Structural basis for the autoinhibition of focal adhesion kinase

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. 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.     

Crystal structure of the FERM domain of focal adhesion kinase

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that localizes to focal adhesions in adherent cells. Through phosphorylation of proteins assembled at the cytoplasmic tails of integrins, FAK promotes signaling events that modulate cellular growth, survival, and migration. The amino-terminal region of FAK contains a region of sequence homology with band 4.1 and ezrin/radixin/moesin (ERM) proteins termed a FERM domain. FERM domains are found in a variety of signaling and cytoskeletal proteins and are thought to mediate intermolecular interactions with partner proteins and phospholipids at the plasma membrane and intramolecular regulatory interactions. Here we report two crystal structures of an NH2-terminal fragment of avian FAK containing the FERM domain and a portion of the regulatory linker that connects the FERM and kinase domains. The tertiary folds of the three subdomains (F1, F2, and F3) are similar to those of known FERM structures despite low sequence conservation. Differences in the sequence and relative orientation of the F3 subdomain alters the nature of the interdomain interface, and the phosphoinositide binding site found in ERM family FERM domains is not present in FAK. A putative protein interaction site on the F3 lobe is masked by the proximal region of the linker. Additionally, in one structure the adjacent Src SH3 and SH2 binding sites in the linker associate with the surfaces of the F3 and F1 lobes, respectively. These structural features suggest the possibility that protein interactions of the FAK FERM domain can be regulated by binding of Src kinases to the linker segment.     

FAK nuclear export signal sequences

Ubiquitously expressed focal adhesion kinase (FAK), a critical component in transducing signals from sites of cell contacts with extracellular matrix, was named after its typical localization in focal adhesions. A nuclear localization of FAK has been also reported and its scaffolding role in nucleus and requirement for p53 ubiquitination were only recently described. Whereas FAK nuclear localization signal (NLS) was found in F2 lobe of FERM domain, nuclear export signal (NES) sequences have not been yet determined. Here we demonstrate that FAK has two NES sequences, NES1 in F1 lobe of FERM domain and NES2 in kinase domain. Although, both NES1 and NES2 are evolutionary conserved, and present as well in FAK-related protein kinase Pyk2, only NES2 demonstrates full biological nuclear export activity.     

FERM domain interaction with myosin negatively regulates FAK in cardiomyocyte hypertrophy

Focal adhesion kinase (FAK) regulates cellular processes that affect several aspects of development and disease. The FAK N-terminal FERM (4.1 protein-ezrin-radixin-moesin homology) domain, a compact clover-leaf structure, binds partner proteins and mediates intramolecular regulatory interactions. Combined chemical cross-linking coupled to MS, small-angle X-ray scattering, computational docking and mutational analyses showed that the FAK FERM domain has a molecular cleft (~998 ?(2)) that interacts with sarcomeric myosin, resulting in FAK inhibition. Accordingly, mutations in a unique short amino acid sequence of the FERM myosin cleft, FP-1, impaired the interaction with myosin and enhanced FAK activity in cardiomyocytes. An FP-1 decoy peptide selectively inhibited myosin interaction and increased FAK activity, promoting cardiomyocyte hypertrophy through activation of the AKT-mammalian target of rapamycin pathway. Our findings uncover an inhibitory interaction between the FAK FERM domain and sarcomeric myosin that presents potential opportunities to modulate the cardiac hypertrophic response through changes in FAK activity.     

