Jul 5, 2015 - accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 4 Clinical Investigator of ..... I Ca/itam I I C~AKMP. I c a p 1. II Hr. Hydrolysis.
THEJOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 264, No. 19, Issue of July 5, pp. 1146&11474,1989 Printed in U.S.A.
Phospholamban Phosphorylationin Intact Ventricles PHOSPHORYLATION OF SERINE 16AND @-ADRENERGICSTIMULATION*
THREONINE 17 INRESPONSETO
(Received for publication, September 27, 1988)
Adam D. Wegener, Heather K. B. Simmerman, Jon P. LindemannS, and LarryR. Jones From the Departmentof Medicine and the Krannert Instituteof Cardiology, Indiana University Schoolof Medicine and the 46202 Richard L. Roudebush Veterans Administration Medical Center, Indianapolis, Indiana
Phospholamban is the major membrane protein of cardiac SR’ (1-5) which has been postulated to regulate the the heart phosphorylated in response to &adrenergic activity of the Ca2+pump of this intracellular membrane (6). stimulation. In cell-freesystems, CAMP-dependent Phospholamban is rapidly phosphorylated in isolated cardiac protein kinase catalyzes exclusive phosphorylationof SR vesicles by both soluble CAMP-dependent protein kinase serine 16 of phospholamban, whereas Ca2+/calmodu- and an intrinsic Ca2+/calmodulin-dependentprotein kinase, lin-dependent protein kinase gives exclusive phosphorand this phosphorylation has been correlated with increased ylation of threonine 17 (Simmerman, H. K. B., Collins, Ca2+sequestration activity (7-14). In experimental systems J. H., Theibert, J. L., Wegener, A. D., and Jones,L. R. of intact myocardium, phospholamban phosphorylation (as (1986) J. Biol. Chem. 261,13333-13341). Inthis assessed by 32Pincorporation) occurs after P-adrenergic stimwork we have localized the sites of phospholamban ulation or other treatments which elevate cAMP (15). Inophosphorylation in intact ventricles treated with the tropic agents which actthrough elevation of intracellular &adrenergic agonist isoproterenol. Isolation of phos- cAMP and activation of CAMP-dependent protein kinase phorylated phospholamban from 32P-perfused guinea cause changes in cardiac relaxation thought to be mediated, pig ventricles, followed by partial acid hydrolysis and at least in part, by phosphorylation of phospholamban (6,15). phosphoamino acid analysis, revealed phosphorylation The role of Ca2+/calmodulin-dependentprotein kinase in of both serine and threonineresidues. At steady state phosphorylation of phospholamban in intact myocardium has after isoproterenolexposure,phospholambanconremained ambiguous, however. Phosphorylation of phosphotained approximately equimolar amountsof these two lamban has been shown not to occur after maneuvers which phosphoamino acids. Two major tryptic phosphopep- elevate intracellular Ca2+independent of cAMP (16). tides containing>90%of the incorporated radioactiv- Thus, itis well established that B-adrenergic stimulation of ity were obtained from phospholamban labeled in in- intact hearts results inphospholamban phosphorylation, pretact ventricles. The amino acid sequences of these two sumably via activation of CAMP-dependent protein kinase tryptic peptides corresponded exactly to residues14- (15-19). If a physiologic role for phospholamban phosphoryla25 and 15-25 of canine cardiac phospholamban, thus tion by Ca2+/calmodulin-dependentprotein kinase exists, it localizing the sitesof in situ phosphorylation to serine might be expected to occur during activation of CAMP-de16 and threonine 17. pendent protein kinase, when intracellular Ca2+ levels are elevated. Consistent with this, previous studies have suggested Phosphorylation of phospholamban at two sites in heart perfused with isoproterenol was supported by both proteinkinases participate in phospholamban phosphordetection of 11 distinct mobility forms of the penta- ylation during perfusion of hearts with isoproterenol. Lindemeric protein by use of the Western blotting method, mann and Watanabe (16) showed inhibition of phospholamban phosphorylation in intact heartsby lowextracellular Ca2+ consistentwitheachphospholambanmonomercontaining two phosphorylation sites, and with each pen- concentrations, with minimal inhibition of cAMP generation. tamercontainingfrom 0 to 10 incorporated phos- Karczewski et al. (20) used the “back phosphorylation technique” (21) to demonstrate that incardiac SR vesicles isolated phates. Our results localize the sites of in situ phospholam- from dogstreated with isoproterenol, in uitro phosphorylation Ca2+/calmodulinban phosphorylation to serine 16 and threonine17 and, of phospholamban by bothCAMP-and furthermore, are consistent with the phosphorylations dependent protein kinases was attenuated. However, these of these 2 residues being catalyzed by CAMP-and Ca2+/ investigators concluded that in intact myocardium, phosphorylation by CAMP-dependent protein kinase was predomcalmodulin-dependent protein kinases, respectively. inant. A problem with all of these studiesexamining phospholamban phosphorylation in intact cardiac tissue is that specific Phospholamban is a small pentameric phosphoprotein of sites of phosphorylation have not yet been analyzed, which renders Enambiguous identification of the relevant protein kinases difficult. Recently, the complete amino acid sequence * This work was supported by Grants HL28556 and HL06308 from of phospholamban has been determined and the sites of in the National Institutes of Health, by the Veterans Administration, and by the Herman C. Krannert Fund. The costs of publication of uitro phosphorylation localized (2, 22) (Fig. 1).Phospholamthis article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 4 Clinical Investigator of the Veterans Administration Research Career Development Program.
The abbreviations used are: SR, sarcoplasmic reticulum; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraaceticacid; PIPES, piperazineN,N’-bis(2-ethanesulfonic acid); SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis.
