle homogenates (lane 2 and 3). A virtually identical phosphopeptide pattern was also seen for purified rat cardiac calsequestrin following phosphorylation by casein kinase 11 (lane 4). A different phosphopeptide pattern was observed for rat skeletal muscle calsequestrin in which the major phosphopeptide(s) were too small to be

283

ADULT MUSCLE IMMUNOPRECIPITATES

ADULT MUSCLE HOMOGENATES ~ORIGIN

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Fig. 4. "~2P-autoradiographyshowing endogenousand casein kinase ll-stimulated phosphorylation in adult rat skeletal muscle homogenates. Samplesof adult rat skeletal muscle homogenate(20 gg) were phosphor,'luted in 100 #.t. Half of the sample was analyzeddirectlyby SDS-PAGE (fight panel), and half was immunoprecipilatedby a mixture of anti-skeletal CSQ and anti-cardiac CSQ as described (left panel). Incubations contained, in additionto the standard (control)reaction conditions(C). 0A prg/mi poiylysine(Ply)or 0.2 #g/ml heparin(Hep). Where indicated, incubations included80 ng of casein kinase Ii. The autoradiograph shown in the left panel was exposed for twice as long as that shown in the righl panel.

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Heparin (p~ml) Fig, 5. ~2P-autoradiographshowingendogenousphosphor~lation in rat muscle homogenalesand the effects of heparin on phosphoryla. lion. Homogenat~samples (12/zg) were phosphorylaledusing standard reaction conditionsin the presence of either 0. 1 or l0 p.g/ml heparin, as indicated.The arrows indicate the mobilitiesof skeletal muscle(S) and cardiac(C) calsequestrinisoforms.

resolved using this gel system and migrated with the dye front. This pattera was seen for both skeletal muscle calsequestrin phosphorylated in adult rat muscle homogenates (lane 5) and for purified skeletal muscle calsequestrin phosphorylated by casein kinase 11 (lane 6). Phosphopeptide mapping by this method was sufficient to distinguish cardiac calsequestrin from other unrelated phosphoproteins (lanes 8-10). The specific SAP-phosphopeptide patterns for cardiac and skeletal muscle calsequestrins were always observed and we did not observe any differences using 10 •g of SAP, 10 mA current for SDS-PAGE, or electrophoresis temperatures between 10 °C and room temperature (data not shown). SAP-peptide maps have previously been shown to yield unique patterns for cardiac versus skeletal muscle calsequestrins from rabbit [35], We compared SAP-phosphopeptide maps of rat calsequestrins with those from dog and rabbit tissues since the sites of phosphorylation have previously been determined for dog cardiac and rabbit skeletal muscle calsequestrins. In Fig. 8 (upper panel), the protein staining of the iab~.led calsequestrins is shown next to

284

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Fig. 6. ': P-autoradiograph shov,ing phospht~D'lation of cardiac calscqueslrin in cultured rat myotubes. Rat myotube cultures ~ere metabolically labeled in phosphate-free media containing 2 mCi of [~ZP]orthophosphate for 5 h and then extracted with cold buffer containing reagents to inhibit further changes in phosphorylation 4see Materials and Methods). lmmunoprecipitation from this extract ~as perfi~rmed using anti-skeletal CSQ (lane !). anti-cardiac CSQ Ilane 21. or anti-cardiac CSQ that was preincubated in 1 mg/ml of purified rat cardiac calseque.qrin for I h at 4 ° C, Lane 4 contains 11.5 g.g of purified cardiac ealsequestrin that was phosphoD'lated in vitro ~- casein kinase I1.

the resulting autoradiogram (middle panel). 3keletal muscle calsequestrins were phosphorylated for longer period of time in order to achieve comparable levels of ~-'P, incorporation. Rabbit cardiac calsequestrin purified as a protein doublet of which only the upper of the two bands was phosphorylated. The reason for this calsequestrin doublet was not further investigated. The predominant phosphoprotein in the purified dog skeletal ealsequestrin sample was due to the copuri~ing cardiac isoform. Similar SAP-phosphopeptide maps were produced from dog. rat and rabbit cardiac calsequestrins, yielding the < 1000 Da peptide band which, in this case, was not resolved as a doublet (cf Fig. 7). Phosphopeptide maps from the three skeletal muscle calsequestrins were also similar and did not contain the SAP phosphopeptide characteristic of cardiac calsequestrins. Discussion

In this study, we have demonstrated that cardiac calsequestrin, an intralumenal SR protein, is phospho-

Fig. 7. -~-'P-autoradiograph of SAP digests showing similar calsequeslrin phosphop~ptides among in vh'o and in vitro labeled ~mpies. PhosphoD-lated proteins bands were excised from dried gels and elcctrophoresed in the presence of 5 ttg SAP according to Cleveland el al. [M]. Phosphot$'lated samples were as folkm's: 1, cardiac cal~qu~slrin immunoprecipitated from 3-'P-labeled myotubes: ! cardiac calsequesltin phosphoDlaled in fetal rat skeletal muscle homogenates ~" endogenous kinase: 3, cardiac calsequestfin phtz~phoDrlated in adult rat skeletal mu~ie homogenates by endogenous kinase; 4. purified cardiac calsequeslrin phosphoD'lated by casein kinaL~: Ih 5, skeletal muscle calsequeslrin phosphorylaled in adult rat skeletal muscle by endogenous kina.~; 6, purified skeletal mu~le calsequestfin phosphofflated by casein kinase ii; 7. same as lane 3:8-10 non-calscqucstrin phesphoproteins included for comparison (Mrs = 90000, 70000 and 1100O0)

rylated in vivo, by a kinase that is similar or identical to casein kinase 11. The site of phosphorylation of rat cardiac calsequestrin in vivo appeared to be the same site phosphorylated in vitro by casein kinase li based on SAP phosphopeptide mapping. A single, small SAP phosphopeptide (< 1000 Do) was generated from rat cardiac calsequestrin phosphorylated in vivo or, in vitro by protein kinase(s) present in tissue homogenates and by purified casein kinase II. This phosphopeptide from rat cardiac calsequestrin co-migrated on SDS-gels with SAP phosphopeptides generated from both dog and rabbit cardiac calsequestrins phosphorylated by casein kinase II. For dog cardiac calsequestrin, the site of phosphorylation has been localized to a group of three serine residues near the COOH terminus of the protein. The small size of the SAP phosphopeptide indicates that the SAP phosphopeptide was fully contained within the cardiac-specific portion of dog cardiac caisequestrin. Homologous serine residues within a cardiac-specific COOH-terminus (and casein kinase 11 consensus sequence) have also been identified in rabbit cardiac calsequestrin (Dr. Muthu Periasamy, personal commumcation). These data strongly suggest that rat cardiac calsequestrin was also phosphorylated on a cardiac-specific COOH-terminal tail in vivo and in vitro and further suggest that casein kinase I! may be involved in the phosphorylation of cardiac calsequestrin in vivo. The cardiac specificity of this < 1000Da SAP phosphopeptide is verified by the absence of

285 Cardiac CSQ

Skeletal

CSQ

i

Coomassie Blue

~ S .6.- C

Auto-

radiogram ~

~

~

~

S

-I-- C

SAP Map

~x

Fig. 8. Protein stain and ~2P-autoradiograph of phospho~lated caisequeslrins and SAP-phosphopeptidcs showing similarity among species. Skeletal mu~le (S) and cardiac (C) catseque.~Irins (2 ~-g) were phusphorylated for 20 rain and 5 rain. respectively, using 11~)ng casein kinase I!. Samples ~ere analyzed by SDS-PAGE followed by peptide mapping. The Cooma~ie blue protein stain (upper panel) and autoradiograph (middle panel) of intact proteins a~'e shown. T h e lower panel depicts the autoradiograph of the SAP map after the labeled protein bands were trealed with proteinase. Identical mobility phosphopeptides appeared in each of the three ~-ardiac calsequestrin samples (single arrow), whereas lhe phosphopeplides from the three skeletal mu~le cal.~questrin samples were not resolved using this gel system and migrated with the dye front (double arrow).

this phosphopeptide in any of the skeletal calsequestrin samples. Skeletal muscle calsequestrin did not appear to be phosphorylated in cultured myotubes, though it was phosphorylated in vitro by casein kinase II. The absence of detectable phosphorylation of skeletal muscle calsequestrin in myotubes might be, in large part, due to reduced level of expression. We would expect, nonetheless, to have detected even a very low level of radiolabeled protein based on our ability to quantitatively immunopreeipitate skeletal muscle calsequestrin from in vitro labeled samples (ef Fig. 4). Phosphorylation of an intralumenal SR protein in vivo raises a question of the cellular localization of the phosphorylation reaction. While it appears that most cellular casein kinase I1 activity is localized to the cytoplasm and nucleus [40], it has also been reported within the Golgi complex [41] and in clathrin-coated

