Focal adhesions (FAs) are multiprotein complexes that link integrins attached to the extracellular matrix with the actin cytoskeleton. Assembly and maintenance of these complexes is regulated by Rho family GTPases. Tyrosine phosphorylation of FA proteins such as paxillin and focal adhesion kinase(FAK) also seems to play a part. This provides docking sites for other proteins (e.g. Src), but its role in regulation of FA assembly/turnover is unclear. Benjamin Geiger and co-workers have therefore developed a novel FA-phosphorylation assay, in which they use a construct comprising yellow fluorescent protein and two phosphotyrosine-binding SH2 domains from Src(YFP-dSH2) to monitor the appearance of phosphorylated tyrosine at FAs dynamically in living cells (see ). The authors observe that, following stimulation of SV80 cells by the microtubule-depolymerizing drug nocodazole, vinculin, paxillin and FA kinase (FAK) are rapidly recruited to growing FAs. Phosphotyrosine residues,however, do not appear until a few minutes later. This indicates that tyrosine phosphorylation is unlikely to drive focal adhesion formation but is a secondary event that might instead be involved in FA turnover or signalling.
