In Vitro Phosphorylation of the Focal Adhesion Targeting Domain of Focal Adhesion Kinase by Src Kinase

Abstract

Focal adhesion kinase (FAK), a key regulator of cell adhesion and migration, is overexpressed in many types of cancer. The C-terminal focal adhesion targeting (FAT) domain of FAK is necessary for proper localization of FAK to focal adhesions and subsequent activation. Phosphorylation of Y926 in the FAT domain by the tyrosine kinase Src has been shown to promote metastasis and invasion in vivo by linking the FAT domain to the MAPK pathway via its interaction with Grb2. Several groups have reported that inherent conformational dynamics in the FAT domain likely regulate phosphorylation of Y926; however, what regulates these dynamics is unknown. In this paper, we demonstrate that there are two sites of in vitro Src-mediated phosphorylation in the FAT domain: Y926, which has been shown to affect FAK function in vivo, and Y1008, which has no known biological role. The phosphorylation of these two tyrosine residues is pH dependent, but this does not reflect the pH dependence of Src kinase activity. CD and NMR data indicate that the stability and conformational dynamics of the FAT domain are sensitive to changes in pH over a physiological pH range. In particular, regions of the FAT domain previously shown to regulate phosphorylation of Y926 as well as regions near Y1008 show pH-dependent dynamics on the μs-ms time scale.

Conformational Dynamics of the Focal Adhesion Targeting Domain Control Specific Functions of Focal Adhesion Kinase in Cells

“…Tyr 925 is an example of a phosphorylation site that has a favorable consensus sequence but a poor conformation for phosphorylation by Srcfamily kinases (34). Our results indicate that the H1 opening allows the helix region surrounding Tyr 925 to unfold and adopt conformations compatible with kinase interactions, in agreement with a previous report exploring the consequences of the hydrophobic core residue mutations V955A/L962A (32).…”

“…Intriguingly, despite the strain in the H1-H2 hinge region, only 0.1% FAT Y925E molecules were in the open conformation (33) and only 2.5% of total FAT were stable, presumably arm-exchanged dimers in size exclusion chromatography analysis (15). The low probability of this transition is consistent with the observation that Tyr 925 in the native FAT domain is a much poorer substrate for SFK phosphorylation than the unstructured peptide mimics of the region around Tyr 925 or FAT domains with destabilized cores (27,34,35). Moreover, by severely destabilizing the FAT bundle structure, H1 opening is also predicted to affect other FAT functions, such as paxillin binding and FAK dimerization.…”

“…H1 opening has subsequently been observed in several studies (15,32,33). The conformational strain in this hinge region was experimentally apparent (34,35) and relieved in the open or H1-swapped dimeric conformation of Pro 944 -APP (27,35). The open state would allow Tyr 925 phosphorylation and subsequent binding to Grb2.…”

“…Previous in vitro and modeling studies of FAT showed that FAT H1 can spontaneously dissociate from the four-helix bundle (15,27,(32)(33)(34)(35). This structural rearrangement allows the formation of FAT dimers in vitro through H1 swapping (27).…”

“…Partners of FAK at FAs that could promote its opening are yet to be identified. Other factors, such local pH changes, could play a role because phosphorylation and conformational dynamics of the FAT domain show some level of sensitivity to changes in pH over a physiological pH range (34). Alternatively, spontaneous FAT opening dynamics may function as a probabilistic switch mechanism, tuned to modulate a sufficiently large FAK subpopulation at sites where FAK is enriched, thus promoting Ras-MAPK signaling and FA disassembly in a timedelayed manner after FAK enrichment (56).…”