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J. Biol. Chem. 279 (17): 17250-17259

© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

Ouabain Assembles Signaling Cascades through the Caveolar Na+/K+-ATPase*

Haojie Wang{ddagger}, Michael Haas{ddagger}, Man Liang{ddagger}, Ting Cai{ddagger}, Jiang Tian{ddagger}, Shengwen Li§, , and Zijian Xie{ddagger}

{ddagger}Departments of Pharmacology and Medicine, Medical College of Ohio, Toledo, Ohio 43614 and §Allergan, Inc., Pharmaceuticals R&D, Irvine, California 92623-9534

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.

Received for publication December 4, 2003. Revision received February 9, 2004.

* This work was supported by National Institutes of Health Grants HL-36573, HL-63238, and HL-67963, awarded by the National Heart, Lung and Blood Institute, United States Public Health Service, Department of Health and Human Services, and a pre-doctoral grant, awarded by American Heart Association-Ohio Valley Affiliate. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pharmacology, Medical College of Ohio, 3035 Arlington Ave., Toledo, OH 43614-5804. Tel.: 419-383-4182; Fax: 419-383-2871; E-mail: zxie{at}mco.edu.

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