Phosphorylation of cardiac and skeletal muscle calsequestrin isoforms by casein kinase II. Demonstration of a cluster of unique rapidly phosphorylated sites in cardiac calsequestrin.
Calsequestrin is an acidic Ca2(+)-binding protein of sarcoplasmic reticulum existing as different gene products in cardiac muscle and skeletal muscle. A unique feature of cardiac calsequestrin is a 31-amino acid-long COOH-terminal tail (Scott, B. T., Simmerman, H. K. B., Collins, J. H., Nadal-Ginard, B., and Jones, L. R. (1988) J. Biol. Chem. 263, 8958-8964), which is highly acidic and contains several consensus phosphorylation sites for casein kinase II. In the work described here, we tested whether this cardiac-specific sequence is a substrate for casein kinase II. Both cardiac and skeletal muscle calsequestrins were phosphorylated by casein kinase II, but cardiac calsequestrin was phosphorylated to a higher stoichiometry and at least 50 times more rapidly. The site of rapid phosphorylation of cardiac calsequestrin was localized to the distinct COOH terminus, where a cluster of three closely spaced serine residues are found (S378DEESN-DDSDDDDE-COOH). The slower phosphorylation of skeletal muscle calsequestrin occurred at its truncated COOH terminus, at threonine residue 363 (I351NTEDDDDDE-COOH). The similar sequence in cardiac calsequestrin (I351NTEDDDNEE) was not phosphorylated. Cardiac calsequestrin, as isolated, already contained 1.2 mol of Pi/mol of protein, whereas skeletal muscle calsequestrin contained only trace levels of Pi. The endogenous Pi of cardiac calsequestrin was also localized to the distinct COOH terminus. Our results indicate that the cardiac isoform of calsequestrin is the preferred substrate for casein kinase II both in vivo and in vitro.
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Krannert Institute of Cardiology, Indiana University School of Medicine, Indianapolis 46202.
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D000595:Amino Acid Sequence; D000818:Animals; D002155:Calsequestrin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D047388:Casein Kinases; D004285:Dogs; D006352:Heart Ventricles / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism*; D009206:Myocardium / Q000201:enzymology / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis; D010748:Phosphopeptides / Q000302:isolation & purification; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D012689:Sequence Homology, Nucleic Acid
D002155:Calsequestrin; D010455:Peptides; D010748:Phosphopeptides; D011494:Protein Kinases; D047388:Casein Kinases