Domain specific regulation of Src kinase by Na/K ATPase alpha1 subunit

Abstract

Invitro binding assay suggests that the α1 subunit of Na/K ATPase interacts with Src via two separate domain interactions‐ the nucleotide binding domain and second cytosolic domain (CD2) binds kinase and SH2 domain of Src respectively. The aim of this work was to assess the functionality of the putative CD2‐SH2 interaction in cellular signal transduction. LLC‐PK1 cells were transfected with either YFP or YFP‐CD2, several clones were generated and characterized. We found that expression of YFP‐CD2 significantly increased basal Src activity and consequently overall protein phosphorylation. YFP‐CD2 disrupted the interaction between Na/K ATPase and Src thereby inhibiting ouabain mediated signal transduction in cells. It also effectively attenuated other Src signaling pathways like cell attachment induced FAK activation. Consistently it inhibited cell spreading and proliferation, both of which require Src signaling. Finally, the C terminal half of CD2 was sufficient in eliciting the inhibitory effects of CD2 on Src kinase. These observations therefore support our notion that CD2 of α1 subunit interacts with Src SH2 domain and is critical for formation of Na/K ATPase/Src receptor complex for ouabain mediated cell signaling. Thus, CD2 acting as a dominant negative mutant can block Src activation dependent signaling pathways via a mechanism different from general Src inhibitors. NIH grant HL36573, GM78565