Phosphorylation of the α-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 species investigated. Phosphorylation of the beta-subunit was not observed. The 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, respectively. Proteolytic fingerprinting of the pig alpha 1-subunits phosphorylated by PKG using specific antibodies raised against N-terminus or C-terminus reveals that phosphorylation sites are located within the intracellular loop of the alpha-subunit between the 35 kDa N-terminal and 27 kDa C-terminal fragments. Phosphorylation sites within the alpha 1-subunit of the purified Na+/K+-ATPase do not appear to be easily accessible for PKG since incorporation 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%. Administration of 50 mu M of cGMP or dbcGMP in yolk-free homogenates of Xenopus oocytes leads to stimulation of ouabain-dependent ATPase activity by 130-198% and to incorporation of P-33 into the alpha-subunit without the detergent. Hence, PKG plays regulatory role in active transmembraneous transport of Na+ and K+ via phosphorylation of the catalytic subunit of the Na+/K+-ATPase.