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Na+/K++-ATPase; Phosphorylation sites; Protein kinases; Site-directed mutagenesis; Xenopus oocytes
Abstract:
Protein-kinase-mediated phosphorylation of the Na+/K+-ATPase has been studied in enzymes purified from pig, dog, sheep, and rat kidneys and in Xenopus oocytes. None of the α subunits from mammalian kidney ATPase is phosphorylated by casein kinase II and Ca2+/calmodulin-dependent protein kinase. For the purified enzymes, rat protein kinase C (PKC) phosphorylates only the α subunit of the Na+/K+-ATPase from rat kidney. Rat α1 subunits mutated in the putative phosphorylation sites Ser-23 and Ser-16 by Ala were co-expressed in Xenopus oocytes with β subunits, and modulation of transport activity as well as phosphorylation of endogenous Xenopus and of expressed rat pumps by PKC was investigated. Activation of endogenous PKC from the Xenopus oocytes by phorbol ester produces inhibition of ouabain-sensitive 86Rb uptake of Xenopus pumps by 40 ± 6%. Microinjection into the oocytes of exogenous rat PKC or its catalytic domain PKM leads to an increase in 86Rb uptake by 41 ± 5 and 25 ± 2%, respectively. On the other hand, in rat α1 isoform expressed in Xenopus oocytes, the rat PKC inhibits ouabain-sensitive 86Rb uptake by 79 ± 4%. Both endogenous Xenopus α subunits and expressed wild-type rat α1 sub-units are phosphorylated by rat PKC. In contrast to its effect on the expressed rat wild-type pump, rat PKC does not inhibit transport activity and does not phosphorylate the α subunit of the S23A mutant. If Ser-16 instead of Ser-23 is substituted by Ala, the mutated α subunits are still phosphorylated, but transport activity of mutated pumps is not modulated by PKC. The data demonstrate that Ser-23 is the actual site of regulatory phosphorylation of rat PKC in the rat kidney α1 subunit, but Ser-16 is, nevertheless, important to mediate the effects of PKC-induced phosphorylation to the changes in transport activity. Substitution of both Ser-23 and Ser-16 by Ala does not influence the inhibition of transport activity and does not abolish phosphorylation of mutated pumps if endogenous Xenopus PKC is activated by PMA. This suggests that additional sites in the rat α1 subunit may be phosphorylated during PMA-dependent activation of the Xenopus PKC
