Protein kinase C phosphorylates a 15 kDa protein but not phospholamban in intact rat cardiac myocytes.
In the present study the effects of the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) as well as the alpha- and beta-adrenoceptor agonists methoxamine and isoproterenol on protein phosphorylation of intact rat cardiac myocytes were investigated. TPA, isoproterenol and methoxamine were shown to stimulate phosphorylation of a 15 kDa protein. EC50 for TPA and isoproterenol were 4 x 10(-8) M and 5 x 10(-9) M respectively. The time course of phosphorylation by TPA and isoproterenol greatly differed, revealing a maximal phosphorylation (2.9-fold) after 10 min and 1 min respectively. Cell fractionation showed a significant enrichment of the 15 kDa protein in a crude membrane preparation. While the 15 kDa protein was the only phosphoprotein stimulated by TPA and methoxamine, isoproterenol additionally enhanced the 32Pi incorporation into four proteins corresponding to 6 kDa (phospholamban), 28 kDa, 97 kDa and 140 kDa. Furthermore, dephosphorylation of a 21 kDa substrate upon beta-adrenoceptor stimulation was observed. Phospholamban phosphorylation was effectively (max. 9.1-fold) stimulated by isoproterenol (EC50 of 5 x 10(-9) M), reaching a maximal phosphorylation state within 1 min. The present study clearly demonstrates: (1) TPA stimulates the phosphorylation of a membrane-localized 15 kDa protein and this effect can be mimicked by both isoproterenol and methoxamine; (2) TPA, in contrast to isoproterenol, does not change the phosphorylation state of phospholamban. Whilst phospholamban under in vitro conditions is known to be a substrate for protein kinase C, it does not appear to be accessible for the enzyme in intact cardiac myocytes.
Department of Physiology, University of Düsseldorf, Germany.
M
M
Hartmann
J
J
Schrader
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D001345:Autoradiography; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006321:Heart / Q000187:drug effects; D066298:In Vitro Techniques; D009124:Muscle Proteins / Q000378:metabolism*; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D012508:Sarcolemma / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D000099211:Phospholamban
D000316:Adrenergic alpha-Agonists; D000318:Adrenergic beta-Agonists; D002135:Calcium-Binding Proteins; D009124:Muscle Proteins; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D000099211:Phospholamban