Focal adhesion kinase controls actin assembly via a FERM ... - Nature

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Focal adhesion kinase controls actin assembly via a FERM-mediated interaction with the Arp2/3 complex Bryan Serrels1, Alan Serrels1, Valerie G. Brunton1, Mark Holt2, Gordon W. McLean1,3, Christopher H. Gray1, Gareth E. Jones2 and Margaret C. Frame1,4 Networks of actin filaments, controlled by the Arp2/3 complex, drive membrane protrusion during cell migration. How integrins signal to the Arp2/3 complex is not well understood. Here, we show that focal adhesion kinase (FAK) and the Arp2/3 complex associate and colocalize at transient structures formed early after adhesion. Nascent lamellipodia, which originate at these structures, do not form in FAK-deficient cells, or in cells in which FAK mutants cannot be autophosphorylated after integrin engagement. The FERM domain of FAK binds directly to Arp3 and can enhance Arp2/3-dependent actin polymerization. Critically, Arp2/3 is not bound when FAK is phosphorylated on Tyr 397. Interfering peptides and FERM-domain point mutants show that FAK binding to Arp2/3 controls protrusive lamellipodia formation and cell spreading. This establishes a new function for the FAK FERM domain in forming a phosphorylation-regulated complex with Arp2/3, linking integrin signalling directly with the actin polymerization machinery. Migration requires the coordinated and dynamic regulation of integrinmediated focal adhesion complexes and the actin structures with which they associate, such as lamellipodia and stress fibres. In particular, integrin engagement during the cell attachment/detachment cycle regulates local actin assembly and membrane protrusion at the leading edge. However, the manner in which integrins signal to the actin polymerization machinery is not yet well understood. Actin filament production at lamellipodia is controlled by the Arp2/3 complex1, which promotes the addition of actin monomers at filament branchpoints through its actin nucleation function2. This leads to formation of a branched dendritic actin network, providing the force for membrane protrusion2,3. The Arp2/3 complex is itself activated by upstream signalling from RhoGTPases (particularly Rac1 and Cdc42) through activation of the Scar/WAVE or WASP adaptor proteins4–7. Although it is not known how integrins signal to the actin polymerization pathway, focal adhesion proteins may be conduits of adhesioninduced signals that control actin assembly. Purified integrin complexes have associated actin polymerization activity8, and the focal adhesion protein vinculin is known to associate transiently with the Arp2/3 complex when cells are stimulated with epidermal growth factor, or when cells attach to fibronectin9. However, vinculin-null fibroblasts can still generate lamellipodia (albeit differently to control cells) and these cells actually move faster10,11, implying that there are other ways in which integrins can signal to actin. In contrast with vinculin deficiency, cells

lacking FAK display impaired integrin-dependent cell migration12, whereas expression of the dominant-negative protein FRNK (FAKrelated non-kinase) suppresses the ability of cells to spread on fibronectin and to elicit integrin-induced signals13. However, it is not known how FAK transduces signals from integrins to the actin polymerization machinery and leads to the formation of new actin structures. RESULTS FAK autophosphorylation is required for lamellipodia, stress fibres and early adhesion assembly To examine the effects of FAK function on cellular actin structures, adherent cells were stained with phalloidin. Mouse embryonic fibroblasts that were deficient in FAK (FAK–/–) were less well spread, and displayed few lamellipodia and reduced numbers of cytoplasmic stress fibres when compared to wild-type FAK-expressing cells (Fig. 1a). In both FAK–/– cells and cells re-expressing FAKY397F (data not shown) to the same level as endogenous wild-type FAK (Fig. 1b), cortical actin was observed around the cell periphery, but no protrusive lamellipodia structures were observed (lamellipodia shown for wild-type FAK-expressing cells in Fig. 1a). Quantification of peripheral lamellipodia (which is tightly linked to the spread phenotype) in FAK-deficient cells, and FAK-deficient cells re-expressing wild-type FAK or FAKY397F, is shown in the Supplementary Information, Fig. S1. These data implicate FAK, and integrin-induced tyrosine phosphorylation of FAK, in lamellipodia production and cell spreading, as previously observed14,15.

The Beatson Institute for Cancer Research, Cancer Research UK Beatson Laboratories, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BD, UK. Randall Division of Cell & Molecular Biophysics, King’s College London, Guy’s Campus, London SE1 1UL, UK. 3Current address: Queen’s Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4SA, UK. 4 Correspondence should be addressed to M.C.F. (e-mail: [email protected]) 1 2

Received 3 July 2007; accepted 30 July 2007; published online 26 August 2007; DOI: 10.1038/ncb1626

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Figure 1 Formation of early spreading adhesions requires FAK and FAK Tyr 397 phosphorylation. (a) FAK–/– cells (mouse embryonic fibroblasts) or FAK–/– cells re-expressing wild-type FAK, were grown on glass cover slips, fixed and stained with phalloidin–TRITC. The red line depicts where protrusive lamellipodia have formed. Arrows indicate stress fibres. (b) Immunoblot showing FAK expression in control and reconstituted cells. An uncropped image of the scan is shown in the Supplementary Information, Fig. S6. (c, d) Suspended control cells

(FAK +/+), FAK-deficient cells re-expressing FAK Y397F (FAK–/– + FAKY397F) and FAK-deficient cells (FAK–/–) were plated on to fibronectin for 30 min and visualized. Cells were stained with anti-RACK1 (red) and anti-FAK (green) in c and with anti-FAK (red) and anti-Arp3 (green) in d. For c and d, solid arrows indicate the presence of peripheral spikes whereas arrowheads indicate their absence. Fluorescently labelled second antibodies were used as described in Methods and cells were examined by confocal microscopy. The scale bars represent 70 µm.

