The Na/K-ATPase alpha 1 and c-Src form signaling complex under native condition: A crosslinking approach

The protein-protein interactions amongst the Na/K-ATPase alpha 1 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 alpha 1 subunit and c-Src in same protein complex, as well as a direct interaction between the alpha 1 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 alpha 1 subunit with proteins, but depletion of cav-1 did not affect the interaction of c-Src with the alpha 1 subunit. The data indicated that there are direct interactions between the alpha 1 subunit and c-Src as well as between the alpha 1 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 alpha 1 subunit and c-Src, which might have different signaling functions.