Structural homology and functional similarity: Cas family proteins, NEDD9 and p130Cas are functionally distinct focal adhesion molecules

Abstract

The majority of work to date pertaining to the Cas family proteins has focused on specifically on p130Cas. Thus, given the strong sequence and structural homologies shared between NEDD9 and p130Cas, similar roles have been attributed to NEDD9. This project focused on the regulatory roles of Src kinase and FAK in determining the molecular exchange of NEDD9 at focal adhesions. The direct interaction of FAK and NEDD9 recruited NEDD9 to focal adhesions, while confocal FRAP analysis revealed Src kinase phosphorylation of NEDD9 stabilised NEDD9 at focal adhesions and regulated cell migration, a result that is in stark contrast to that described for p130Cas. The importance of the structurally homologous C-terminal Focal Adhesion Targeting (FAT) domains of NEDD9, p130Cas and FAK in regulating focal adhesion mechanotransduction and mechanosensing were also studied. Using overlap-extension PCR, FAT domain exchanged constructs were generated and subjected to numerous microscopy based assays. Distinct, rigidity-specific functional roles for each FAT domain were subsequently identified. The p130Cas FAT domain was identified as a novel rigidity-dependent mechanosensor, while the NEDD9 and FAK FAT domains were found to be functionally analogous. Taken together, these findings contribute to the understanding of the molecular mechanisms that underpin focal adhesion biology and cell migration, and importantly highlight the functional differences of structurally homologous proteins.