As FAK is an integrin effector protein, cells were plated on fibronectin and stained for early spreading structures at the cell periphery. After 30 min, newly forming peripheral ‘spikes’ were visible at the edges of control cells (Fig. 1c) and were absent from FAK-deficient cells or from FAK-deficient cells re-expressing FAKY397F (Fig. 1c). FAK-deficient cells, in particular, remained small and round. The peripheral spike structures may be similar to the previously described spreading initiation centres (SICs), as they contained RACK1 (receptor for active C kinase 1; previously described as a marker of SICs16; Fig. 1c). However, they were

somewhat different in appearance and were longer lived in the mouse embryo fibroblasts used here; therefore, we have named these early spreading adhesions. These structures may represent the ‘first pointof-contact’ adhesion sites as cells attach to extracellular matrix. These sites contained FAK (Fig. 1c) and were not visible at the edges of FAKdeficient cells or in cells expressing only FAKY397F (Fig. 1c). The early spreading adhesions also contained components of the Arp2/3 actin nucleation complex, with colocalization of Arp3 with FAK observed at the tips in control cells (Fig. 1d; arrows). RACK1 and Arp3 were


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Arp3 and N-WASP associate with FAK and the complex is destabilized by FAK Tyr 397 phosphorylation As FAK and Arp3 colocalized at early spreading adhesions, Arp3 was immunoprecipitated from lysates of suspended and adherent control cells, FAK-deficient cells and FAK-deficient cells re-expressing FAKY397F or kinase-deficient FAK (FAKKD). A small amount of wild-type FAK associated with endogenous Arp3 in adherent cells (Fig. 2a). However, more FAK was associated with Arp3 in suspended cells when compared to adherent cells (Fig. 2b), and was inversely correlated with FAK phosphorylation on Tyr 397 (data not shown). Association of FAK with Arp3 was increased when FAKY397F or FAKKD was expressed in adherent FAK–/– cells (Fig. 2b), implying that FAK mutants that cannot be autophosphorylated on Tyr 397 associate more stably with Arp3. In a suspension–re-plating time course experiment, FAK that coimmunoprecipitated with Arp3 was not phosphorylated on Tyr 397 (Fig. 2c), even though phosphorylated FAK was present in the cells. This implies that FAK Tyr 397 phosphorylation and Arp3 binding to FAK are mutually exclusive, suggesting that phosphorylation of FAK at Tyr 397 may be a trigger for dissociation of a dynamic FAK–Arp3 complex. We also noted that there are further, as yet unknown, events in addition to FAK Tyr 397 phosphorylation that contribute to FAK–Arp3 complex dynamics, as even FAKY397F binding to Arp3 is further enhanced in suspended cells (Fig. 2b), and these events may involve other FAK phosphorylation events. Quantification indicated that approximately 0.5% of total endogenous FAK was associated with Arp3 30 min after plating cells on fibronectin, and there was more than a 20-fold increase in FAKY397F binding to Arp3 in adherent cells when compared to wild-type FAK (data not shown). We did not find altered FAK Tyr 397 phosphorylation, or altered binding of Arp3 to FAK in the embryonic fibroblasts used here when the concentration of fibronectin to which cells were adhered was modulated (data not shown). As less wild-type FAK bound stably to Arp3 in adherent cells (Fig. 2a, b), it is likely that the complex between wild-type FAK and Arp3 is dynamic, but is stabilized when FAK cannot be phosphorylated on Tyr 397 — as would occur either when cells are in suspension or when FAK is mutated so that Tyr 397 cannot be phosphorylated (FAKY397F or FAKKD). If this hypothesis is true, integrin-induced auto-phosphorylation of FAK Tyr 397 would promote dissociation of the FAK–Arp3 complex, explaining why the FAKY397F–Arp3 complex is more stable. As FAK is reported to bind N-WASP17, and N-WASP can associate with and activate the Arp2/3 complex18, we also examined whether the FAK–NWASP complex was similarly regulated by FAK Tyr 397 phosphorylation. Bacterially produced GST–N-WASP-VCA domain fusion protein readily associated with wild-type FAK in lysates of suspended cells, whereas association with wild-type FAK in adherent cells was only just detectable (see Supplementary Information, Fig. S2a). However, FAKY397F and FAKKD associated more robustly with GST–N-WASP-VCA from lysates of adherent cells (see Supplementary Information, Fig. S2a). As controls for the FAK–N-WASP binding experiments, association between GST-N–WASP and p34ARC (or p21ARC; data not shown), other components of the

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differently localized in FAK-deficient cells and in cells expressing only FAKY397F (Fig. 1c, d), indicating that assembly of new integrindependent adhesions (at which components of the actin polymerization machinery are present), requires integrin-induced phosphorylation of FAK Tyr 397. Our data raised the question of whether, and if so how, FAK regulates the Arp2/3 complex and actin assembly downstream.

Figure 2 FAK binds to Arp3 when FAK Tyr 397 is not phosphorylated. (a) Arp3 was immunoprecipitated (IP) from adherent control (+/+) or FAK– /– (–/–) cell lysates, transferred to nitrocellulose and probed with anti-FAK. Expression levels of FAK and Arp3 in extracts are shown by blotting with the relevant antibodies in the lower two panels. (b) Extracts from control cells (FAK+/+; lanes 1 and 5), cells without FAK–/– (lanes 2 and 6), FAK–/– cells re-expressing FAKY397F (lanes 3 and 7) or FAKKD (lanes 4 and 8), which were either adherent (lanes 1–4) or suspended in PBS for 2 h at 4 °C to cause dephosphorylation of FAK (lanes 5–8), were immunoprecipitated with an anti-Arp3 antibody or with normal IgG (nIgG; lane 9), transferred to nitrocellulose and blotted using anti-FAK. Expression levels of FAK and Arp3 in extracts are shown by blotting with the relevant antibodies (lower two panels). (c) Endogenous wild-type FAK-expressing cells were either adhered (lane 1), suspended in PBS for 2 h at 4 °C (lane 2), or suspended and replated on fibronectin-coated dishes for 30 min (lane 3) or 1 hour (lane 4). Extracts were separated by SDS–PAGE, blotted and probed with anti-phospho-FAK Tyr 397 or general anti-FAK, or immunoprecipitated with an Arp3 antibody and probed with anti-FAK or anti-phospho-FAK Tyr 397. Uncropped images of the scans in b and c are shown in the Supplementary Information, Fig. S6.

