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Src激酶和小窝蛋白是否直接与钠钾ATP酶相互作用?

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

作者信息

Yosef Eliyahu, Katz Adriana, Peleg Yoav, Mehlman Tevie, Karlish Steven J D

机构信息

From the Department of Biological Chemistry.

the Israel Structural Proteomics Center, and.

出版信息

J Biol Chem. 2016 May 27;291(22):11736-50. doi: 10.1074/jbc.M116.721084. Epub 2016 Mar 28.

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.

摘要

大量证据表明钠钾ATP酶在哇巴因依赖的信号转导中发挥作用。基于对不同细胞系和天然组织膜的实验,目前的一种假说假定钠钾ATP酶与Src激酶(非受体酪氨酸激酶)之间存在直接相互作用。有人提出钠钾ATP酶与Src激酶结合并抑制其活性,而哇巴因,即钠钾ATP酶的特异性抑制剂,结合并稳定E2构象,从而暴露Src激酶结构域及其活性位点酪氨酸-418以使其激活。哇巴因依赖的信号传导被认为是由钠钾ATP酶、小窝蛋白和Src激酶组成的复合物在小窝内介导的。在当前的研究中,我们利用纯化的重组钠钾ATP酶(人α1β1FXYD1或猪α1D369Nβ1FXYD1)、纯化的人Src激酶和人小窝蛋白1来寻找直接相互作用,或者在从兔肾分离的天然膜囊泡中寻找这些蛋白质之间的相互作用。通过几种独立的标准和技术,未检测到钠钾ATP酶与纯化的Src激酶之间存在稳定的相互作用。发现钠钾ATP酶是Src激酶在酪氨酸-144处磷酸化的底物。尽管摩尔化学计量比很低(1:15 - 30小窝蛋白1/钠钾ATP酶),但确实获得了纯化的人钠钾ATP酶与人类小窝蛋白之间存在直接相互作用的明确证据。在天然肾膜中,发现一种特异性的小窝蛋白1 4 - 5寡聚体(95 kDa)与钠钾ATP酶直接相互作用。我们推断肾钠钾ATP酶分子的一小部分与小窝蛋白1 4 - 5寡聚体形成约1:1的复合物。因此,总体而言,虽然小窝蛋白1/钠钾ATP酶的直接相互作用得到了证实,但Src激酶/钠钾ATP酶缺乏直接结合需要重新评估哇巴因依赖信号传导的机制。

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