vesicles [42]. ~ ialysis of highly purified dog cardiac SR (Cala, S.E. attd Jones, L.R., unpublished observations) suggests that casein kinase !I activity is not intrinsic to SR, although a membrane-bound activity is present in impure tractions of SR membrane vesicles. Thus, the cellular site of calsequestrin phosphorylation may not be the junctional SR, where calsequestrin deposition occurs. This would imply that calsequestrin is phosphorylated prior to its localization within the SR. Recently, calsequestrin was found to be transported to junctional SR by a Golgi-derived clathrin-coated membrane vesicle [43]. Interestingly, Capasso et al. [44] demonstrated that the Golgi complex is permeable to ATP and contains an intralumenal casein kinase I1 activity that phosphorylates several intralumenal proteins. Whether calsequestrin is phosphorylated within SR or in the course of its movement through cellular pathways of membrane trafficking is the subject of current investigation. Calsequestrin phosphorylatic, n was also a prominent biochemical reaction in vitro in rat striated muscle, where phosphorylation of both calsequestrin isoforms was observed in muscle homogenates. The endogenous calsequestrin kinase in homogenates for both isoforms was probably casein kinase ii as it was inhibited by heparin and polylysine, both of which exhibited similar inhibitory effects on phosphorylation catalyzed by added casein kinase II. Moreover, endogenously phosphorylated calsequestrin isoforms generated peptides having molecular masses similar to those generated from samples phosphorylated by casein kinase !1. Within adult rat heart, cardiac calsequestrin was the predominant heparin-sensitive phosphoprotein. In all preparations that were examined, cardiac calsequestrin exhibited a higher level of phosphorylation than did skeletal muscle calsequestrin. This apparent prevalence of cardiac calsequestrin phosphorylation is consistent with relative rates of phosphorylation for the two calsequestrin isoforms as previously determined using the purified components of the reaction [26]. Calsequestrin phosphorylation in fetal tissue homogenates appeared less sensitive to inhibition by heparin, The reason for this was not clear. It is possible that there exists an additional calsequestrin kinase activity in fetal rat muscle that is less sensitive to heparin, However, no additional sites of phosphorylation on cardiac calsequestrin from fetal tissue were evident from SAP phosphopeptide maps. Furthermore, measurements of casein kinase I1 activity, using the casein kinase ll-specific peptide RRREEETEEE as substrate, showed that fetal rat muscle tissues contained a 4-5-fold higher level of casein kinase !i activity than did adult tissues and this activity was less sensitive to heparin than was activity in the adult (Cala, S.E., unpublished observations), These findings might suggest that some factor exists in fetal muscle 11o-

286 moge~ates that renders endogenous casein kinase !1 activity less sensitive to hcparin. Cardiac calsequestrin was the predominant form expressed in the fetal rat hindleg muscle and in cultured myotubes. This conclusion was supported by three observations. First. the predominant fetal rat isoform exhibited the immunoreactivity characteristic of rat cardiac calsequestrin. Second, the fetal isoform exhibited an apparent molecular mass on SDS-PAGE that was indistinguishable from that of rat cardiac calsequestrin. And finally, the fetal isoform was phosphorylated by casein kinase !1 and exhibited a SAP phosphopeptide map that was indistinguishable from rat cardiac calsequestrin, suggesting that it contains the cardiac-specific COOH-terminal tail characteristic of cardiac isoforms from rats. rabbits and dogs. As thc existence of a cardiac form of calsequestrin was only recently reported, Iittle data exist concerning the relative levels of the two protein isoforms. Scott et al. [!5] showed that cardiac calscquestrin mRNA was present in heart and slow-twitch skeletal muscle from dogs, an isoform distribution characteristic of some contractile proteins {for review see Ref. 45). Rabbit slow-twitch skeletal muscle was also shown to contain and express both calsequestrin isoforms [46]. Such coexistence of isoforms in slow-twitch muscle is generally not characteristic of contractile proteins, nor of the SR Ca-'+-ATPase, for which slow-twitch muscle expresses only the cardiac isoform [47]. Expression of cardiac calsequestrin during development has not been previously exami,ed to our knowledge. While slow-twitch skeletal muscle was shown here to undergo a change in calsequestrin isoform expression during development, we found no significant change in isoform expression during rat heart development (S. Cala, unpublished observations). A presumed neonatal form of calsequestrin was previously described in rabbit fast-twitch skeletal muscle by MacLennan and co-workers [25] having the identical sequence as does the adult skeletal muscle isoform except for a 7-residue COOH-terminal extension. Though described over 10 years ago, the exact nature and relationship to the adult form remains uncertain [48]. Thus, it appears that developmental control of calsequestrin expression is complex and much remains to be learned. Increased levels of cardiac calsequestrin relative to skeletal muscle calsequestrin in fetal slow-twitch skeletal muscle might indicate differences in ftmction for the two protein isoforms. Such differences might include a difference in the cellular processing of calsequcs'rin which is better suited to rapid growth. Since increased levels of casein kinase ii activity have been associated with growth (indeed, fetal skeletal muscle compared to adult had 5-fold higher levels of activity), and since cardiac calsequestrin is such an outstanding substrate for casein kinase 11, it would be expected that phosphorylation of

