Protein kinase-dependent phosphorylation of cardiac sarcolemmal Ca2+-ATPase, as studied with a specific monoclonal antibody

Phosphorylation of the Ca2+-pump ATPase of cadiac sarcolemmal vesicles by exogeneously added protein kinases was examined to elucidate the molecular basis for its regulation. The Ca2+-pump ATPase was isolated from protein kinase-treated sarcolemmal vesicles using a monoclonal antibody raised against the erythrocyte Ca2+-ATPase. Protein kinase C (C-kinase) was found to phosphorylate the Ca2+-ATPase. The stoichiometry of this phosphor-ylation was about 1 mol per mol of the ATPase molecule. The C-kinase activation resulted in up to twofold acceleration of Ca2+ uptake by sarcolemmal vesicles due to its effect on the affinity of the Ca2+ pump for Ca2+ in both the presence and absence of calmodulin. Both the phosphorylation and stimulation of ATPase activity by C kinase were also observed with a highly-purified Ca2+-ATPase preparation isolated from cardiac sarcolemma with cal-modulin-Sepharose and a high salt-washing procedure. Thus, C-kinase appears to stimu-late the activity of the sarcolemmal Ca2+ pump through its direct phosphorylation. In contrast to these results, neither cAMP-dependent protein kinase, cGMP-dependent protein kinase nor Ca2+/calmodulin-dependent protein kinase II phosphorylated the Ca2+-ATPase in the sarcolemmal membrane or the purified enzyme preparation, and also they exerte