Focal adhesion kinase (FAK) binds RET kinase via its FERM domain, priming a direct and reciprocal RET-FAK transactivation mechanism

Whether RET is able to directly phosphorylate and activate downstream targets independently of the binding of proteins that contain Src homology 2 or phosphotyrosine binding domains and whether mechanisms in trans by cytoplasmic kinases can modulate RET function and signaling remain largely unexplored. In this study, oligopeptide arrays were used to screen substrates directly phosphorylated by purified recombinant wild-type and oncogenic RET kinase domain in the presence or absence of small molecule inhibitors. The results of the peptide array were validated by enzyme kinetics, in vitro kinase, and cell-based experiments. The identification of focal adhesion kinase (FAK) as a direct substrate for RET kinase revealed (i) a RET-FAK transactivation mechanism consisting of direct phosphorylation of FAK Tyr-576/577 by RET and a reciprocal phosphorylation of RET by FAK, which crucially is able to rescue the kinase-impaired RET K758M mutant and (ii) that FAK binds RET via its FERM domain. Interestingly, this interaction is abolished upon RET phosphorylation, indicating that RET binding to the FERM domain of FAK is a priming step for RET-FAK transactivation. Finally, our data indicate that FAK inhibitors could be used as potential therapeutic agents for patients with multiple endocrine neoplasia type 2 tumors because both, treatment with the FAK kinase inhibitor NVP-TAE226 and FAK down-regulation by siRNA reduced RET phosphorylation and signaling as well as the proliferation and survival of tumor and transfected cell lines expressing oncogenic RET.     

FAK dimerization controls its kinase‐dependent functions at focal adhesions

Focal adhesion kinase (FAK) controls adhesion‐dependent cell motility, survival, and proliferation. FAK has kinase‐dependent and kinase‐independent functions, both of which play major roles in embryogenesis and tumor invasiveness. The precise mechanisms of FAK activation are not known. Using x‐ray crystallography, small angle x‐ray scattering, and biochemical and functional analyses, we show that the key step for activation of FAK's kinase‐dependent functions—autophosphorylation of tyrosine‐397—requires site‐specific dimerization of FAK. The dimers form via the association of the N‐terminal FERM domain of FAK and are stabilized by an interaction between FERM and the C‐terminal FAT domain. FAT binds to a basic motif on FERM that regulates co‐activation and nuclear localization. FAK dimerization requires local enrichment, which occurs specifically at focal adhesions. Paxillin plays a dual role, by recruiting FAK to focal adhesions and by reinforcing the FAT:FERM interaction. Our results provide a structural and mechanistic framework to explain how FAK combines multiple stimuli into a site‐specific function. The dimer interfaces we describe are promising targets for blocking FAK activation.     

FERM control of FAK function: Implications for cancer therapy

Integrins are transmembrane receptors that bind to extracellular matrix proteins and convey anchorage-dependent signals regulating normal cell proliferation. Integrin signals within the tumor micro-environment also impact cancer cell survival and invasion during tumor progression. These integrin-associated signaling events are transduced in part through the activation of non-receptor protein-tyrosine kinases. Focal adhesion kinase (FAK) is activated by β-subunit integrins in both normal and transformed cells. As genetic inactivation of β1 integrin or FAK yield early embryonic lethal phenotypes associated with decreased cell proliferation, and dominant-negative inhibition of FAK can cause increased cell apoptosis, there is a concern that FAK inhibition may have cytotoxic effects on cell growth or survival.However, FAK-specific small molecule inhibitors do not directly impact cell growth in culture, but yet show potent anti-tumor growth effects in vivo. Additionally, recent studies have shed new insight into the FAK kinase-independent regulation of cell proliferation and survival mediated by the FAK N-terminal FERM (band 4.1, ezrin, radixin, moesin homology) domain.Herein, we review the role of the FAK FERM domain in both the intrinsic regulation of FAKkinase activity and how FERM-mediated nuclear localization of FAK promotes enhanced cell survival through the inhibition of tumor suppressor p53 activation during development and under conditions of cellular stress. As we find that FAK FERM-mediated regulation of p53 occurs in human carcinoma cells, elevated FAK expression in tumors may promote both kinase-dependent and –independent survival mechanisms. We discuss how the pharmacological inhibition of FAK kinase activity may impact tumor progression through combined effects of blocking both tumor- and stromal-associated signaling regulating neovascularization.     

