Lowndes J M, Hokin-Neaverson M, Bertics P J
Department of Physiological Chemistry, University of Wisconsin School of Medicine, Madison.
Biochim Biophys Acta. 1990 Apr 9;1052(1):143-51. doi: 10.1016/0167-4889(90)90069-p.
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 alpha-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 (Ka approximately 1.0 microM) and was stimulated by tumor-promoting phorbol ester (12-O-tetradecanoylphorbol 13-acetate) in the presence of a low concentration of calcium (0.1 microM). PKC phosphorylation of Na+/K(+)-ATPase was rapid and plateaued within 30 min. The apparent Km of PKC for Na+/K(+)-ATPase as a substrate was 0.5 microM for dog kidney enzyme and 0.3 microM 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 microM. Phosphorylation of purified kidney and salt-gland Na+/K+ ATPases occurred at both serine and threonine residues. The 32P-phosphopeptide pattern on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis after hydroxylamine cleavage of pure 32P-phosphorylated alpha 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.
在体外对钙和磷脂依赖性蛋白激酶C（PKC）使一种整合膜酶——钠钾ATP酶磷酸化的动力学特性进行了表征。PKC介导的磷酸化作用发生在来自犬肾和鸭盐腺的纯化酶制剂以及鸭盐腺微粒体制剂中钠钾ATP酶的催化α亚基上。磷酸化作用需要钙离子（Ka约为1.0微摩尔），并且在低浓度钙离子（0.1微摩尔）存在的情况下，可被促肿瘤佛波酯（12 - O - 十四酰佛波醇13 - 乙酸酯）所刺激。PKC对钠钾ATP酶的磷酸化作用迅速，在30分钟内达到平台期。以钠钾ATP酶作为底物时，PKC对犬肾酶的表观Km为0.5微摩尔，对鸭盐腺酶的表观Km为0.3微摩尔。当纯化的盐腺钠钾ATP酶浓度大于1.0微摩尔时，观察到PKC活性存在明显的底物抑制现象。纯化的肾和盐腺钠钾ATP酶的磷酸化发生在丝氨酸和苏氨酸残基上。对纯的32P - 磷酸化α亚基进行羟胺裂解后，在15%十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳上的32P - 磷酸肽图谱对于这两种酶来源是相同的，这表明磷酸化位点相似。结果表明，钠钾ATP酶可能作为完整细胞中PKC磷酸化的底物，并且钠钾ATP酶可能是PKC与整合膜蛋白相互作用的一种有用的体外模型底物。
