Purification and Complete Sequence Determination of the Major Plasma Membrane Substrate for CAMP-dependent Protein Kinase and Protein Kinase C in Myocardium*

A protein of apparent M, = 15,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is the major plasma membrane substrate for CAMP-dependent protein kinase (PK-A) and protein kinase C (PK-C) in several different tissues. In the work described here, we purified, cloned, and sequenced the canine cardiac sarcolemmal “15-kDa protein.” The amino terminus of the purified protein was not blocked, allowing determination of 50 consecutive residues by standard Ed- man degradation. Overlapping proteolytic phospho- peptides yielded 22 additional residues at the carboxyl terminus. Dideoxy sequencing of the full-length cDNA confirmed that the 15-kDa protein contains 72 amino acids, plus a 20-residue signal sequence. The mature protein has a calculated M, = 8409. There is one hy- drophobic membrane-spanning segment composed of residues 18-37. The acidic amino-terminal end (resi- dues 1-17) of the protein is oriented extracellularly, whereas the basic carboxyl-terminal end (residues 38- 72) projects into the cytoplasm. The positively charged carboxyl terminus contains the phosphorylation sites for PK-A and PK-C. In the transmembrane region, the 15-kDa protein exhibits 52% amino acid identity with the “7” subunit of Na,K-ATPase. High stringency Northern blot analysis revealed that 15-kDa mRNA is present in heart, skeletal muscle, smooth muscle, and liver but absent from brain and kidney.

From the Krannert Institute of Cardioloev and the Deoartments of Medicine and Pharmacology, Indiana University School of membranes from skeletal (6, 7) and smooth (8-10) muscle, liver ( l l ) , and adrenal tumor cells (12). Stimulation of these tissues with different agonists leads to phosphorylation of the 15-kDa protein by Ca2+and CAMP-dependent mechanisms (8)(9)(10)(11)(12)(13)(14). In cardiac muscle, phosphorylation of t h e 15-kDa protein occurs after activation of either CY-or ,&adrenergic receptors, and correlates with an increase in contractility (13,14). In spite of its prominence as a major plasma membrane phosphoprotein, the precise function of the 15-kDa protein remains undefined. No sequence information on the protein has yet been reported nor has the protein been purified.
In the work described here, we report on the purification and complete amino acid sequence of the cardiac sarcolemmal 15-kDa protein. The protein is quite small and contains a single transmembrane domain. A highly basic carboxyl-terminal tail projects into the cytoplasm, which contains several protein kinase phosphorylation sites. Knowledge of the protein structure gives some clues regarding 15-kDa protein function, which are briefly discussed.

EXPERIMENTAL PROCEDURES
Isolation of "P-Labeled 15-kDa Protein from Canine Cardiac Sarcolemmal Vesicles-Sarcolemmal vesicles were isolated from dog left ventricles as described previously (15). By omitting NaCl from the tained which exhibited &fold greater phosphorylation of the 15-kDa homogenization buffer and gradient solutions, membranes were obprotein compared with our earlier study (5). Protein concentrations were determined by the method of Lowry et al. (16).
Sarcolemmal vesicles were permeabilized by freeze-thaw shock and phosphorylated by endogenous PK-C (5). Freeze-thaw-treated sarcolemmal vesicles were preincubated for 2 min at 30 "C in buffer containing 75 mM Pipes-Tris (pH 6.8), 7.5 mM MgC12, 0.75 mM EGTA, and 0.88 mM CaCI2 (1.0 mg of protein/2 ml). Phosphorylation was initiated by adding 80 PM [y-3ZP]ATP (500 pCi/mg protein) and incubating at 30 "C for 2 min. Reaction mixtures were then centrifuged at 500,000 X g for 7 min at 4 "C. Pellets were solubilized in electrophoresis sample buffer (5), and SDS-PAGE was performed according to the method of Laemmli (17) using three-well 15% polyacrylamide gels (16 cm X 18 cm X 1.5 mm). Each well was loaded with 325 p g of solubilized sarcolemmal membranes containing approximately 125 pmol of "P-labeled 15-kDa protein. Following electrophoresis, sample lanes were cut horizontally into 2-mm slices and analyzed for labeled 15-kDa protein by Cerenkov counting. Radioactive protein was electroeluted using a Bio-Rad Mini Protean I1 apparatus and then concentrated using Centricon-10 Microconcentrators (Amicon) and precipitated (18). Protein precipitates were solubilized in 70% formic acid containing 1 mg/ml CNBr and incubated in absence of light at 25 "C for 16 h and then dried in a Savant Speed-Vac and solubilized in electrophoresis sample buffer. SDS-PAGE was performed, and sample lanes were cut and analyzed by Cerenkov counting as above. The 15-kDa protein was electroeluted, concentrated, and precipitated as before. Some fractions were further purified by high pressure liquid chromatography using a C, reversephase column equilibrated with 0.1% trifluoroacetic acid in water and developed with a linear gradient o f 0.1"; trifluoroacetic arid in propanol-I. Column fractions were monitorrrl hy al~sorl)ance at 214 nrn and hv Cerenkov counting.
Amino Acid .Sryuc.ncc, Annlysis-Protein and phosphopeptide sequences were determined using an Applied Hiosystems model for nucleic arid sequence analvsis. D N A sequencing was performed in both directions by the dideoxy method f2:1) using synthetic oligonucleotide primers and ' 1' 7 I)NA polvmerase.