Direct interaction of focal adhesion kinase (FAK) with Met is required for FAK to promote hepatocyte growth factor-induced cell invasion

Focal adhesion kinase (FAK) has been implicated to be a point of convergence of integrin and growth factor signaling pathways. Here we report that FAK directly interacts with the hepatocyte growth factor receptor c-Met. Phosphorylation of c-Met at Tyr-1349 and, to a lesser extent, Tyr-1356 is required for its interaction with the band 4.1 and ezrin/radixin/moesin homology domain (FERM domain) of FAK. The F2 subdomain of the FAK FERM domain alone is sufficient for Met binding, in which a patch of basic residues (216KAKTLRK222) are critical for the interaction. Met-FAK interaction leads to FAK activation and subsequent contribution to hepatocyte growth factor-induced cell motility and cell invasion. Our results provide evidence that constitutive Met-FAK interaction may be a critical determinant for tumor cells to acquire invasive potential.     

Identification of Tyr-397 as the primary site of tyrosine phosphorylation and pp60src association in the focal adhesion kinase, pp125FAK.

A number of cellular processes, such as proliferation, differentiation, and transformation, are regulated by cell-extracellular matrix interactions. Previous studies have identified a novel tyrosine kinase, the focal adhesion kinase p125FAK, as a component of cell adhesion plaques. p125FAK was identified as a 125-kDa tyrosine-phosphorylated protein in cells transformed by the v-src oncogene. p125FAK is an intracellular protein composed of three domains: a central domain with homology to protein tyrosine kinases, flanked by two noncatalytic domains of 400 amino acids which bear no significant homology to previously cloned proteins. p125FAK is believed to play an important regulatory role in cell adhesion because it localizes to cell adhesion plaques and because its phosphorylation on tyrosine residues is regulated by binding of cell surface integrins to the extracellular matrix. Recent studies have shown that Src, through its SH2 domain, stably associates with pp125FAK and that this association prevents dephosphorylation of pp125FAK in vitro by protein tyrosine phosphatases. In this report, we identify Tyr-397 as the primary in vivo and in vitro site of p125FAK tyrosine phosphorylation and association with Src. Substituting phenylalanine for tyrosine at position 397 significantly reduces p125FAK tyrosine phosphorylation and association with Src but does not abolish p125FAK kinase activity. In addition, p125FAK kinase is able to trans-phosphorylate Tyr-397 in vitro in a kinase-deficient p125FAK variant. Phosphorylation of Tyr-397 provides a site [Y(P)AEI] that fits the consensus sequence for the binding of Src.     

Protein-tyrosine kinase CAKbeta/PYK2 is activated by binding Ca2+/calmodulin to FERM F2 alpha2 helix and thus forming its dimer

CAKbeta (cell adhesion kinase beta)/PYK2 (proline-rich tyrosine kinase 2) is the second protein-tyrosine kinase of the FAK (focal adhesion kinase) subfamily. It is different from FAK in that it is activated following an increase in cytoplasmic free Ca2+. In the present study we have investigated how Ca2+ activates CAKbeta/PYK2. Calmodulin-agarose bound CAKbeta/PYK2, but not FAK, in the presence of CaCl2. An alpha-helix (F2-alpha2) present in the FERM (band four-point-one, ezrin, radixin, moesin homology) F2 subdomain of CAKbeta/PYK2 was the binding site of Ca2+/calmodulin; a mutant of this region, L176A/Q177A (LQ/AA) CAKbeta/PYK2, bound to Ca2+/calmodulin much less than the wild-type. CAKbeta/PYK2 is known to be prominently tyrosine phosphorylated when overexpressed from cDNA. The enhanced tyrosine phosphorylation was inhibited by W7, an inhibitor of calmodulin, and by a cell-permeable Ca2+ chelator and was almost defective in the LQ/AA-mutant CAKbeta/PYK2. CAKbeta/PYK2 formed a homodimer on binding of Ca2+/calmodulin, which might then induce a conformational change of the kinase, resulting in transphosphorylation within the dimer. The dimer was formed at a free-Ca2+ concentration of 8-12 muM and was stable at 500 nM Ca2+, but dissociated to a monomer in a Ca2+-free buffer. The dimer formation of CAKbeta/PYK2 FERM domain was partially defective in the LQ/AA-mutant FERM domain and was blocked by W7 and by a synthetic peptide with amino acids 168-188 of CAKbeta/PYK2, but not by a peptide with its LQ/AA-mutant sequence. It is known that the F2-alpha2 helix is found immediately adjacent to a hydrophobic pocket in the FERM F2 lobe, which locks, in the autoinhibited FAK, the C-lobe of the kinase domain. Our results indicate that Ca2+/calmodulin binding to the FERM F2-alpha2 helix of CAKbeta/PYK2 releases its kinase domain from autoinhibition by forming a dimer.     

