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 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 Pi per mol alpha-subunit, respectively. Proteolytic fingerprinting of the pig alpha1-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 alpha1-subunit of the purified Na+/K+-ATPase do not appear to be easily accessible for PKG since incorporation of Pi 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 microm 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 33P 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.
Fotis
H
H
Max-Planck-Institute for Biophysics, Frankfurt/Main, Germany.
Tatjanenko
L V
LV
Vasilets
L A
LA
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D004285:Dogs; D004789:Enzyme Activation; D007668:Kidney / Q000201:enzymology*; D009865:Oocytes / Q000201:enzymology; D010766:Phosphorylation; D051381:Rats; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013552:Swine; D014981:Xenopus
D017869:Cyclic GMP-Dependent Protein Kinases; D000251:Adenosine Triphosphatases; D000254:Sodium-Potassium-Exchanging ATPase