Src is a non-receptor cytoplasmic tyrosine kinase activated by integrins and receptor tyrosine kinases . In normal cells, Src is involved in a vast range of physiological functions, including cell proliferation, cytoskeletal regulation, cell shape control, cell-matrix adhesion dynamics and motility [2, 3]. In many types of human cancer, Src is overexpressed or hyperactivated [4, 5]. The prominent role of Src in regulating cytoskeletal dynamics and cell motility makes the study of Src indispensable in understanding cancer cell migration and invasion.
Initially identified as a tyrosine-phosphorylated protein in v-Src infected chicken embryo fibroblasts , cortactin is a direct substrate of cellular Src kinase . It is phosphorylated by Src at three tyrosine residues (Tyr421, 466, 482 of murine cortactin) in vitro. The phosphorylation of Tyr475 was identified by a mass spectrometry study . These tyrosine phosphorylation sites reside in the proline-rich region, which is the least conserved domain in cortactin from different species .
Many studies have suggested that cortactin and its tyrosine phosphorylation regulate lamellipodial protrusion, cell spreading, intercellular adhesion and cell motility [11–13]. Src-catalyzed cortactin tyrosine phosphorylation is involved in integrin-mediated cell adhesion and spreading . Cortactin knockdown in murine fibroblasts impairs both random and directional cell migration . The expression of cortactin mutated at Src phosphorylation sites (Y421/466/482F) decreases cell motility in ECV304 endothelial cells . The impaired cell motility in cortactin knockdown gastric cancer cell lines, with a low cortactin phosphorylation level, can be rescued by the ectopic expression of wild-type cortactin, but not by the mutant cortactin (Y421/466/482F) .
Early studies revealed that cortactin colocalizes with F-actin in the cortical structures of adherent cells [7, 17]. It associates with the F-actin cytoskeleton through the F-actin binding tandem cortactin repeats and the N-terminal acidic domain that interacts with the actin-related protein (Arp) 2/3 complex for dendritic actin nucleation [10, 18, 19]. At the cell periphery, the F-actin cytoskeleton forms a highly organized meshwork that controls membrane protrusion and regulates cell motility [20, 21]. During cell migration, the propelling force is generated by membrane protrusions and by membrane-matrix adhesions, called focal adhesions, at which transmembrane integrins link the extracellular matrix to the intracellular actin cytoskeleton .
In contrast to the cortactin that colocalizes with F-actin at cortical regions, tyrosine phosphorylated cortactin (pTyr421, 466, 482) is almost exclusively localized at focal adhesions [16, 23]. It is colocalized with paxillin and vinculin at the ends of F-actin stress fibers [16, 23]. At focal adhesions, the clustered integrins recruit FAK and facilitate its activation, forming an active FAK-Src complex that initiates many intracellular signaling events [24–27]. The autophosphorylation of FAK at Tyr397 creates a high affinity binding site for the Src-homology 2 domain of Src kinase . The binding of Src to FAK leads to the formation of an active FAK-Src complex in which the active Src kinase trans-phosphorylates FAK at Tyr576, 577 for maximal FAK catalytic activity .
FAK recruits adaptor proteins and signaling molecules into focal adhesions, at which many are phosphorylated by the FAK-Src complex. The N-terminal FERM domain binds to growth factor receptors, the C-terminal FAT domain interacts with talin and paxillin, and the proline-rich regions recruit Src-homology 3 (SH3) domain-containing proteins, such as p130Cas, GRAF and ASAP1 [26, 30].
In this study, we report that cortactin interacted with FAK at focal adhesions at which it is phosphorylated by the FAK-Src complex. Cortactin helps mediate the association of F-actin with focal adhesions. Its N-terminus interacts with F-actin, and its C-terminus associates with FAK. The tyrosine phosphorylation of cortactin by the FAK-Src complex reduces its interaction with FAK, most likely to increase its turnover at focal adhesions to promote cell motility.