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

  • Gress Kadaré
    Affiliations
    INSERM, UMR-S 839, F-75005 Paris, France

    Université Pierre & Marie Curie (UPMC), Sorbonne Universités, F-75005 Paris, France

    Institut du Fer à Moulin, F-75005 Paris, France
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  • Nicolas Gervasi
    Affiliations
    INSERM, UMR-S 839, F-75005 Paris, France

    Université Pierre & Marie Curie (UPMC), Sorbonne Universités, F-75005 Paris, France

    Institut du Fer à Moulin, F-75005 Paris, France
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  • Karen Brami-Cherrier
    Footnotes
    Affiliations
    INSERM, UMR-S 839, F-75005 Paris, France

    Université Pierre & Marie Curie (UPMC), Sorbonne Universités, F-75005 Paris, France

    Institut du Fer à Moulin, F-75005 Paris, France
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  • Heike Blockus
    Footnotes
    Affiliations
    INSERM, UMR-S 839, F-75005 Paris, France

    Université Pierre & Marie Curie (UPMC), Sorbonne Universités, F-75005 Paris, France

    Institut du Fer à Moulin, F-75005 Paris, France
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  • Said El Messari
    Footnotes
    Affiliations
    INSERM, UMR-S 839, F-75005 Paris, France

    Université Pierre & Marie Curie (UPMC), Sorbonne Universités, F-75005 Paris, France

    Institut du Fer à Moulin, F-75005 Paris, France
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  • Stefan T. Arold
    Affiliations
    King Abdullah University of Science and Technology (KAUST), Division of Biological and Environmental Sciences and Engineering, Computational Bioscience Research Center (CBRC), Thuwal, Saudi Arabia

    Centre de Biochimie Structurale, CNRS UMR5048, INSERM U1054, Université de Montpellier I & II, Montpellier, France
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  • Jean-Antoine Girault
    Correspondence
    To whom correspondence should be addressed: Institut du Fer à Moulin, Inserm UPMC UMR-S 839, 17 rue du Fer à Moulin, 75005 Paris, France. Tel.: 33-1-45-87-61-52; Fax: 33-1-45-87-61-32
    Affiliations
    INSERM, UMR-S 839, F-75005 Paris, France

    Université Pierre & Marie Curie (UPMC), Sorbonne Universités, F-75005 Paris, France

    Institut du Fer à Moulin, F-75005 Paris, France
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  • Author Footnotes
    * This work was supported in part by Agence Nationale de la Recherche Grant ANR-05-2_42589, Association pour la Recherche sur le Cancer (ARC) Grant A05/3/3138, Fondation pour la Recherche Médicale, European Research Council, Inserm, UPMC, and King Abdullah University of Science and Technology (KAUST).
    1 Present address: University of California at Irvine, Irvine, CA 92697.
    2 Undergraduate funded by the Paris School of Neuroscience (ENP). Present address: Institut de la Vision, UMR-S 968 Inserm/UPMC/CNRS 7210, 75012 Paris, France.
    3 Present address: University of Franche-Comté, UFR Sciences et Techniques, 25030 Besançon, France.
Open AccessPublished:November 12, 2014DOI:https://doi.org/10.1074/jbc.M114.593632
      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 Tyr925 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, Tyr925 phosphorylation, paxillin binding, and FA targeting and turnover. Phosphorylation of Tyr861, located between the kinase and FAT domains, was also enhanced by the mutation that opened the FAT bundle. Similarly phosphorylation of Ser910 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.Focal adhesion kinase (FAK) is enriched at focal adhesions through its focal adhesion targeting (FAT) domain, a four-helix bundle.

      Results

      Mutations that facilitate or prevent opening of the first helix have profound consequences on the biochemical properties of FAK and its function in cells.

      Conclusion

      The ability of FAT to open and close is essential for FAK function.

      Significance

      This provides evidence for the functional importance of the conformational dynamics of FAT.

