Expression of Mutant α1 Na/K-ATPase Defective in Conformational Transition Attenuates Src-mediated Signal Transduction

The alpha 1 Na/K-ATPase possesses both pumping and signaling functions. Using purified enzyme we found that the alpha 1 Na/K-ATPase might interact with and regulate Src activity in a conformation-dependent manner. Here we further explored the importance of the conformational transition capability of alpha 1 Na/K-ATPase in regulation of Src-related signal transduction in cell culture. We first rescued the alpha 1-knockdown cells by wild-type rat alpha 1 or alpha 1 mutants (I279A and F286A) that are known to be defective in conformational transition. Stable cell lines with comparable expression of wild type alpha 1, I279A, and F286A were characterized. As expected, the defects in conformation transition resulted in comparable degree of inhibition of pumping activity in the mutant-rescued cell lines. However, I279A was more effective in inhibiting basal Src activity than either the wild-type or the F286A. Although much higher ouabain concentration was required to stimulate Src in I279A-rescued cells, extracellular K+ was comparably effective in regulating Src in both control and I279A cells. In contrast, ouabain and extracellular K+ failed to produce detectable changes in Src activity in F286A-rescued cells. Furthermore, expression of either mutant inhibited integrin-induced activation of Src/FAK pathways and slowed cell spreading processes. Finally, the expression of these mutants inhibited cell growth, with I279A being more potent than that of F286A. Taken together, the new findings suggest that the alpha 1 Na/K-ATPase may be a key player in dynamic regulation of cellular Src activity and that the capability of normal conformation transition is essential for both pumping and signaling functions of alpha 1 Na/K-ATPase.