11468
Phosphorylation Sites of in Situ Phospholamban
11469
with buffer B. After extensive washing with buffer B, kinase activity was eluted with 20 mM PIPES, pH 6.8, 1 mM dithiothreitol, and 1 mM EGTA. Active fractions were stored at -40 "cafter addition Of 20% glycerol. Protein kinase activity was totally dependent on CaZ+ and calmodulin. Phosphorylation of Synthetic Phospholamban Peptide-A synthetic peptide corresponding to phospholamban residues 2-25 (Fig. 1) was synthesized by Biosearch. The sequence was confirmed with use of an Applied Biosystems model 477A protein sequenator. Phosphorylation of the peptide by CAMP-dependentprotein kinase was performed in 50 pl of 25 mM PIPES, pH 6.8, 10 mM MgCl2, 10 mM EGTA, 1% bovine serum albumin, and 0.1 mM ATP with 5 pCi of [Y-~'P]ATP. Reactions contained 5-10hgof peptide and 10-20 units of the catalytic subunit of CAMP-dependent protein kinase. PhosphorylaEXPERIMENTALPROCEDURES tion of the peptide by liver Ca'+/calmodulin-dependent protein kinase Heart Perfusions-Hearts were obtained from guinea pigs of either was conducted in the same buffer also containing 9.5 mMCaC1' and sex weighing 400-450 g and perfused by the Langendorff technique 1 PM calmodulin. With these conditions, maximal phosphorylation as previously described (15). The composition of the oxygenated (95% of the peptide was achieved in 20 min a t 30 "C and was similar for OB,5% COZ) Krebs-Henseleit buffer was (millimolarity) NaHC03, both protein kinases. Partial Acid Hydrolysis and Phosphoamino Acid Analysis of Syn27.2; NaCI, 118.0; KCl, 4.8; KH2P04,0.231; MgSO,,1.2;CaC12,2.5; and glucose, 11.1. The pH of the buffer was maintained at 7.4 at thetic Phospholamban Peptide-To determine optimalconditions for 30 "C. The diastolic tension was adjusted to 5 g and the hearts phosphoamino acid analysis of phospholamban, the synthetic phoscontinuously stimulated at a rate of 3 Hz. After 10 min of perfusion pholamban peptide was first analyzed. 20-4 aliquots of the peptide by drip-through at a constant flow of 6.0ml/min, the perfusion circuit phosphorylated by CAMP-dependent protein kinase or Ca2+/calmodwas switched to a recirculating system containing 60 ml of the same ulin-dependent protein kinase were added to 1 ml of 6 N HCl and buffer to which 2-20mCiof 32Piwas added. Following 30 min of hydrolysis conducted in evacuated tubes at 105 'C. HC1 was evapoperfusion with the radioactive buffer, the hearts were returned to rated and residues were taken upin water containing 1 mg/ml phosphoserine and phosphothreonine. 5-pl aliquots were applied to nonrecirculating perfusion with nonradioactive buffer for 2-3 min thin layer cellulose plates (Kodak Chromagram) sprayed with 2.5% after which drugs were administered by continuous infusion. At formic acid, 7.5% acetic acid, pH 1.8, and subjected to thin layer indicated times, the hearts were freeze-clamped with clamps cooled electrophoresis for 3 h at 500 V. Standards were visualized with in liquid nitrogen, pulverized in a precooled mortar and pestle, and ninhydrin, and 32P-labeled phosphoamino acids were detected by stored under liquid nitrogen until further analysis. autoradiography. Ninhydrin-stainedspots were scraped from the Membrane Vesicle Preparations-Partially purified SR vesicles plate and 32Pquantitated by liquid scintillation counting. Total were prepared by minor modifications of procedures previously de- phosphorylation of the peptide was quantitated by trichloroacetic scribed (15, 16). Separate membrane vesicle preparations were made acid precipitation of an aliquot of the phosphorylation mixture. from each heart. All procedures were carried out at 4 "C. The powPhosphoamino Acid Analysis of Phospholamban Phosphorylated in dered tissue from each heart was thawed and homogenized simulta- intact Hearts-In order to perform phosphoamino acid analysis of neously in 10 ml of a medium containing 50 mM Na2HP04,pH 7.4, phospholamban phosphorylated in intact hearts, aliquots of SR ves10 mM EDTA, and 25 mM NaF. The tissue was homogenized three icles isolated from 32P-perfusedhearts were first subjected to SDStimes for 30 s with a Polytron PT-10 (Brinkmann Instruments). An PAGE in 8% gels (26). 200 Mg of protein were applied/lane. Aliquots additional 10 ml of homogenization mediumwas added and the of membrane samples in SDS were not boiled prior to electrophoresis, homogenate sedimented twice for 20 min at 14,000 X g,. . The so that phospholamban remained mostly in the pentameric form (3). supernatant from the second spin was then sedimented at 45,000 X After electrophoresis, phospholamban was localized by autoradiogg, for 30 min and theresultingpellet resuspended in homogenization raphy of the unfixed gels. Gel segments containing pentamericphosmedium containing 0.6 M NaCl, pH 7.0. This material was sedimented pholamban were excised and washed for 2 h in 25% isopropyl alcohol for 30 min. The final pellet, enriched in SR vesicles, to remove SDS and solutes. Gel segments were then lyophilized to a t 45,000 X gmaX was resuspended in a small volume of 30 mM histidine/HCl, pH 7.4, dryness and placed in 3 ml of buffer containing 1%NH4HC03,0.1 0.25 M sucrose, 10 mM EDTA, and 10 mM NaF, and storedat -20 "C mM CaC12,and 50 pg/ml trypsin. Proteolysis was conducted overnight until further assay. Protein was measured by the method of Lowry et with shaking. Buffer was then removed and thegel segments shaken al. (23) using bovine serum albumin as a standard.We have previously an additional 2 h with 5 ml of distilled water. The eluates were pooled shown that under these preparative conditions, dephosphorylation of and separated from residual gel fragments by a low speed centrifuphospholamban in SR membranes is less than 15% (15, 16). gation. 