      Introduction

      Focal adhesion kinase (FAK)
      The abbreviations used are: FAK
      focal adhesion kinase
      FA
      focal adhesion
      FAT
      focal adhesion targeting
      FERM
      four-point-one, ezrin, radixin, moesin
      FRAP
      fluorescence recovery after photobleaching
      H1
      first helix of the FAT domain
      H2
      second helix of the FAT domain
      MEF
      mouse embryonic fibroblasts
      R-FAT/FAK
      relaxed FAT/FAK
      SFK
      Src family kinase
      T-FAT/FAK
      tense FAT/FAK
      VSV
      vesicular stomatitis virus
      ANOVA
      analysis of variance
      ERK
      extracellular signal-regulated kinase
      SH2
      Src-homology 2
      VSV
      vesicular stomatitis virus.
      is a non-receptor tyrosine kinase enriched at focal adhesions (FAs) (
      • Hanks S.K.
      • Calalb M.B.
      • Harper M.C.
      • Patel S.K.
      Focal adhesion protein-tyrosine kinase phosphorylated in response to cell attachment to fibronectin.
      ,
      • Schaller M.D.
      • Borgman C.A.
      • Cobb B.S.
      • Vines R.R.
      • Reynolds A.B.
      • Parsons J.T.
      pp125FAK, A structurally distinctive protein-tyrosine kinase associated with focal adhesions.
      ,
      • Arold S.T.
      How focal adhesion kinase achieves regulation by linking ligand binding, localization and action.
      ). FAK plays a major role in transducing signals downstream from integrins and other membrane receptors (
      • Parsons J.T.
      Focal adhesion kinase: the first ten years.
      ,
      • Schaller M.D.
      Cellular functions of FAK kinases: insight into molecular mechanisms and novel functions.
      ,
      • Zachary I.
      • Rozengurt E.
      Focal adhesion kinase (p125FAK): a point of convergence in the action of neuropeptides, integrins, and oncogenes.
      ) and it is involved in the control of cell growth, survival, and migration (
      • Mitra S.K.
      • Hanson D.A.
      • Schlaepfer D.D.
      Focal adhesion kinase: in command and control of cell motility.
      ,
      • Schaller M.D.
      Biochemical signals and biological responses elicited by the focal adhesion kinase.
      ). Embryonic lethality of the FAK null mutation before midgestation underlines its biological importance (
      • Ilić D.
      • Furuta Y.
      • Kanazawa S.
      • Takeda N.
      • Sobue K.
      • Nakatsuji N.
      • Nomura S.
      • Fujimoto J.
      • Okada M.
      • Yamamoto T.
      • Aizawa S.
      Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice.
      ). The overexpression of FAK observed in many tumors correlates with increased tumor invasiveness and metastases, making FAK an attractive therapeutic target (
      • Owens L.V.
      • Xu L.
      • Craven R.J.
      • Dent G.A.
      • Weiner T.M.
      • Kornberg L.
      • Liu E.T.
      • Cance W.G.
      Overexpression of the focal adhesion kinase (p125FAK) in invasive human tumors.
      ).
      Following integrin engagement by extracellular matrix proteins, FAK is recruited to FAs. The C-terminal focal adhesion targeting (FAT) domain mediates this recruitment by association with paxillin (
      • Hildebrand J.D.
      • Schaller M.D.
      • Parsons J.T.
      Identification of sequences required for the efficient localization of the focal adhesion kinase, pp125FAK, to cellular focal adhesions.
      ,
      • Tachibana K.
      • Sato T.
      • D'Avirro N.
      • Morimoto C.
      Direct association of pp125FAK with paxillin, the focal adhesion-targeting mechanism of pp125FAK.
      ) through two binding sites for paxillin LD-motifs, formed by helices H1/H4 and H2/H3 on opposite sides of the molecule (
      • Bertolucci C.M.
      • Guibao C.D.
      • Zheng J.
      Structural features of the focal adhesion kinase-paxillin complex give insight into the dynamics of focal adhesion assembly.
      ,
      • Gao G.
      • Prutzman K.C.
      • King M.L.
      • Scheswohl D.M.
      • DeRose E.F.
      • London R.E.
      • Schaller M.D.
      • Campbell S.L.
      NMR solution structure of the focal adhesion targeting domain of focal adhesion kinase in complex with a paxillin LD peptide: evidence for a two-site binding model.
      ,
      • Hoellerer M.K.
      • Noble M.E.
      • Labesse G.
      • Campbell I.D.
      • Werner J.M.
      • Arold S.T.
      Molecular recognition of paxillin LD motifs by the focal adhesion targeting domain.
      ,
      • Alam T.
      • Alazmi M.
      • Gao X.
      • Arold S.T.
      How to find a leucine in a haystack? Structure, ligand recognition and regulation of leucine-aspartic acid (LD) motifs.
      ). FAT also interacts with talin, which may contribute to recruitment of either partner to FAs (
      • Chen H.-C.
      • Appeddu P.A.
      • Parsons J.T.
      • Hildebrand J.D.
      • Schaller M.D.
      • Guan J.-L.
      Interaction of focal adhesion kinase with cytoskeletal protein talin.
      ,
      • Lawson C.
      • Lim S.T.
      • Uryu S.
      • Chen X.L.
      • Calderwood D.A.
      • Schlaepfer D.D.
      FAK promotes recruitment of talin to nascent adhesions to control cell motility.
