Cyclic GMP-dependent protein kinase stimulates the plasma membrane Ca2+ pump ATPase of vascular smooth muscle via phosphorylation of a 240-kDa protein.
The plasma membrane Ca2+ pump ATPase from porcine aorta was isolated by the calmodulin affinity chromatographic method of Kosk-Kosicka et al. (Kosk-Kosicka, D., Scaillet, S., and Inesi, G. (1986) J. Biol. Chem. 261, 3333-3338). Its activity was restored by adding either phosphatidylcholine or phosphatidylserine. Cyclic GMP-dependent protein kinase (G-kinase) stimulated the enzyme in a concentration-dependent manner. However, phosphatidylinositol kinase (PI-kinase) activity was not detected in the enzyme preparation, and the presence of phosphatidylinositol was not necessary for stimulation by G-kinase. Furthermore, adenosine, a potent PI-kinase inhibitor, did not affect the stimulation. The enzyme preparation contained three major proteins, with molecular masses of 240, 145, and 135 kDa, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 240- and 135-kDa proteins were phosphorylated in association with the stimulation by G-kinase, but only the phosphorylation of the 240-kDa protein was dependent on the G-kinase concentration. A purified enzyme without the 240-kDa protein, prepared by our previous method (Imai, S., Yoshida, Y., and Sun, H.-T. (1990) J. Biochem. (Tokyo) 107, 755-761), was not activated by G-kinase. Immunoblotting with an antibody against the human erythrocyte Ca2+ pump revealed that the 135-kDa protein corresponded to one of the isoforms of the plasma membrane Ca2+ pump. These results suggest that the phosphorylation of the 240-kDa protein is responsible for stimulation of the plasma membrane Ca2+ pump ATPase by G-kinase.
Yoshida
Y
Y
Department of Pharmacology, Niigata University School of Medicine, Japan.
Sun
H T
HT
Cai
J Q
JQ
Imai
S
S
D000241:Adenosine / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D000252:Calcium-Transporting ATPases / Q000502:physiology*; D002147:Calmodulin / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D002846:Chromatography, Affinity; D006152:Cyclic GMP / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D009131:Muscle, Smooth, Vascular / Q000201:enzymology* / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D013552:Swine
D002147:Calmodulin; D010716:Phosphatidylinositols; D011494:Protein Kinases; D000252:Calcium-Transporting ATPases; D006152:Cyclic GMP; D000241:Adenosine