Involvement of Na/K-ATPase in hydrogen peroxide-induced activation of the Src/ERK pathway in LLC-PK1 cells

Free Radic Biol Med. 2014 Jun;71:415-426. doi: 10.1016/j.freeradbiomed.2014.03.036. Epub 2014 Apr 1.


We have shown that Na/K-ATPase interacts with Src. Here, we test the role of this interaction in H2O2-induced activation of Src and ERK. We found that exposure of LLC-PK1 cells to H2O2 generated by the addition of glucose oxidase into the culture medium activated Src and ERK1/2. It also caused a modest reduction in the number of surface Na/K-ATPases and in ouabain-sensitive Rb(+) uptake. These effects of H2O2 seem similar to those induced by ouabain, a specific ligand of Na/K-ATPase, in LLC-PK1 cells. In accordance, we found that the effects of H2O2 on Src and ERK1/2 were inhibited in α1 Na/K-ATPase-knockdown PY-17 cells. Whereas expression of wild-type α1 or the A420P mutant α1 defective in Src regulation rescued the pumping activity in PY-17 cells, only α1, and not the A420P mutant, was able to restore the H2O2-induced activation of protein kinases. Consistent with this, disrupting the formation of the Na/K-ATPase/Src complex with pNaKtide attenuated the effects of H2O2 on the kinases. Moreover, a direct effect of H2O2 on Na/K-ATPase-mediated regulation of Src was demonstrated. Finally, H2O2 reduced the expression of E-cadherin through the Na/K-ATPase/Src-mediated signaling pathway. Taken together, the data suggest that the Na/K-ATPase/Src complex may serve as one of the receptor mechanisms for H2O2 to regulate Src/ERK protein kinases and consequently the phenotype of renal epithelial cells.

Keywords: ERK; Free radicals; Hydrogen peroxide; Mutant; Na/K-ATPase; Src.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cadherins / genetics
  • Cadherins / metabolism
  • Enzyme Activation / drug effects
  • Enzyme Inhibitors / pharmacology
  • Gene Expression Regulation
  • Glucose / metabolism
  • Glucose / pharmacology
  • Hydrogen Peroxide / pharmacology*
  • LLC-PK1 Cells
  • Mitogen-Activated Protein Kinase 1 / genetics*
  • Mitogen-Activated Protein Kinase 1 / metabolism
  • Mitogen-Activated Protein Kinase 3 / genetics*
  • Mitogen-Activated Protein Kinase 3 / metabolism
  • Ouabain / pharmacology
  • Phenotype
  • Protein Subunits / genetics*
  • Protein Subunits / metabolism
  • Signal Transduction
  • Sodium-Potassium-Exchanging ATPase / genetics*
  • Sodium-Potassium-Exchanging ATPase / metabolism
  • Swine
  • src-Family Kinases / genetics*
  • src-Family Kinases / metabolism


  • Cadherins
  • Enzyme Inhibitors
  • Protein Subunits
  • Ouabain
  • Hydrogen Peroxide
  • src-Family Kinases
  • Mitogen-Activated Protein Kinase 1
  • Mitogen-Activated Protein Kinase 3
  • Sodium-Potassium-Exchanging ATPase
  • Glucose