Feschenko M S, Sweadner K J
Laboratory of Membrane Biology, Massachusetts General Hospital, Charlestown 02129.
J Biol Chem. 1994 Dec 2;269(48):30436-44.
Phosphorylation of sodium and potassium ion-activated adenosine triphosphatase (Na,K-ATPase) by protein kinase A (PKA) and protein kinase C (PKC) was investigated in vitro, where substrate conformation, kinase activity, and consequent effects on Na,K-ATPase activity could be controlled. With Na, K-ATPase from rat kidney, optimal stoichiometries were close to 1 mol 32P/mol Na,K-ATPase for both kinases. Addition of Na+, K+, P(i), or ouabain is known to stabilize the Na,K-ATPase in different states and was found to affect phosphorylation by the two kinases in a reciprocal way. This indicates that exposure of the phosphorylation sites varies with conformation and suggests a structural basis for the variable responses to kinase activation in intact cells. Further evidence for the importance of Na,K-ATPase conformation in its interaction with kinase came from the autophosphorylation of PKC, which varied in proportion to both the concentration and conformation of rat Na,K-ATPase. With pig and dog Na,K-ATPase, little phosphorylation by PKC was detected, and yet the PKC phosphorylated itself when the Na,K-ATPase was in the optimal conformation. The location of the PKA phosphorylation site was confirmed to be Ser-938 by sequence analysis of a tryptic peptide. Effects of PKA on Na,K-ATPase activity could not be measured because of inhibition by the Triton X-100 needed to obtain phosphorylation. Phosphorylation by PKC, even in optimal conditions, failed to result in inhibition of Na,K-ATPase activity. This suggests that any physiological role of phosphorylation either entails a subtle modulation of enzyme properties, or requires additional regulatory proteins.
在体外研究了蛋白激酶A（PKA）和蛋白激酶C（PKC）对钠钾离子激活的三磷酸腺苷酶（Na，K - ATPase）的磷酸化作用，在这种情况下，可以控制底物构象、激酶活性以及对Na，K - ATPase活性的后续影响。对于大鼠肾脏来源的Na，K - ATPase，两种激酶的最佳化学计量比均接近1摩尔32P/摩尔Na，K - ATPase。已知添加Na⁺、K⁺、P(i)或哇巴因可使Na，K - ATPase稳定在不同状态，并且发现其以相互的方式影响两种激酶的磷酸化作用。这表明磷酸化位点的暴露随构象而变化，并提示了完整细胞中对激酶激活的可变反应的结构基础。Na，K - ATPase构象在其与激酶相互作用中的重要性的进一步证据来自PKC的自磷酸化，其与大鼠Na，K - ATPase的浓度和构象均成比例变化。对于猪和狗的Na，K - ATPase，未检测到PKC的磷酸化作用，然而当Na，K - ATPase处于最佳构象时，PKC会自身磷酸化。通过胰蛋白酶肽段的序列分析证实PKA磷酸化位点位于Ser - 938。由于获得磷酸化所需的Triton X - 100的抑制作用，无法测量PKA对Na，K - ATPase活性的影响。即使在最佳条件下，PKC的磷酸化也未能导致Na，K - ATPase活性的抑制。这表明磷酸化的任何生理作用要么需要对酶特性进行微妙调节，要么需要额外的调节蛋白。