      ). The molecular basis of the FAK-talin interaction is not known but it seems not to require the structural integrity of FAT (
      • Chen H.-C.
      • Appeddu P.A.
      • Parsons J.T.
      • Hildebrand J.D.
      • Schaller M.D.
      • Guan J.-L.
      Interaction of focal adhesion kinase with cytoskeletal protein talin.
      ,
      • Hayashi I.
      • Vuori K.
      • Liddington R.C.
      The focal adhesion targeting (FAT) region of focal adhesion kinase is a four-helix bundle that binds paxillin.
      ).
      Enrichment of FAK at FAs favors its transient dimerization, which triggers trans-autophosphorylation of Tyr397 (
      • Brami-Cherrier K.
      • Gervasi N.
      • Arsenieva D.
      • Walkiewicz K.
      • Boutterin M.C.
      • Ortega A.
      • Leonard P.G.
      • Seantier B.
      • Gasmi L.
      • Bouceba T.
      • Kadaré G.
      • Girault J.A.
      • Arold S.T.
      FAK dimerization controls its kinase-dependent functions at focal adhesions.
      ). This is essential for FAK function, as shown by the embryonic lethality of mutant mice selectively lacking the autophosphorylation site (
      • Corsi J.M.
      • Houbron C.
      • Billuart P.
      • Brunet I.
      • Bouvrée K.
      • Eichmann A.
      • Girault J.A.
      • Enslen H.
      Autophosphorylation-independent and -dependent functions of focal adhesion kinase during development.
      ). Phosphorylation of Tyr397, which is located in the linker region between the N-terminal FERM (4.1, ezrin, radixin, moesin) (
      • Girault J.A.
      • Labesse G.
      • Mornon J.P.
      • Callebaut I.
      The N-termini of FAK and JAKs contain divergent band 4.1 domains.
      ) and the central kinase domains, allows binding and activation of Src-family kinases (SFKs) (
      • Schaller M.D.
      • Hildebrand J.D.
      • Shannon J.D.
      • Fox J.W.
      • Vines R.R.
      • Parsons J.T.
      Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src.
      ). Subsequent phosphorylation of Tyr925 by SFKs creates a binding site for the SH2 domain of Grb2, linking FAK to the Ras/extracellular signal-regulated kinase (ERK) pathway (
      • Schlaepfer D.D.
      • Hanks S.K.
      • Hunter T.
      • van der Geer P.
      Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase.
      ,
      • Schlaepfer D.D.
      • Hunter T.
      Evidence for in vivo phosphorylation of the Grb2 SH2-domain binding site on focal adhesion kinase by Src-family protein-tyrosine kinases.
      ) and facilitating the release of FAK from FAs (
      • Katz B.Z.
      • Romer L.
      • Miyamoto S.
      • Volberg T.
      • Matsumoto K.
      • Cukierman E.
      • Geiger B.
      • Yamada K.M.
      Targeting membrane-localized focal adhesion kinase to focal adhesions: roles of tyrosine phosphorylation and SRC family kinases.
      ). Tyr925, positioned within H1 of the four-helix bundle, is not accessible for phosphorylation and Grb2 binding (
      • Hayashi I.
      • Vuori K.
      • Liddington R.C.
      The focal adhesion targeting (FAT) region of focal adhesion kinase is a four-helix bundle that binds paxillin.
      ,
      • Arold S.T.
      • Hoellerer M.K.
      • Noble M.E.
      The structural basis of localization and signaling by the focal adhesion targeting domain.
      ,
      • Liu G.
      • Guibao C.D.
      • Zheng J.
      Structural insight into the mechanisms of targeting and signaling of focal adhesion kinase.
      ). This apparent contradiction suggested that the FAT domain can undergo conformational rearrangement in cells. In support of this hypothesis, H1-swapped dimeric FAT was observed in crystallographic studies, suggesting that H1 has the capacity to dissociate from the rest of the bundle (
      • Arold S.T.
      • Hoellerer M.K.
      • Noble M.E.
      The structural basis of localization and signaling by the focal adhesion targeting domain.
      ). Opening of H1 is likely to result from a strain introduced by the Pro944-APP motif that connects H1 and H2 (
      • Arold S.T.
      • Hoellerer M.K.
      • Noble M.E.
      The structural basis of localization and signaling by the focal adhesion targeting domain.
      ), because a similar PXP motif triggers domain swapping in p13suc1 (
      • Bergdoll M.
      • Remy M.H.
      • Cagnon C.
      • Masson J.M.
      • Dumas P.
      Proline-dependent oligomerization with arm exchange.
      ,
      • Bourne Y.
      • Arvai A.S.
      • Bernstein S.L.
      • Watson M.H.
      • Reed S.I.
      • Endicott J.E.
      • Noble M.E.
      • Johnson L.N.
      • Tainer J.A.
      Crystal structure of the cell cycle-regulatory protein suc1 reveals a β-hinge conformational switch.
      ,
      • Rousseau F.
      • Schymkowitz J.W.
      • Wilkinson H.R.
      • Itzhaki L.S.
      Three-dimensional domain swapping in p13suc1 occurs in the unfolded state and is controlled by conserved proline residues.
      ). H1 opening has subsequently been observed in several studies (
      • Hoellerer M.K.
      • Noble M.E.
      • Labesse G.
      • Campbell I.D.
      • Werner J.M.
