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

Mol Biol Cell. 1999 Sep;10(9):2847-59. doi: 10.1091/mbc.10.9.2847.


Phosphorylation of the alpha-subunit of Na+,K(+)-ATPase plays an important role in the regulation of this pump. Recent studies suggest that insulin, known to increase solute and fluid reabsorption in mammalian proximal convoluted tubule (PCT), is stimulating Na+,K(+)-ATPase activity through the tyrosine phosphorylation process. This study was therefore undertaken to evaluate the role of tyrosine phosphorylation of the Na+,K(+)-ATPase alpha-subunit in the action of insulin. In rat PCT, insulin and orthovanadate (a tyrosine phosphatase inhibitor) increased tyrosine phosphorylation level of the alpha-subunit more than twofold. Their effects were not additive, suggesting a common mechanism of action. Insulin-induced tyrosine phosphorylation was prevented by genistein, a tyrosine kinase inhibitor. The site of tyrosine phosphorylation was identified on Tyr-10 by controlled trypsinolysis in rat PCTs and by site-directed mutagenesis in opossum kidney cells transfected with rat alpha-subunit. The functional relevance of Tyr-10 phosphorylation was assessed by 1) the abolition of insulin-induced stimulation of the ouabain-sensitive (86)Rb uptake in opossum kidney cells expressing mutant rat alpha1-subunits wherein tyrosine was replaced by alanine or glutamine; and 2) the similarity of the time course and dose dependency of the insulin-induced increase in ouabain-sensitive (86)Rb uptake and tyrosine phosphorylation. These findings indicate that phosphorylation of the Na+,K(+)-ATPase alpha-subunit at Tyr-10 likely participates in the physiological control of sodium reabsorption in PCT.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Substitution
  • Animals
  • Cells, Cultured
  • Enzyme Activation / drug effects
  • Genistein / pharmacology
  • Insulin / pharmacology*
  • Insulin Antagonists / pharmacology
  • Kidney Tubules, Proximal / cytology
  • Kidney Tubules, Proximal / drug effects
  • Kidney Tubules, Proximal / enzymology*
  • Male
  • Opossums
  • Ouabain / pharmacology
  • Phosphorylation / drug effects
  • Phosphotyrosine / metabolism*
  • Protein Kinase Inhibitors
  • Protein Kinases / metabolism
  • Rats
  • Rats, Wistar
  • Sodium-Potassium-Exchanging ATPase / antagonists & inhibitors
  • Sodium-Potassium-Exchanging ATPase / chemistry
  • Sodium-Potassium-Exchanging ATPase / metabolism*
  • Transfection
  • Tyrosine / genetics
  • Tyrosine / metabolism
  • Vanadates / pharmacology


  • Insulin
  • Insulin Antagonists
  • Protein Kinase Inhibitors
  • Phosphotyrosine
  • Vanadates
  • Tyrosine
  • Ouabain
  • Genistein
  • Protein Kinases
  • Sodium-Potassium-Exchanging ATPase