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 alpha1 subunit of rat renal Na+,K+-ATPase (alpha1 Na+,K+-ATPase). Using in vitro phosphotransferase assays we found that classical PKCs (cPKCs) alpha, betaI, and gamma efficiently phosphorylate alpha1 Na+,K+-ATPase. However, alpha1 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 86Rb+ uptake) in COS-7 cells expressing the rat alpha1 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 nPKCepsilon, 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.
Kazanietz
M G
MG
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104-6160, USA.
Caloca
M J
MJ
Aizman
O
O
Nowicki
S
S
D000818:Animals; D019556:COS Cells; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D007509:Irritants / Q000494:pharmacology; D007527:Isoenzymes; D010042:Ouabain / Q000494:pharmacology; D010449:Peptide Mapping; D010703:Phorbol Esters / Q000494:pharmacology; D010766:Phosphorylation; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism; D021381:Protein Transport; D051381:Rats; D012413:Rubidium / Q000494:pharmacology; D012964:Sodium / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
D018076:DNA, Complementary; D007509:Irritants; D007527:Isoenzymes; D010703:Phorbol Esters; D020033:Protein Isoforms; C029704:12-deoxyphorbolphenylacetate; D010042:Ouabain; D012964:Sodium; D011493:Protein Kinase C; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium