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.     

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.     

Spatial and temporal regulation of focal adhesion kinase activity in living cells

Focal adhesion kinase (FAK) is an essential kinase that regulates developmental processes and functions in the pathology of human disease. An intramolecular autoinhibitory interaction between the FERM and catalytic domains is a major mechanism of regulation. Based upon structural studies, a fluorescence resonance energy transfer (FRET)-based FAK biosensor that discriminates between autoinhibited and active conformations of the kinase was developed. This biosensor was used to probe FAK conformational change in live cells and the mechanism of regulation. The biosensor demonstrates directly that FAK undergoes conformational change in vivo in response to activating stimuli. A conserved FERM domain basic patch is required for this conformational change and for interaction with a novel ligand for FAK, acidic phospholipids. Binding to phosphatidylinositol 4,5-bisphosphate (PIP2)-containing phospholipid vesicles activated and induced conformational change in FAK in vitro, and alteration of PIP2 levels in vivo changed the level of activation of the conformational biosensor. These findings provide direct evidence of conformational regulation of FAK in living cells and novel insight into the mechanism regulating FAK conformation.     

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.     

The FERM domain: organizing the structure and function of FAK

Focal adhesion kinase (FAK) is a scaffold and tyrosine kinase protein that binds to itself and cellular partners through its four-point-one, ezrin, radixin, moesin (FERM) domain. Recent structural work reveals that regulatory protein partners convert auto-inhibited FAK into its active state by binding to its FERM domain. Further, the identity of FAK FERM domain-interacting proteins yields clues as to how FAK coordinates diverse cellular responses, including cell adhesion, polarization, migration, survival and death, and suggests that FERM domains might mediate information transfer between the cell cortex and nucleus. Importantly, the FAK FERM domain might act as a paradigm for the actions of other FERM domain-containing proteins.     

Pyk2 Inhibition of p53 as an Adaptive and Intrinsic Mechanism Facilitating Cell Proliferation and Survival

Pyk2 is a cytoplasmic tyrosine kinase related to focal adhesion kinase (FAK). Compensatory Pyk2 expression occurs upon FAK loss in mice. However, the impact of Pyk2 up-regulation remains unclear. Previous studies showed that nuclear-localized FAK promotes cell proliferation and survival through FAK FERM domain-enhanced p53 tumor suppressor degradation (Lim, S. T., Chen, X. L., Lim, Y., Hanson, D. A., Vo, T. T., Howerton, K., Larocque, N., Fisher, S. J., Schlaepfer, D. D., and Ilic, D. (2008) Mol. Cell 29, 9–22). Here, we show that FAK knockdown triggered p53 activation and G₁ cell cycle arrest in human umbilical vein endothelial cells after 4 days. However, by 7 days elevated Pyk2 expression occurred with a reduction in p53 levels and the release of the G₁ block under conditions of continued FAK knockdown. To determine whether Pyk2 regulates p53, experiments were performed in FAK^−/−p21^−/− mouse embryo fibroblasts expressing endogenous Pyk2 and in ID8 ovarian carcinoma cells expressing both Pyk2 and FAK. In both cell lines, Pyk2 knockdown increased p53 levels and inhibited cell proliferation associated with G₁ cell cycle arrest. Pyk2 FERM domain re-expression was sufficient to reduce p53 levels and promote increased BrdUrd incorporation. Pyk2 FERM promoted Mdm2-dependent p53 ubiquitination. Pyk2 FERM effects on p53 were blocked by proteasomal inhibition or mutational-inactivation of Pyk2 FERM nuclear localization. Staurosporine stress of ID8 cells promoted endogenous Pyk2 nuclear accumulation and enhanced Pyk2 binding to p53. Pyk2 knockdown potentiated ID8 cell death upon staurosporine addition. Moreover, Pyk2 FERM expression in human fibroblasts upon FAK knockdown prevented cisplatin-mediated apoptosis. Our studies demonstrate that nuclear Pyk2 functions to limit p53 levels, thus facilitating cell growth and survival in a kinase-independent manner.     

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.     

Quantitative analysis of the binding of ezrin to large unilamellar vesicles containing phosphatidylinositol 4,5 bisphosphate

The plasma membrane-cytoskeleton interface is a dynamic structure participating in a variety of cellular events. Among the proteins involved in the direct linkage between the cytoskeleton and the plasma membrane is the ezrin/radixin/moesin (ERM) family. The FERM (4.1 ezrin/radixin/moesin) domain in their N-terminus contains a phosphatidylinositol 4,5 bisphosphate (PIP₂) (membrane) binding site whereas their C-terminus binds actin. In this work, our aim was to quantify the interaction of ezrin with large unilamellar vesicles (LUVs) containing PIP₂. For this purpose, we produced human recombinant ezrin bearing a cysteine residue at its C-terminus for subsequent labeling with Alexa488 maleimide. The functionality of labeled ezrin was checked by comparison with that of wild-type ezrin. The affinity constant between ezrin and LUVs was determined by cosedimentation assays and fluorescence correlation spectroscopy. The affinity was found to be ∼5 μM for PIP₂-LUVs and 20-to 70-fold lower for phosphatidylserine-LUVs. These results demonstrate, as well, that the interaction between ezrin and PIP₂-LUVs is not cooperative. Finally, we found that ezrin FERM domain (area of ∼30 nm²) binding to a single PIP₂ can block access to neighboring PIP₂ molecules and thus contributes to lower the accessible PIP₂ concentration. In addition, no evidence exists for a clustering of PIP₂ induced by ezrin addition.     

