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Na+/K+-ATPase and Signal Transduction

Wang, Haojie
http://rave.ohiolink.edu/etdc/view?acc_num=mco1147300366

Abstract Details

2006, Doctor of Philosophy in Medical Sciences (Ph.D.), University of Toledo, College of Graduate Studies.
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.
Zi-Jian Xie, Ph.D. (Advisor)
139 p.
Na+/K+-ATPase; Signal Transduction

Recommended Citations

Citations

  • Wang, H. (2006). Na+/K+-ATPase and Signal Transduction [Doctoral dissertation, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=mco1147300366

    APA Style (7th edition)

  • Wang, Haojie. Na+/K+-ATPase and Signal Transduction. 2006. University of Toledo, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=mco1147300366.

    MLA Style (8th edition)

  • Wang, Haojie. "Na+/K+-ATPase and Signal Transduction." Doctoral dissertation, University of Toledo, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=mco1147300366

    Chicago Manual of Style (17th edition)

mco1147300366
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© 2005, all rights reserved.
This open access ETD is published by University of Toledo Health Science Campus and OhioLINK.