The Na/K-ATPase α1 and c-Src form signaling complex under native condition: A crosslinking approach.

The protein-protein interactions amongst the Na/K-ATPase α1 subunit, c-Src, and caveolin-1 (cav-1) are essential for the Na/K-ATPase signaling functions. However, there are arguments concerning the interaction model. The present study aims to clarify the interactions amongst the endogenous native proteins in live cells under native resting condition. Under native condition, Blue Native-PAGE and Blue Native-PAGE/SDS-PAGE 2D analyses demonstrated co-existence of the α1 subunit and c-Src in same protein complex, as well as a direct interaction between the α1 subunit and c-Src. By comparison of cleavable and non-cleavable cysteine-cysteine crosslinked samples, capillary immunoblotting analysis demonstrated that depletion of Src kinase family members (c-Src, Yes, and Fyn) or cav-1 clearly reduced the interactions of the α1 subunit with proteins, but depletion of cav-1 did not affect the interaction of c-Src with the α1 subunit. The data indicated that there are direct interactions between the α1 subunit and c-Src as well as between the α1 subunit and cav-1, but argued about the interaction between c-Src and cav-1 under the condition. Furthermore, the data also indicated the existence of different protein complexes containing the α1 subunit and c-Src, which might have different signaling functions.