90% of the 32Pincorporated into phospholamban was rePurification of Ca'+/Calmodulin-dependent Protein Kinase-Puricovered from the gel segments by this method. Eluates were lyophification of soluble Ca2+/calmodulin-dependent protein kinase from lized to dryness and then takenup in l ml of 6 N HCl. Samples were rabbit liver was modified from the method of Ahmad et al. (24). 180 often turbid at this point but could be clarified by centrifugation in g of rabbit liver was homogenized at 4 "C in a blender for 60 s in 500 the cold with no loss of 32P.Samples were subjected to partial acid ml of buffer containing 1 mM NH4S04,4 mM EDTA, 1 mM dithio- hydrolysis and phosphoamino acid analysis as described above. threitol, 0.25 mg/ml leupeptin, 0.1 mM tosyl-L-lysine chloromethylZmmunoaffinity Purification and Trypsinolysis of Phospholamban ketone, and 0.5 mM phenylmethylsulfonyl fluoride, pH 7.0. The Phosphorylated in intact Hearts-Three guinea pig ventricles perhomogenate was centrifuged for 40 min a t 13,000 X g., . The super- fused with 32P,and stimulated for 2 min with M isoproterenol natant was acidified to pH 6.0 with 5 ml of3.5 N acetic acid. The were quickfrozen in liquid N, and powdered with a mortarand pestle. resulting precipitatewas pelleted and resuspended in 150 ml of buffer The frozen powder from the three hearts was combined and homogA (20 mM PIPES, pH 6.8, 1 mM EGTA, 1 mM dithiothreitol) plus enized at 4 "C in 30 ml of buffer containing 1%Triton X-100, 0.4% protease inhibitors, as above. The suspension was centrifuged for 90 deoxycholate, 50 mM Na2HP04, pH 7.6, 10 mM Na,EDTA, and 10 min at 100,000 X g, and the supernatantloaded on a 70-ml column mM NaF, using a glass tissue homogenizer. The homogenate was of phosphocellulose, preequilibrated with buffer A. The column was warmed to room temperature and was spun a t 45,000 x gmex for 30 washed extensively with buffer A and thekinase eluted with a gradient min. The supernatant was decanted and added to 1 ml of canine of NaCl from 0 to 0.8 M in buffer A. cardiac phospholamban monoclonal (IgM) antibody cross-linked to Ca2+/calmodulin-dependent protein kinase activity of phosphocel- Affi-Gel-Hydrazide (Bio-Rad) according to the manufacturer's speclulose column fractions was assayed using a water-soluble synthetic ifications. 5 mg of monoclonal antibody were covalently linked/ml of peptide comprised of residues 2-25 of canine cardiac phospholamban gel. The monoclonal antibody to phospholamban was produced as (Fig. 1).Phosphorylation of the peptide was conducted as described recently described (27) and was purified from ascites fluid by protein in the next section. Reaction products were resolved using paper A-Sepharose affinity chromatography. chromatography (25). Active fractions were pooled and diluted 1:1 Antibody gel and detergentextractcontaining phosphorylated into buffer B (20 mM PIPES, pH 6.8, 10% glycerol, 0.2 M NaC1, and phospholamban were incubated 3 h at room temperature with gentle 1 mM dithiothreitol) plus 5 mM CaCl,. The crude kinase preparation rotation. The beads were then allowed to sediment, followed by was then applied to 10 ml of calmodulin-Sepharose, preequilibrated extensive washing in the above homogenization buffer containing 0.4
ban is a pentamer of identical 52 amino acid monomers, and at each monomer, serine 16 is exclusively phosphorylated by CAMP-dependent protein kinase, whereas threonine 17 is specifically phosphorylated by Ca*+/calmodulin-dependent protein kinase (2). In thepresent study, we have localized the sites of in situ phospholamban phosphorylation using Langendorff perfused guinea pig hearts. Taking advantage of the known phosphorylation site specificity, we provide evidence that is consistent with a major role for Ca2+/calmodulindependent protein kinase in phospholamban phosphorylation during @-adrenergicstimulation.
11470
Phosphorylation Sites of in Situ Phospholamban
M NaCI. Purified phospholamban was eluted in 6 ml of buffer condependent protein kinase for catalyzing serine phosphorylataining 50 mM glycine, pH 2.4, and 0.1% Triton X-100. The pH of tion of phospholamban in canine cardiac SR vesicles; Ca2+/ the eluate was adjusted to 7.4 by adding 0.3 ml of 2 M Tris base, p H 8.6. The sample was then concentrated in an Amicon Centricon to calmodulin-dependent protein kinase catalyzed only threoapproximately 40 pg (6.7 nmol) in200 pl. 88 pg of trypsin wasadded, nine phosphorylation (1). These residues were subsequently and the mixture was incubated overnight. Consistent with previous localized to serine 16 and threonine17 (2) with respect to the results (1).SDS-PAGE followed by autoradiography revealed that all amino acid sequence (22) (Fig. 1).In the present work, with of the incorporated"P was liberated duringtrypsinolysis. The digest guinea pig SR membranes phosphorylated in vitro, we again was spun in a Centricon, and radioactive peptides passing through observed only serine phosphorylation by CAMP-dependent the concentrating membrane were collected in the filtrate with a protein kinase and only threonine phosphorylation by Caz+/ recovery of >98%. Peptides were fractionated by reverse-phase chrocalmodulin-dependent protein kinase (data notshown). matography and analyzedas described below. In order to apply phosphoamino acid analysis to phosphoReverse-phase Chromatography, Sequence Analysis, and Phosphoamino Acid Analysis of Phosphopeptides-Tryptic peptides generated lamban isolated from intact "Pi-perfused ventricles, we first from in situ phosphorylated phospholamban purified by immunoaf- optimized conditionsfor partial acid hydrolysis. For this finitychromatography were fractionatedusing a Pharmacia