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Phosphorylation of the Catalytic Subunit of Rat Renal Na+,K+-ATPase by Classical PKC Isoforms

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Abstract

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.

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  • Cited by (0)

    1

    M.G.K. and M.J.C. equally contributed to this work.

    2

    S.N. is a Senior investigator at Consejo Nacional de Investigaciones Científicas y Técnicas.

    3

    To whom correspondence and reprint requests should be addressed at Centro de Investigaciones Endocrinológicas, CONICET, Hospital de Niños “R. Gutierrez,” Gallo 1330, (1425) Buenos Aires, Argentina. Fax: + 54 11 4963 5930. E-mail: [email protected].

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