Arp2/3 complex19, was also examined (see Supplementary Information, Fig. S2a). These associated with GST–N-WASP-VCA, but, in contrast with FAK, their association was not affected by whether the cells were adhered or suspended, the presence of FAK, or its ability to be autophosphorylated on Tyr 397 (see Supplementary Information, Fig. S2a). Endogenous N-WASP and FAK were also coimmunoprecipitated, and the amount of FAK associated with N-WASP was increased when FAKY397F was expressed in FAKdeficient cells (see Supplementary Information, Fig. 2b). These data indicate that, similarly to Arp3 binding to FAK, N-WASP binding to FAK could be strongly influenced by FAK phosphorylation; however, N-WASP binding to the Arp2/3 complex was independent of FAK phosphorylation. nature cell biology volume 9 | number 9 | SEPTEMBER 2007

© 2007 Nature Publishing Group

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Figure 3 Arp3 no longer colocalizes with FAK as lamellipodia form beyond early spreading adhesions. (a) Control cells (FAK+/+) were plated on to fibronectin for 30 min, 1 h, 2 h or 17 h, fixed and stained with anti-FAK (red) and anti-Arp3 (green). In the enlarged single cell edge (30 min, lower panel) arrows indicate regions of colocalization between Arp3 and FAK evident in structures along, or at the tips of, nascent adhesive structures. Solid arrows indicate the presence of Arp3, whereas dotted arrows indicate

FAK at the cell periphery. In cells plated on to fibronectin for 30 min, 35–50 cells were examined per experiment and the experiment was repeated four times. In all cells (100%) in which new spreading adhesions had just formed, there was visible evidence of Arp3 along the length, or at the tip, of early adhesion structures. (b) FAK-deficient cells re-expressing FAKY397F were treated and stained as described in a. Solid arrows indicate lack of peripheral adhesion structures. The scale bars represent 70 µm.


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Figure 4 Arp3 association with the FAK FERM domain is regulated by FAK Tyr 397 phosphorylation. (a) Fusion proteins produced in bacteria were separated by SDS–PAGE. (b) Partially purified preparation of the Arp2/3 complex was separated by SDS–PAGE. Components of the complex are indicated. (c) GST–FERM domain fusion, GST–FERMY397, GST–FERMY397F and GST alone, coupled to GSH–agarose, were incubated with Arp2/3 complex for 30 min, washed extensively, and transferred to nitrocellulose for immunoblotting using anti-Arp3 as probe. (d) GST–FERM, GST–FERMY397

or GST–FERMY397F were incubated with purified Arp2/3 complex, and bound proteins immunoblotted using anti-Arp3 and anti-phospho-FAK Tyr 397, as indicated. (e) Far-western blots on Arp2/3 complex preparations were performed by SDS–PAGE, blotting on to nitrocellulose, incubating with GST–FERM domain fusion protein or GST alone, washed and probed with anti-GST. A ‘no-prey’ control (Arp2/3 run on a gel and probed with only anti-GST-HRP) is also shown. Uncropped images of the scans in c and d are shown in the Supplementary Information, Fig. S6.

Release of Arp3 from complex with FAK is associated with nascent lamellipodia formation initiated at early spreading adhesions The dynamics of association and dissociation of the FAK–Arp2/3 complex after cell plating on fibronectin (Fig. 2c), particularly the finding that more FAK is present in anti-Arp3 coimmunoprecipitations at 30 min, before apparent dissociation, prompted closer examination of the timing of localization of FAK and Arp3. At 30 min after plating, Arp3 was present either along, or at the tips of, early spreading adhesions in all control cells that had made such adhesion structures (Fig. 3a). In the enlarged image of a typical single cell edge soon after spreading, small Arp3-containing structures were observed along or at the tips of the much larger FAK-containing early spreading adhesions (Fig. 3a). After 1 h, Arp3 was predominantly located at, or just beyond, the tips

of FAK-containing early spreading adhesions in many cases, and was occasionally observed in structures that seemed to ‘loop out’ away from the early spreading centres. After 2 h, the newly formed lamellipodia structures containing Arp3 more obviously looped out from where the early spreading adhesions had formed (Fig. 3a and see Supplementary Information, Fig. S3a), and Arp3 no longer colocalized with FAK, which remained in focal adhesion structures back from the newly forming lamellipodia (Fig. 3a and see Supplementary Information, Fig. S3a). In fully spread cells at 17 h, Arp3 was present in mature lamellipodia and not in focal adhesions (Fig. 3a and see Supplementary Information, Fig. S3b). In contrast, cells expressing only FAKY397F did not form early spreading adhesions, and although these cells did flatten at later times, their morphologies were different as they had uniform cell edges with

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Figure 5 Identification of Arp2/3 peptide binding sequences within FAK FERM domain. (A) GST–FERM domain, GST–FERM13–125 and GST alone were incubated with purified Arp2/3 complex for 30 min, washed extensively and transferred to nitrocellulose for immunoblotting using anti-Arp3 as probe. (B) Ten-mer peptides spanning FAK residues 13–125, displaced one amino acid at a time, were spotted on to nitrocellulose,

incubated with Arp2/3 complex, washed extensively and probed with antiArp3. Peptides a and b, which consistently bound to Arp3, are boxed. (C) The core binding sequences common to peptides a or b are shown in red, with the charged residues Lys 38 and Arg 86 (that were subsequently mutated) underlined. An uncropped image of the scan in a is shown in the Supplementary Information, Fig. S6.

no protrusive lamellipodia (Fig. 3b). These findings imply that Arp3 and FAK may be corecruited to transient adhesion structures as cells begin to spread on extracellular matrix. Arp3 then becomes dissociated from FAK with time and localizes in new lamellipodia as they form from the tips of early adhesions. As early spreading adhesions and protrusive lamellipodia do not form in cells expressing FAKY397F, this implies that release of the Arp2/3–N-WASP complex from FAK is likely to be important for lamellipodia formation beyond early spreading adhesions. Arp3 only colocalized and complexed with FAK during the very early stages of spreading as early transient adhesions formed, but not at mature focal adhesions that sit back from protrusive lamellipodia. Together with the spreading and actin impairments of FAK-deficient cells (Fig. 1 and see Supplementary Information, Fig. S1), our data suggest that FAK and Arp2/3 need to be corecruited to the correct cellular structures during cell spreading and lamellipodia formation. Given that stress fibres were also reduced in FAK-deficient cells (Fig. 1) and cells expressing FAKY397F (data not shown), it is likely that the cycle of binding and release of