Fast purpose, a synthetic peptide (residues 2-25) corresponding to Protein Liquid Chromatography system. Peptides were injected directly onto a reverse-phase CIS column (Pep RPC) equilibrated with the NH2-terminal (cytoplasmic) domain of phospholamban 0.1% trifluoroacetic acid in water (solvent A). Peptides were eluted was utilized (Fig. 1). This peptideretained the specificity with solvent B (0.1% trifluoroacetic acid in acetonitrile) a t a flow requirements for differentialphosphorylation of serine by rate of 0.5 ml/min according to the following program: 0-5 min, 0% CAMP-dependent protein kinase and threonine by Ca2+/calB; 5-15 min, 0-2076 B; 15-55 min, 20-40% B; 55-65 min, 40-100% modulin-dependent protein kinase(Fig. 2). Recoveries of total R. Absorbance of the effluent was monitored at 214 nm. Fractions "P incorporation at 4, 8, and 16 h of hydrolysis were 50, 29, were collected in 500-p1 aliquots a t I-min intervals, and radioactive and 14%, respectively, for phosphoserine and 22,39, and 31%, peaks were identified by sampling 100 p1 of each fraction for liquid scintillation counting. The radioactive fractions were dried separately respectively, for phosphothreonine. For the experiments asin a Savant Speed-Vac, taken up in 50pl of 50% acetonitrile/water, sessing phospholamban phosphorylation in perfused hearts and aliquots were applied to polybrene-treated precycled glass fiber described below, the two hydrolysis times of 4 and 8 h were filters. Automated sequence analysis was doneusing an Applied used for phosphoamino acid analysis, and results were corBiosystems model 477A protein sequencer and a n Applied Biosystems rected for differences in recovery of phosphoserine and phosmodel 120A high pressure liquid chromatograph for on-line analysis phothreonine based on theabove peptide results. of phenylthiohydantoin-aminoacid derivatives. To identify the sequence location of phosphorylated residues in the major radioactive fraction isolated by reverse-phase chromatog10 Met-Asp-Lvs-Val-Gln-Wr-Leu-Thr-Ara-Serraphy, the filter cutting method of automated Edman degradation 2 0 was used (28). An aliquot of the sample was applied to a Polybrene* * Ala-Ile-Ara-Ara-Ala-Ser-Thr-Ile-Glu-Mettreated, precycled glass fiber filter and the filter was then quartered. 30 The four pieces were reassembled in the sequencer cartridge and the Pro-Gln-Gln-Ala-Arg-Gln-Asn-Leu-Gln-Asnsequencing run proceeded normally for one wash cycle and five 40 Leu-Phe-Ile-Asn-Phe-Cys-Leu-Ile-Leu-Ilecleavage cycles. At theconclusion of each cleavage cycle for cycles250 5, the instrument was paused for removal of one piece of filter from Cys-Leu-Leu-Leu-Ile-Cys-Ile-Ile-Val-Metthe cartridge. "2P-Labeled peptides and 32Piwere quantitatively extracted from each of the filterpieces by ultrasonication in 50% formic Leu-Leu acid. The extractswere dried in a Savant Speed-Vac, taken up in 50 FIG. 1. Amino acid sequence of caninecardiac phospholamp1 of water, and 5-pl aliquots were examined by thin layer electrophoresis at pH 1.8 (500 V, 75 min) followed by autoradiography. The ban (from Refs. 2 and 22). Asterisks denote serine 16 and threonine amount of "P associated with peptides and 32Piwas determined by 17 phosphorylated by CAMP- and Ca2'/calmodulin-dependent proscraping the spots from the thin layer plate and liquid scintillation tein kinases, respectively. The underlined sequence corresponds to the synthetic peptide used in this study. In native phospholamban, counting. methionine 1 is acetylated (22). To further characterize the peptide composition and phosphorylated residues of the majorradioactive fraction,thesample was subjected to thinlayer electrophoresis a t p H1.8 (500 V, 75 min), and the radioactive spots were scraped from the plate for phosphoamino acid analysis. Peptides were extracted from thecellulose matrix into acetonitrile/water/trifluoroaceticacid (50500.1), dried, and taken up in 6 N hydrochloric acid for hydrolysis at 110 "C for 4 h. Phosphorylated residueswereidentified by thin layer electrophoresis as deP-thrscribed above. Western Blotting-SR vesicles prepared from intact heartsexposed to isoproterenol were electrophoresed in 10-15% polyacrylamide gradient gels (3,26) with20 pg of protein applied/gel lane. T o maximize resolving distance, gels were run a t 20 mA constant current for twice the time required for the dye front to elute from the bottom of the gel. Proteins were transferred to nitrocellulose as described previously (19). After incubation with a 1:500 dilution of phospholamban polyclonal antiserum(4),phospholambanwas visualized using horseCAP",' ~ c ; Ca/itam C ~ A K M Pc a p radish peroxidase-coupled goat-anti-rabbit IgG (19). Materials-Catalytic subunit of CAMP-dependent protein kinase, II Hr. Hydrolysis 8 Hr. Hydrolysis 16 Hr. Hydrolysis bovine brain calmodulin, bovineserum albumin, protein A-Sepharose, and calmodulin-Sepharose were obtained from Sigma. Gel electroFIG. 2. Autoradiograph depicting phosphoamino acid analysis of synthetic phospholamban peptide. Synthetic peptide phoresis reagents and Affi-Gel Hydrazide were purchased from BioRad. [-y-"'PIATP and "P, were purchased from Du Pont-New Engof phospholamban containing residues 2-25 was phosphorylated by land Nuclear. CAMP-dependent protein kinase (CAMP P K ) or Ca'+/calmodulindependent protein kinase (&/Cam P K ) and subjected to acid hydrolysis for the times indicated. Shown is the autoradiograph of thin RESULTS layer electrophoresis of hydrolysates. P-ser, phosphoserine; P-thr, Phosphorylation of Synthetic Phospholamban Peptide-In phosphothreonine; Origin, sample application; Peptides,incompletely a prior report, we showed a high degree of specificity of CAMP- hydrolyzed peptide.