Protein-tyrosine phosphatase PTPD1 regulates focal adhesion kinase autophosphorylation and cell migration

PTPD1 is a cytosolic nonreceptor tyrosine phosphatase and a positive regulator of the Src-epidermal growth factor transduction pathway. We show that PTPD1 localizes along actin filaments and at adhesion plaques. PTPD1 forms a stable complex via distinct molecular modules with actin, Src tyrosine kinase, and focal adhesion kinase (FAK), a scaffold protein kinase enriched at adhesion plaques. Overexpression of PTPD1 promoted cell scattering and migration, short hairpin RNA-mediated silencing of endogenous PTPD1, or expression of PTPD1 mutants lacking either catalytic activity (PTPD1(C1108S)) or the FERM domain (PTPD1(Delta1-325)) significantly reduced cell motility. PTPD1 and Src catalytic activities were both required for epidermal growth factor-induced FAK autophosphorylation at its active site and for downstream propagation of ERK1/2 signaling. Our findings demonstrate that PTPD1 is a component of a multivalent scaffold complex nucleated by FAK at specific intracellular sites. By modulating Src-FAK signaling at adhesion sites, PTPD1 promotes the cytoskeleton events that induce cell adhesion and migration.     

Nuclear FAK promotes cell proliferation and survival through FERM-enhanced p53 degradation

FAK is known as an integrin- and growth factor-associated tyrosine kinase promoting cell motility. Here we show that, during mouse development, FAK inactivation results in p53- and p21-dependent mesodermal cell growth arrest. Reconstitution of primary FAK-/-p21-/- fibroblasts revealed that FAK, in a kinase-independent manner, facilitates p53 turnover via enhanced Mdm2-dependent p53 ubiquitination. p53 inactivation by FAK required FAK FERM F1 lobe binding to p53, FERM F2 lobe-mediated nuclear localization, and FERM F3 lobe for connections to Mdm2 and proteasomal degradation. Staurosporine or loss of cell adhesion enhanced FERM-dependent FAK nuclear accumulation. In primary human cells, FAK knockdown raised p53-p21 levels and slowed cell proliferation but did not cause apoptosis. Notably, FAK knockdown plus cisplatin triggered p53-dependent cell apoptosis, which was rescued by either full-length FAK or FAK FERM re-expression. These studies define a scaffolding role for nuclear FAK in facilitating cell survival through enhanced p53 degradation under conditions of cellular stress.     

FERM domain interaction promotes FAK signaling

From the results of deletion analyses, the FERM domain of FAK has been proposed to inhibit enzymatic activity and repress FAK signaling. We have identified a sequence in the FERM domain that is important for FAK signaling in vivo. Point mutations in this sequence had little effect upon catalytic activity in vitro. However, the mutant exhibits reduced tyrosine phosphorylation and dramatically reduced Src family kinase binding. Further, the abilities of the mutant to transduce biochemical signals and to promote cell migration were severely impaired. The results implicate a FERM domain interaction in cell adhesion-dependent activation of FAK and downstream signaling. We also show that the purified FERM domain of FAK interacts with full-length FAK in vitro, and mutation of this sequence disrupts the interaction. These findings are discussed in the context of models of FAK regulation by its FERM domain.     

