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King Abdullah University of Science and Technology (KAUST), Division of Biological and Environmental Sciences and Engineering, Computational Bioscience Research Center (CBRC), Thuwal, Saudi ArabiaCentre de Biochimie Structurale, CNRS UMR5048, INSERM U1054, Université de Montpellier I & II, Montpellier, France
* 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.
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.
Mutations that facilitate or prevent opening of the first helix have profound consequences on the biochemical properties of FAK and its function in cells.
The ability of FAT to open and close is essential for FAK function.
This provides evidence for the functional importance of the conformational dynamics of FAT.
). 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 (
). 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 (
). 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 (
). 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.
Previous in vitro and modeling studies of FAT showed that FAT H1 can spontaneously dissociate from the four-helix bundle (
). 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 (
). 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 (
) (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 (
), 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 (
). 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 (
). 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 (
) 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) (
). 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 (
). 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 (
), 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 (
). 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 (
). 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 (
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.
Focal adhesion protein-tyrosine kinase phosphorylated in response to cell attachment to fibronectin.