      • Arold S.T.
      Molecular recognition of paxillin LD motifs by the focal adhesion targeting domain.
      ,
      • Dixon R.D.
      • Chen Y.
      • Ding F.
      • Khare S.D.
      • Prutzman K.C.
      • Schaller M.D.
      • Campbell S.L.
      • Dokholyan N.V.
      New insights into FAK signaling and localization based on detection of a FAT domain folding intermediate.
      ,
      • Zhou Z.
      • Feng H.
      • Bai Y.
      Detection of a hidden folding intermediate in the focal adhesion target domain: implications for its function and folding.
      ). The conformational strain in this hinge region was experimentally apparent (
      • Cable J.
      • Prutzman K.
      • Gunawardena H.P.
      • Schaller M.D.
      • Chen X.
      • Campbell S.L.
      In vitro phosphorylation of the focal adhesion targeting domain of focal adhesion kinase by Src kinase.
      ,
      • Prutzman K.C.
      • Gao G.
      • King M.L.
      • Iyer V.V.
      • Mueller G.A.
      • Schaller M.D.
      • Campbell S.L.
      The focal adhesion targeting domain of focal adhesion kinase contains a hinge region that modulates tyrosine 926 phosphorylation.
      ) and relieved in the open or H1-swapped dimeric conformation of Pro944-APP (
      • Arold S.T.
      • Hoellerer M.K.
      • Noble M.E.
      The structural basis of localization and signaling by the focal adhesion targeting domain.
      ,
      • Prutzman K.C.
      • Gao G.
      • King M.L.
      • Iyer V.V.
      • Mueller G.A.
      • Schaller M.D.
      • Campbell S.L.
      The focal adhesion targeting domain of focal adhesion kinase contains a hinge region that modulates tyrosine 926 phosphorylation.
      ). The open state would allow Tyr925 phosphorylation and subsequent binding to Grb2. Intriguingly, despite the strain in the H1-H2 hinge region, only 0.1% FAT Y925E molecules were in the open conformation (
      • Zhou Z.
      • Feng H.
      • Bai Y.
      Detection of a hidden folding intermediate in the focal adhesion target domain: implications for its function and folding.
      ) and only 2.5% of total FAT were stable, presumably arm-exchanged dimers in size exclusion chromatography analysis (
      • Hoellerer M.K.
      • Noble M.E.
      • Labesse G.
      • Campbell I.D.
      • Werner J.M.
      • Arold S.T.
      Molecular recognition of paxillin LD motifs by the focal adhesion targeting domain.
      ). The low probability of this transition is consistent with the observation that Tyr925 in the native FAT domain is a much poorer substrate for SFK phosphorylation than the unstructured peptide mimics of the region around Tyr925 or FAT domains with destabilized cores (
      • Arold S.T.
      • Hoellerer M.K.
      • Noble M.E.
      The structural basis of localization and signaling by the focal adhesion targeting domain.
      ,
      • Cable J.
      • Prutzman K.
      • Gunawardena H.P.
      • Schaller M.D.
      • Chen X.
      • Campbell S.L.
      In vitro phosphorylation of the focal adhesion targeting domain of focal adhesion kinase by Src kinase.
      ,
      • Prutzman K.C.
      • Gao G.
      • King M.L.
      • Iyer V.V.
      • Mueller G.A.
      • Schaller M.D.
      • Campbell S.L.
      The focal adhesion targeting domain of focal adhesion kinase contains a hinge region that modulates tyrosine 926 phosphorylation.
      ). 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. Thus, although these data establish that the opening of H1 occurs occasionally in vitro and hence might provide a functional switch for FAT, its biological role remains highly questionable.
      Here, we investigated the biological importance of FAT dynamics using mutant forms of the isolated FAT domain and full-length FAK in which the FAT H1-H2 hinge region was modified to increase or decrease its propensity to open (Fig. 1A). Our results demonstrate that these mutations have profound consequences on specific FAK functions in vitro and in vivo. 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.
      Figure thumbnail gr1
      FIGURE 1Mutations in the H1-H2 hinge of FAT alter its self-association. A, mutations designed to relax (R) or increase (T) the tension in the HI-H2 FAT hinge region. B, pull-down assays with immobilized GST-FAT or GST and purified FAT domain: WT-FAT (lanes 2 and 3), R-FAT (lane 4), and T-FAT (lane 5). Input (lane 1): 1 μg of soluble FAT equivalent to the total amount used in the pull-down assays. Molecular mass markers positions are indicated in kDa. C, quantification of bound FAT in three independent pull-down experiments (mean ± S.E., a.u., arbitrary units). One-way ANOVA, F2,6 = 37.7, p = 0.0004, Tukey's test **, p < 0.01; ***, p < 0.001; ns, not significant. D, representative surface plasmon resonance sensorgrams showing the binding curves of purified FAT domains to a sensor chip covalently coated with purified GST-FAT. RU, resonance units. Differential response was obtained by subtracting the signal in the blank channel from that in the experimental channel. Estimated Kd: FAT, 23 ± 8.6 μm; T-FAT, 1.3 ± 0.8 μm; R-FAT, 24 ± 5.5 μm (mean ± S.E., n = 3).

