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 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.
Walaas
S I
SI
Neurochemical Laboratory, Institute for Basic Medical Sciences, University of Oslo, Norway.
Czernik
A J
AJ
Olstad
O K
OK
Sletten
K
K
Walaas
O
O
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D000242:Cyclic AMP / Q000494:pharmacology*; D004285:Dogs; D004837:Epinephrine / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D013820:Thermolysin / Q000378:metabolism
D007328:Insulin; D008565:Membrane Proteins; D010750:Phosphoproteins; C069141:phospholemman; D010768:Phosphoserine; D000242:Cyclic AMP; D011494:Protein Kinases; D011493:Protein Kinase C; D013820:Thermolysin; D004837:Epinephrine