Diversity of Regulatory Phosphorylation of the Na<sup>+</sup>/K<sup>+</sup>-ATPase from Mammalian Kidneys and <i>Xenopus</i>Oocytes by Protein Kinases: Characterization of the Phosphorylation Site for Protein Kinase C

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 <i>Xenopus </i>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 <i>Xenopus </i>oocytes with β subunits, and modulation of transport activity as well as phosphorylation of endogenous <i>Xenopus </i>and of expressed rat pumps by PKC was investigated. Activation of endogenous PKC from the <i>Xenopus </i>oocytes by phorbol ester produces inhibition of ouabain-sensitive ⁸⁶Rb uptake of <i>Xenopus </i>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 <i>Xenopus </i>oocytes, the rat PKC inhibits ouabain-sensitive ⁸⁶Rb uptake by 79 ± 4%. Both endogenous <i>Xenopus </i>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 <i>Xenopus </i>PKC is activated by PMA. This suggests that additional sites in the rat α1 subunit may be phosphorylated during PMA-dependent activation of <i>the Xenopus PKC.</i>