title: Do Src Kinase and Caveolin Interact Directly with Na,K-ATPase?
abstract: Much evidence points to a role of Na,K-ATPase in ouabain-dependent signal transduction. Based on experiments with different cell lines and native tissue membranes, a current hypothesis postulates direct interactions between the Na,K-ATPase and Src kinase (non-receptor tyrosine kinase). Na,K-ATPase is proposed to bind Src kinase and inhibit its activity, whereas ouabain, the specific Na,K-ATPase inhibitor, binds and stabilizes the E2 conformation, thus exposing the Src kinase domain and its active site Tyr-418 for activation. Ouabain-dependent signaling is thought to be mediated within caveolae by a complex consisting of Na,K-ATPase, caveolin, and Src kinase. In the current work, we have looked for direct interactions utilizing purified recombinant Na,K-ATPase (human α1β1FXYD1 or porcine α1D369Nβ1FXYD1) and purified human Src kinase and human caveolin 1 or interactions between these proteins in native membrane vesicles isolated from rabbit kidney. By several independent criteria and techniques, no stable interactions were detected between Na,K-ATPase and purified Src kinase. Na,K-ATPase was found to be a substrate for Src kinase phosphorylation at Tyr-144. Clear evidence for a direct interaction between purified human Na,K-ATPase and human caveolin was obtained, albeit with a low molar stoichiometry (1:15-30 caveolin 1/Na,K-ATPase). In native renal membranes, a specific caveolin 14-5 oligomer (95 kDa) was found to be in direct interaction with Na,K-ATPase. We inferred that a small fraction of the renal Na,K-ATPase molecules is in a ∼1:1 complex with a caveolin 14-5 oligomer. Thus, overall, whereas a direct caveolin 1/Na,K-ATPase interaction is confirmed, the lack of direct Src kinase/Na,K-ATPase binding requires reassessment of the mechanism of ouabain-dependent signaling.
authors.affiliation: From the Department of Biological Chemistry.
authors.forename: Eliyahu
authors.initials: E
authors.lastname: Yosef
authors.affiliation: From the Department of Biological Chemistry.
authors.forename: Adriana
authors.initials: A
authors.lastname: Katz
authors.affiliation: the Israel Structural Proteomics Center, and.
authors.forename: Yoav
authors.initials: Y
authors.lastname: Peleg
authors.affiliation: the Biological Services Department-Mass Spectrometry unit, Weizmann Institute of Science, Rehovot 7610001, Israel.
authors.forename: Tevie
authors.initials: T
authors.lastname: Mehlman
authors.affiliation: From the Department of Biological Chemistry, Steven.Karlish@weizmann.ac.il.
authors.forename: Steven J D
authors.initials: SJ
authors.lastname: Karlish
mesh_terms: D000818:Animals; D015153:Blotting, Western; D021941:Caveolae / Q000378:metabolism; D051242:Caveolin 1 / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D022162:Cytoplasmic Vesicles / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D007679:Kidney Medulla / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011817:Rabbits; D015398:Signal Transduction; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013552:Swine; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: D051242:Caveolin 1; D019061:src-Family Kinases; D000254:Sodium-Potassium-Exchanging ATPase
keywords: Na+/K+-ATPase; Src; caveolin; protein-protein interaction; signaling