Arp2/3 by FAK as adhesion complexes form is not only linked to lamellipodia formation, but also to the assembly of stress fibres in fully spread cells. These findings raised questions about the nature of the amino acid sequences in FAK that bound the Arp2/3 complex. Arp3 and Arp2 can associate with FAK FERM sub-domain 1 directly and this is regulated by FAK Tyr 397 phosphorylation As FAK Tyr 397 phosphorylation is an important regulator of the association of FAK with the Arp2/3 complex, we examined whether the FAK FERM domain, which is amino-terminal to FAK Tyr 397, could mediate Arp3 binding. GST–FAK-FERM domain fusion proteins, with and without sequences at the carboxyl terminus that contained the adjacent Tyr 397, or FAKY397F (Fig. 4a), were generated and incubated with partially purified Arp2/3 complex (Fig. 4b). GST–FERM domain bound tightly to the Arp2/3 complex (Fig. 4c). When sequences encoding FAK Tyr 397 were included, the GST–FERM domain was unable to bind to Arp3 (Fig. 4c); however, when FAK Tyr 397 was mutated to the


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Figure 6 Introduction of FERM domain Arp2/3 binding peptides reduces cell spreading and stress fibres. (a) GST–FERM + DMSO vehicle, GST– FERM + binding peptides a and b, GST–FERM domain + control peptide and GST alone + binding peptides a and b, were incubated with purified Arp2/3 complex for 30 min, washed and transferred to nitrocellulose for immunoblotting using anti-Arp3 as probe. GST-fusion proteins are shown in the lower panel. Peptide a, VLKVF; peptide b, YGLRL; control peptide, YRGYSLGNWK. (b) Control cells were plated in the presence of DMSO, control peptide or Arp2/3 binding peptides a and b at a total concentration of 100 µM. The peptides were fluorescein- and Penetratin-coupled, and the morphologies of cells that had taken up the peptides as they were

plated were visualized by fluorescence microscopy (green in top panels). Phalloidin staining revealed the reduction in stress fibres and protrusive lamellipodia in cells treated with interfering peptides a and b, when compared to DMSO or control peptide. Red lines indicate areas where protrusive lamellipodia have formed in the presence of control peptide. (c) Cells were scored as to whether or not they had protrusive lamellipodia or cytoplasmic actin cables, which correlate with the degree of spreading. Quantification of the percentage of cells is shown for a representative experiment in which more than 100 cells were counted. The scale bars represent 70 µm in b. An uncropped image of the top panel in a is shown in the Supplementary Information, Fig. S6.

non-phosphorylatable phenylalanine residue, binding to Arp3 was restored (Fig. 4c), suggesting that loss of tyrosine phosphorylation at FAK Tyr 397 stabilized binding between the FAK FERM domain and Arp3 in vitro. This was a surprising result, as we did not expect a

tyrosine kinase to be present in this in vitro binding assay. Confirmation that modulation of FAK FERM-domain binding by presence of FAK Tyr 397 coincided with phosphorylation was provided by showing that the GST–FERM-FAK-Y397 fusion protein was indeed phosphorylated

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Figure 7 Introduction of a FERM-domain effector mutant reduces lamellipodia production and cell spreading. (a) Arp3 was immunoprecipitated from cell extracts of FAK–/– cells re-expressing FAKY397F or FAKY397F,K38A,R86A and probed with anti-FAK. Expression levels of FAK in lysates are shown in the lower panel by blotting with an anti-FAK antibody. (b, c) FAK–/– cells re-expressing wild type (WT) FAK or WT-FAKK38A,R86A were plated overnight, fixed and stained with anti-FAK (b, red), anti-Arp3 (b, green) or TRITC-labelled phalloidin (c), and examined by confocal microscopy. Arrows indicate lamellipodia at the periphery of wild-type FAK-expressing cells. (d) Cells were scored as to whether or not they had protrusive lamellipodia or cytoplasmic actin cables, which correlate with the degree of spreading. Quantification of the percentage of cells is shown for a representative experiment in which more than 100 cells were counted. The scale bars in b and c represent 70 µm. Uncropped images of the scans in a are shown in the Supplementary Information, Fig. S6.

on Tyr 397 (Fig. 4d). These in vitro data are consistent with the FAK FERM domain binding Arp2/3 and phosphorylation on the adjacent FAK Tyr 397 causing release, or precluding the binding interaction between FAK and Arp2/3. An auto-kinase reaction after addition of 32 P-ATP to a partially purified Arp2/3 complex preparation confirmed that a kinase was present and was able to phosphorylate some, but not all, subunits of the Arp2/3 complex (data not shown). To address whether the binding between the FAK FERM domain and Arp2/3 complex was direct, and to identify the nature of the binding subunits, far-western blots were performed. Arp2/3 components were separated by SDS–PAGE, blotted onto membrane, incubated with GST–FERM or GST alone and probed with anti-GST (Fig. 4e). The GST– FERM domain bound directly to Arp3, and also, although consistently to a lesser extent, to Arp2. To map the sites of Arp2/3 binding, we first showed that GST–FERM sub-domain 1 (FAK residues 13–125) contained Arp3 binding sequences (Fig. 5a), and then performed peptide array binding analysis. An array of 10-mer peptides spanning FAK FERM sub-domain 1 (from FAK residues 13–125) was created, incubated with purified Arp2/3 complex and probed with anti-Arp3 (Fig. 5b). Two predominant binding peptides were consistently identified — designated a (VLKVF) and b (YGLRL) — and the core binding sequences present in all of these peptides were identified (Fig. 5c). As these peptides are not adjacent in the FAK primary amino-acid sequence, they were modelled onto the FAK FERM domain structure and were positioned close to each other in the threedimensional structure (data not shown). Interfering with Arp2/3 binding to FAK impairs spreading and actin assembly Peptides a and b were synthesized from the FAK FERM sub-domain 1 and coupled to fluorescein and Penetratin to facilitate monitoring and entry into cells. Combination of peptides a and b blocked the binding of Arp3 to GST–FAK FERM domain when compared with DMSO vehicle or control peptides (Fig. 6a). When cells were incubated with Penetratincoupled peptides a and b as they were plated overnight, cells were smaller and assumed a less well spread morphology than those incubated with control peptides (judged by morphology of fluorescein-labelled cells after 17 h; Fig. 6b). This was consistent with the Arp2/3–FAK complex being necessary for normal cell spreading. Introduction of peptides a and b visibly reduced cytoplasmic stress fibres, and these cells lacked protrusive lamellipodia evident at the periphery of control DMSO-treated cells or cells containing control peptides (Fig. 6b). Quantification of the effects of the competing peptides a and b on the spreading/actin phenotype is shown (Fig. 6c), implying that inhibition of the association between FAK and Arp2/3 causes loss of lamellipodia and reduced stress fibres. A FAK FERM-domain effector mutant inhibits Arp3 binding, lamellipodia and spreading Based on knowledge of the Arp3 binding sequences in the FAK FERM domain, mutant FAK proteins (FAKK38A,R86A) were generated that had a charged residue in each of the two binding sequences changed to alanine (Lys 38 in a and Arg 86 in b). This was done in the background of both wild-type FAK and FAKY397F, the latter to allow the effects on binding Arp3 to be determined biochemically. The double point mutant impaired stable association of FAKY397F with Arp3, demonstrated by coimmunoprecipitation (Fig. 7a). Introduction of these


