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

Gress Kadaré,Nicolas Gervasi,Karen Brami-Cherrier,Heike Blockus,Said El Messari,Stefan T Arold,Jean-Antoine Girault

doi:10.1074/jbc.m114.593632

Abstract

Focal adhesion (FA) kinase (FAK) regulates cell survival and motility by transducing signals from membrane receptors. The C-terminal FA targeting (FAT) domain of FAK fulfils multiple functions, including recruitment to FAs through paxillin binding. Phosphorylation of FAT on Tyr(925) facilitates FA disassembly and connects to the MAPK pathway through Grb2 association, but requires dissociation of the first helix (H1) of the four-helix bundle of FAT. We investigated the importance of H1 opening in cells by comparing the properties of FAK molecules containing wild-type or mutated FAT with impaired or facilitated H1 openings. These mutations did not alter the activation of FAK, but selectively affected its cellular functions, including self-association, Tyr(925) phosphorylation, paxillin binding, and FA targeting and turnover. Phosphorylation of Tyr(861), located between the kinase and FAT domains, was also enhanced by the mutation that opened the FAT bundle. Similarly phosphorylation of Ser(910) by ERK in response to bombesin was increased by FAT opening. Although FAK molecules with the mutation favoring FAT opening were poorly recruited at FAs, they efficiently restored FA turnover and cell shape in FAK-deficient cells. In contrast, the mutation preventing H1 opening markedly impaired FAK function. Our data support the biological importance of conformational dynamics of the FAT domain and its functional interactions with other parts of the molecule.

Highlights

Focal adhesion kinase (FAK) is enriched at focal adhesions through its focal adhesion targeting (FAT) domain, a four-helix bundle
A Mutation Designed to Open the H1-H2 Hinge Region Increases FAT Self-association—We reasoned that if H1 opening of the FAT domain is relevant for selected FAK functions, these functions would be enhanced by a FAT mutant with enhanced propensity to open H1, and abrogated by a FAT mutant with defective H1 opening, whereas wild-type FAK would show an intermediate phenotype
If the conformational dynamics of FAT were irrelevant for FAK function, WT FAK would behave like the FAK mutant with abrogated H1 opening

Summary

Background

Focal adhesion kinase (FAK) is enriched at focal adhesions through its focal adhesion targeting (FAT) domain, a four-helix bundle. We investigated the importance of H1 opening in cells by comparing the properties of FAK molecules containing wild-type or mutated FAT with impaired or facilitated H1 openings These mutations did not alter the activation of FAK, but selectively affected its cellular functions, including self-association, Tyr925 phosphorylation, paxillin binding, and FA targeting and turnover. Present address: Institut de la Vision, UMR-S 968 Inserm/UPMC/CNRS 7210, 75012 Paris, France. Subsequent phosphorylation of Tyr925 by SFKs creates a binding site for the SH2 domain of Grb, linking FAK to the Ras/extracellular signal-regulated kinase (ERK) pathway [24, 25] and facilitating the release of FAK from FAs [26]. Tyr925, positioned within H1 of the four-helix bundle, is not accessible for phosphorylation and Grb binding [19, 27, 28] This apparent contradiction suggested that the FAT domain can undergo conformational rearrangement in cells. Comparative analysis of the wild-type (WT) and mutant phenotypes strongly supports that the conformational dynamics of FAT are an essential regulator for the cellular function of wt FAK at FAs

EXPERIMENTAL PROCEDURES

RESULTS

Root mean square deviations