Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official. Federal government websites often end in . gov or VSports app下载. mil. Before sharing sensitive information, make sure you’re on a federal government site. .

Https

The site is secure V体育官网. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely. .

. 2007 Jun 15;129(6):1177-87.
doi: 10.1016/j.cell.2007.05.041.

VSports app下载 - Structural basis for the autoinhibition of focal adhesion kinase

Daniel Lietha  1 , Xinming Cai, Derek F J Ceccarelli, Yiqun Li, Michael D Schaller, Michael J Eck
Affiliations

Structural basis for the autoinhibition of focal adhesion kinase

Daniel Lietha et al. Cell. .

Abstract

Appropriate tyrosine kinase signaling depends on coordinated sequential coupling of protein-protein interactions with catalytic activation. Focal adhesion kinase (FAK) integrates signals from integrin and growth factor receptors to regulate cellular responses including cell adhesion, migration, and survival VSports手机版. Here, we describe crystal structures representing both autoinhibited and active states of FAK. The inactive structure reveals a mechanism of inhibition in which the N-terminal FERM domain directly binds the kinase domain, blocking access to the catalytic cleft and protecting the FAK activation loop from Src phosphorylation. Additionally, the FERM domain sequesters the Tyr397 autophosphorylation and Src recruitment site, which lies in the linker connecting the FERM and kinase domains. The active phosphorylated FAK kinase adopts a conformation that is immune to FERM inhibition. Our biochemical and structural analysis shows how the architecture of autoinhibited FAK orchestrates an activation sequence of FERM domain displacement, linker autophosphorylation, Src recruitment, and full catalytic activation. .