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two mutations in the background of wild-type FAK (WT-FAKK38A,R86A) caused inhibition of protrusive Arp3-containing lamellipodia that were evident in wild-type FAK-expressing cells (Fig. 7b). Furthermore, phalloidin staining showed that WT-FAKK38A,R86A-expressing cells were less well spread and displayed few lamellipodia and cytoplasmic stress fibres compared to wild-type FAK-expressing cells (Fig. 7c). Thus, re-expression of the FAK FERM domain effector mutant protein that was impaired in Arp3 binding was not able to restore cell spreading or lamellipodia and stress fibre assembly to FAK-deficient cells, when compared to re-expression of wild-type FAK (Fig. 7d).

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DISCUSSION This is the first demonstration that FAK and FAK Tyr 397 phosphorylation mediate the formation of nascent spreading adhesions, probably analogous to previously described, RACK1-containing, SICs that form transiently after integrin engagement16 (Fig.1). FAK binds directly to the Arp2/3 complex through sequences in the first FERM subdomain, and FAK phosphorylated on Tyr 397 does not complex with Arp2/3 (Figs 2 and 4). In the recently solved crystal structure, it seems that FAK Tyr 397 binds to the FERM domain to maintain the auto-inhibited FAK state20. Although it is not yet known how phosphorylation at FAK Tyr 397 affects the binding of proteins to FERM-domain sequences at a structural level, it is possible that phosphorylation of FAK Tyr 397 destabilizes existing interactions, including binding to Arp3. Close examination revealed that Arp3 is located at the tips, or along the length, of FAK-containing early spreading adhesions as they form at the periphery of cells, and that Arp3 is ‘released’ beyond maturing adhesion structures into nascent lamellipodia that emanate from the early transient adhesions (Figs 1 and 3). FAK-deficient cells, cells that only express FAKY397F, and cells in which the FAK–Arp2/3 complex has been disrupted by interfering peptides or by mutation of binding sequences within the FAK FERM domain, all display impaired lamellipodia formation and a spreading defect (Figs 1, 3, 6 and 7). Surprisingly, FAK deficiency, or disruption of the FAK–Arp2/3 complex, is also visibly linked to reduced numbers of cytoplasmic stress fibres and poor spreading (Figs 1, 6 and 7). This implies that the FAK–Arp3 complex is also important for stress-fibre assembly, and fluorescence recovery after photobleaching (FRAP) showed that recruitment of actin into stress

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The FAK FERM domain has some actin polymerization promoting activity Our data raised the question of whether FAK was simply acting in a capacity to bind to the complex, or whether binding via the FERM domain may also be contributing to Arp2/3 function by influencing actin polymerization. Pyrene–actin polymerization assays were performed in vitro, and addition of the FAK FERM domain modestly promoted Arp2/3-mediated actin nucleation activity (Fig. 8a), and could also promote actin polymerization induced by the N-WASP-VCA domain (shown for addition of 2.5 µM FAK FERM domain in Fig. 8b; quantified in Fig. 8c). Importantly, this was suppressed by the FERM mutations (K38A and R86A) that inhibit Arp3 binding (Fig. 8b, c), implying that binding of the FAK FERM domain to Arp2/3 can promote actin polymerization, and this may contribute to efficient lamellipodia initiation at the point of FAK–Arp3 contact. The dose-response relationship of increased actin polymerization at various concentrations of FERM domain is shown in the Supplementary Information, Fig. S4a.

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Figure 8 FAK FERM domain enhances Arp2/3-mediated actin polymerization. (a) The effects of adding the FAK FERM domain or the mutant FAKK38A,R86A (both at 2.5 µM) on actin polymerization in the presence of Arp2/3 complex and the N-WASP-VCA domain, were determined by using in vitro pyrene–actin polymerization assays, as described in Methods. Actin alone is also shown. (b) Relative fluorescence units at the 10 min time point of a representative experiment were plotted to quantify the effects of the FERM domains.

fibres is impaired when FAK is missing or cannot be phosphorylated on Tyr 397 (data not shown). Although stress-fibre assembly is not generally thought to be controlled by the Arp2/3 complex, there is now evidence that subsets of stress fibres may require Arp2/3 function21, and that lamellipodia and stress fibre assembly may be coordinately regulated during spreading22. One possibility is that an indirect consequence of interfering with the FAK–Arp2/3 complex is suppression of stress fibres that depend on prior lamellipodia formation. Although direct connections between integrin signalling and actin polymerization have been proposed, few studies have addressed the mechanism by which this might occur. The Arp2/3 complex binds transiently to vinculin, and although this is required for optimal lamellipodia formation and cell spreading9, it is unclear how Arp2/3-mediated nature cell biology volume 9 | number 9 | SEPTEMBER 2007

