Abstract
A protein of apparent Mr = 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 Edman degradation. Overlapping proteolytic phosphopeptides 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 Mr = 8409. There is one hydrophobic membrane-spanning segment composed of residues 18-37. The acidic amino-terminal end (residues 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 "gamma" 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. We propose the name "phospholemman" for the 15-kDa protein, which denotes the protein's location within the plasma membrane and its characteristic multisite phosphorylation.
Highlights
A protein of apparent M, = 15,000on sodium dodecyl membranes from skeletal [6, 7] and smooth [8,9,10] muscle, sulfate-polyacrylamide gel electrophoresiiss the major liver ( l l ), and adrenal tumor cells [12]. Stimulation of these plasma membrane substrate foCr AMP-dependent pro- tissues withdifferent agonists leads to phosphorylation of the teinkinase (PK-A) and protein kinase C(PK-C) in 15-kDa protein by Ca2+-and CAMP-dependent mechanisms several different tissues
In cardiacmuscle,phosphorylation of t h e 15-kDa we purified, cloned, and sequenced the canine cardiac protein occurs after activation of either CY- or,&adrenergic sarcolemmal “15-kDa protein.”
Dideoxy sequencingof the full-lengthcDNA confirmed that the 15-kDa protein contains 72 amino
Summary
All thrrr rlones were similar in size and suhsequentlv shown to coda for the same protein. 1.5-kDa Protrin Isolation-The 15-kDa protein is the major T h e nucleotide sequence and deduced amino acid sequence substrate phosphorylated in sarcolemmal vesicles hy endoge- are shown inFig. 2 8. The cDNA was 6 5 6 nrlclrotitles Irrnz, nous PK-C ( 5 ) (Fig. 1, SI,). Phosphorylated 15-kDa protein consisting of a single open reading framr o f 279 h s p~airs was purified from sarcolemmal vesicles by serial electropho- flanked hv 102 and 27.5 hase pairs of 5 ' - and : ~ ' - r ~ ntransl : ~ t e d resis and elect.roelution, providing essentially complete recovs-equence, respectively. The initial electroe- at position +4, t.ypical fortranslationinitiation I%). Surlutedsamplecontained a broadhand of CoomassieBlue rounded the initiating AT(; codon. $0 nucleotides,suggestingthat a full-leng.. h co1)yofthc - cleave the 15-kDa protein, hut shifted other proteins in this mKNA was sequenced
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