PubMed Disclaimer

Figures

Figure 1
Figure 1. Structure of autoinhibited FAK
(A) Domain structure of FAK. Key tyrosine phosphorylation sites are indicated. (B) Overall structure of autoinhibited FAK including the FERM, linker, and kinase regions. In the autoinhibited state, the FERM domain (blue ribbon representation) binds the kinase domain (red), primarily through an interaction between the FERM F2 lobe and the kinase C-lobe. A section of the linker that contains the autophosphorylation site Tyr 397 (yellow) is located between the FERM F1 lobe and the kinase N-lobe. The FERM domain also blocks access to the active site cleft and to the kinase activation loop (A-loop, green). Disordered segments are indicated as dashed lines. The staurosporine analogue AFN941 is bound to the active site of the kinase and is shown in stick representation. (C) Sequence alignment of the FERM, linker and kinase regions of avian FAK (cFAK1), human FAK (hFAK1) and human Pyk2 (hPyk2). cFAK1 shares 94% sequence identity with hFAK1, and hFAK1 43% with hPyk2. Secondary structure elements are indicated and the sequence is shaded to correspond to the colors in panel b. Residues involved in the FERM F2 lobe/kinase C-lobe interaction are indicated by an asterisk and regulatory tyrosines are colored magenta.
Figure 2
Figure 2. Structure of the active kinase domain of FAK
(A) The structure of the FAK kinase domain phosphorylated by Src is shown in ribbon representation (green) with the activation loop in blue. The side chains of phosphotyrosines 576 and 577 and AMP-PNP, which is bound to the active site, are shown in stick representation. A Mg2+ ion at the active site is shown as a yellow sphere. (B) Close up view of the activation loop with the side chains of pY576, pY577, R569, R545 and main chains of A579 and S580 shown in stick representation. A network of hydrogen bonds (orange dashed lines) involving the phosphate group of pY577 stabilizes the conformation of the activation loop. (C) Superposition of active and inactive FAK kinases. The autoinhibited structure is shown with the FERM domain as a surface representation (light blue), and the linker and kinase domains in a ribbon representation colored yellow and red, respectively. The structure of the active kinase domain (green ribbon and blue activation loop) is superimposed based on the kinase C-lobes. The side chain of pY576 and the main chain carbonyl of A579 in the active kinase (both residues are shown in space filling representations) clash with the FERM domain in the autoinhibited structure.
Figure 3
Figure 3. Phosphorylation of the activation loop overrides FERM domain inhibition
The in vitro kinase activity of the FAK kinase (FAK411−686) and single-chain FERM+kinase (FAK31−686) proteins is plotted for proteins that are unphosphorylated (n) or phosphorylated on Y576 and Y577 (y). Phosphorylated proteins were obtained by treatment with Src and subsequent repurification of phosphorylated FAK proteins. Note the reduced activity of the FERM+kinase protein compared to that of the isolated kinase domain prior to Src phosphorylation. Phosphorylation of the FERM+kinase protein by Src on Tyr 576 and Tyr 577 (shown for Y397F mutant, see text) yields an activity equal to that of the isolated kinase domain in the phosphorylated state.
Figure 4
Figure 4. The FERM/kinase interface inhibits FAK
(A) Detailed view of the interaction between the FERM F2 lobe and the kinase C-lobe. Sidechains that participate in the interaction are shown in stick form and are labeled. At the center of the interface F596 on the kinase domain inserts into a hydrophobic pocket on the FERM domain formed by Y180, M183, V196, L197. The periphery of the interaction is predominantly polar. Interdomain hydrogen bonds are shown as yellow dashed lines. (B) Detailed view of the linker region, which forms a b-sheet interaction with the FERM F1 lobe and contacts the kinase N-lobe. Side chains of the autophosphorylation site Y397 and residues at the linker/kinase contact point are shown. (C) Surface representation of separated FERM (right), linker (center) and kinase (left) domains colored according to sequence conservation. Viewing angles are indicated relative to Figure 1B. Conservation is indicated with a color scale from red (most highly conserved) to green (most variable). Note that the interacting surfaces of the FERM F2 lobe and kinase domain are highly conserved. Additionally, a patch of residues at the bottom of the F2 lobe (a potential binding site for a competitive activator, see text) and Trp266 and surrounding residues on the FERM F3 lobe are also highly conserved. Interestingly, Trp 266 and adjacent residues on the FERM domain form the same two-fold symmetric “dimer” interaction in every crystal structure containing the FERM domain, even though these FAK proteins appear to be monomeric in solution. The analysis was performed with ClustalW (Chenna et al., 2003) and ConSurf (Glaser et al., 2003) servers, and the figure was generated with Pymol (DeLano, 2002). (D) In vitro kinase activity of purified wt and mutant FERM+kinase proteins are plotted as negative slopes of NADH depletion (see methods). Disruption of the FERM F2 lobe/kinase C-lobe interface (Y180A, M183A and F596D mutations) activates the autoinhibited form of FAK. (E) Size exclusion chromatography of purified wild-type and mutant single-chain FERM+kinase proteins. Stokes radii were calculated by comparison with standards (Catalase, Aldolase, BSA, Ovalbumin, Chymotrypsinogen and Ribonuclease)(Siegel and Monty, 1966). Mutations that activate FAK result in a larger stokes radius, indicating an ‘open’ conformation, compared to the ‘closed’ autoinhibited form.
Figure 5
Figure 5. Analysis of wild type and mutant FAK in HEK 293 cells
