Isoform specificity of Na-K-ATPase-mediated Ouabain signaling

Dept. of Physiology and Pharmacology, Univ. of Toledo College of Medicine, 3035 Arlington Ave., Toledo, OH 43614-5804, USA.
American journal of physiology. Renal physiology (Impact Factor: 3.25). 05/2008; 294(4):F859-66. DOI: 10.1152/ajprenal.00089.2007
Source: PubMed


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.

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Available from: Sandrine V Pierre, May 20, 2015
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    • "All human α-subunit isoforms contain a conserved ouabain-binding motif [56], thus it is expected that CTS-sensitivity is similar for all cell lines used in this work. However, ouabain-induced ERK signaling is affected differently by α-isoform expression, with α1, α3 and α4 allowing signal transduction, and α2 having no effect on ERK activation [57]. Furthermore, two plasma membrane pools of Na,K-ATPase α-subunits has been proposed, in which the caveolar fraction contains a non-pumping pool of α-subunits which functions in signal transduction, and the non-caveolar fraction contains a pumping pool which functions in ion homeostasis [58]. "
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    • "Overall, there were no consistent differences between the nonouabain and ouabain inhibited activities of total ATPase. It is known that there are various isoforms of the a subunit of Na 1 , K 1 -ATPase that are species specific, and have varying degrees of sensitivity to ouabain inhibition (Anner et al., 1992; Pierre et al., 2007). It has also been shown that certain regions of the a subunit of Na 1 , K 1 -ATPase, when mutated, will reduce ouabain sensitivity (Croyle et al., 1997). "
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