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 P-32(i) 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.