FIGURE 1.

Interaction between the α1 Na/K-ATPase and Src. A, GST pulldown analyses showing the concentration-dependent interaction between Src and two domains of the α1 subunit. The indicated amounts of GST-fused proteins were incubated with 100 ng of purified His-Src, and the bound His-Src in the pulldown pellets was detected by Western blotting with anti-His antibody shown in the upper panel. The lower panel shows the Coomassie Blue staining of GST, GST-CD2 (amino acid residues 152–288), and GST-ND1 (amino acid residues 379–435, first 57 amino acids of the N domain). n = 3. B, modeling of α1 Na/K-ATPase–Src interaction. Modeling of E1 and E2 Na/K-ATPase is based on SERCA1a (Protein Data Bank ID code 1SU4) and Na/K-ATPase (Protein Data Bank ID code 2ZXE). Three-dimensional structure was generated using the SPDBView V3.7 program. The A domain in the α1 subunit is in blue, N domain in black. The SH2 domain of Src is in orange, kinase domain in cyan, activation loop and Tyr-418 in red. In E1-like conformation, the kinase domain (in cyan) is tightly bound with the N domain (in black), and Tyr-418 is buried (boxed area), therefore Src activity is suppressed; whereas in E2-like conformation, the kinase domain is moved away from the N domain and Tyr-418 is exposed (boxed area), resulting in Src activation. C, purified Na/K-ATPase was incubated with either His-Src buffer or His-Src (at molar ratio of 1:1). Ouabain-sensitive Na/K-ATPase activities were measured as described under “Experimental Procedures.” Activity was calculated and is expressed as % of control. Quantitative data from three independent experiments are presented as mean ± S.E. (error bars).