Ouabain stimulates protein kinase B (Akt) phosphorylation in opossum kidney proximal tubule cells through an ERK-dependent pathway

Syed J. Khundmiri, Vishal Amin, Jeff Henson, John Lewis, Mohamed Ameen, Madhavi J. Rane, Nicholas A Delamere

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

Endogenous cardiotonic glycosides bind to the inhibitory binding site of the plasma membrane sodium pump (Na+/K+-ATPase). Plasma levels of endogenous cardiotonic glycosides increase in several disease states, such as essential hypertension and uremia. Low concentrations of ouabain, which do not inhibit Na+/K+-ATPase, induce cell proliferation. The mechanisms of ouabain-mediated response remain unclear. Recently, we demonstrated that in opossum kidney (OK) proximal tubular cells, low concentrations of ouabain induce cell proliferation through phosphorylation of protein kinase B (Akt) in a calcium-dependent manner. In the present study, we identified ERK as an upstream kinase regulating Akt activation in ouabain-stimulated cells. Furthermore, we provide evidence that low concentrations of ouabain stimulate Na+/K+-ATPase-mediated 86Rb uptake in an Akt-, ERK-, and Src kinase-dependent manner. Ouabain-mediated ERK phosphorylation was inhibited by blockade of intracellular calcium release, calcium entry, tyrosine kinases, and phospholipase C. Pharmacological inhibition of phosphoinositide-3 kinase and Akt failed to inhibit ouabain-stimulated ERK phosphorylation. Ouabain-mediated Akt phosphorylation was inhibited by U0126, a MEK/ERK inhibitor, suggesting that ouabain-mediated Akt phosphorylation is dependent on ERK. In an in vitro kinase assay, active recombinant ERK phosphorylated recombinant Akt on Ser 473. Moreover, transient transfection with constitutively active MEK1, an upstream regulator of ERK, increased Akt phosphorylation and activation, whereas overexpression of constitutively active Akt failed to stimulate ERK phosphorylation. Ouabain at low concentrations also promoted cell proliferation in an ERK-dependent manner. These findings suggest that ouabain-stimulated ERK phosphorylation is required for Akt phosphorylation on Ser473, cell proliferation, and stimulation of Na+/K +-ATPase-mediated 86Rb uptake in OK cells.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume293
Issue number3
DOIs
StatePublished - Sep 2007

Fingerprint

Opossums
Proto-Oncogene Proteins c-akt
Proximal Kidney Tubule
Phosphorylation
MAP Kinase Signaling System
Ouabain
Cell proliferation
Adenosine Triphosphatases
Cell Proliferation
Cardiotonic Agents
Phosphotransferases
Glycosides
Calcium
Chemical activation
Kidney
Sodium-Potassium-Exchanging ATPase
1-Phosphatidylinositol 4-Kinase
src-Family Kinases
Uremia
Mitogen-Activated Protein Kinase Kinases

Keywords

  • Cell proliferation
  • Extracellular signal-regulated kinase
  • Opossum kidney cells
  • Sodium/potassium adenosine triphosphatase

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Cell Biology
  • Physiology

Cite this

Ouabain stimulates protein kinase B (Akt) phosphorylation in opossum kidney proximal tubule cells through an ERK-dependent pathway. / Khundmiri, Syed J.; Amin, Vishal; Henson, Jeff; Lewis, John; Ameen, Mohamed; Rane, Madhavi J.; Delamere, Nicholas A.

In: American Journal of Physiology - Cell Physiology, Vol. 293, No. 3, 09.2007.

Research output: Contribution to journalArticle

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abstract = "Endogenous cardiotonic glycosides bind to the inhibitory binding site of the plasma membrane sodium pump (Na+/K+-ATPase). Plasma levels of endogenous cardiotonic glycosides increase in several disease states, such as essential hypertension and uremia. Low concentrations of ouabain, which do not inhibit Na+/K+-ATPase, induce cell proliferation. The mechanisms of ouabain-mediated response remain unclear. Recently, we demonstrated that in opossum kidney (OK) proximal tubular cells, low concentrations of ouabain induce cell proliferation through phosphorylation of protein kinase B (Akt) in a calcium-dependent manner. In the present study, we identified ERK as an upstream kinase regulating Akt activation in ouabain-stimulated cells. Furthermore, we provide evidence that low concentrations of ouabain stimulate Na+/K+-ATPase-mediated 86Rb uptake in an Akt-, ERK-, and Src kinase-dependent manner. Ouabain-mediated ERK phosphorylation was inhibited by blockade of intracellular calcium release, calcium entry, tyrosine kinases, and phospholipase C. Pharmacological inhibition of phosphoinositide-3 kinase and Akt failed to inhibit ouabain-stimulated ERK phosphorylation. Ouabain-mediated Akt phosphorylation was inhibited by U0126, a MEK/ERK inhibitor, suggesting that ouabain-mediated Akt phosphorylation is dependent on ERK. In an in vitro kinase assay, active recombinant ERK phosphorylated recombinant Akt on Ser 473. Moreover, transient transfection with constitutively active MEK1, an upstream regulator of ERK, increased Akt phosphorylation and activation, whereas overexpression of constitutively active Akt failed to stimulate ERK phosphorylation. Ouabain at low concentrations also promoted cell proliferation in an ERK-dependent manner. These findings suggest that ouabain-stimulated ERK phosphorylation is required for Akt phosphorylation on Ser473, cell proliferation, and stimulation of Na+/K +-ATPase-mediated 86Rb uptake in OK cells.",
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AU - Lewis, John

AU - Ameen, Mohamed

AU - Rane, Madhavi J.

AU - Delamere, Nicholas A

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