      DISCUSSION

      Previous in vitro and modeling studies of FAT showed that FAT H1 can spontaneously dissociate from the four-helix bundle (
      • Hoellerer M.K.
      • Noble M.E.
      • Labesse G.
      • Campbell I.D.
      • Werner J.M.
      • Arold S.T.
      Molecular recognition of paxillin LD motifs by the focal adhesion targeting domain.
      ,
      • Arold S.T.
      • Hoellerer M.K.
      • Noble M.E.
      The structural basis of localization and signaling by the focal adhesion targeting domain.
      ,
      • Dixon R.D.
      • Chen Y.
      • Ding F.
      • Khare S.D.
      • Prutzman K.C.
      • Schaller M.D.
      • Campbell S.L.
      • Dokholyan N.V.
      New insights into FAK signaling and localization based on detection of a FAT domain folding intermediate.
      ,
      • Zhou Z.
      • Feng H.
      • Bai Y.
      Detection of a hidden folding intermediate in the focal adhesion target domain: implications for its function and folding.
      ,
      • Cable J.
      • Prutzman K.
      • Gunawardena H.P.
      • Schaller M.D.
      • Chen X.
      • Campbell S.L.
      In vitro phosphorylation of the focal adhesion targeting domain of focal adhesion kinase by Src kinase.
      ,
      • Prutzman K.C.
      • Gao G.
      • King M.L.
      • Iyer V.V.
      • Mueller G.A.
      • Schaller M.D.
      • Campbell S.L.
      The focal adhesion targeting domain of focal adhesion kinase contains a hinge region that modulates tyrosine 926 phosphorylation.
      ). This structural rearrangement allows the formation of FAT dimers in vitro through H1 swapping (
      • Arold S.T.
      • Hoellerer M.K.
      • Noble M.E.
      The structural basis of localization and signaling by the focal adhesion targeting domain.
      ). In addition, available in vitro data suggest that partial detachment of H1 is required for Tyr925 phosphorylation by SFKs and subsequent association of Tyr(P)925 with Grb2 (
      • Arold S.T.
      • Hoellerer M.K.
      • Noble M.E.
      The structural basis of localization and signaling by the focal adhesion targeting domain.
      ,
      • Prutzman K.C.
      • Gao G.
      • King M.L.
      • Iyer V.V.
      • Mueller G.A.
      • Schaller M.D.
      • Campbell S.L.
      The focal adhesion targeting domain of focal adhesion kinase contains a hinge region that modulates tyrosine 926 phosphorylation.
      ). However, H1 opening in vitro is a very rare event, occurring only in 2.5% or less of the total FAT population (
      • Hoellerer M.K.
      • Noble M.E.
      • Labesse G.
      • Campbell I.D.
      • Werner J.M.
      • Arold S.T.
      Molecular recognition of paxillin LD motifs by the focal adhesion targeting domain.
      ). It is therefore unclear if these conformational dynamics of FAT have a biological importance. To address this question, we engineered mutations in the hinge between H1 and H2 of FAT, designed to stabilize (R) or destabilize (T) the FAT four-helix bundle, applying a strategy previously used to modulate domain-opening dynamics in p13suc1 (
      • Bergdoll M.
      • Remy M.H.
      • Cagnon C.
      • Masson J.M.
      • Dumas P.
      Proline-dependent oligomerization with arm exchange.
      ,
      • Bourne Y.
      • Arvai A.S.
      • Bernstein S.L.
      • Watson M.H.
      • Reed S.I.
      • Endicott J.E.
      • Noble M.E.
      • Johnson L.N.
      • Tainer J.A.
      Crystal structure of the cell cycle-regulatory protein suc1 reveals a β-hinge conformational switch.
      ,
      • Rousseau F.
      • Schymkowitz J.W.
      • Wilkinson H.R.
      • Itzhaki L.S.
      Three-dimensional domain swapping in p13suc1 occurs in the unfolded state and is controlled by conserved proline residues.
      ). We reasoned that any biological effect linked to FAT dynamics would be abrogated in R-FAK and enhanced in T-FAK. Conversely, if FAT dynamics plays only a minor role in vivo, comparable with their low occurrence rate in vitro, then R-FAK would behave like WT FAK and not produce opposing effects to T-FAK.
      In cells, T-FAK displayed increased Tyr925 phosphorylation but decreased paxillin binding, whereas opposite effects were observed for R-FAT. These results were in agreement with enhanced opening of the four-helix bundle in T-FAT and lost the ability to open in R-FAT. Importantly, the hinge mutations did not impair FAK expression, stability, or activation (as indicated by normal levels of phosphorylation on Tyr397 and Tyr576), showing their specific consequences on FAT functions. The T mutation also increased the ability of FAT to self-associate in vitro. Several potential mechanisms could account for FAT-FAT interactions, including H1-swapping and binding of a flexible FAT extension (residues 895–915) to FAT H1-H4 (
      • Arold S.T.
      • Hoellerer M.K.
      • Noble M.E.
      The structural basis of localization and signaling by the focal adhesion targeting domain.
      ) (see Fig. 2). Deletion of residues 895–915 did not prevent the FAT-FAT interaction, suggesting that interactions of isolated FAT domains result from H1 swapping, although we cannot formally exclude other modes of interaction. We have recently observed that FAK can dimerize through a combination of FERM-FERM and FERM-FAT interactions (
      • Brami-Cherrier K.
      • Gervasi N.
      • Arsenieva D.
      • Walkiewicz K.
      • Boutterin M.C.
      • Ortega A.
      • Leonard P.G.
      • Seantier B.
      • Gasmi L.
      • Bouceba T.
      • Kadaré G.
      • Girault J.A.
      • Arold S.T.
      FAK dimerization controls its kinase-dependent functions at focal adhesions.