1.5-kDa
Protrin Isolation-The 15-kDa protein is the major substrate phosphorylated in sarcolemmal vesicles hy endogenous PK-C ( 5 ) (Fig. 1, SI,).  was purified from sarcolemmal vesicles by serial electrophoresis and elect.roelution, providing essentially complete recovery of radioact.ive protein at. each step. The initial electroeluted sample contained a broad hand of Coomassie Blue staining material with a rnohilky corresponding to that of the 15-kDa protein (Fig. 1,  E l ) . A suhstantial purification was achieved hy treating sample E l with CNHr, which did not cleave the 15-kDa protein, hut shifted other proteins in this 31 -
Amino Acid Sryucncr I~rtrrminalir~n-Sam~,lc E2 was further purified hy reverse-phase chromatography and srlhierted to automated Edman degradation. The single radioactive peak from the reverse phase column, containing 380 pmol o f incorporated "P, yielded a single sequence n f 50 amino acid residues ( Fig. 'W, linr 0). In other experiments. four prnteolytic phosphopeptides (100-200 pmol each) were isolated and sequenced. Two of the tr.yptic phosphopepticles werr limit pept,ides ( Fig. 'W, lints h and c ) , whereas one was the prodtlct o f incomplete digestion ( Fig. 2A, linr d ) . A singlr phosphopeptide was isolated from the V8 digest, whosr sequence overlapped all three tryptic phosphopeptidrs (   Protein Sequence Analysis-The mature 15-kDa protein contained 72 amino acid residues with a calculated molecular weight of 8409. The deduced amino acid sequence (Fig. 2B) corresponded exactly to the protein sequence ( Fig. 2 A ) , demonstrating that we had sequenced the entire protein by standard Edman degradation techniques. cDNA sequencing also revealed that the protein contained a typical signal sequence (residues -20 through -l), with 2 basic residues located amino-terminally, followed by a hydrophobic core and a small aliphatic residue (alanine) a t position -1 (30). The presence of a cleaved signal sequence is consistent with the amino terminus of the mature protein being unblocked. Likewise, the absence of methionine in the mature protein explained the lack of effect of CNBr on its mobility in SDS gels.

G C T A C C C T G G G C G G C G G G G G G A G G A G M G C O l Y C
A distinguishing feature of the 15-kDa protein is its highly basic nature, with a calculated isoelectric point of 9.7. Most of the basic residues are concentrated at the carboxyl-terminal region, where consensus phosphorylation sites for PK-A and PK-C are found (31). The phosphorylated forms of the protein  have neutral or slightly acidic isoelectric points (6). 2 Hydropathy analysis (Fig. 2C) revealed the presence of a single hydrophobic domain of 20 uncharged amino acids (residues 18-37), sufficient to cross the sarcolemmal membrane, which separated the acidic amino-terminal end of the protein from the basic carboxyl-terminal end. A stop-transfer sequence, Arg-Arg-Cys-Arg-Cys-Lys (residues 38-43), was immediately adjacent to the transmembrane segment on the carboxyl side, consistent with the protein positioned in the membrane with its 35 carboxyl-terminal residues facing the cytoplasm (30). The 15-kDa protein can thus be defined as a class I, bitopic integral membrane protein by nature of its cleavable signal peptide and the stop-transfer sequence (30). A cartoon of the membrane topology of the 15-kDa protein is presented in Fig. 3.

15-kDa Q E H D P F T Y D Y O S L R I G G L I I
Nucleic Acid and Protein Sequence Data Bank Comparisons-Comparison of the amino acid sequence of the cardiac 15-kDa protein with proteins in the National Biomedical Research Foundation Protein Identification Resources data base revealed sequence similarity with the "7 subunit," or "proteolipid," of Na,K-ATPase isolated from sheep kidney (32). Residues 4-36 of the 15-kDa protein exhibited 52% identity with the partial sequence of the y subunit (Fig. 4). Search of a recent release of the GenBank nucleic acid data base revealed no nucleic acid sequences homologous with the 15-kDa clone.
Tissue Distribution of Cardiac 15-kDa mRNA-High-stringency Northern blot analysis was performed with the fulllength antisense mRNA. A major hybridizing RNA species of about 700 nucleotides, approximating the size of the cDNA insert of the sequenced clone, was observed (Fig. 5, arrow). Alter n ten-times longer :~utorntliographic exposure. a faint l.5-kI)a mltNA signal W:IS d r t c~t c~l in t h c~ brain sample h u t was insignificant compartd with t h a t i n t ht, muscle and liver snmples.
15-kDa mRNA was detected at high levels in heart as well a s in aorta, esophagus, stomach, skeletal muscle (triceps), and liver. No significant 15-kDa mRNA was detected in kidney or brain.

