Protein kinase C-dependent phosphorylation of sarcolemmal Ca2(+)-ATPase isolated from bovine aortic smooth muscle.
We examined the effect of protein kinase C (PKC)-dependent phosphorylation on Ca2+ uptake and ATP hydrolysis by microsomal as well as purified sarcolemmal Ca2(+)-ATPase preparations isolated from bovine aortic smooth muscle. The phosphorylation was performed by treating these preparations with PKC and saturating concentrations of ATP (or ATP-gamma S), Ca2+, and 12-O-tetradecanoyl phorbol-13-acetate (TPA) at 37 degrees C for 10 min. In microsomes, treatment with PKC enhanced a portion of the Ca2+ uptake activity inhibitable by 10 microM vanadate, by up to about 30%. On the other hand, Ca2(+)-dependent ATPase activity in the purified Ca2(+)-ATPase preparation was stimulated by up to twofold. Up to twofold stimulation by PKC was also observed for the Ca2+ uptake by proteoliposomes reconstituted from purified sarcolemmal Ca2(+)-ATPase and phospholipids. Since these effects were evident only at Ca2+ concentrations between 0.1 to 1.0 microM, we concluded that it was the affinity of the Ca2(+)-ATPase for Ca2+ that was increased by the PKC treatment. Under conditions in which PKC increased Ca2+ pump activity, the sarcolemmal Ca2(+)-ATPase was phosphorylated to a level of about 1 mol per mol of the enzyme. There was good parallelism between the ATPase phosphorylation and the extent of enzyme activation. These results strongly suggest that the activity of the sarcolemmal Ca2+ pump in vascular smooth muscle is regulated through its direct phosphorylation by PKC.
Fukuda
T
T
Department of Molecular Physiology, National Cardiovascular Center Research Institute, Osaka.
Ogurusu
T
T
Furukawa
K
K
Shigekawa
M
M
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D000818:Animals; D001011:Aorta / Q000187:drug effects / Q000201:enzymology; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification*; D002417:Cattle; D004789:Enzyme Activation / Q000187:drug effects; D006868:Hydrolysis; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000494:pharmacology*; D012508:Sarcolemma / Q000187:drug effects / Q000201:enzymology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D015220:Calcium Channels; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D000252:Calcium-Transporting ATPases; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium