SH3 domain of c-Src governs its dynamics at focal adhesions and the cell membrane
SH3 domain of c-Src governs its dynamics at focal adhesions and the cell membrane
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DOI：
10.1111/febs.13404
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发表时间：
2015-10-01
期刊：
影响因子：
5.4
通讯作者：
Fujita, Hideaki
中科院分区：
文献类型：
--
作者：
Machiyama, Hiroaki;Yamaguchi, Tomoyuki;Fujita, Hideaki
我们利用定点突变和活细胞成像技术研究了Src SH3结构域在细胞膜动力学中的作用。生理上，细胞的增殖和迁移需要Src家族蛋白的表达。在多种癌细胞中都检测到了Src分子的过度激活。虽然对Src的激活机制已经进行了深入的研究，但其在细胞膜上的动态变化仍不清楚。虽然Src分子也存在于不同的细胞位置，但我们发现激活的Src分子主要定位于外周细胞黏附部位。SRC的磷酸化状态和亚区构象被认为调节了Src的激活和易位。在本研究中，我们分析了野生型和SH2和SH3突变的Src在细胞膜上的单分子动力学。在SH3区域引入突变导致细胞膜上的Src运动性降低，无论是在局灶性粘连的内部还是外部。肌动蛋白细胞骨架的破坏导致Src在细胞膜上的扩散运动减少。我们证明，在局灶性粘连中，SH3结构域促进了Src从粘连部位的解离，并且SH3结构域的破坏改变了Src在细胞膜上的分布。在局灶性粘连中，在SH3结构域突变导致的Src迁移率下降中，Src的活性是必不可少的，这表明在SH3结构域介导的局灶性粘连中，Src的快速迁移率是催化活性依赖的。这些发现表明，Src的SH3结构域控制着细胞膜上Src的动态变化，可能参与了细胞内的快速信号转导。
We studied the role of the Src SH3 domain in its dynamics at the cell membrane using site-directed mutagenesis and live cell imaging. Physiologically, cell proliferation and migration require the expression of Src family kinases. Hyperactivation of Src molecules has been detected in various cancer cells. Although the activation mechanism of Src has been intensively studied, the dynamics of Src at the cell membrane are still unclear. Although Src molecules also exist at various cellular locations, we found that activated Src molecules are mainly localized at peripheral cell adhesion sites. Src phosphorylation status and subdomain conformations are thought to regulate Src activation and translocation. In this study, we analyzed the single-molecule dynamics of wild-type Src and SH2- and SH3-mutated Src at the cell membrane. Introducing mutations in the SH3 domain resulted in reduced Src motility at the cell membrane, both inside and outside of focal adhesions. Disruption of the actin cytoskeleton resulted in less diffusive Src movement at the cell membrane. We demonstrate that, inside focal adhesions, the SH3 domain enhanced dissociation of Src from the adhesion site and disruption of the SH3 domain altered the distribution of Src at the cell membrane. Inside focal adhesions, kinase activity of Src was essential for the Src mobility reduction by SH3 domain mutation, suggesting that rapid mobility of Src at focal adhesions mediated by the SH3 domain is catalytic-activity-dependent. These findings show that the SH3 domain of Src governs the dynamics of Src at the cell membrane and may be involved in rapid signal transduction in cells.