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A RT I C L E S actin polymerization is affected by transient vinculin binding, or how the complex might be spatially linked to nascent integrin-dependent adhesion structures. In another study, FAK bound and phosphorylated N-WASP17; however, the authors of this study found that FAKY397F binding to N-WASP was similar to wild-type FAK. This difference with our results, in which the complex is stabilized by the Y397F mutation, is unexplained, but may be due to effects caused by the presence of endogenous FAK in the NIH3T3 cells used, or to levels of overexpression17. We have identified a new function for the FAK FERM domain — previous reported functions include binding to the tyrosine kinase Etk (thought to activate Etk kinase and promote cell migration23). The FAK FERM domain, particularly a basic surface-exposed region in subdomain 2, has also been implicated in adhesion-dependent signalling downstream of FAK24. In addition, the FAK FERM domain negatively regulates the catalytic activity of FAK through an intra-molecular interaction with the kinase domain20,25. Interestingly, FAK Lys 38 of FERM sub-domain 1, one of the residues we identified within the core Arp3 binding sequences (Fig. 5, peptide a), has been implicated in negatively regulating the kinase activity of FAK26. Arp2/3 binding to FAK may, therefore, modulate, or be modulated by, an intra-molecular FERM-kinase domain interaction that normally suppresses the catalytic activity of FAK. Such displacement may be part of the integrincontrolled cycle of FAK-mediated Arp2/3 binding, catalytic activation leading to autophosphorylation on FAK Tyr 397, and subsequent release of Arp2/3 from FAK to promote lamellipodia that protrude beyond early spreading adhesions (see Supplementary Information, Fig. S5). Although we believe this the most likely explanation, we have been unable to prove ‘recruitment and release’ by FAK of the Arp2/3 complex using direct real-time imaging of the complex at new adhesion sites for technical reasons. It is possible that FAK and N-WASP could bind simultaneously to the Arp2/3 complex, for the following reasons: first, the FERM domain promotes N-WASP-VCA-induced actin polymerization; second, N-WASP and FAK bind to different Arp2/3 subunits; and third, the N-WASP–FAK interaction is regulated in a similar manner to the Arp3–FAK interaction with respect to adhesion and integrin-induced phosphorylation (see Supplementary Information, Fig. S2). It is likely that the N-WASP– Arp2/3 complex will still require activation by the normal upstream regulators (such as the Rho GTPases Rac1 or Cdc42), either while bound to FAK or after it is released. What is clear is that FAK provides an important link between integrin engagement and the mechanics of the cell spreading process, by binding to the Arp2/3 complex. The recruitment of Arp3 to peripheral lamellipodia requires FAK FERM-domain binding to the Arp2/3 complex, and the positioning of Arp2/3 into protrusive lamellipodia structures is visibly perturbed in cells expressing the Arp3binding impaired FAK mutant (FAK FERMK38A,R86A; see Supplementary Information, Fig. S4b). Thus, integrin-induced regulation of FAK– Arp2/3 binding controls organized lamellipodia that protrude beyond initial contacts with the extracellular matrix. Methods

Cells. FAK null (FAK–/–) MEFs (control cells; kind gift from D. Ilic, StemLifeLine Inc., CA)12 were cultured in Dulbecco’s modified Eagles medium supplemented with 10% fetal calf serum, 2 mM l-glutamine and non-essential amino acid supplements at 37 °C. FAKY397F and FAKKD reconstituted FAK–/– cells were maintained in 1 mg ml–1 hygromycin B. FAK–/– cells expressing FAK proteins were generated using the pWZL retroviral expression system as follows: FAK was subcloned into

the retroviral expression vector pWZL (a close derivative of pBABE) as previously described27. It was cloned as a BamH1–Snab1 fragment into the polylinker. Phoenix ecotropic cells were then transfected with either of the pWZL–FAK constructs, the virus supernatant collected and used to infect FAK–/– cells. The cells were selected in hygromycin b and single clones isolated. Clones expressing similar levels of FAK to those in control cells expressing endogenous FAK were chosen for investigation. Antibodies and reagents. Antibodies for immunoblotting, immunoprecipitation and immunocytochemistry were as follows: anti-FAK (Transduction Laboratories, Oxford, UK); anti-Arp3 for immunoprecipitation (Transduction Laboratories); anti-Arp3 for immunocytochemistry (Upstate, NY); anti-p21Arc (Transduction Laboratories); anti-p34Arc (Upstate); anti-actin (Sigma, Poole, UK); anti-N-WASP (kind gift from M. W. Kirschner, Harvard University, Boston, MA); anti-phospho-FAK-Y397 (Upstate); anti-vinculin (Sigma); anti-RACK1 (Transduction Laboratories). Anti-mouse and anti-rabbit IgG-peroxidase conjugated secondary antibodies were from New England Biolabs (Hertfordshire, UK) and TRITC–phalloidin was obtained from Sigma. Immunoblotting and immunoprecipitation. Cells were washed twice with PBS and lysed in RIPA lysis buffer (50 mM Tris–HCL at pH 7.4, 150 mM sodium chloride, 5 mM EGTA, 0.1% SDS, 1% NP40 and 1% deoxycholate) with inhibitors (10 mM pyrophosphate, 100 mM sodium fluoride, 1 mM PMSF, 10 µg ml–1 aprotinin, 100 µM sodium orthovanadate, 10 µg ml–1 leupeptin and 10 µg ml–1 benzamidine; all purchased from Sigma). Clarification was by high speed centrifugation (16,000g at 4 °C for 15 min). Cell lysates (10–20 µg as measured by Micro BCA Protein Assay Kit; Pierce Ltd., Northumberland, UK) was supplemented with SDS sample buffer (Tris at pH 6.8, 20% glycerol, 5% SDS, β-mercaptoethanol and bromo-phenol blue) separated by SDS–PAGE, transferred to nitrocellulose and immunoblotted with specific antibodies. For immunoprecipitation experiments, 0.5–1 mg of cell lysate was immunoprecipitated with 2 µg of antibody and immune complexes collected on anti-mouse IgG agarose beads (Sigma) or protein A–Sepharose beads (from Cancer Research UK Central Services, London, UK). Beads were washed three times with RIPA buffer and once with 0.6 M lithium chloride and added to SDS sample buffer. Pulldown experiments with GST–N-WASP VCA domain (Cytoskeleton Inc., Peterborough, UK) were performed as per manufacturer’s instructions with the exception that 1 mg of cell lysate was used. Immunocytochemistry. For immunocytochemistry, cells were grown on glass coverslips, rinsed in PBS, fixed in 3.7% formaldehyde–PBS, permeabilized in 0.5% Triton X100 (Sigma), 0.1% BAS (Sigma) in PBS, blocked in 10% FCS in PBS and incubated with primary antibody overnight. Primary antibody incubation was followed by several washes with PBS containing 0.5% Tween 20 (Sigma) and subsequent incubation with FITC-labelled or Texas Red-labelled secondary antibodies (Jackson Immunoresearch Laboratories, Luton, UK). Actin stress fibres were visualized after staining with tetramethyl-rhodamine B isothiocyanate (TRITC) –phalloidin (Sigma). Immunostaining of cells was visualized by confocal microscopy. Arp3 immunocytochemistry was performed as previously described28. For the analysis of spreading initiation centres, cells were trypsinized, washed and resuspended in PBS, and then incubated with rotation at 4 °C for 2 h before being plated on fibronectin coated (0.01 mg ml–1 human fibronection; Becton Dickinson (Oxford, UK) coverslips for the indicated times in serum-free DMEM. For in vivo peptide competition experiments, Penetratin- and fluorescein-coupled peptides (Thistle Scientific, Uddingston, UK) were incubated with the cells at the time of plating on glass coverslips at a total concentration of 100 µM, and left overnight before fixation and staining. FAK FERM domain GST-pulldown experiments. Avian FAK sequences encoding residues 37–378 and residues 37–413 were amplified by PCR and cloned into pGEX 6P3 vector (Amersham, Buckinghamshire, UK) in frame with GST coding sequences. FAK FERM 32–125 was generated from a pGEX–FAK-FERM domain construct by restriction digest with BamH1–EcoR1 and subsequent cloning back into pGEX 6P3 in frame with GST. All clones were sequenced using a Beckman CEQ2000XL sequencer. Constructs were then transformed into BL21 cells (Stratagene, Amsterdam, Netherlands) for protein expression. Expression of GST fusion proteins was induced for 3 h in the presence of 1 mM IPTG. Bacteria were pelleted, resuspended in lysis buffer containing 50 mM Tris–HCl at pH 8,


