Phospholemman, a transmembrane, 72 residue protein enriched in striated 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 study is aimed at identifying the phosphorylation sites of this protein. A synthetic peptide, GTFRSS63IRRLS68TRRR (in the single letter code) and consisting of phospholemman residues 58-72, is a substrate for both protein kinase C and cyclic AMP (cAMP)-dependent protein kinase, with Km values of 6-7 microM for both enzymes. Amino acid sequencing of the phosphopeptide shows that protein kinase C phosphorylates both Ser-63 and Ser-68, while cAMP-dependent protein kinase phosphorylates Ser-68. Thermolytic phosphopeptide mapping of 32P-labelled phospholemman from rat diaphragms shows that treatment with insulin results in labelling of phosphopeptides containing both Ser-63 and Ser-68, whereas treatment with adrenaline results in labelling of the phosphopeptide containing Ser-68. Hence, insulin and adrenaline regulate the phosphorylation of phospholemman, presumably through protein kinase C and cAMP-dependent protein kinase, respectively, on partly overlapping phosphorylation sites.

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The metabolism of uterine muscle is still incompletely known. GRACBARD and P r~c v s (1940) found high concentration of cytochrome Qxidase in uterine tissue, and MCSHAN, ERTVAY and MEYER (1946) demonstrated the occurrence of succinic dehydrogenase. GRAUBARD and PINCUS could inhibit the metabolism of uterus either by cyanide or by iodoacetate, and therefore concluded that oxidative and glycolytic processes were involved in the total respiration of uterine tissue.The striking changes produced by estrogenic hormones in the motility of uterine muscle must probably be related to effects on the fundamental metabolic processes, but investigations in this field are still few.Several investigators have reported an increase in oxygen consumption and an increased glycolysis in the isolated uterus after estrogenic treatment (MAC LOED and REYNOLDS 1938, KERLY 1940, CARROLL 1942. Unfortunately most of these investigations were made using whole uterus and no direct comparison has been made of the overall metabolism of the uterine muscle in castrates and estrogenic treated animals. This may seriously affect the results, as GRAUBARD and PINCUS found the oxygen uptake for endometrium 2.5 times higher than for myonietrium. Therefore it seems of iniportance to perform metabolic studies on the uterine muscle separately.

Calcium/phospholipid-dependent protein kinase activity (protein kinase C) was identified in rat diaphragm membrane and cytosol fractions by means of in vitro phosphorylation either of histones or of a specific 87 kDa protein substrate, combined with phosphopeptide-mapping techniques. Both insulin and tumor-promoting phorbol ester treatment of the diaphragm preparations led to increased protein kinase C activity in the membrane fractions. In contrast to the phorbol ester, however, insulin did not induce a concomitant decrease in cytosolic activity, indicating that translocation of the enzyme had not taken place. Thus, insulin appears to increase specifically membrane protein kinase C activity in rat skeletal muscle, possibly through a mechanism not identical to that induced by phorbol esters.Insulin; Protein kinase C; 87 kDa protein; (Rat diaphragm, Sarcolenna)

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