      ). Interestingly, the present results showed that the T mutation increased the FAT-FAT interaction without interfering with the FAT-FERM interaction.
      Despite the impaired recruitment of T-FAT to FAs, T-FAK preserved its phosphorylation on Tyr397, the autophosphorylation site (
      • Schaller M.D.
      • Hildebrand J.D.
      • Shannon J.D.
      • Fox J.W.
      • Vines R.R.
      • Parsons J.T.
      Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src.
      ), and on Tyr576, a residue of the activation loop phosphorylated by SFKs (
      • Calalb M.B.
      • Polte T.R.
      • Hanks S.K.
      Tyrosine phosphorylation of focal adhesion kinase at sites in the catalytic domain regulates kinase activity: a role for Src family kinases.
      ). Because autophosphorylation and SFK recruitment are promoted by FAK dimerization (
      • Brami-Cherrier K.
      • Gervasi N.
      • Arsenieva D.
      • Walkiewicz K.
      • Boutterin M.C.
      • Ortega A.
      • Leonard P.G.
      • Seantier B.
      • Gasmi L.
      • Bouceba T.
      • Kadaré G.
      • Girault J.A.
      • Arold S.T.
      FAK dimerization controls its kinase-dependent functions at focal adhesions.
      ), it is possible that the increased FAT H1 opening in T-FAT favored the H1-swapped dimerization of the mutant protein and thus contributed to the persistence of normal FAK activation despite decreased FA recruitment. In cells, formation of H1-swapped WT FAK dimers is likely to be a rare event, although it could conceivably complement the FERM-FERM interaction and replace the FAT-FERM interaction under some circumstances.
      In full-length FAK as in purified FAT, Tyr925 phosphorylation was increased by the T mutation and tended to be decreased by the R mutation, a contrast enhanced by Fyn co-transfection. Tyr925 is an example of a phosphorylation site that has a favorable consensus sequence but a poor conformation for phosphorylation by Src-family kinases (
      • Cable J.
      • Prutzman K.
      • Gunawardena H.P.
      • Schaller M.D.
      • Chen X.
      • Campbell S.L.
      In vitro phosphorylation of the focal adhesion targeting domain of focal adhesion kinase by Src kinase.
      ). Our results indicate that the H1 opening allows the helix region surrounding Tyr925 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 (
      • Dixon R.D.
      • Chen Y.
      • Ding F.
      • Khare S.D.
      • Prutzman K.C.
      • Schaller M.D.
      • Campbell S.L.
      • Dokholyan N.V.
      New insights into FAK signaling and localization based on detection of a FAT domain folding intermediate.
      ). Thus, the conformational dynamics of FAT appear to be a biologically important mechanism to control FAK Tyr925 phosphorylation and hence FAK interactions with the Ras-MAPK pathway (
      • Schlaepfer D.D.
      • Hanks S.K.
      • Hunter T.
      • van der Geer P.
      Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase.
      ,
      • Schlaepfer D.D.
      • Hunter T.
      Evidence for in vivo phosphorylation of the Grb2 SH2-domain binding site on focal adhesion kinase by Src-family protein-tyrosine kinases.
      ) as well as the release of FAK from FAs (
      • Katz B.Z.
      • Romer L.
      • Miyamoto S.
      • Volberg T.
      • Matsumoto K.
      • Cukierman E.
      • Geiger B.
      • Yamada K.M.
      Targeting membrane-localized focal adhesion kinase to focal adhesions: roles of tyrosine phosphorylation and SRC family kinases.
      ).
      The T-FAT mutation also enhanced phosphorylation of Tyr861, another major substrate residue for SFKs in the FAK C-terminal region (
      • Schlaepfer D.D.
      • Hunter T.
      Evidence for in vivo phosphorylation of the Grb2 SH2-domain binding site on focal adhesion kinase by Src-family protein-tyrosine kinases.
      ,
      • Calalb M.B.
      • Polte T.R.
      • Hanks S.K.
      Tyrosine phosphorylation of focal adhesion kinase at sites in the catalytic domain regulates kinase activity: a role for Src family kinases.
      ). Tyr861 is located in a presumably flexible region of the kinase-FAT linker, more than 50 residues upstream of FAT and about 10 residues upstream of the third Pro-rich motif (PR3). Enhanced Tyr861 phosphorylation may be a direct structural consequence of altered conformational FAT dynamics and/or it may result indirectly from increased Tyr925 phosphorylation, as the latter has been reported to influence Tyr861 phosphorylation (
      • Deramaudt T.B.
      • Dujardin D.
      • Hamadi A.
      • Noulet F.
      • Kolli K.
      • De Mey J.
      • Takeda K.
      • Rondé P.
      FAK phosphorylation at Tyr-925 regulates cross-talk between focal adhesion turnover and cell protrusion.
      ). FAK is also phosphorylated on several serine residues (
      • Ma A.
      • Richardson A.
      • Schaefer E.M.
      • Parsons J.T.
      Serine phosphorylation of focal adhesion kinase in interphase and mitosis: a possible role in modulating binding to p130Cas.
      ,
      • Grigera P.R.
      • Jeffery E.D.
      • Martin K.H.
      • Shabanowitz J.
      • Hunt D.F.
      • Parsons J.T.
      FAK phosphorylation sites mapped by mass spectrometry.
      ) that regulate cell spreading and migration (
      • Jiang X.
      • Sinnett-Smith J.
      • Rozengurt E.
      Differential FAK phosphorylation at Ser-910, Ser-843 and Tyr-397 induced by angiotensin II, LPA and EGF in intestinal epithelial cells.
      ). The major such site in the C-terminal region of FAK is Ser910, a substrate of ERK (
