Do Src Kinase and Caveolin Interact Directly with Na,K-ATPase?

Abstract

Much evidence points to a role of Na,K-ATPase in ouabaindependent 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 ␣1␤1FXYD1 or porcine ␣1D369N␤1FXYD1) 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,KATPase 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 1 4 -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 ϳ1:1 complex with a caveolin 1 4 -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.

Involvement of the Na+,K+‐ATPase isoforms in control of cerebral perfusion

“…There is substantial evidence for physical interaction between the Na + ,K + -ATPase and Src that is disturbed by ouabain but does not depend on ATPase activity (for details, see Cui & Xie, 2017). This is in disagreement with cell-free assay-based studies, which suggested that Src is activated by changes in ATP consumption (Yosef, Katz, Peleg, Mehlman, & Karlish, 2016).…”

Targeting Na/K-ATPase Signaling: A New Approach to Control Oxidative Stress

“…A second model is that c-Src activation is primarily a consequence of an ATP-sparing effect (observed in a cell-free system) [45, 46]. A third model proposes that c-Src transiently interacts with a complex formed between the Na/K-ATPase α1 subunit and caveolin-1[47]. A common charateristic in these models is that the E2-P conformational state of the Na/K-ATPase is favored, and stablized by Na/K- ATPase inhibitors (ouabain, vanadate, oligomycin) and energy status (ATP/ADP ratio).…”

Sodium potassium adenosine triphosphatase (Na/K-ATPase) as a therapeutic target for uremic cardiomyopathy

“…There are some data that are opposite with our model. It has been shown that c-Src activation was not involved in ouabain induced Na/K-ATPase endocytosis in non-small cell lung cancer cells [86] and no stable interactions were detected between purified recombinant Na/K-ATPase and purified human Src kinase [87]. We would like to point out that these observations are different from what we have reported using purified pig kidney Na/K-ATPase, LLC-PK1 cell, human HK-2, human dermal fibroblasts, renal fibroblast cell line, and rat cardiac fibroblasts cell line [84,88,89].…”