Focal Adhesion Kinase FERM Domain Research Articles

Phosphohistidine signaling promotes FAK-RB1 interaction and growth factor-independent proliferation of esophageal squamous cell carcinoma.

Current clinical therapies targeting receptor tyrosine kinases including focal adhesion kinase (FAK) have had limited or no effect on esophageal squamous cell carcinoma (ESCC). Unlike esophageal adenocarcinomas, ESCC acquire glucose in excess of their anabolic need. We recently reported that glucose-induced growth factor-independent proliferation requires the phosphorylation of FAKHis58. Here, we confirm His58 phosphorylation in FAK immunoprecipitates of glucose-stimulated, serum-starved ESCC cells using antibodies specific for 3-phosphohistidine and mass spectrometry. We also confirm a role for the histidine kinase, NME1, in glucose-induced FAKpoHis58 and ESCC cell proliferation, correlating with increased levels of NME1 in ESCC tumors versus normal esophageal tissues. Unbiased screening identified glucose-induced retinoblastoma transcriptional corepressor 1 (RB1) binding to FAK, mediated through a "LxCxE" RB1-binding motif in FAK's FERM domain. Importantly, in the absence of growth factors, glucose increased FAK scaffolding of RB1 in the cytoplasm, correlating with increased ESCC G1→S phase transition. Our data strongly suggest that this glucose-mediated mitogenic pathway is novel and represents a unique targetable opportunity in ESCC.

PKCθ-mediated serine/threonine phosphorylations of FAK govern adhesion and protrusion dynamics within the lamellipodia of migrating breast cancer cells.

The cytoskeleton and cell-matrix adhesions constitute a dynamic network that controls cellular behavior during development and cancer. The Focal Adhesion Kinase (FAK) is a central actor of these cell dynamics, promoting cell-matrix adhesion turnover and active membrane fluctuations. However, the initial steps leading to FAK activation and subsequent promotion of cell dynamics remain elusive. Here, we report that the serine/threonine kinase PKCθ participates in the initial steps of FAK activation. PKCθ, which is strongly expressed in aggressive human breast cancers, controls the dynamics of cell-matrix adhesions and active protrusions through direct FAK activation, thereby promoting cell invasion and lung metastases. Using various tools for in vitro and live cell studies, we precisely decipher the molecular mechanisms of FAK activation. PKCθ directly interacts with the FAK FERM domain to open FAK conformation through PKCθ's specific V3 domain, while phosphorylating FAK at newly identified serine/threonine residues within nascent adhesions, inducing cell dynamics and aggressive behavior. This study thus places PKCθ-directed FAK opening and phosphorylations as an original mechanism controlling dynamic, migratory, and invasive abilities of aggressive breast cancer cells, further strengthening the emerging oncogenic function of PKCθ.

Focal adhesion kinase plays a dual role in TRAIL resistance and metastatic outgrowth of malignant melanoma

Despite remarkable advances in therapeutic interventions, malignant melanoma (MM) remains a life-threating disease. Following high initial response rates to targeted kinase-inhibition metastases quickly acquire resistance and present with enhanced tumor progression and invasion, demanding alternative treatment options. We show 2nd generation hexameric TRAIL-receptor-agonist IZI1551 (IZI) to effectively induce apoptosis in MM cells irrespective of the intrinsic BRAF/NRAS mutation status. Conditioning to the EC50 dose of IZI converted the phenotype of IZI-sensitive parental MM cells into a fast proliferating and invasive, IZI-resistant metastasis. Mechanistically, we identified focal adhesion kinase (FAK) to play a dual role in phenotype-switching. In the cytosol, activated FAK triggers survival pathways in a PI3K- and MAPK-dependent manner. In the nucleus, the FERM domain of FAK prevents activation of wtp53, as being expressed in the majority of MM, and consequently intrinsic apoptosis. Caspase-8-mediated cleavage of FAK as well as FAK knockdown, and pharmacological inhibition, respectively, reverted the metastatic phenotype-switch and restored IZI responsiveness. FAK inhibition also re-sensitized MM cells isolated from patient metastasis that had relapsed from targeted kinase inhibition to cell death, irrespective of the intrinsic BRAF/NRAS mutation status. Hence, FAK-inhibition alone or in combination with 2nd generation TRAIL-receptor agonists may be recommended for treatment of initially resistant and relapsed MM, respectively.

Computational-based discovery of FAK FERM domain chemical probes that inhibit HER2-FAK cancer signaling.

