Kinetics of phosphorylation of Na+/K(+)-ATPase by protein kinase C.
Kinetics of phosphorylation of Na+/K(+)-ATPase by protein kinase C.
复制标题
蛋白激酶 C 磷酸化 Na /K( )-ATPase 的动力学。
DOI：
10.1016/0167-4889(90)90069-p
复制
发表时间：
1990
期刊：
影响因子：
--
通讯作者：
Bertics,PJ
中科院分区：
文献类型：
--
作者：
Lowndes,JM;Hokin-Neaverson,M;Bertics,PJ
在体外研究了钙和磷脂依赖性蛋白激酶C（PKC）对膜酶Na+-K+-ATP酶磷酸化动力学的影响。在狗肾和鸭盐腺Na+-K+-ATP酶纯化液及鸭盐腺微粒体中，Na+-K+-ATP酶催化α-亚基被PKC磷酸化。磷酸化需要钙（Ka ≤ 1.0 μM），在低浓度钙（0.1 μM）存在下，促肿瘤佛波酯（12-O-十四烷酰基佛波13-乙酸酯）可刺激磷酸化。Na+ K+-ATPase的PKC磷酸化迅速，在30 min内达到平台，PKC对Na+ K+-ATPase的表观Km值，狗肾酶为0.5 μM，鸭盐腺酶为0.3 μM。当纯化的盐腺Na+ K+-ATP酶浓度大于1.0 μM时，观察到PKC活性的明显底物抑制。纯化的肾脏和盐腺Na+ K+-ATP酶的磷酸化发生在丝氨酸和苏氨酸残基。经羟胺裂解后，32 P-磷酸化α亚基在15%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上的32 P-磷酸肽图谱与两种来源的酶相同，表明磷酸化位点相似。结果表明，Na+ K+-ATP酶可能作为一个底物的PKC磷酸化在完整的细胞和Na+ K+-ATP酶可能是一个有用的体外模型底物PKC与膜蛋白的相互作用。
The kinetics of phosphorylation of an integral membrane enzyme, Na+ K+-ATPase, by calcium-and phospholipid-dependent protein kinase C (PKC) were characterized in vitro. The phosphorylation by PKC occurred on the catalytic α-subunit of Na+ K+-ATPase in preparations of purified enzyme from dog kidney and duck salt-gland and in preparations of duck salt-gland microsomes. The phosphorylation required calcium (K a≈ 1.0 μM) and was stimulated by tumor-promoting phorbol ester (12-O-tetradecanoylphorbol 13-acetate) in the presence of a low concentration of calcium (0.1 μM). PKC phosphorylation of Na+ K+-ATPase was rapid and plateaued within 30 min. The apparent K m of PKC for Na+ K+-ATPase as a substrate was 0.5 μM for dog kidney enzyme and 0.3 μM for duck salt-gland enzyme. Apparent substrate inhibition of PKC activity was observed at concentrations of purified salt-gland Na+ K+-ATPase greater than 1.0 μM. Phosphorylation of purified kidney and salt-gland Na+ K+-ATPase occurred at both serine and threonine residues. The 32 P-phosphopeptide pattern on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis after hydroxylamine cleavage of pure 32 P-phosphorylated α subunit was the same for the two sources of enzyme, which suggests that the phosphorylation sites are similar. The results indicate that Na+ K+-ATPase may serve as a substrate for PKC phosphorylation in intact cells and that the Na+ K+-ATPase could be a useful in vitro model substrate for PKC interaction with integral membrane proteins.