Novel FRET-Based Src Biosensor Reveals Mechanisms of Src Activation and Its Dynamics in Focal Adhesions

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 very compact structure stabilized by inhibitory intramolecular interactions. Taking advantage of this spatial regulation, we constructed a FRET-based Src biosensor and analyzed conformational changes of Src following Src activation and the spatio-temporal dynamics of Src activity in cells. We found that activatory mutations either in regulatory or kinase domains induce opening of Src structure. Surprisingly we discovered that Src inhibitors differ in their effect on the Src structure, some counterintuitively inducing opened conformation. Finally, we analyzed the dynamics of Src activity in focal adhesions by FRET imaging and found that Src is rapidly activated during focal adhesions assembly, its activity remains steady and high throughout the focal adhesions life cycle and decreases during focal adhesions disassembly.