Heterogeneity of signal transduction by Na-K-ATPase α-isoforms: role of Src interaction

Of the four Na/K-ATPase α isoforms, the ubiquitous α1 Na/K-ATPase possesses both ion transport and Src-dependent signaling functions. Mechanistically, we have identified two putative pairs of domain interactions between α1 Na/K-ATPase and Src that are critical for α1 signaling. Our subsequent report that α2 Na/K-ATPase lacks these putative Src-binding sites and fails to carry on Src-dependent signaling further supported our proposed model of direct interaction between α1 Na/K-ATPase and Src, but fell short of providing evidence for a causative role. This hypothesis was specifically tested here by introducing key residues of the putative Src-interacting domains present on α1 but not α2 sequence into the α2 polypeptide, generating stable cell lines expressing this mutant, and comparing its signaling properties to those of α2-expressing cells. The mutant α2 was fully functional as a Na/K-ATPase. In contrast to wild-type α2, the mutant gained α1-like signaling function, capable of Src interaction and regulation. Consistently, the expression of mutant α2 redistributed Src into caveolin-1-enriched fractions and allowed ouabain to activate Src-mediated signaling cascades, unlike wild-type α2 cells. Finally, mutant α2 cells exhibited a growth phenotype similar to the α1 cells, and proliferated much faster than wild-type α2 cells. These findings reveal the structural requirements for the Na/K-ATPase to function as a Src-dependent receptor and provide strong evidence of isoform-specific Src interaction involving the identified key amino acids. The sequences surrounding the putative Src-binding sites in α2 are highly conserved across species, suggesting that the lack of Src-binding may play a physiologically important and isoform-specific role.