Phosphorylation of the catalytic subunit of rat renal Na+,K+-ATPase by classical PKC isoforms

In this study we have evaluated the specificity of different PKC isozymes for the phosphorylation of the catalytic alpha (1), subunit of rat renal Na+, K+-ATPase (alpha (1) Na+, K+-ATPase). Using in vitro phosphotransferase assays we found that classical PKCs (cPKCs) alpha, beta1, and gamma efficiently phosphorylate alpha (1) Na+, K+-ATPase. However, alpha (1) Na+, K+-ATPase was a poor substrate for the novel PKCs (nPKCs) delta and epsilon. Two-dimensional phosphopeptide mapping revealed a similar pattern of phosphorylation by all cPKCs. The functional significance of this finding was evaluated by measuring Na+, K+-ATPase activity (assessed by Rb-86(+) uptake) in COS-7 cells expressing the rat alpha (1) Na+, K+-ATPaSe. 1-oleoyl-2-acetayl-sn-glyceroI (OAG;), a nonselective PKC activator, inhibited Na+, K+-ATPase activity in this system. On the other hand, 12-deoxyphorbol-13-phenylacetate (DPP), which preferentially activates nPKC epsilon, did not affect Rb-86(+) uptake. These results indicate a differential pattern of phosphorylation and regulation of rat renal Na+, K+-ATPase activity by PKC isoforms and suggest an important role for cPKCs in the physiological regulation of the pump, (C) 2001 Academic Press.