Focal Adhesion Kinase Regulatory Interactions Quantified By Tirf-Fret

Focal adhesion kinase related non-kinase (FRNK) is an inhibitor of focal adhesion kinase (FAK)-dependent cell growth, survival, and migration pathways. In vascular smooth muscle cells, both FAK and FRNK are upregulated after vascular injury, suggesting balanced stimulation and inhibition of growth during recovery. Here we propose a novel mechanism by which FRNK can inhibit FAK: direct binding of FRNK to FAK forming an inhibitory complex within focal adhesions. This hypothesis is supported by co-immunoprecipitation of FAK and FRNK. In addition, total internal reflection fluorescence (TIRF)-mode fluorescence resonance energy transfer (FRET) measurements suggest that FRNK binds directly to FAK within focal adhesions of live vascular smooth muscle cells. We hypothesized that this interaction might be regulated by serine phosphorylation of FRNK S217, a site we found to be basally highly phosphorylated at S217 by extracellular signal-regulated kinase 1/2 (ERK). To determine the significance of S217 phosphorylation, we generated a non-phosphorylatable mutant FRNK by mutating S217 to alanine. S217A-FRNK exhibited significantly higher amounts of FRET compared to wild type FRNK, suggesting increased interaction with FAK. Progressive acceptor photobleaching analysis revealed that the FRNK-FAK complex consisted of FRNK and multiple FAK proteins in a high order hetero-oligomer. To determine the functional role of the proposed regulatory site we performed an apoptosis assay using flow cytometry. S217A FRNK was a much more potent inducer of apoptosis in vascular smooth muscle cells compared to wild type FRNK. Additionally, we determined that S217A FRNK was a more potent inhibitor of FAK signaling by examining FAK Y397 phosphorylation. We conclude that dephosphorylation of FRNK at S217 during response to injury results in increased FRNK-FAK complex formation, thereby inhibiting FAK signaling.