DISCUSSION
T h e molecular weight of the 15-kDa protein determined from the deduced amino acid sequence (8409) was suhstantially less than that estimated by SDS-PAGE. It is unlikely that the protein is a sulfhydryl-linked dimer, since its mobility in SDS-gels is not changed by boiling in the presence or absence of sulfhydryl-reducing agents. The nomenclature of "15-kDa protein" (5,6,13) or "16-kDa protein" (8,9,11) used previously for this entity is inappropriate when, in fact, the actual molecular mass is approximately one-half this value. Therefore, we propose the name "phospholemman" for this protein, indicat.ing a major phosphoprotein substrate localized to the plasmalemma.
The amino acid sequence of phospholemman was consistent with previous studies suggest.ing multiple, separate sites phosphorylated by at least two protein kinases. The carhoxylterminal region of phospholemman contained predicted phosphorylation sites for I'K-A and PK-C, a s well as for cGM1'dependent and Ca"+/calmodulin-dependent protein kinases (31). Our own studies,:' however, revealed that the protein was not a subst.rate for the latter two kinases, which is consistent with the results of Walaas ct ai. (6,7), who examined the phosphoprotein in skeletal muscle plasma membranes. We ohserved only serine phosphorylation of phospholemman using I'K-A, whereas I'K-C phosphorylated both serine and threonine residues (data not shown).
The mRNA transcript of phospholemman was not limited to heart hut was present in all muscle t?ipes a s well a s in liver. An interesting finding was t he lack of significant phospholemman mRNA in dog kidney and brain, even though phospholemman shares sequence similarity with the y subunit of Na,K-ATPase isolated from sheep kidney. Brain and kidney contain relatively high levels of Na.K-ATPase and so would be predicted t o contain high levels of y subunit. The inability of the probe to hybridize with an mHNA species in brain and kidney could mean that the y suhunit and phospholemman are products of different genes, an idea supported by comparison of our cDNA sequence to the kidney y su1)unit c I ) S A sequence.' The y subunit of Na,K-ATPase is not known to be phosphorylated, and it may be that phospholemman has evolved specifically t o allow regulation by multisite phosphorylation (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14).
Phospholemman shows many similarities t o the regulatory protein, phospholamhan, which is localized to sarcoplasmic reticulum in heart and modulates the activity of the sarcoplasmic reticulum calcium pump (33,34). Both proteins are small, highly basic, and possess single membrane-spanning domains consisting entirely of uncharged residues (X5). Both proteins are oriented with their most positively charged regions directed toward the cytoplasm, where the sites nt phosphorylation are localized (35). Multisite phosphorylation of both proteins occurs in intact myocardium 1)y protein kinases which are activated by CAMP and calcium (13,14, :3f), and phosphorylation of both proteins produces substantial changes in charge, with isoelectric points changing from approximately 10 to 5 or f ( 3 7 ) . A short region of' sequence similarity between phospholamhan and phospholemman. where 7 out of 9 residues are identical, is especially intriguing. Ser''' and Thr" of phospholamhan in this region are phosphorylated exclusively hv PK-A and multifunctional Cn.'+/ calmodulin-dependent protein kinase, respectively (3.5). Consensus phosphorylation site data (31) suggest that Ser"' o f phospholemman is a prime target for PK-A phosphorylation.
Although phospholemman is not a substrate for C'a"'/calmoddin-dependent protein kinase, the position of 3 consecutive carboxyl-terminal arginine residues (residues 70-72). as well as arginine residues 61,65, and 66, suggest that serine residues 62, 63, and 68 and threonine residue 69 are all potential substrates for PK-C. Several peptides encompassing residues 61-72 were isolated as phosphopeptides, demonstrating that serine and threonine residues in this region of the molecule are phosphorylated. As suggested for phospholamhan (:38), it is possihle that alteration of membrane surface charge secondary to phospholemman phosphorylation may play a role in phospholemman function.
I'hosphorylation-indrlccd perturbation of sarcolemmal surface charge could alter the local calcium concentration, with resultant effects on activities o f co-localized channels, pumps, and/or antiporters. Hecently, a cardiac delayed rectifier 1 . k channel has heen cloned and sequenced (39). which shows some structural similarity t o phospholemman. Both proteins are small and traverse the sarcolemmal membrane once. The membrane-spanning regions of both proteins are enriched in glycine and contain no charged residues. The possibility that phospholemman is a channel should also be considered.