The N-terminal FERM domain of focal adhesion kinase (FAK) contributes to FAK scaffolding and interacts with HER2, an oncogene and receptor tyrosine kinase. The interaction between HER2 and FAK drives resistance to FAK-kinase domain inhibitors through FAK Y397 transphosphorylation and FAK re-activation upon inhibition. As such, FAK FERM remains an attractive drug discovery target. In this report, we detail an alternative approach to targeting FAK through virtual screening-based discovery of chemical probes that target FAK FERM. We validated the binding interface between HER2 and FAK using site-directed mutagenesis and GST pull-down experiments. We assessed the ligandability of key-binding residues of HER2 and FAK utilizing computational tools. We developed a virtual screening method to screen ~200,000 compounds against the FAK FERM domain, identifying 20 virtualchemical probes. We performed GST pull-down screening on these compounds, discovering two hits, VS4 and VS14, with nanomolar IC50 s in disrupting HER2-FAK. We performed further testing, including molecular docking, immunofluorescence, phosphorylation, and cellular invasion assays to evaluate the compounds' biological effects. One probe, VS14, was identified with the ability to block both auto- and transphosphorylation of Y397. In all, these studies identify two new probes that target FAK FERM, enabling future investigation of this domain.

Beta-arrestins operate an on/off control switch for focal adhesion kinase activity.

Focal adhesion kinase (FAK) regulates key biological processes downstream of G protein-coupled receptors (GPCRs) in normal and cancer cells, but the modes of kinase activation by these receptors remain unclear. We report that after GPCR stimulation, FAK activation is controlled by a sequence of events depending on the scaffolding proteins β-arrestins and G proteins. Depletion of β-arrestins results in a marked increase in FAK autophosphorylation and focal adhesion number. We demonstrate that β-arrestins interact directly with FAK and inhibit its autophosphorylation in resting cells. Both FAK-β-arrestin interaction and FAK inhibition require the FERM domain of FAK. Following the stimulation of the angiotensin receptor AT1AR and subsequent translocation of the FAK-β-arrestin complex to the plasma membrane, β-arrestin interaction with the adaptor AP-2 releases inactive FAK from the inhibitory complex, allowing its activation by receptor-stimulated G proteins and activation of downstream FAK effectors. Release and activation of FAK in response to angiotensin are prevented by an AP-2-binding deficient β-arrestin and by a specific inhibitor of β-arrestin/AP-2 interaction; this inhibitor also prevents FAK activation in response to vasopressin. This previously unrecognized mechanism of FAK regulation involving a dual role of β-arrestins, which inhibit FAK in resting cells while driving its activation at the plasma membrane by GPCR-stimulated G proteins, opens new potential therapeutic perspectives in cancers with up-regulated FAK.

Conformational Dynamics of FERM-Mediated Autoinhibition in Pyk2 Tyrosine Kinase.

Pyk2 is a non-receptor tyrosine kinase that evolved from gene duplication of focal adhesion kinase (FAK) and subsequent functional specialization in the brain and hemopoietic cells. Pyk2 shares a domain organization with FAK, with an N-terminal regulatory FERM domain adjoining the kinase domain. FAK regulation involves integrin-mediated membrane clustering to relieve autoinhibitory interactions between FERM and kinase domains. Pyk2 regulation remains cryptic, involving Ca2+ influx and protein scaffolding. While the mechanism of the FAK FERM domain in autoinhibition is well-established, the regulatory role of the Pyk2 FERM is ambiguous. We probed the mechanisms of FERM-mediated autoinhibition of Pyk2 using hydrogen/deuterium exchange mass spectrometry and kinase activity profiling. The results reveal FERM-kinase interfaces that are responsible for autoinhibition. Pyk2 autoinhibition impacts the activation loop conformation. In addition, the autoinhibitory FERM-kinase interface exhibits allosteric linkage with the FERM basic patch conserved in both FAK and Pyk2.

The Atypical Kinesin KIF26A Facilitates Termination of Nociceptive Responses by Sequestering Focal Adhesion Kinase

Kinesin superfamily proteins (KIFs) are molecular motors that typically alter the subcellular localization of their cargos. However, the atypical kinesin KIF26A does not serve as a motor but can bind microtubules and affect cellular signaling cascades. Here, we show that KIF26A maintains intracellular calcium homeostasis and negatively regulates nociceptive sensation. Kif26a-/- mice exhibit intense and prolonged nociceptive responses. In their primary sensory neurons, excessive inhibitory phosphorylation of plasma membrane Ca2+ ATPase (PMCA) mediated by focal adhesion kinase (FAK) rendered the Ca transients resistant to termination, and the peripheral axonal outgrowth was significantly enhanced. Upstream, KIF26A is directly associated with a FERM domain of FAK and antagonizes FAK function in integrin-Src family kinase (SFK)-FAK signaling, possibly through steric hindrance and localization to cytoplasmic microtubules.

