Insulin-induced stimulation of Na+,K+-ATPase activity in kidney proximal tubule cells depends on phosphorylation of the alpha-subunit at Tyr-10

Phosphorylation of the alpha-subunit of Na+,K+-ATPase plays an important ro le in the regulation of this pump. Recent studies suggest that insulin, kno wn to increase solute and fluid reabsorption in mammalian proximal convolut ed tubule (PCT), is stimulating Na+,K+-ATPase activity through the tyrosine phosphorylation process. This study was therefore undertaken to evaluate t he role of tyrosine phosphorylation of the Na+,K+-ATPase alpha-subunit in t he action of insulin. In rat PCT, insulin and orthovanadate (a tyrosine pho sphatase inhibitor) increased tyrosine phosphorylation level of the alpha-s ubunit more than twofold. Their effects were not additive, suggesting a com mon mechanism of action. Insulin-induced tyrosine phosphorylation was preve nted by genistein, a tyrosine kinase inhibitor. The site of tyrosine phosph orylation was identified on Tyr-10 by controlled trypsinolysis in rat PCTs and by site-directed mutagenesis in opossum kidney cells transfected with r at alpha-subunit. The functional relevance of Tyr-10 phosphorylation was as sessed by 1) the abolition of insulin-induced stimulation of the ouabain-se nsitive Rb-86 uptake in opossum kidney cells expressing mutant rat alpha 1- subunits wherein tyrosine was replaced by alanine or glutamine; and 2) the similarity of the time course and dose dependency of the insulin-induced in crease in ouabain-sensitive Rb-86 uptake and tyrosine phosphorylation. Thes e findings indicate that phosphorylation of the Na+,K+-ATPase alpha-subunit at Tyr-10 likely participates in the physiological control of sodium reabs orption in PCT.