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A RT I C L E S 300 mM NaCl2, 1 mM EDTA, 3 mM DTT and 0.1% Triton X100 (Sigma), then sonicated. Fusion proteins were incubated with glutathione–agarose beads (Sigma) overnight at 4 °C with constant mixing and washed three times with PBS. Fusion proteins were incubated with purified Arp2/3 complex (Cytoskeleton Inc.) in binding buffer (20 mM Tris–HCl at pH 7.5, 25 mM KCl, 1 mM MgCl2, 50 mM NaCl2) for 30 min at 4 °C while mixing, subsequently washed three times with binding buffer and supplemented with SDS sample buffer. Samples were separated by gel electrophoresis, transferred and immunoblotted using specific antibodies. For peptide competition assays, 1 µg of purified Arp2/3 complex was incubated with the peptides at a total concentration of 200 µM in binding buffer with mixing for 1 h at 4 °C before the addition of GST-fusion proteins. Far-western blotting. Purified Arp2/3 complex was separated by SDS–PAGE, transferred to nitrocellulose membrane and blocked in 5% BSA in PBS. GSTbound fusion proteins were resuspended in elution buffer (50 mM Tris–HCl at pH 7.5, 250 mM NaCl2, 0.03% Brij35, 0.1 mM EGTA, 20 mM glutathione, 50 µM PMSF, 10 µg ml–1 benzamidine, 14.3 mM β-mercaptoethanol) at room temperature for 30 min with rotation. Beads were spun down and the supernatant was collected and protein concentration assayed using a Bradford protein assay (Perbio, Northumberland, UK). FERM-domain fusion proteins were incubated with the membrane at a concentration of 1 mg ml–1 in 5% BSA–PBS for 2 h and binding was detected using an anti-GST HRP antibody (Perbio). Peptide arrays. Peptide arrays of the FAK FERM-domain amino acids 32–125 were generated by Cancer Research UK Core Services. The peptides spotted onto the array are 10 amino-acids long, moving along the sequence by a single amino acid per peptide. The array consists of control peptides that are comprised of polyaspartic acids, polyarginines and polyphenylalanines. The array was blocked in 5% Marvel in PBS for 1 h, incubated with purified Arp2/3 complex at a concentration of 1 mg ml–1 in 5% milk in PBS for 2 h, then subjected to the westernblotting protocol (see above). Generation of FAK-FERM domain effector mutant. FAK mutants were generated by site directed mutagenesis. Template DNA (20 ng) and 125 ng of each primer was added to PFU Ultra Hotstart DNA polymerase (Stratagene, Amsterdan, Netherlands) in a total volume of 50 µl. Samples were preheated at 95 °C for 10 min before standard PCR. PCR reactions were digested with DPN1 for 1 h at 37 °C and transformed into chemically competent TOP10 bacteria. Transformations were plated on ampicillin (1 mg ml–1) agar plates to select for positive colonies. Colonies were picked and sequenced to check for presence of the mutation. The primers were as follows: K38A Primer 1, 5'-GGAGCGAGTCC TAGCGGTTTTTCACTAC-3'; K38A Primer 2, 5'-GTAGTGAAAAACCGCTAG GACTCGCTCC-3'; R86A Primer 1, 5'-GCTATGGGTTGGCACTCAGTCATCT G-3'; R86A Primer 2, 5'-CAGATGACTGAGTGCCAACCCATAGC-3'. Actin polymerization assays. Actin polymerization was measured using a pyrene actin assay (Cytoskeleton) in which the rate of pyrene-labelled G-actin conversion into F-actin is monitored. The pyrene conjugated G-actin stock was depolymerized (as per the manufacturer’s instruction) and spun at 100,000g for 2 h at 4 °C to remove any oligomers that may have formed during storage before use in the assay. GST–VCA domain (final concentration of 100 nM) and Arp2/3 complex (final concentration of 13 nM) were incubated either in the presence or absence of FAK FERM-domain fusion protein (final concentration of 2.5 µM) for 10 min before the addition of actin. Fluorescence was read immediately after the addition of actin using a Flex Station (Molecular Devices, Berkshire, UK) set on kinetic mode to read every 1 min for the duration of the assay. Flex station settings were as follows: Excitation, 350 nM; emission, 410 nM; and sensitivity, maximum. The assay was performed in black-walled clear-bottom plates. Conversion is accompanied by enhanced fluorescence, and represented by relative fluorescence units (RFU). Note: Supplementary Information is available on the Nature Cell Biology website. ACKNOWLEDGEMENTS This work was supported by Cancer Research UK Program Grant and the Medical Research Council (G.E.J.). We would like to thank M. Kirschner for anti-WASP antibody and D. Ilic for FAK-deficient mouse cells.

