Department of Biomedical Sciences, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV, 25755, USA.
Department of Medicine, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV, 25755, USA.
Sci Rep. 2020 Apr 7;10(1):6006. doi: 10.1038/s41598-020-61920-4.
The protein-protein interactions amongst the Na/K-ATPase α1 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 α1 subunit and c-Src in same protein complex, as well as a direct interaction between the α1 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 α1 subunit with proteins, but depletion of cav-1 did not affect the interaction of c-Src with the α1 subunit. The data indicated that there are direct interactions between the α1 subunit and c-Src as well as between the α1 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 α1 subunit and c-Src, which might have different signaling functions.
钠钾泵α1 亚基、原癌基因 c-Src 和 caveolin-1（cav-1）之间的蛋白-蛋白相互作用对于钠钾泵的信号转导功能至关重要。然而，关于相互作用模型存在争议。本研究旨在阐明在天然静息状态下活细胞中内源性天然蛋白之间的相互作用。在天然条件下，Blue Native-PAGE 和 Blue Native-PAGE/SDS-PAGE 2D 分析表明α1 亚基和 c-Src 存在于同一蛋白质复合物中，并且α1 亚基和 c-Src 之间存在直接相互作用。通过比较可裂解和不可裂解的半胱氨酸-半胱氨酸交联样品，毛细管免疫印迹分析表明，Src 激酶家族成员（c-Src、Yes 和 Fyn）或 cav-1 的耗竭明显减少了α1 亚基与蛋白质的相互作用，但 cav-1 的耗竭不影响 c-Src 与α1 亚基的相互作用。数据表明α1 亚基与 c-Src 以及α1 亚基与 cav-1 之间存在直接相互作用，但在该条件下 c-Src 与 cav-1 之间的相互作用存在争议。此外，数据还表明存在含有α1 亚基和 c-Src 的不同蛋白质复合物，它们可能具有不同的信号转导功能。