Mechanism of Focal Adhesion Kinase Mechanosensing

Mechanosensing at focal adhesions regulates vital cellular processes. Here, we present results from molecular dynamics (MD) and mechano-biochemical network simulations that suggest a direct role of Focal Adhesion Kinase (FAK) as a mechano-sensor. Tensile forces, propagating from the membrane through the PIP₂ binding site of the FERM domain and from the cytoskeleton-anchored FAT domain, activate FAK by unlocking its central phosphorylation site (Tyr576/577) from the autoinhibitory FERM domain. Varying loading rates, pulling directions, and membrane PIP₂ concentrations corroborate the specific opening of the FERM-kinase domain interface, due to its remarkably lower mechanical stability compared to the individual alpha-helical domains and the PIP₂-FERM link. Analyzing downstream signaling networks provides further evidence for an intrinsic mechano-signaling role of FAK in broadcasting force signals through Ras to the nucleus. This distinguishes FAK from hitherto identified focal adhesion mechano-responsive molecules, allowing a new interpretation of cell stretching experiments.     

Regulation of focal adhesion kinase by a novel protein inhibitor FIP200

Focal adhesion kinase (FAK) is a major mediator of integrin signaling pathways. The mechanisms of regulation of FAK activity and its associated cellular functions are not very well understood. Here, we present data suggesting that a novel protein FIP200 functions as an inhibitor for FAK. We show the association of endogenous FIP200 with FAK, which is decreased upon integrin-mediated cell adhesion concomitant with FAK activation. In vitro- and in vivo-binding studies indicate that FIP200 interacts with FAK through multiple domains directly. FIP200 bound to the kinase domain of FAK inhibited its kinase activity in vitro and its autophosphorylation in vivo. Overexpression of FIP200 or its segments inhibited cell spreading, cell migration, and cell cycle progression, which correlated with their inhibition of FAK activity in vivo. The inhibition of these cellular functions by FIP200 could be rescued by coexpression of FAK. Last, we show that disruption of the functional interaction between endogenous FIP200 with FAK leads to increased FAK phosphorylation and partial restoration of cell cycle progression in cells plated on poly-L-lysine, providing further support for FIP200 as a negative regulator of FAK. Together, these results identify FIP200 as a novel protein inhibitor for FAK.     

The Pyk2 FERM regulates Pyk2 complex formation and phosphorylation

The focal adhesion kinase Pyk2 integrates signals from cell adhesion receptors, growth factor receptors, and G-protein-coupled receptors leading to the activation of intracellular signaling pathways that regulate cellular phenotypes. The intrinsic mechanism for the activation of Pyk2 activity remains to be fully defined. Previously, we reported that mutations in the N-terminal FERM domain result in loss of Pyk2 activity and expression of the FERM domain as an autonomous fragment inhibits Pyk2 activity. In the present study, we sought to determine the mechanism that underlies these effects. Utilizing differentially epitope-tagged Pyk2 constructs, we observed that Pyk2 forms oligomeric complexes in cells and that complex formation correlates positively with tyrosine phosphorylation. Similarly, when expressed as an autonomous fragment, the Pyk2 FERM domain formed a complex with other Pyk2 FERM domains but not the FAK FERM domain. When co-expressed with full-length Pyk2, the autonomously expressed Pyk2 FERM domain formed a complex with full-length Pyk2 preventing the formation of Pyk2 oligomers and resulting in reduced Pyk2 phosphorylation. Deletion of the FERM domain from Pyk2 enhanced Pyk2 complex formation and phosphorylation. Together, these data indicate that the Pyk2 FERM domain is involved in the regulation of Pyk2 activity by acting to regulate the formation of Pyk2 oligomers that are critical for Pyk2 activity.     