0
e
1
I I
IC?~
a
I
0
I I
I
1
Phosphorylation Sites of in Situ Phospholamban Phosphoamino Acid Analysis of Phospholamban Phosphorylated in Intact Ventricles-Membrane vesicles enriched in SR were prepared from 32Pi-perfusedhearts exposed to lo" M isoproterenol, and radiolabeled phospholamban was detected by SDS-PAGE followed by autoradiography (15, 19). Gel segments containing radiolabeled phospholamban pentamers were excised and incubated overnight with trypsin, and liberated peptides were then subjected to partialacid hydrolysis and phosphoamino acid analysis. Fig. 3 shows averaged results of three separate phosphoamino acid analyses from a set of perfused hearts. During exposure of hearts to isoproterenol, phosphorylation of serine in phospholambanoccurred rapidly (within 15-30 s) andthen plateaued. Phosphorylation of threonine inphospholamban occurred a t a lower rate, peaking a t 60 to 180 s of isoproterenol exposure. At this later time, phosphate detected in serine and threonineresidues was about equal (Fig. 3, legend). Although Fig. 3 shows a decrease in serine phosphorylation a t 45 s of isoproterenol exposure, this was not routinely observed in other perfusions. Sequence Analysis of in Situ Phosphorylation Sites-It was important tolocalize the sitesof phospholamban phosphorylated in intact ventricles in relation to the amino acid sequence. Fig. 1 shows that there are four potential phosphorylation sites (threonine 8, serine 10, serine 16, and threonine 17). For sequence analysis of phosphorylation sites, we first purified 32P-labeledphospholamban by immunoaffinity chroM matography from guinea pig ventricles perfused with isoproterenol for 2 min. Under these perfusion conditions the protein is maximally phosphorylated. Fig. 4 shows the high specificity of the monoclonal antibody used to purify phospholamban from the crude ventricular homogenate. For hearts exposed to isoproterenol, phospholamban can be identified in
CRUDEEXTRACTS
11471
El purified
Boil
Mr 200
66
43
-
. . . . . .
- hP.P
FIG. 4. Autoradiographdepictingpurification of "P-labeled phospholamban from intact ventricles by monoclonal antibody affinity chromatography. Lanes 1-4 show results of SDS-PAGE followed by autoradiography of crude homogenates from control ( C O N ) hearts and hearts exposed to M isoproterenol ( I S O ) . Lanes 5 and 6 show "P-labeled phospholamban purified by monoclonal antibody affinity chromatography from the IS0 extract. f indicates whether samples were boiled in SDS prior to electrophoresis. PLBH and PLBL designate the high (pentameric) and low (monomeric) M,forms of phospholamban, respectively.
crude homogenates subjected to SDS-PAGEby its characteristic dissociation into subunits induced by boiling in SDS (Fig. 4, ISO, PLBH, and PLBL) (1-4). Of the radiolabeled proteins, only phospholamban in the crude homogenate is bound by the affinity matrix (Fig. 4, Ab-purified). The antibody-purified, 32P-labeled phospholamban depicted in Fig. 4 was incubatedwithtrypsin, andtryptic peptides were resolved using reverse-phase chromatography. 89% of the applied 32P was recovered ina single major radioactive peak (Fig. 5, bars B and C ) ,and 6%of the applied radioactivity was recovered in a second minor peak of radioactivity (Fig. 5, bar A). Amino acid sequencing of fraction B revealed two phenylthiohydantoin amino acids in each cycle, which were found at the end of the sequencing run to form I I I the limit and nonlimit tryptic peptides of the same segment 11 IO .I o: -io of phospholamban, uiz. residues 14-25 and 15-25 (Table I, ISOPROTERENOL EXPOSURE [ w a d s ) FIG. 3. Phosphoaminoacidanalysis of phospholamban Fig. 1).The limit tryptic peptides 15-25 and 14-25 comprised phosphorylated in intact ventricles. Duplicate ventricles were 520% and 280% of the sample, respectively (Table I). Fracperfused with lo-' M isoproterenol (ZSO) for the times indicated and tion C , eluting 1 min after fraction B and comprising 15% of SR vesicles prepared. The upper panel shows the autoradiograph of the recovered 32P (Fig. 5), was also sequenced and found phosphoamino acid analysis of 32P-labeledphospholamban isolated likewise to contain exclusively a mixture of peptides 14-25 by SDS-PAGE andsubjected to trypsinolysis followed by partial acid and 15-25 (not shown). hydrolysis. For this typical example, acid hydrolysis time was 8 h. To identify the phosphorylation site(s) of these peptides, PS, phosphoserine; PT,phosphothreonine. The lowerpanel plots 32P incorporation into phosphoserine and phosphothreonine of phospho- fraction B was examined for release of 32Piby thin layer electrophoresis (see next paragraph) following the cycles of lamban uersus duration of isoproterenol exposure. Results are the averages (+) standard errors from three separate hydrolysates using Edman degradation (Fig. 6). Little 32Piwas released before one 4-h and two 8-h hydrolysis times. Results are plotted as percent cycle 3. At cycle 3, a burst of 32Piwas observed (Fig. 6, lane "P incorporation, taking the incorporation detected at 3-min isopro- 3), constituting 45% of the total 32Pand indicating that the terenol exposure as 100% for each amino acid. Counts per minute (cpm) were corrected for recovery of phosphoserine and phosphothre- third residue is phosphorylated. 55% of the radioactivity onine, as described under "Results." Corrected cpm/5-pl aliquot for remained in peptides after cycle 3, but following cycle4,81% 3 min (100%) isoproterenol perfusion samples were 830 k 124 for of 32Pwas found in inorganic phosphate,indicating that phosphoserine and 898 k 167 for phosphothreonine. residue 4 is also a phosphorylated site (Fig. 6, lane 4 ) . These
Phosphorylation Sitesof in SituPhospholamban
11472
CycleNumber
-
Frac. 8
35
-
2
3
9
5
30
L
D
fi
z
-10
4 E
-15
- -P 10
I
0
I
I
I
J
10
10
30
PO
4- Pi
N U M B E R
F R A C T I O N
