Isoform specificity of Na-K-ATPase-mediated ouabain signaling.
The ion transporter Na-K-ATPase functions as a cell signal transducer that mediates ouabain-induced activation of protein kinases, such as ERK. While Na-K-ATPase composed of the alpha(1)-polypeptide is involved in cell signaling, the role of other alpha-isoforms (alpha(2), alpha(3), and alpha(4)) in transmitting ouabain effects is unknown. We have explored this using baculovirus-directed expression of Na-K-ATPase polypeptides in insect cells and ERK phosphorylation as an indicator of ouabain-induced signaling. Ouabain addition to Sf-9 cells coexpressing Na-K-ATPase alpha(1)- and beta(1)-isoforms stimulated ERK phosphorylation. In contrast, expression of the alpha(1)- and beta(1)-polypeptides alone resulted in no effect, indicating that the alphabeta-complex is necessary for Na-K-ATPase signaling. Moreover, the ouabain effect was sensitive to genistein, suggesting that Na-K-ATPase-mediated tyrosine kinase activation is a critical event in the intracellular cascade leading to ERK phosphorylation. In addition, the Na-K-ATPases alpha(3)beta(1)- and alpha(4)beta(1)-isozymes, but not alpha(2)beta(1), responded to ouabain treatment. In agreement with the differences in ouabain affinity of the alpha-polypeptides, alpha(1)beta(1) required 100- to 1,000-fold more ouabain to signal than did alpha(4)beta(1) and alpha(3)beta(1), respectively. These results confirm the role of the Na-K-ATPase in ouabain signal transduction, show that there are important isoform-specific differences in Na-K-ATPase signaling, and demonstrate the suitability of the baculovirus expression system for studying Na-K-ATPase-mediated ouabain effects.
Pierre
Sandrine V
SV
Dept. of Physiology and Pharmacology, Univ. of Toledo College of Medicine, 3035 Arlington Ave., Toledo, OH 43614-5804, USA. sandrine.pierre@utoledo.edu
Sottejeau
Yoann
Y
Gourbeau
Jean-Michel
JM
Sánchez
Gladis
G
Shidyak
Amjad
A
Blanco
Gustavo
G
D000818:Animals; D016367:Baculoviridae / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D048049:Extracellular Signal-Regulated MAP Kinases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D010042:Ouabain / Q000494:pharmacology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera / Q000201:enzymology
D007527:Isoenzymes; D010042:Ouabain; D048049:Extracellular Signal-Regulated MAP Kinases; D000254:Sodium-Potassium-Exchanging ATPase