Abstract A64: Human epidermal growth factor receptor 2 (HER2) directly binds and activates focal adhesion kinase (FAK) to promote oncogenesis

Abstract Introduction: Human epidermal growth factor receptor 2 (HER2) is known to require focal adhesion kinase (FAK) for cellular transformation, tumor growth, invasion, and metastasis. FAK is overexpressed in nearly all types of human cancer, serving as a molecular scaffold protein to integrate oncogenic growth factor receptors with focal adhesion proteins. We have identified a novel direct protein-protein interaction between HER2 and FAK. In this study, we investigated whether the HER2-FAK direct interaction is required for HER2-dependent oncogenesis and its potential as a novel drug target. Methods: We have taken a multidisciplinary approach (biophysics, biochemistry, molecular modeling, structural biology, and molecular biology) to map the HER2-FAK binding interface and to study the functional aspects of the interaction. Results: Our data indicated that the N-lobe of the HER2 kinase domain directly interacts with the F1 lobe of the FAK FERM domain. In addition, molecular modeling studies have identified a putative HER2-FAK interface suitable for the binding of small molecule inhibitors. Site-directed mutagenesis studies validated this binding interface. Functional data indicated that HER2 directly transphosphorylated FAK at key tyrosine residues, Y397, Y861, and Y925, irrespective of FAK kinase activity. Additionally, kinome reprogramming analysis revealed upregulation of HER2 signaling and maintenance of FAK phosphorylation after FAK-kinase inhibitor treatment. Finally, cellular data suggested that loss of the HER2-FAK interaction, but not FAK-kinase activity, resulted in a defect in Heregulin β1-stimulated cell migration/invasion. Conclusion: We have identified a direct interaction between HER2 and FAK that is required for HER2-dependent oncogenic signaling and is amenable to targeting by small molecule therapeutics. In addition, this interaction mediated a novel drug resistance mechanism whereby HER2 rescued and maintained FAK activation under FAK-kinase inhibition. Citation Format: Timothy Marlowe, Sheila Figel, Felicia Lenzo, Vita Golubovskaya, Elena Kurenova, Alexander Tropsha, William Cance. Human epidermal growth factor receptor 2 (HER2) directly binds and activates focal adhesion kinase (FAK) to promote oncogenesis. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A64.

Fenretinide Perturbs Focal Adhesion Kinase in Premalignant and Malignant Human Oral Keratinocytes. Fenretinide's Chemopreventive Mechanisms Include ECM Interactions.

The membrane-associated protein, focal adhesion kinase (FAK), modulates cell-extracellular matrix interactions and also conveys prosurvival and proliferative signals. Notably, increased intraepithelial FAK levels accompany transformation of premalignant oral intraepithelial neoplasia (OIN) to oral squamous cell carcinoma (OSCC). OIN chemoprevention is a patient-centric, optimal strategy to prevent OSCC's comorbidities and mortality. The cancer chemopreventive and synthetic vitamin A derivative, fenretinide, has demonstrated protein-binding capacities, for example, mTOR- and retinol-binding protein interactions. These studies used a continuum of human oral keratinocytes (normal-HPV E6/E7-transduced-OSCC) to assess potential fenretinide-FAK drug protein interactions and functional consequences on cellular growth regulation and motility. Molecular modeling studies demonstrated that fenretinide has approximately 200-fold greater binding affinity relative to the natural ligand (ATP) at FAK's kinase domain. Fenretinide also shows intermediate binding at FAK's FERM domain and interacts at the ATP-binding site of the closest FAK analogue, PYK2. Fenretinide significantly suppressed proliferation via induction of apoptosis and G2-M cell-cycle blockade. Fenretinide-treated cells also demonstrated F-actin disruption, significant inhibition of both directed migration and invasion of a synthetic basement membrane, and decreased phosphorylation of growth-promoting kinases. A commercially available FAK inhibitor did not suppress cell invasion. Notably, although FAK's FERM domain directs cell invasion, FAK inhibitors target the kinase domain. In addition, FAK-specific siRNA-treated cells showed an intermediate cell migration capacity; data which suggest cocontribution of the established migrating-enhancing PYK2. Our data imply that fenretinide is uniquely capable of disrupting FAK's and PYK2's prosurvival and mobility-enhancing effects and further extend fenretinide's chemopreventive contributions beyond induction of apoptosis and differentiation.

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.

Scaffold Protein Tethering Ability under Load: FAK's FERM Domain Mechanical Properties V. Binding Site

Mechanotransductive scaffolding proteins are tethered and thus subjected to mechanical loads that potentially induce partial or total unfolding. Focal adhesion kinase (FAK) is regulated by mechanical stimulation through extracellular matrix (ECM) proteins and actin cytoskeleton contractility. FAK is composed of three major domains: two of which putatively perform tethering (the FERM and FAT domains) while the central kinase domain is catalytically active in a wide variety of cell motility/invasion pathways upon activation. The so-called “basic patch” is the ligand-binding site on FERM's F2 subdomain, which is connected, via an unstructured loop, directly to FERM's F3 subdomain and distally to the kinase. As a mechanically competent tether, the FERM domain must carry loads between the basic patch and the F3 subdomain's C-terminal. A key question is whether these subdomains lose their tertiary structure under load_and therefore unwrap into a “beads on a string” configuration_and, if so, what consequences this has for ligand-binding subdomain stability. Towards an understanding of the FERM domain's ability to tether a mechanically competent FAK (pdb: 2al6), FERM's unfolding pathways are studied via Steered Molecular Dynamics (SMD). SMD simulations of the unfolding process reveal force peaks, extended conformations of intermediate states, and intra-molecular load pathways. Loads are applied to FERM's C-terminal and a set of residues in the basic patch known to bind to both phospholipids and phosphopeptides. By differentially applying loads to the basic patch's secondary structures, unfolding behavior, including both force levels and intermediate states, is revealed. Pulling-mode simulations_mimicking AFM_identify unfolding intermediate states; constant-force-mode simulations probe the structural behavior of identified intermediates. Given the diversity of ligands known to bind to the basic patch, mechanical behavior as a function of binding-site secondary structure is crucial for understanding mechanotransduction.

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 Å(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.

A Complex between FAK, RACK1, and PDE4D5 Controls Spreading Initiation and Cancer Cell Polarity

A fundamental question in cell biology concerns how cells respond to their environment by polarizing after sensing directional cues. This requires the differential localization of protein complexes in cells, and it is important to identify and understand how these complexes function. Here we describe a novel "direction-sensing" pathway that links the integrin effector focal adhesion kinase (FAK), the molecular scaffold protein RACK1, and activity of one of its client proteins, PDE4D5, a cAMP-degrading phosphodiesterase. The complex is recruited to nascent adhesions and promotes cell polarity. We identify FAK FERM domain residues whose mutation impairs RACK1 binding. When re-expressed in cancer cells in which endogenous fak is deleted by Cre-lox-mediated recombination, the RACK1-binding-impaired FAK mutant protein does not support formation of nascent actin adhesion structures as cells spread. These cancer cells, like FAK-deficient cells, cannot undergo directional responses, including wound-induced polarization or chemotactic invasion into three-dimensional matrix gels. We show that RACK1 serves as the molecular bridge linking FAK to the recruitment of PDE4D5. FAK/RACK1/PDE4D5 is a novel 'direction-sensing' complex that acts to recruit specific components of the cAMP second-messenger system to nascent integrin adhesions and to the leading edge of polarizing cells.

The Role of JAK3 mutations in Adult T-Cell Leukemia/Lymphoma.