FIG.5. Purification of phospholamban tryptic phosphopeptides. Tryptic phosphopeptides from 40 pl of phospholamban phosphorylated in intact hearts were injected on a Pharmacia reversephase C,Scolumn (PepRPC) andeluted with a gradient of acetonitrile. Fractions of 500 pl were collected at 1-min intervals, and 100-pl aliquots were measured for radioactivity. Fractions A, B, and C contained 6, 74, and 15%, respectively, of the radioactivity eluting from the column. All of the radioactivity applied to the column was recovered. The inset (left) shows an autoradiograph of thin layer electrophoresis of fraction B, resolving peptides 1-3. An autoradiograph of phosphoamino acid analysis (P-AAA) of peptides 1-3 is displayed on the right. Nonradioactive phosphoamino acid standards were visualizedwith ninhydrin. OR, sample origin; PS, phosphoserine; PT, phosphothreonine. (+) and (-), anode and cathode, respectively. TABLE I Sequence analysis of fractions A and B peptides of phospholamban The radioactive fractions A and B separated by reverse-phase chromatography were dried and redissolved in 50 pl of 50% acetonitrile/water. 30 pl of fraction A and 15 pl of fraction B were subjected to automated sequence analysis, with 40% of the sample from each cycle analyzed on-line to identify the products. Amino acids (AA) are given along with yields (pmol) identified in each cleavagecycle. Assignment of the residue numbers of the sequenced peptides with respect to the complete sequence of phospholamban is shown (PLB Res no.). Fraction A Fraction B Cycle
1 2 3 4 5 6 7 8 9 10 11 12
PLB Res. no.:
C O R
AA
pmol
AA
pmol
AA
pmol
AA
pmol
Val Gln Tyr Leu Thr Arg
437 328 9 226 137 47
Ala Ser Thr Ile Glu Met Pro Gln Gln Ala
58 16 15 12 25 4 13 7 13 7
Arg Ala Ser Thr Ile Glu Met Pro Gln Gln Ala Arg
26 186 12 28 107 53 95 67 39 60 38 9
Ala Ser Thr Ile Glu Met Pro Gln Gln Ala Arg
41 11 14 22 13 9 14 8 7 2
4-9 15-25 14-25 15-25
results suggest that bothserine 16andthreonine17are phosphorylated within the peptide 14-25, which constitutes most of the sequenced sample. Note that following two cleavage cycles, peptides 2 and 3 of fraction B resolved by thin layer electrophoresis (Fig. 6, lune 1 )are less positively charged
FIG.6. Autoradiograph showing location of phosphorylated residues in fraction B. The major radioactive fraction B isolated by reverse-phase chromatography was dried and redissolved in 50 pl of 50% acetonitrile/water. 15 pl was subjected to automated Edman degradation and “Pi phosphate released a t cycles 2-5 was analyzed as described under “ExperimentalProcedures.” In the autoradiograph obtained after thin layer electrophoresis, pH 1.8,lane 1 shows the original fraction B containing peptides 1, 2, and 3 before sequential degradation. Lanes 2-5 show the products resulting after 2-5 cycles of Edman degradation, respectively. OR, sample origin; Pi, radioactive inorganic phosphate. + and -, anode and cathode, respectively.
(Fig. 6, lune Z ), consistent with the loss of an NH2-terminal arginine from both of these peptides. To determinewhetherserine 16 and threonine 17 were simultaneously phosphorylated in themajor component peptide 14-25 of fraction B, or whether distinct populations of serine-phosphorylated and threonine-phosphorylated peptides were present, the sample was subjected to thin layer electrophoresis and the resulting peptide spots analyzed for phosphoamino acids (Fig. 5, inset). Upon thin layer electrophoresis, fraction B was resolved into one major phosphopeptide (Fig. 5, inset, spot Z),and two minor phosphopeptides (Fig. 5, inset, spots 1 and 3). The results show that whereas peptide 2 contained both phosphoserine and phosphothreonine, peptide 3 contained only phosphoserine (Fig. 5, inset, P-AAA). As both these peptides probably have the same sequence (see above paragraph), beginning with arginine 14 and continuing to arginine 25 ofthe complete phospholamban sequence, these electrophoretic results indicatethat peptide 3 is phosphorylated exclusively at serine 16, and that themajor product peptide, peptide 2, probably represents a single population of phospholambanpeptide 14-25, which is dually phosphorylated at serine 16 and threonine 17. The slowest migrating peptideresolved by thin layer electrophoresis, peptide 1, also contained both phosphothreonine and phosphoserine (Fig. 5, inset, P-AAA) and probably represents the dually phosphorylated limit tryptic peptide 15-25 lacking the NH2-terminal arginine 14. This interpretation of the combined results is consistent with the yields of each peptide observed by sequencing (Table I), with electrophoresis results of fraction B (Fig. 5), with the shift inelectrophoretic mobilities observed following Edman degradation and the removal of positive charge, arginine 14, from the NH2 termini of peptides 2 and 3 (Fig. 6), and with the generation of 32Pi
Phosphorylation Sites
of in Situ Phospholamban
during consecutive Edman degradation cycles (Fig. 6). Fraction A, containing 6% of the radioactivity eluting from the reverse phase column (Fig. 5), was also sequenced. Two phenylthiohydantoinamino acids were observed ineach cleavagecycle, but the discretepeptides from which they originated were easily distinguished based on the recovery yields of the residues (Table 1).The peptide correspondingto residues 4-9 of phospholamban was the major component of fraction A, comprising 290% of the sample, whereas the peptide containing residues 15-25 formed the remainder of the sample, as estimated from yields of sequencing. Phosphoamino acid analysis of fraction A indicated approximately equal amounts of phosphoserine and phosphothreonine (not shown), suggesting that the origin of the radioactivity is predominantly from peptide 15-25 (containing phosphorylationsitesserine 16 andthreonine17), with littleor no radioactivity associated with peptide 4-9 (containing threonine 8). Western BlotAnalysis of Phospholamban Pentamers Phosphorylated