Abstract
Based on the observation that the Na(+)/K(+)-ATPase alpha subunit contains two conserved caveolin-binding motifs, we hypothesized that clustering of the Na(+)/K(+)-ATPase and its partners in caveolae facilitates ouabain-activated signal transduction. Glutathione S-transferase pull-down assay showed that the Na(+)/K(+)-ATPase bound to the N terminus of caveolin-1. Significantly, ouabain regulated the interaction in a time- and dose-dependent manner and stimulated tyrosine phosphorylation of caveolin-1 in LLC-PK1 cells. When added to the isolated membrane fractions, ouabain increased tyrosine phosphorylation of proteins from the isolated caveolae but not other membrane fractions. Consistently, ouabain induced the formation of a Na(+)/K(+)-ATPase-Src-caveolin complex in the isolated caveolae preparations as it did in live cells. Finally, depletion of either cholesterol by methyl beta-cyclodextrin or caveolin-1 by siRNA significantly reduced the caveolar Na(+)/K(+)-ATPase and Src. Concomitantly, cholesterol depletion abolished ouabain-induced recruitment of Src to the Na(+)/K(+)-ATPase signaling complex. Like depletion of caveolin-1, it also blocked the effect of ouabain on ERKs, which was restored after cholesterol repletion. Clearly, the caveolar Na(+)/K(+)-ATPase represents the signaling pool of the pump that interacts with Src and transmits the ouabain signals.
Highlights
Naϩ/Kϩ-ATPase, or the sodium pump, is a ubiquitous transmembrane pump that transports Naϩ and Kϩ across the plasma membrane by hydrolyzing ATP [1,2,3]
To begin addressing the role of the caveolar Naϩ/Kϩ-ATPase in ouabaininduced signal transduction, we first determined how the Naϩ/ Kϩ-ATPase is distributed in these cells
We demonstrated that the signaling Naϩ/ Kϩ-ATPase interacts with caveolin-1 in a ligand-dependent manner and that the interaction is important for the signaling pump to transmit the ouabain signal
Summary
Naϩ/Kϩ-ATPase, or the sodium pump, is a ubiquitous transmembrane pump that transports Naϩ and Kϩ across the plasma membrane by hydrolyzing ATP [1,2,3]. Binding of ouabain to the pump activates multiple signal transduction pathways and regulates transcription and translation of many genes in cardiac myocytes and other cell types [5,6,7]. Some of these ouabain effects were independent of changes in intracellular ion concentrations (8 –10). Realization that Naϩ/Kϩ-ATPase has to interact with Src, EGFR, and other proteins to transmit the ouabain signal has prompted us to propose that the signaling pump is pre-assembled with its partners in membrane microdomains such as caveolae. This study aims to determine whether Naϩ/Kϩ-ATPase directly interacts with caveolin-1 and if caveolae are involved in formation of a signaling module for components of the ouabainactivated Naϩ/Kϩ-ATPase signaling complex
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