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Degree

Doctor of Philosophy in Medical Sciences (Ph.D.), University of Toledo Health Science Campus, College of Graduate Studies, 2006.

Abstract

Based on the observation that the Na+/K+-ATPase á1 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. GST 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. 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-1 complex in the isolated caveolae preparations as it did in live cells. Finally, depletion of either cholesterol by methyl â-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. To test our hypothesis that Na+/K+-ATPase may be involved in caveolae biogenesis and/or maintenance, we studied caveolin-1 expression, distribution and caveolae cholesterol content in the á1 knockdown cell lines developed in our laboratory. In the caveolae fractionation study with the á1 knockdown A4-11 cells, the caveolin-1 protein was no longer concentrated in the caveolae fractions but widely distributed throughout all the medium and heavy fractions. In the á1 knockdown cells, both the endogenous and exogenous caveolin-1 protein was displaced from the cell membrane to the intracellular space. Reintroduction of either wild type rat á1 or the D376E mutant rat á1 (DPP) reversed the EGFP- and ECFP-caveolin-1 distribution to the cell membrane. FRET analysis showed that in the rescued cells, both rat á1 and DPP directly interacted with caveolin-1 on the cell membrane and in the perinuclear region. Cavevolin-1 expression and caveolar cholesterol content also were regulated by the Na+/K+-ATPase á1 expression level. Our data suggest a role of the Na+/K+-ATPase in regulating caveolin-1 expression, intracellular transport and targeting as well as the caveolae composition.

Keywords

Na+/K+-ATPase; Signal Transduction

Advisor

Zi-Jian Xie, Ph.D

Pages

139p.

Document number: mco1147300366
Permalink: http://rave.ohiolink.edu/etdc/view?acc_num=mco1147300366

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