in Intact Ventricles-We have proposed that phospholamban is a pentamer of identical phosphorylatable monomers (1-4). Additional evidence for this pentameric model of phospholamban has been obtained in other laboratories. For example, phosphorylation of each site in the pentamer produces a stepwise decrease in its electrophoretic mobility during SDS-PAGE; up to 10 32P-labeledmobility forms have been observed (5, 29). By using gradient gels for SDS-PAGE in combination with the sensitive Western blotting method, we were able to discern 11 discrete mobility forms (forms 010) of phospholamban pentamers afterexposure of hearts to lo-' M isoproterenol, a concentration chosen to yield a submaximally phosphorylated protein (Fig. 7). In control hearts (Fig. 7, time 0), phospholamban was mainly in its highest mobility, dephosphorylated state (form 0),although some phospholamban inmobility state 1was also observed (form I, Fig. 7). Exposure of hearts to M isoproterenol allowed 11 different mobility states to be observed, which is consistent with phospholamban pentamers containing 0-10 incorporatedphosphates. These mobility states changed with increasing time of isoproterenol exposure. At steady state, 3-5 min after isoproterenol perfusion was started, ventricles incubated with 1.0 mM Ca2+contained phospholamban pentamers mainly in mobility states 3-7, whereas those incubated with 2.5 mM Ca2+ contained phospholamban pentamers mainly in mobility states 4-10. Since each phospholamban monomer contains only two phosphorylation sites (serine 16 and threonine 17), some of the phospholamban monomers comprising pentameric phospholamban were probably dually phosphorylated at thelonger times
:zJ 0
of incubation. This latter interpretation is consistent with the results presented in Figs. 5 and 6. DISCUSSION
In this studywe have identified serine 16 and threonine 17 as thetwo predominant amino acids of phospholamban phosphorylated in intact ventricles in response to @-adrenergic stimulation. Previous work with in vitro membrane systems and purified phospholamban has shown that CAMP-dependent protein kinase catalyzes the exclusive phosphorylation of serine 16 of phospholamban, whereas Ca2+/calmodulin-dependent protein kinase gives phosphorylation of only threonine 17 (2). Serine 16 and threonine 17 are ideally situated for phosphorylation by these two protein kinases,in that they are positioned on theCOOH-terminal side of the two nearby basic residues, arginine 13 andarginine 14 (Fig. 1) (2). Knowledge of the sitespecificities of these two protein kinases acting onphospholamban,incombinationwithidentification of serine 16 and threonine 17 as theonly residues of phospholamban significantly phosphorylated during @-adrenergic stimulation, allows us to conclude that these twoprotein kinases are probably responsible in large part for phospholamban phosphorylation in intact ventricles. In support of this, 10 mobility forms of phosphorylated pentameric phospholamban were detected after @-adrenergicstimulation of ventricles. Since phosphorylation of phospholamban by CAMP-dependent protein kinase alone could produce only five of these mobility forms (forms one to five) (Ref. 5), the additional mobility forms of phospholamban (forms6-10) probably originated from simultaneous phosphorylation by a second protein kinase, most likely Ca2+/calmodulin-dependentprotein kinase. Although protein kinase C has been shown to phosphorylate phospholamban in isolated membrane preparations (30, 31), this protein kinase probably does not do so in intact cardiac tissueduring @- (or CY-) adrenergicstimulation. First,the specificity of protein kinase Cfor phospholamban is low; large amounts of added proteinkinase are required in in vitro systems for significant phosphorylationof phospholamban to occur (32). Second, a-adrenergic stimulation of rodent ventricles causes phosphorylation of a 15-kDa sarcolemmal protein (33), an excellent substrate of protein kinase C (32),but under this condition of presumed protein kinase C activation, there is no significant phospholamban phosphorylation. And third, peptide mapping studies reveal that protein kinase C phosphorylates other residues of phospholamban (1, 30), in addition to serine 16 and threonine 17 (2). Only 32P-labeled serine 16 and threonine 17 were detected at significant levels in the presentwork. The results reported here in conjunction with previous work (16) are consistent with Ca2+/calmodulin-dependentprotein kinase phosphorylating phospholamban in intact ventricles but only after elevation of CAMP and activation of CAMPdependent protein kinase.There could be several mechanisms favoring dual site phosphorylation of phospholamban after @adrenergic stimulation. 1)Although not expected from in vitro experiments, CAMP-dependent protein kinase might phosphorylate (and therebydirectly activate) Ca2+/calmodulindependent protein kinase. 2) CAMP-dependent protein kinase could inhibit phosphatase activities (directly or indirectly), allowing net phosphorylation at threonine 17 to become manifest. 3) An increase in intracellular Ca2+concentration could occur after @-adrenergic stimulation, which could become large enough to activate Ca2+/calmodulin-dependentprotein kinase. Relevant to 3) above, upon first consideration it seems
" 2.5rnM ~ a * +
!
.-. 4 0
1
3
5
u
1
MINUTESAFTERISOPROTERENOLSTIMULATION
FIG. 7. Western blotting of phospholamban phosphorylated in intact ventricles. SR vesicles were isolatedfrom ventricles exposed to M isoproterenolfor the times indicated and subjected to SDS-PAGE followed by Western blotting. The blot was incubated with phospholamban polyclonal antiserum and developed colorimetrically. 11 discrete mobility steps (0-10) of the high molecular weight pentameric form of phospholamban are resolved. 1 mM Ca2+ and2.5 mM Ca2+ designate hearts perfused in low and high Ca" buffers, respectively.