Mutations were introduced into full-length FAK to disrupt the interaction of the FERM F2 lobe with the kinase C-lobe (Y180A, M183A and V196D, L197D on the FERM domain and F596D on the kinase domain) or to disrupt the contact of the linker with the kinase N-lobe (S463Y). (A) Full-length wild-type or mutant FAK proteins were transiently expressed in HEK 293 cells and cell lysates were analyzed by Western blotting with the indicated phospho-specific antibodies or with the 4.47anti-FAK antibody. (B) In vitro kinase assay for paxillin phosphorylation. Wild type and mutant full-length FAK proteins were immunoprecipitated from HEK 293 lysates using BC4. The immune complex was incubated in a kinase assay utilizing recombinant GST-paxillin-N-C3 as an exogenous substrate (Lyons et al., 2001). Phosphorylation of paxillin was detected using the phosphospecific antibodies pY31 and pY118, as well as the 4G10 phosphotyrosine antibody. Equal amounts of substrate were verified by blotting for paxillin using a polyclonal antiserum. Equal recovery of FAK proteins in the immune complex was verified by blotting for FAK. (C) HEK 293 cells transiently expressing FAK proteins were lysed and analyzed for cellular phosphotyrosine by Western blotting cell lysates with 4G10. (D) Paxillin and FAK were transiently co-expressed in HEK 293 cells and paxillin phosphorylation analyzed by Western blotting lysates using the pY31 antibody (upper panel). Lysates were blotted for paxillin using a monoclonal antibody as a loading control (middle panel) and for FAK to verify comparable expression of the wild type and mutant FAK proteins (bottom panel). (E) Wild type or mutant full-length FAK was transiently expressed in FAK null cells and adherent cells (Ad) or cells incubated in suspension at 37°C for 1 hour (sus) were lysed. FAK was immunoprecipitated using BC4 and the immune complexes analyzed by Western blotting for phosphotyrosine using 4G10 (top panel). Equal amounts of FAK in the immune complexes were verified by blotting for FAK (bottom panel).
Figure 6
Figure 6. Tyrosine phosphorylation sites are protected in autoinhibited FAK
(A) Time course of autophosphorylation at Tyr 397 was carried out for wild type and mutant purified single-chain FERM+kinase proteins (upper bands, labeled F-L-K for FERM-Linker-Kinase). FERM+linker protein (Y180A, M183A mutant), which has Y397 exposed, was included in the reactions as a control substrate (lower bands, labeled F-L for FERM-Linker). Tyr 397 phosphorylation was monitored by Western blotting using a specific anti-pY397 antibody (left panels). As loading controls equivalent gels were stained by Coomassie (right panels). Note that autoinhibited FERM+kinase protein (wt and S463Y mutant) autophosphorylate on Y397 much less efficiently than they phosphorylate Y397 of the control substrate, but that this discrimination is lost on the ‘open’ mutants (Y180A, M183A and F596D), which autophosphorylate as rapidly as they phosphorylate the control substrate. Also, note the overall increase in phosphorylation in the open mutants, consistent with their increased catalytic activity (as shown in Figure 4C). (B) and (C) Time course of Src phosphorylation of wt and mutant FERM+kinase on Tyr 576 and Tyr 577. Phosphorylation was monitored by Western blotting using specific anitibodies against pY576 (b, left panels) or pY577 (c, left panels) and equivalent gels were Coomassie stained as loading controls (right panels). Note that Src phosphorylation of the activation loop tyrosines is diminished in the intact, autoinhibited protein.
Figure 7
Figure 7. Schematic of autoinhibited FAK and a sequential model of activation
In the inactive state (left), the FERM domain blocks the kinase active site and sequesters the Tyr 397 and activation loop phosphorylation sites. Although not observed in the present structure, we indicate the docking of the PxxP motif in the linker to the F3 lobe of the FERM domain based on previous work (Ceccarelli et al., 2006). We propose that FAK activation will be initiated by displacement of the FERM domain by competitive binding of an activating protein (orange star) to the FERM F2 surface. Such interactions have not been structurally characterized, but candidate activating proteins include the cytoplasmic regions of b-integrins or growth factor receptors EGFR, PDGFR, c-Met or EphA2. Disassembly of the autoinhibited conformation allows rapid autophosphorylation of the linker residue Tyr 397, and exposes the Src docking sites in the linker (center panel). In a subsequent step, Src is recruited and activated via SH2 binding to pTyr 397 and SH3 binding to the PxxP sequence in the linker region. Localized Src then phosphorylates the activation loop residues Tyr 576 and Tyr 577 of FAK (right panel). Phosphorylation of the activation loop yields full catalytic activity, and even in the event of activator dissociation will not allow kinase inhibition by the FERM domain. Note that for simplicity we have depicted FAK autophosphorylation in cis, it may, however, also occur in trans.
See this image and copyright information in PMC

Comment in (V体育平台登录)

References

    1. Arold ST, Hoellerer MK, Noble ME. The structural basis of localization and signaling by the focal adhesion targeting domain. Structure (Camb) 2002;10:319–327. - PubMed
    1. Bershadsky AD, Balaban NQ, Geiger B. Adhesion-dependent cell mechanosensitivity. Annu Rev Cell Dev Biol. 2003;19:677–695. - PubMed
    1. Boggon TJ, Eck MJ. Structure and regulation of Src family kinases. Oncogene. 2004;23:7918–7927. - PubMed
    1. Boggon TJ, Li Y, Manley PW, Eck MJ. Crystal structure of the Jak3 kinase domain in complex with a staurosporine analog. Blood. 2005;106:996–1002. - PMC - PubMed
    1. Calalb MB, Polte TR, Hanks SK. Tyrosine phosphorylation of focal adhesion kinase at sites in the catalytic domain regulates kinase activity: a role for Src family kinases. Mol Cell Biol. 1995;15:954–963. - PMC (VSports app下载) - PubMed

Publication types

MeSH terms

Substances