Phosphorylation of the alpha-subunits of the Na+/K+-ATPase from mammalian kidneys and Xenopus oocytes by cGMP-dependent protein kinase results in stimulation of ATPase activity

Phosphorylation of Na+/K+-ATPase by cGMP-dependent protein kinase (PKG) has been studied in enzymes purified from pig, dog, sheep and rat kidneys, and in Xenopus oocytes. PKG phosphorylates the alpha-subunits of all animal sp ecies investigated. Phosphorylation of the beta-subunit was not observed. T he stoichiometry of phosphorylation estimated for pig, sheep and dog renal Na+/K+-ATPase is 3.5, 2.2 and 2.1 mol P-i per mol alpha-subunit, respective ly. Proteolytic fingerprinting of the pig alpha 1-subunits phosphorylated b y PKG using specific antibodies raised against N-terminus or C-terminus rev eals that phosphorylation sites are located within the intracellular loop o f the alpha-subunit between the 35 kDa N-terminal and 27 kDa C-terminal fra gments. Phosphorylation sites within the alpha 1-subunit of the purified Na +/K+-ATPase do not appear to be easily accessible for PKG since incorporati on of P-i requires 0.2% of Triton X-100. Administration of cGMP and PKG in the presence of 5 mM ATP, which prevents inactivation of the Na+/K+-ATPase by detergent, leads to stimulation of hydrolytic activity by 61%. Administr ation of 50 mu M of cGMP or dbcGMP in yolk-free homogenates of Xenopus oocy tes leads to stimulation of ouabain-dependent ATPase activity by 130-198% a nd to incorporation of P-33 into the alpha-subunit without the detergent. H ence, PKG plays regulatory role in active transmembraneous transport of Na and K+ via phosphorylation of the catalytic subunit of the Na+/K+-ATPase.