11473
11474
Phosphorylation Sitesof in Situ Phospholamban
unlikely that the change in cytosolic Ca2+induced by cAMP is of itself adequate to allow phosphorylation of phospholamban by Ca2+/calmodulin-dependent protein kinase. Other agents which give comparable elevations of intracellular Ca2+ concentration by cyclic nucleotide-independent mechanisms do not induce phospholamban phosphorylation (16). In the experiment of Fig. 7 , for example, hearts perfused with 2.5 mM Ca2+in the absence of isoproterenol attained higher peak tensions (reflectingcytosolic Ca2+transients) thanthose perfused with 1.0 mM Ca2+ and stimulated with isoproterenol (data notshown), but only in the lattercondition did significant phospholamban phosphorylation occur. However, it remains possible that intracellular cAMP selectively increases cytosolic Ca2+concentration in a specific compartment (SR localized ?), and that this local increase in Ca2+is required for activation of the endogenous Ca2+/calmodulin-dependent protein kinase. If this were the case, those agents elevating intracellular Ca2+concentration independently of cAMP production would not be expected to induce phosphorylation of phospholamban. Although our results demonstrate directly that serine 16 and threonine l7 Of phospholamban are phosphoqlated in intact ventricles in response to /3-adrenergic stimulation, further experiments will be required to elucidate the precise regulatory mechanisms controlling the selective phosphorylation of these two residues. Acknowledgment-The excellent secretarial assistance of Teresa C. Fajfer is greatly appreciated.
1. 2. 3. 4. 5.
6. 7.
REFERENCES Wegener, A. D., Simmerman, H. K. B., Liepnieks, J., and Jones, L. R. (1986) J. Biol. Chem. 2 6 1 , 5154-5159 Simmerman, H. K. B., Collins, J. H., Theibert, J. L., Wegener, A. D., and Jones, L. R. (1986) J. Biol. Chem. 261,13333-13341 Wegener, A. D., andJones, L. R. (1984) J. Biol. Chem. 2 5 9 , 1834-1841 Jones, L. R., Simmerman, H. K. B., Wilson, W. W., Gurd, F. R. N., and Wegener, A. D. (1985) J. Bid. Chem. 2 6 0 , 7721-7730 Imagawa,T., Watanabe, T., andNakamura, T. (1986) J. Biochem. (Tokyo) 99,41-53 Tada, M., and Inui, M. (1983) J. Mol. Cell. Cardiol. 15, 565-575 La Raia, P. J., and Morkin, E. (1974) Circ. Res. 3 5 , 298-306
8. Kirchberger, M.A., Tada, M., and Katz, A.M. (1974) J. Biol. Chem. 249,6166-6173 9. Le Peuch, C. J., Haiech, J., and Demaille, J. G. (1979) Biochemistry 18,5150-5157 10. Jones, L. R., Maddock, S. W., and Hathaway, D. R. (1981) Biochim. Biophys. Acta 6 4 1 , 242-253 11. Kranias, E. G. (1985) Biochim. Biophys. Acta 8 4 4 , 193-199 12. Tada, M., Inui, M., Yamada, M., Kadoma, M., Kuzuya, T., Abe, H., and Kakiuchi, S. (1983) J. Mol. Cell. Cardiol. 1 5 , 335-346 13. Plank, B., Wyskovsky, W., Hellmann, G., and Suko, J. (1983) Biochim. Biophys. Acta 732,99-109 14. Kirchberger, M. A., and Antonetz, T. (1982) J. Biol. Chem. 257, 5685-5691 15. Lindemann, J. P., Jones, L. R., Hathaway, D. R., Henry, B. G., and Watanabe, A. M. (1983) J. Biol. Chem. 258,464-471 16. Lindemann, J. P., and Watanabe, A.M. (1985) J. Biol. Chem. 260,4516-4525 17. Le Peuch, C. J., Guilleux, J. C., and Demaille, J. G. (1980) FEBS Lett. 114, 165-168 18. Huggins, J. P., and England, P. J. (1983) FEBS Lett. 1 6 3 , 297302 19. Presti, C. F., Jones, L. R., and Lindemann, J. P. (1985) J . Biol. Chem. 260,3860-3867 20. Karczewskl P., Bartel, S., Haase, H., and Krause, E.-G. (1987) Bwmed. Biochim. Acta 46, S433-S439 21. Forn, J., and Greengard, P. (1978) Proc. Natl. Acad. Sei. U. S. A. 75,5195-5199 22. Fujii, J., Ueno, A,, Kitano, K., Tanaka, S., Kadoma, M., and Tada, M. (1987) J. Clin. Znuest. 79,301-304 23. Lowry, 0.H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193. 265-275 24. Ahmad,’Z., DePaoli-Roach, A. A,, and Roach, P. J. (1982) J. Biol. Chem. 257,8348-8355 25. Li, H.-C., and Felmly, D. A. (1973) Anal. Biochem. 52, 300-304 26. Porzio, M. A., and Pearson, A. M. (1977) Biochim. Biophys. Acta 490,27-34 27. Adunyah, S. E., Jones, L. R., and Dean, W.L. (1988) Biochim. Biophys. Acta 941,63-70 28. Wang, Y., Bell, A. W., Hermodson, M. A., and Roach, P. J. (1986) J . Biol. Chem. 261,16909-16915 29. Gasser, J. T.,Chiesi, M. P., and Carafoli, E. (1986) Biochemistry 25,7615-7623 30. Movsesian, M. A., Nishikawa, M., and Adelstein, R. S. (1984) J. Biol. Chem. 259,8029-8032 31. Iwasa, Y., and Hosey, M. M. (1984) J. Biol. Chem. 259,534-540 32. Presti, C. F., Scott, B. T., and Jones, L.R. (1985) J. Bid. Chem. 2 6 0 , 13879-13889 33. Lindeman, J. P. (1986) J. Biol. Chem. 261,4860-4867