FERM domain promotes resveratrol-induced apoptosis in endothelial cells via inhibition of NO production

Focal adhesion kinase (FAK) consists of an N-terminal band 4.1; ezrin, radixin, moesin (FERM) domain; tyrosine kinase domain; and C-terminal FA targeting domain. Here we show that ectopically expressed FERM is largely located in the cytosolic fraction under quiescent conditions. We further found that this ectopically expressed FERM domain aggravates endothelial cell apoptosis triggered by 100 μM resveratrol, whereas FERM had no effect on apoptosis induced by TNF-α. We determined that resveratrol at low doses (<20 μM) promotes phosphorylation (S1177) of eNOS via an AMPK-dependent pathway. The presence of the FERM domain blocked this resveratrol-stimulated eNOS phosphorylation and NO production. Thus, the pro-apoptotic activity of cytosolic FERM domain is at least partially mediated by down-regulation of NO, a critical cell survival factor. Consistently, we found that the apoptosis induced by cytosolic FERM in the presence of resveratrol was reversed by an NO donor, SNAP. In conclusion, FERM located in the cytosolic fraction plays a pivotal role in aggravating cell apoptosis through diminishing NO production.     

GIT1 utilizes a focal adhesion targeting-homology domain to bind paxillin

The GIT proteins, GIT1 and GIT2, are GTPase-activating proteins for the ADP-ribosylation factor family of small GTP-binding proteins, but also serve as adaptors to link signaling proteins to distinct cellular locations. One role for GIT proteins is to link the PIX family of Rho guanine nucleotide exchange factors and their binding partners, the p21-activated protein kinases, to remodeling focal adhesions by interacting with the focal adhesion adaptor protein paxillin. We here identified the C-terminal domain of GIT1 responsible for paxillin binding. Combining structural and mutational analyses, we show that this region folds into an anti-parallel four-helix domain highly reminiscent to the focal adhesion targeting (FAT) domain of focal adhesion kinase (FAK). Our results suggest that the GIT1 FAT-homology (FAH) domain and FAT bind the paxillin LD4 motif quite similarly. Since only a small fraction of GIT1 is bound to paxillin under normal conditions, regulation of paxillin binding was explored. Although paxillin binding to the FAT domain of FAK is regulated by tyrosine phosphorylation within this domain, we find that tyrosine phosphorylation of the FAH domain GIT1 is not involved in regulating binding to paxillin. Instead, we find that mutations within the FAH domain may alter binding to paxillin that has been phosphorylated within the LD4 motif. Thus, despite apparent structural similarity in their FAT domains, GIT1 and FAK binding to paxillin is differentially regulated.     

Focal adhesion kinase controls actin assembly via a FERM-mediated interaction with the Arp2/3 complex

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.     

Activation of Endogenous FAK via Expression of Its Amino Terminal Domain in Xenopus Embryos

Background

The Focal Adhesion Kinase is a well studied tyrosine kinase involved in a wide number of cellular processes including cell adhesion and migration. It has also been shown to play important roles during embryonic development and targeted disruption of the FAK gene in mice results in embryonic lethality by day 8.5.

Principal Findings

Here we examined the pattern of phosphorylation of FAK during Xenopus development and found that FAK is phosphorylated on all major tyrosine residues examined from early blastula stages well before any morphogenetic movements take place. We go on to show that FRNK fails to act as a dominant negative in the context of the early embryo and that the FERM domain has a major role in determining FAK’s localization at the plasma membrane. Finally, we show that autonomous expression of the FERM domain leads to the activation of endogenous FAK in a tyrosine 397 dependent fashion.

Conclusions

Overall, our data suggest an important role for the FERM domain in the activation of FAK and indicate that integrin signalling plays a limited role in the in vivo activation of FAK at least during the early stages of development.