Na/K-ATPase, A Signaling Receptor
Abstract (Summary)In this study, we tested if the Na/K-ATPase and Src can form a functional signaling complex. We also examined the cellular function of the signaling Na/K-ATPase. In LLC-PK1 cells the Na/K-ATPase and Src colocalized in the plasma membrane. FRET analysis indicated that both proteins were in close proximity, suggesting a direct interaction. GST pull-down assay showed a direct, ouabain-regulated, and multi-focal interaction between the á1 subunit of Na/K-ATPase and Src. While the interaction between the Src kinase domain and the third cytosolic domain (CD3) of á1 is regulated by ouabain, the Src SH3SH2 domain binds to the second cytosolic domain constitutively. Functionally, binding of Src to either the Na/K-ATPase or GST-CD3 inhibited Src activity. The Na/K-ATPase-bound Src can be activated by ouabain treatment. Concomitantly, ouabain treatment also stimulated tyrosine phosphorylation of the proteins that are associated with the Na/K-ATPase. These new findings illustrate a novel molecular mechanism of signal transduction involving the interaction of a P-type ATPase and a non-receptor tyrosine kinase. We also showed here that ouabain differentially regulates cell growth in different cell lines. It stimulated LLC-PK1 cells proliferation at 10 to 50 nM, but induced cell growth inhibition at the same concentrations in BT20 cells and DU145 cells. Meanwhile, low concentration of ouabain up-regulated the Na/K-ATPase expression in LLC-PK1 cells but dramatically decreased Na/K-ATPase amount in BT20 cells and DU145 cells. Using the recently established Na/K-ATPase knock-down cell lines, we further showed that reducing of Na/K-ATPase amount not only slowed down cell growth but also sensitized the cells to ouabain-induced cell growth inhibition. More importantly, our results also showed that the major components of Na/K-ATPase signaling cascade, caveolin-1 and Src, are critical for up-regulation of Na/K-ATPase abundance and activation of the Akt survival pathway in LLC-PK1 cells during ouabain treatment. We then conclude that the capacity of Na/K-ATPase repletion after ouabain treatment is closely linked with the ouabain-induced cell growth inhibition, and the regulation of Na/K-ATPase repletion relies on the integrity of the signaling cascade of Na/K-ATPase. The present study also provides a new insight of how the signaling function of Na/K-ATPase works as a sensor for its self-regulation and therefore regulate other cellular functions.
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:na k atpase src signal transduction ouabain
Date of Publication:01/01/2007