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首页> 外文期刊>Cell Regulation >A Highlights from MBoC Selection: Src-dependent phosphorylation of caveolin-1 Tyr-14 promotes swelling and release of caveolae
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A Highlights from MBoC Selection: Src-dependent phosphorylation of caveolin-1 Tyr-14 promotes swelling and release of caveolae

机译：MBoC选择的亮点：小孔蛋白1 Tyr-14的Src依赖性磷酸化促进小孔肿胀和释放

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Caveolin 1 (Cav1) is a required structural component of caveolae, and its phosphorylation by Src is associated with an increase in caveolae-mediated endocytosis. Here we demonstrate, using quantitative live-cell 4D, TIRF, and FRET imaging, that endocytosis and trafficking of caveolae are associated with a Cav1 Tyr-14 phosphorylation-dependent conformational change, which spatially separates, or loosens, Cav1 molecules within the oligomeric caveolar coat. When tracked by TIRF and spinning-disk microscopy, cells expressing phosphomimicking Cav1 (Y14D) mutant formed vesicles that were greater in number and volume than with Y14F-Cav1-GFP. Furthermore, we observed in HEK cells cotransfected with wild-type, Y14D, or Y14F Cav1-CFP and -YFP constructs that FRET efficiency was greater with Y14F pairs than with Y14D, indicating that pY14-Cav1 regulates the spatial organization of Cav1 molecules within the oligomer. In addition, albumin-induced Src activation or direct activation of Src using a rapamycin-inducible Src construct (RapR-Src) led to an increase in monomeric Cav1 in Western blots, as well as a simultaneous increase in vesicle number and decrease in FRET intensity, indicative of a Src-mediated conformational change in CFP/YFP-tagged WT-Cav1 pairs. We conclude that phosphorylation of Cav1 leads to separation or “spreading” of neighboring negatively charged N-terminal phosphotyrosine residues, promoting swelling of caveolae, followed by their release from the plasma membrane.
机译：小窝蛋白1（Cav1）是小窝蛋白的必需结构成分，其被Src磷酸化与小窝蛋白介导的内吞作用增加有关。在这里，我们证明了使用定量活细胞4D，TIRF和FRET成像，小窝的内吞作用和运输与Cav1 Tyr-14磷酸化依赖的构象变化有关，它在空间上分离或疏松寡聚小窝内的Cav1分子涂层。当通过TIRF和旋转盘显微镜进行追踪时，表达磷酸化Cav1（Y14D）突变体的细胞形成的囊泡，其数量和体积都比Y14F-Cav1-GFP大。此外，我们在与野生型，Y14D或Y14F Cav1-CFP和-YFP构建体共转染的HEK细胞中观察到，与Y14D相比，Y14F对的FRET效率更高，这表明pY14-Cav1调节了Cav1分子在细胞内的空间组织。低聚物。此外，白蛋白诱导的Src激活或使用雷帕霉素诱导的Src构建体（RapR-Src）直接激活Src导致Western印迹中单体Cav1的增加，以及囊泡数的同时增加和FRET强度的降低，指示CFP / YFP标签的WT-Cav1对中Src介导的构象变化。我们得出的结论是，Cav1的磷酸化会导致相邻的带负电荷的N末端磷酸酪氨酸残基分离或“扩散”，从而促进小窝肿胀，然后从质膜释放出来。

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