PROTEIN-KINASE-C AND CYCLIC-AMP-DEPENDENT PROTEIN-KINASE PHOSPHORYLATE PHOSPHOLEMMAN, AN INSULIN AND ADRENALINE-REGULATED MEMBRANE PHOSPHOPROTEIN, AT SPECIFIC SITES IN THE CARBOXY-TERMINAL DOMAIN

Phospholemman, a transmembrane, 72 residue protein enriched in striate d muscle and heart [Palmer, Scott and Jones (1991) J. Biol. Chem. 266, 11126-11130], is phosphorylated in response to insulin [Walaas, Horn and Walaas (1991) Biochim. Biophys. Acta 1094, 92-102]. The present st udy is aimed at identifying the phosphorylation sites of this protein. A synthetic peptide, GTFRSS(63)IRRLS(68)TRRR (in the single letter co de) and consisting of phospholemman residues 58-72, is a substrate for both protein kinase C and cyclic AMP (cAMP)-dependent protein kinase, with K-m values of 6-7 mu M for both enzymes. Amino acid sequencing o f the phosphopeptide shows that protein kinase C phosphorylates both S er-63 and Ser-68, while cAMP-dependent protein kinase phosphorylates S er-68. Thermolytic phosphopeptide mapping of P-32-labelled phospholemm an from rat diaphragms shows that treatment with insulin results in la belling of phosphopeptides containing both Ser-63 and Ser-68, whereas treatment with adrenaline results in labelling of the phosphopeptide c ontaining Ser-68. Hence, insulin and adrenaline regulate the phosphory lation of phospholemman, presumably through protein kinase C and cAMP- dependent protein kinase, respectively, on partly overlapping phosphor ylation sites.