cardiac calscqucstrin would be an important biochemical feature of this fetal tissue. In conclusion, we have shown that rat calsequestrin, an intralumenal SR Ca2+-binding protein, is phosphorylated in vivo and in vitro by casein kinase II. The sites of cal~qucstrin phosphorylation and the isoform specificity of this reaction in rat muscles and ,'nyotubes corroborate and extend previous work performed with purified proteins. These results suggest that casein kina.~e II-dependent phosphorylation of cardiac calsequestrin reflects an. as yet, unknown aspect of SR biochemistry and its possible physiological role awaits further study.

AckJrmwledgements We are grateful to Dr. Paul Greengard, in whose laboratory, this work was carried out. This work was supported by a Neuromuscular Deseasc Research Grant from the Muscular Dystrophy Association. S.E.C. was supported by Postdoctoral Fellowship Grant NF07607 from the National Institutes of Health. References l MacLennan. D.H. and Wong. P.T.S. (19711 Proc. Natl. Acad. Sci. USA 68. IZ'~1-1~5. 2 I[emoto. N., Bhatnagar. G.M. and Gergell,'. J. (19711 Biochem. Biophys. Res. Commun. 44, 1510-1517. 3 Meissner. G. {1975) Biochim. Bioph)~. Acta 389, 51-68. 4 Jorgen~n, A.O., Kalnins. V. and MacLcnnan. D.H. (19791 J. Cell. Biol. 80. 372-384. 5 Campbell, K.P., Franzini-Armstrong, C. and Shamoo. A.E. (1980) Biochim. Biophys. Acta 6tlZ 97-116. 6 Franzini-Armstrong. C. (19gO) Fed. Proc., Fed. Am. Soc. Exp. Biol. 39. 2403-2409. 7 Jones L.R. and Cala S.E, ( 198! ) L Biol. Chem. 256, 11809- I 1818. Campbell, K.P., MacLcnnan, D.H., Jorgensen A.O. and Mintzer, M.C. (19831J. Biol. Chem. ~ 8 , i 197-120~. 9 Cala S.E. and Jones L.R. (1983) J. Biol. Chem. ~ 8 . 11932-1193fi. 1{I MacLennan, D.H.. Campbell ILP. and Reithmeier. R.A.F. (1983) in Calcium and C¢1[ Function (Chung, W.Y., ed.), 4th Edn., pp. 151-173. Academic Press. New YorL ii Cain S.E., Scou, B.T. and Jones L.R. (199{I) Seminars in C¢11 Biolo~, I. 265-275. 12 lkemolo. N.. Bhatnagar. G.M. and Gergely J. (19721 I. Biol. Chem. 247. 7835-7837. 13 lkcmoto. N., Nagy, B.. Bhatnagar. G.M, and Gergely J. (19741J. Biol, Chem. 249, ~ 5 7 - ~ 6 5 . 14 Mitchell. R.D.. Simm~rman, H.K.B. and Jones, L.R. (19881 J. Biol, Chem. 263, 1376-1381. 15 Scott, B.T.. Simmerman. I].K.B., Collins, J.H., NadaI-Ginard, B. and Jones, L.R. (19881 J. Biol, Chem. 263. 8958-8964. 16 Franzini-Armstrong. C., Kenne~'. L.J, and Varriano-Marston, E. (19871J. Cell. Biol. 105, 49-56. 17 Collins. J.H., Tarcsafalvi, A. and lkemoto. N. (19901 Biochem. Biophys. Res. Commun. 167, 189-193. 18 Michalak, M., Campbell. K.P. and MacLennan, D.H. 0980) J. Biol. Chem. ~ 2 . 4626-4632. 19 Hofmann S.L, Brown. M.S.. Lee, E., Pathak, R.K., Anderson, R.G.W. and Goldstein, J,L. (19891J. Biol. Chem. 264. 8260-8270.

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Phosphorylation of the cardiac isoform of calsequestrin in cultured rat myotubes and rat skeletal muscle.

Calsequestrin is a high-capacity Ca(2+)-binding protein and a major constituent of the sarcoplasmic reticulum (SR) of both skeletal and cardiac muscle...
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