TY - JOUR
T1 - Ouabain stimulates protein kinase B (Akt) phosphorylation in opossum kidney proximal tubule cells through an ERK-dependent pathway
AU - Khundmiri, Syed J.
AU - Amin, Vishal
AU - Henson, Jeff
AU - Lewis, John
AU - Ameen, Mohamed
AU - Rane, Madhavi J.
AU - Delamere, Nicholas A.
PY - 2007/9
Y1 - 2007/9
N2 - 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.
AB - 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.
KW - Cell proliferation
KW - Extracellular signal-regulated kinase
KW - Opossum kidney cells
KW - Sodium/potassium adenosine triphosphatase
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U2 - 10.1152/ajpcell.00535.2006
DO - 10.1152/ajpcell.00535.2006
M3 - Article
C2 - 17634416
AN - SCOPUS:34548710397
SN - 0363-6143
VL - 293
SP - C1171-C1180
JO - American Journal of Physiology - Cell Physiology
JF - American Journal of Physiology - Cell Physiology
IS - 3
ER -