SH 3 domain of c‐Src governs its dynamics at focal adhesions and the cell membrane

We studied the role of the Src SH 3 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 SH 2‐ and SH 3‐mutated Src at the cell membrane. Introducing mutations in the SH 3 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 SH 3 domain enhanced dissociation of Src from the adhesion site and disruption of the SH 3 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 SH 3 domain mutation, suggesting that rapid mobility of Src at focal adhesions mediated by the SH 3 domain is catalytic‐activity‐dependent. These findings show that the SH 3 domain of Src governs the dynamics of Src at the cell membrane and may be involved in rapid signal transduction in cells.