Abstract 1940Poster Board I-963Adult T-cell leukemia/lymphoma (ATLL) is an aggressive and incurable cancer caused by infection from a retrovirus, Human T-cell Leukemia Virus type-1 (HTLV-1). HTLV-1 infects T cells and induces cell transformation through the expression of viral oncogenes such as tax. In newly infected cells, tax activates the expression of Interleukin-2 and the alpha subunit of its receptor (IL2RA) resulting in autocrine signaling that drives proliferation. In ATLL cells, tax is frequently deleted or silenced by methylation but there is constitutive activation of pathways downstream of IL-2 and related cytokines. These data led us to hypothesize that somatic mutations in this cytokine pathway may play a role in ATLL tumor progression. Encouraged by the recent discovery of activating mutations in JAK3 in diverse hematologic malignancies, we sequenced the exons of JAK3 in a small cohort of ATLL patients and found three missense mutations in the amino terminal FERM domain which were not found in Single Nucleotide Polymorphism databases. IL-2 signaling recapitulated in Human Embryonic Kidney 293T cells showed that these missense mutations caused increased tyrosine phosphorylation of the JAK3 substrate, STAT5A. Furthermore, enforced expression of these mutant JAK3s caused cytokine independent growth of BaF3 cells. The FERM domain of JAK3 is similar to the FERM domain of Focal Adhesion Kinase (FAK), which has a solved crystal structure. Interestingly, the FAK FERM domain contacts and autoinhibits its own kinase domain. Our homology-based model suggests that the JAK3 missense mutations disrupt the secondary structure needed for this intramolecular contact thereby perturbing autoregulation of JAK3 kinase activity. Our findings suggest that JAK3 gain of function may play an important role in ATLL disease progression. Disclosures:No relevant conflicts of interest to declare.

Dynamic conformational changes in the FERM domain of FAK are involved in focal-adhesion behavior during cell spreading and motility

Focal adhesion kinase (FAK) controls cellular adhesion and motility processes by its tight link to integrin- and extracellular-matrix-mediated signaling. To explore the dynamics of the regulation of FAK, we constructed a FRET-based probe that visualizes conformational rearrangements of the FERM domain of FAK in living cells. The sensor reports on an integrin-mediated conformational change in FAK following cellular adhesion. The perturbation is kinase-independent and involves the polybasic KAKTLR sequence in the FERM domain. It is manifested by an increased FRET signal and is expressed primarily in focal adhesions, and to a lesser extent in the cytoplasm. The conformational change in the FERM domain of FAK is observed in two consecutive phases during spreading - early and late - and is enriched in fully adhered motile cells at growing and sliding peripheral focal-adhesion sites, but not in stable or retracting focal adhesions. Inhibition of the actomyosin system indicates the involvement of tension signaling induced by Rho-associated kinase, rather than by myosin light-chain kinase, in the modulation of the FERM response. We conclude that the heterogeneous conformation of the FERM domain in focal adhesions of migrating cells reflects a complex regulatory mechanism for FAK that appears to be under the influence of cellular traction forces.

FAK Goes Nuclear

Under normal growth conditions, the tumor suppressor protein p53 is ubiquitinated and degraded, and cells proliferate. However, when cells are under stress, p53 accumulates and induces the expression of the gene encoding p21, which triggers cell cycle arrest. Signals from integrins, transmembrane receptors involved in cell adhesion, can counteract the activation of p53, but the mechanism involved is unclear. Lim et al . investigated whether focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase stimulated by integrin activation, might regulate p53 activity. They generated fak –/– mice, which were embryonic lethal due to a block in mesodermal cell proliferation. Western blotting analyses showed that the abundance of p53 was higher in fak –/– embryos than in wild-type embryos. Whereas cells taken from fak –/– embryos did not proliferate in culture, those taken from fak –/– p53 –/– or fak –/– p21 –/– embryos did. Expression of FAK, a kinase-inactive FAK, or the FAK FERM domain (which regulates kinase activity) in fak –/– p21 –/– fibroblasts reduced the abundance of p53 compared to that in control fak –/– p21 –/– fibroblasts. The effects of FAK and FAK FERM were blocked by a proteasomal inhibitor. Immunofluorescence and Western blotting studies demonstrated that FAK was localized to the nucleus (an effect dependent on FERM) and that it mediated the ubiquitination and degradation of p53. Coimmunoprecipitation studies showed the formation of a complex between FAK, p53, and the E3 ubiquitin ligase Mdm2, which was also FERM-dependent. Depletion of FAK from human fibroblasts resulted in increased abundance of p53 and p21 and an increased susceptibility to cisplatin-induced apoptosis. Together, these data suggest a kinase-independent, FERM-dependent role for FAK as a nuclear scaffold protein that targets p53 for degradation and enhances cell proliferation. S.-T. Lim, X. L. Chen, Y. Lim, D. A. Hanson, T.-T. Vo, K. Howerton, N. Larocque, S. J. Fisher, D. D. Schlaepfer, D. Ilic, Nuclear FAK promotes cell proliferation and survival through FERM-enhanced p53 degradation. Mol . Cell 29 , 9-22 (2008). [PubMed]

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.

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.

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.