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author contributions B.S. and A.S. performed the experimental work. M.H. performed FRAP analysis. V.G.B., G.W.M., G.E.J. and M.C.F. provided extensive scientific input. C.H.G. provided structural biology support. M.C.F. carried out the scientific writing. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Published online at http://www.nature.com/naturecellbiology/ Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/ 1. Welch, M. D., Mallavarapu, A., Rosenblatt, J. & Mitchison, T. J. Actin dynamics in vivo. Curr. Opin. Cell Biol. 9, 54–61 (1997). 2. Svitkina, T. M. & Borisy, G. G. Arp2/3 complex and actin depolymerizing factor/cofilin in dendritic organization and treadmilling of actin filament array in lamellipodia. J. Cell Biol. 145, 1009–1026 (1999). 3. Mullins, R. D., Heuser, J. A. & Pollard, T. D. The interaction of Arp2/3 complex with actin: nucleation, high affinity pointed end capping, and formation of branching networks of filaments. Proc. Natl Acad. Sci. USA 95, 6181–6186 (1998). 4. Machesky, L. M. & Insall, R. H. Scar1 and the related Wiskott-Aldrich syndrome protein, WASP, regulate the actin cytoskeleton through the Arp2/3 complex. Curr. Biol. 8, 1347–1356 (1998). 5. Miki, H., Suetsugu, S. & Takenawa, T. WAVE, a novel WASP-family protein involved in actin reorganization induced by Rac. EMBO J. 17, 6932–6941 (1998). 6. Robinson, R. C. et al. Crystal structure of Arp2/3 complex. Science 294, 1679–1684 (2001). 7. Rohatgi, R. et al. The interaction between N-WASP and the Arp2/3 complex links Cdc42-dependent signals to actin assembly. Cell 97, 221–231 (1999). 8. Butler, B., Gao, C., Mersich, A. T. & Blystone, S. D. Purified integrin adhesion complexes exhibit actin-polymerization activity. Curr. Biol. 16, 242–251 (2006). 9. DeMali, K. A., Barlow, C. A. & Burridge, K. Recruitment of the Arp2/3 complex to vinculin: coupling membrane protrusion to matrix adhesion. J. Cell Biol. 159, 881–891 (2002). 10. Coll, J. L. et al. Targeted disruption of vinculin genes in F9 and embryonic stem cells changes cell morphology, adhesion, and locomotion. Proc. Natl Acad. Sci. USA 92, 9161–9165 (1995). 11. Xu, W., Baribault, H. & Adamson, E. D. Vinculin knockout results in heart and brain defects during embryonic development. Development 125, 327–337 (1998). 12. Ilic, D. et al. Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice. Nature 377, 539–544 (1995). 13. Richardson, A., Malik, R. K., Hildebrand, J. D. & Parsons, J. T. Inhibition of cell spreading by expression of the C-terminal domain of focal adhesion kinase (FAK) is rescued by coexpression of Src or catalytically inactive FAK: a role for paxillin tyrosine phosphorylation. Mol. Cell Biol. 17, 6906–6914 (1997). 14. Ren, X. D. et al. Focal adhesion kinase suppresses Rho activity to promote focal adhesion turnover. J. Cell Sci. 113, 3673–3678 (2000). 15. Westhoff, M. A., Serrels, B., Fincham, V. J., Frame, M. C. & Carragher, N. O. SRC-mediated phosphorylation of focal adhesion kinase couples actin and adhesion dynamics to survival signaling. Mol. Cell Biol. 24, 8113–8133 (2004). 16. de Hoog, C. L., Foster, L. J. & Mann, M. RNA and RNA binding proteins participate in early stages of cell spreading through spreading initiation centers. Cell 117, 649–662 (2004). 17. Wu, X., Suetsugu, S., Cooper, L. A., Takenawa, T. & Guan, J. L. Focal adhesion kinase regulation of N-WASP subcellular localization and function. J. Biol. Chem. 279, 9565– 9576 (2004). 18. Pollard, T. D. & Beltzner, C. C. Structure and function of the Arp2/3 complex. Curr. Opin. Struct. Biol. 12, 768–774 (2002). 19. Millard, T. H., Sharp, S. J. & Machesky, L. M. Signalling to actin assembly via the WASP (Wiskott-Aldrich syndrome protein)-family proteins and the Arp2/3 complex. Biochem. J. 380, 1–17 (2004). 20. Lietha, D. et al. Structural basis for the autoinhibition of focal adhesion kinase. Cell 129, 1177–1187 (2007). 21. Hotulainen, P. & Lappalainen, P. Stress fibers are generated by two distinct actin assembly mechanisms in motile cells. J. Cell Biol. 173, 383–394 (2006). 22. Guo, F., Debidda, M., Yang, L., Williams, D. A. & Zheng, Y. Genetic deletion of Rac1 GTPase reveals its critical role in actin stress fiber formation and focal adhesion complex assembly. J. Biol. Chem. 281, 18652–18659 (2006). 23. Chen, R. et al. Regulation of the PH-domain-containing tyrosine kinase Etk by focal adhesion kinase through the FERM domain. Nature Cell Biol. 3, 439–444 (2001). 24. Dunty, J. M. et al. FERM domain interaction promotes FAK signaling. Mol. Cell Biol. 24, 5353–5368 (2004). 25. Jacamo, R. O. & Rozengurt, E. A truncated FAK lacking the FERM domain displays high catalytic activity but retains responsiveness to adhesion-mediated signals. Biochem. Biophys. Res. Commun. 334, 1299–1304 (2005). 26. Cohen, L. A. & Guan, J. L. Residues within the first subdomain of the FERM-like domain in focal adhesion kinase are important in its regulation. J. Biol. Chem. 280, 8197–8207 (2005). 27. Avizienyte, E. et al. Src-induced de-regulation of E-cadherin in colon cancer cells requires integrin signalling. Nature Cell Biol. 4, 632–638 (2002). 28. Yarrow, J. C., Lechler, T., Li, R. & Mitchison, T. J. Rapid de-localization of actin leading edge components with BDM treatment. BMC Cell Biol. 4, 5 (2003).


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