Src kinase plays an important role in a multitude of fundamental cellular processes and is often found deregulated in tumors. Active Src adopts an open conformation, whereas inactive Src is characterized by a very compact structure stabilized by inhibitory intramolecular interactions. Taking advantage of this spatial regulation, we constructed a fluorescence resonance energy transfer (FRET)-based Src biosensor and analyzed conformational changes of Src following Src activation and the spatiotemporal dynamics of Src activity in cells. We found that activatory mutations either in regulatory or kinase domains induce opening of the Src structure. Surprisingly, we discovered that Src inhibitors differ in their effect on the Src structure, some counterintuitively inducing an open conformation. Finally, we analyzed the dynamics of Src activity in focal adhesions by FRET imaging and found that Src is rapidly activated during focal adhesion assembly, and its activity remains steady and high throughout the life cycle of focal adhesion and decreases during focal adhesion disassembly.

中文翻译：

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基于FRET的新型Src生物传感器揭示了Src活化的机理及其在粘着斑中的动力学。

Src激酶在许多基本的细胞过程中起着重要作用，并且经常在肿瘤中被发现失调。活性Src采用开放构象，而无活性Src的特征是通过抑制性分子内相互作用而稳定的非常紧凑的结构。利用这种空间调节，我们构建了基于荧光共振能量转移（FRET）的Src生物传感器，并分析了Src激活后Src的构象变化以及细胞中Src活性的时空动态。我们发现，调节域或激酶域中的激活性突变均可诱导Src结构的打开。出人意料的是，我们发现Src抑制剂对Src结构的作用不同，有些是违反直觉的诱导开放构象。最后，