Chibalin A V, Vasilets L A, Hennekes H, Pralong D, Geering K
Institut de Pharmacologie et de Toxicologie de l'Université, Lausanne, Switzerland.
J Biol Chem. 1992 Nov 5;267(31):22378-84.
The phosphorylation of the alpha-subunit of Na+/K(+)-transporting ATPase (Na,K-ATPase) by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) was characterized in purified enzyme preparations of Bufo marinus kidney and duck salt gland and in microsomes of Xenopus oocytes. In addition, we have examined cAMP and phorbol esters, which are stimulators of PKA and PKC, respectively, for their ability to provoke the phosphorylation of alpha-subunits of Na,K-ATPase in homogenates of Xenopus oocytes. In the enzyme from the duct salt gland, phosphorylation by PKA and PKC occurs on serine and threonine residues, whereas in the enzyme from B. marinus kidney and Xenopus oocytes, phosphorylation by PKA occurs only on serine residues. Phosphopeptide analysis indicates that a site phosphorylated by PKA resides in a 12-kDa fragment comprising the C terminus of the polypeptide. Studies of phosphorylation performed on homogenates of Xenopus oocytes show that not only endogenous oocyte Na,K-ATPase but also exogenous Xenopus Na,K-ATPase expressed in the oocyte by microinjection of cRNA can be phosphorylated in response to stimulation of oocyte PKA and PKC. In conclusion, these data are consistent with the possibility that the alpha-subunit of Na,K-ATPase can serve as a substrate for PKA and PKC in vivo.
在海蟾蜍肾脏和鸭盐腺的纯化酶制剂以及非洲爪蟾卵母细胞的微粒体中，对环磷酸腺苷（cAMP）依赖性蛋白激酶（PKA）和蛋白激酶C（PKC）使钠钾转运ATP酶（Na,K-ATPase）α亚基磷酸化的情况进行了表征。此外，我们还分别检测了作为PKA和PKC刺激剂的cAMP和佛波酯，观察它们引发非洲爪蟾卵母细胞匀浆中Na,K-ATPaseα亚基磷酸化的能力。在盐腺导管的酶中，PKA和PKC介导的磷酸化发生在丝氨酸和苏氨酸残基上，而在海蟾蜍肾脏和非洲爪蟾卵母细胞的酶中，PKA介导的磷酸化仅发生在丝氨酸残基上。磷酸肽分析表明，PKA磷酸化的位点位于一个包含多肽C末端的12 kDa片段中。对非洲爪蟾卵母细胞匀浆进行的磷酸化研究表明，不仅卵母细胞内源性的Na,K-ATPase，而且通过显微注射cRNA在卵母细胞中表达的外源性非洲爪蟾Na,K-ATPase，在受到卵母细胞PKA和PKC刺激时都能被磷酸化。总之，这些数据与Na,K-ATPaseα亚基在体内可作为PKA和PKC底物的可能性一致。
