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 α1 subunit of rat renal Na+,K+-ATPase (α1 Na+,K+-ATPase). Using in vitro phosphotransferase assays we found that classical PKCs (cPKCs) α, βI, and γ efficiently phosphorylate α1 Na+,K+-ATPase. However, α1 Na+,K+-ATPase was a poor substrate for the novel PKCs (nPKCs) δ and ϵ. 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 86Rb+ uptake) in COS-7 cells expressing the rat α1 Na+,K+-ATPase. 1-oleoyl-2-acetoyl-sn-glycerol (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 nPKCϵ, did not affect 86Rb+ 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.