      • Hunger-Glaser I.
      • Salazar E.P.
      • Sinnett-Smith J.
      • Rozengurt E.
      Bombesin, lysophosphatidic acid, and epidermal growth factor rapidly stimulate focal adhesion kinase phosphorylation at Ser-910: requirement for ERK activation.
      ). Phosphorylation of Ser910 in response to bombesin-induced ERK activation (
      • Hunger-Glaser I.
      • Salazar E.P.
      • Sinnett-Smith J.
      • Rozengurt E.
      Bombesin, lysophosphatidic acid, and epidermal growth factor rapidly stimulate focal adhesion kinase phosphorylation at Ser-910: requirement for ERK activation.
      ) was increased in T-FAK, suggesting that destabilization of the bundle also increased Ser910 accessibility. Ser910 is located 15 residues upstream from Tyr925, in an N-terminal extension of FAT that can bind back to the FAT surface formed between H1 and H4 (see Fig. 2B). This interaction between the N-terminal extension and FAT H1/H4 restricts the accessibility of Ser910. Opening of H1 would therefore increase exposure of Ser910. Additionally, it is possible that phosphorylation of Tyr925 hinders back-binding of the N-terminal extension and thus promotes exposure of Ser910. These observations underscore a strong interaction between the FAT domain and the upstream region of the C-terminal moiety of FAK.
      Paxillin binding to full-length FAK was impaired by the T mutation and enhanced by the R mutation. These effects were expected because H1 opening and subsequent rearrangement of the FAT structure affect both paxillin binding sites (on H1/H4 and H2/H3) (
      • Gao G.
      • Prutzman K.C.
      • King M.L.
      • Scheswohl D.M.
      • DeRose E.F.
      • London R.E.
      • Schaller M.D.
      • Campbell S.L.
      NMR solution structure of the focal adhesion targeting domain of focal adhesion kinase in complex with a paxillin LD peptide: evidence for a two-site binding model.
      ,
      • Hoellerer M.K.
      • Noble M.E.
      • Labesse G.
      • Campbell I.D.
      • Werner J.M.
      • Arold S.T.
      Molecular recognition of paxillin LD motifs by the focal adhesion targeting domain.
      ). Accordingly, R-FAK accumulation at FAs was increased, whereas it was reduced for T-FAK. Interestingly, T-FAK was still found at FAs, although its binding to paxillin was dramatically decreased. This remaining enrichment may result from the residual paxillin binding as well as from paxillin-independent mechanisms (
      • Cooley M.A.
      • Broome J.M.
      • Ohngemach C.
      • Romer L.H.
      • Schaller M.D.
      Paxillin binding is not the sole determinant of focal adhesion localization or dominant-negative activity of focal adhesion kinase/focal adhesion kinase-related nonkinase.
      ). Indeed, consistent with previous data showing that integrity of the four-helix bundle was not required for talin binding (
      • Chen H.-C.
      • Appeddu P.A.
      • Parsons J.T.
      • Hildebrand J.D.
      • Schaller M.D.
      • Guan J.-L.
      Interaction of focal adhesion kinase with cytoskeletal protein talin.
      ,
      • Hayashi I.
      • Vuori K.
      • Liddington R.C.
      The focal adhesion targeting (FAT) region of focal adhesion kinase is a four-helix bundle that binds paxillin.
      ), the FAT hinge mutations did not alter the FAK interaction with talin.
      FAK is involved in the disassembly of FAs (
      • Ilić D.
      • Furuta Y.
      • Kanazawa S.
      • Takeda N.
      • Sobue K.
      • Nakatsuji N.
      • Nomura S.
      • Fujimoto J.
      • Okada M.
      • Yamamoto T.
      • Aizawa S.
      Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice.
      ) and phosphorylation of Tyr925 is implicated in the exclusion of FAK from FAs and promotion of their turnover (
      • Katz B.Z.
      • Romer L.
      • Miyamoto S.
      • Volberg T.
      • Matsumoto K.
      • Cukierman E.
      • Geiger B.
      • Yamada K.M.
      Targeting membrane-localized focal adhesion kinase to focal adhesions: roles of tyrosine phosphorylation and SRC family kinases.
      ,
      • Hamadi A.
      • Deramaudt T.B.
      • Takeda K.
      • Ronde P.
      Hyperphosphorylated FAK delocalizes from focal adhesions to membrane ruffles.
      ). Despite its strongly decreased presence at FAs, T-FAK restored an elongated shape to transfected FAK−/− cells and increased global FA turnover as well as or more efficiently than WT FAK. In contrast, R-FAK was inefficient despite its higher enrichment at FAs. The phenotype of R-FAK in these assays was similar to that of Y925F-FAK, previously reported to impair FAK function (
      • Deramaudt T.B.
      • Dujardin D.
      • Hamadi A.
      • Noulet F.
      • Kolli K.
      • De Mey J.
      • Takeda K.
      • Rondé P.
      FAK phosphorylation at Tyr-925 regulates cross-talk between focal adhesion turnover and cell protrusion.
      ), suggesting that decreased phosphorylation of this residue is an important aspect in R-FAK properties. Remarkably, loss of R-FAK function was very apparent in cells although the in vitro properties of R-FAK were not very different from those of the wild-type, as expected because it stabilized the closed conformation that is likely to be predominant. This contrast clearly underlines the functional importance of FAT dynamics.
      Our study provides strong evidence that the structural transitions of FAT are physiologically relevant and specifically implicated in key functions of FAK (Fig. 10 summarizes the working model of the dynamics of FAT based on the current study and previous works). It also supports the proposed role of the Pro944-APP motif in the H1-H2 hinge, as the driving force behind H1 opening (
      • Arold S.T.
      • Hoellerer M.K.
      • Noble M.E.
      The structural basis of localization and signaling by the focal adhesion targeting domain.
      ). Interestingly, this motif is conserved among vertebrate FAK, but not in other species (
      • Corsi J.M.
      • Rouer E.
      • Girault J.A.
      • Enslen H.
      Organization and post-transcriptional processing of focal adhesion kinase gene.
      ) or other proteins with FAT-related domains (
      • Hayashi I.
      • Vuori K.
      • Liddington R.C.
      The focal adhesion targeting (FAT) region of focal adhesion kinase is a four-helix bundle that binds paxillin.
      ). H1 dynamics may therefore be an evolutionarily recent property of FAK. Our results also reveal a functional connection between FAT and the rest of the FAK C-terminal region. An important open question is whether specific factors regulate H1 opening in physiological conditions. 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 (
      • Cable J.
      • Prutzman K.
      • Gunawardena H.P.
      • Schaller M.D.
      • Chen X.
      • Campbell S.L.
      In vitro phosphorylation of the focal adhesion targeting domain of focal adhesion kinase by Src kinase.
      ). 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 time-delayed manner after FAK enrichment (
      • Ladbury J.E.
      • Arold S.T.
      Noise in cellular signaling pathways: causes and effects.
      ).
      Figure thumbnail gr10
      FIGURE 10Model for the role of FAT conformational dynamics in FAK function at focal adhesions. The FAT four-helix bundle exists in closed (1) and open (2) conformations in a dynamic equilibrium that is strongly shifted toward the closed state. By decreasing or increasing the propensity of the H1-H2 hinge to open, R-FAK and T-FAK mutants favor the closed and open FAT domain conformation, respectively. The scheme depicts the proposed role of these forms in FAK function at FAs. 1, the closed conformation of FAT has a strong affinity for paxillin LD motifs (two binding sites in FAT) and FAK can be strongly recruited to FAs through this interaction. However, in this configuration, Tyr925 is buried in the four-helix bundle. Ser910 and Tyr861 are also poorly accessible, possibly because of their masking by intramolecular interactions. FAT may also interact with FERM (
      • Brami-Cherrier K.
      • Gervasi N.
      • Arsenieva D.
      • Walkiewicz K.
      • Boutterin M.C.
      • Ortega A.
      • Leonard P.G.
      • Seantier B.
      • Gasmi L.
      • Bouceba T.
      • Kadaré G.
      • Girault J.A.
      • Arold S.T.
      FAK dimerization controls its kinase-dependent functions at focal adhesions.
      ) (not shown in the scheme). 2, local accumulation of FAK at FAs promotes FAK dimerization through FERM-FERM interactions (
      • Brami-Cherrier K.
      • Gervasi N.
      • Arsenieva D.
      • Walkiewicz K.
      • Boutterin M.C.
      • Ortega A.
      • Leonard P.G.
      • Seantier B.
      • Gasmi L.
      • Bouceba T.
      • Kadaré G.
      • Girault J.A.
      • Arold S.T.
      FAK dimerization controls its kinase-dependent functions at focal adhesions.
      ) (only the FERM domain of the second FAK molecule is drawn in dark gray). Possibly with the help of co-activators, the association between FERM and kinase domains in the dimer is loosened, which promotes autophosphorylation by intermolecular transphosphorylation of Tyr397. The Src homology 3 and 2 domains of SFK bind to a proline-rich motif 1 region and Tyr(P)397, respectively, in the FERM:kinase linker peptide. Either spontaneously (in a stochastic manner) or in response to unknown factors, H1 dissociates from the rest of the bundle. In the open conformation of FAT, the affinity of paxillin for the H1/H4 binding site on FAT is completely lost. Opening of FAT H1 is also expected to alter the stability and disposition of the other three helices, compromising paxillin binding to the H2/H3 site (
      • Dixon R.D.
      • Chen Y.
      • Ding F.
      • Khare S.D.
      • Prutzman K.C.
      • Schaller M.D.
      • Campbell S.L.
      • Dokholyan N.V.
      New insights into FAK signaling and localization based on detection of a FAT domain folding intermediate.
      ,
      • Zhou Z.
      • Feng H.
      • Bai Y.
      Detection of a hidden folding intermediate in the focal adhesion target domain: implications for its function and folding.
      ); Tyr925 is exposed and can be phosphorylated by SFKs and then bind Grb2, which activates the ERK pathway. Through unfolding and/or unmasking of the linker region between FAT and the kinase domain, Ser910 and Tyr861 become exposed and accessible to ERK and SFKs, respectively. Together with the loss of paxillin affinity, phosphorylation of the above sites contributes to detachment of FAK from FAs for degradation or recycling.

      Acknowledgments

      We thank Dr. Dusko Ilic (Department of Cell and Tissue Biology, University of California San Francisco) for the gift of Ptk2−/− fibroblasts, Dr. Monique Arpin (Curie Institute, Paris) for providing anti-VSV antibodies and Dr. Alexandre Maucuer for the gift of His6-U2AF. We acknowledge the European Synchrotron Radiation Facility for provision of synchrotron radiation facilities and thank the user support team of beamline ID14-2 for assistance with data recording. J. A. Girault's team is affiliated with the ENP and the Bio-Psy laboratory of excellence.

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