Open Conformation of Ezrin Bound to Phosphatidylinositol 4,5-Bisphosphate and to F-actin Revealed by Neutron Scattering

Ezrin is a member of the ezrin-radixin-moesin family (ERM) of adapter proteins that are localized at the interface between the cell membrane and the cortical actin cytoskeleton, and they regulate a variety of cellular functions. The structure representing a dormant and closed conformation of an ERM protein has previously been determined by x-ray crystallography. Here, using contrast variation small angle neutron scattering, we reveal the structural changes of the full-length ezrin upon binding to the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP₂) and to F-actin. Ezrin binding to F-actin requires the simultaneous binding of ezrin to PIP₂. Once bound to F-actin, the opened ezrin forms more extensive contacts with F-actin than generally depicted, suggesting a possible role of ezrin in regulating the interfacial structure and dynamics between the cell membrane and the underlying actin cytoskeleton. In addition, using gel filtration, we find that the conformational opening of ezrin in response to PIP₂ binding is cooperative, but the cooperativity is disrupted by a phospho-mimic mutation S249D in the 4.1-ezrin/radixin/moesin (FERM) domain of ezrin. Using surface plasmon resonance, we show that the S249D mutation weakens the binding affinity and changes the kinetics of 4.1-ERM to PIP₂ binding. The study provides the first structural view of the activated ezrin bound to PIP₂ and to F-actin.     

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.     

The Phosphatidylinositol (3,4,5)-Trisphosphate-dependent Rac Exchanger 1·Ras-related C3 Botulinum Toxin Substrate 1 (P-Rex1·Rac1) Complex Reveals the Basis of Rac1 Activation in Breast Cancer Cells

The P-Rex (phosphatidylinositol (3,4,5)-trisphosphate (PIP₃)-dependent Rac exchanger) family (P-Rex1 and P-Rex2) of the Rho guanine nucleotide exchange factors (Rho GEFs) activate Rac GTPases to regulate cell migration, invasion, and metastasis in several human cancers. The family is unique among Rho GEFs, as their activity is regulated by the synergistic binding of PIP₃ and Gβγ at the plasma membrane. However, the molecular mechanism of this family of multi-domain proteins remains unclear. We report the 1.95 Å crystal structure of the catalytic P-Rex1 DH-PH tandem domain in complex with its cognate GTPase, Rac1 (Ras-related C3 botulinum toxin substrate-1). Mutations in the P-Rex1·Rac1 interface revealed a critical role for this complex in signaling downstream of receptor tyrosine kinases and G protein-coupled receptors. The structural data indicated that the PIP₃/Gβγ binding sites are on the opposite surface and markedly removed from the Rac1 interface, supporting a model whereby P-Rex1 binding to PIP₃ and/or Gβγ releases inhibitory C-terminal domains to expose the Rac1 binding site.     

pHocal adhesion kinase regulation is on a FERM foundation

Increases in intracellular pH (pHi) occur upon integrin receptor binding to matrix proteins and in tumor cells. In this issue, Choi et al. (2013. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201308034) show that pHi increase activates FAK by causing deprotonation of histidine 58 in its FERM (band 4.1, ezrin, radixin, moesin) homology domain, which exposes a region important for FAK autophosphorylation. This model of FAK activation could contribute to motility of tumor cells by promoting focal adhesion turnover.Integrins are transmembrane receptors for extracellular matrix that mediate physical cell attachment and also control cell shape, growth, and survival. Integrin signals are generated by the recruitment and activation of protein tyrosine kinases (PTKs) such as Src, Abl, Syk, and FAK that initiate protein phosphorylation signaling cascades. These sites of integrin signal initiation and cell attachment are generally termed focal adhesions. Despite identification of adhesion protein constituents, our understanding of the molecular mechanisms of PTK activation at focal adhesions remains rudimentary. FAK, Src, and Abl signaling contribute to tumor growth and metastasis, and small molecule drugs targeting these PTKs have been approved or are undergoing clinical trials. However, for FAK we really do not fully understand the contributing factors that lead to its elevated activation, and although FAK is an amplified gene in cancer, mutations that increase FAK activation are uncommon.In this issue, Choi et al. (2013) elucidate a novel connection between increased intracellular pH (pHi) and FAK activation. In the early 1990s, transient pHi elevation upon matrix binding was one of the first integrin-associated signals identified (Schwartz et al., 1991). Pharmacological inhibitors pointed to the importance of sodium–proton antiporters in mediating increased pHi. NHE-1 (sodium-hydrogen antiporter 1) is part of a larger family (NHE1-9) and a ubiquitously expressed transmembrane protein that actively extrudes protons from inside the cell to counter balance acidity and maintain cytosolic pHi (Malo and Fliegel, 2006). NHE-I can be found at focal adhesions (Grinstein et al., 1993) and can connect to the actin cytoskeleton via binding to ezrin (Denker et al., 2000). NHE-1 point mutations disrupting either ion translocation or its binding to ezrin prevent cell migration (Denker and Barber, 2002). NHE-1 overexpression in cancer cells elevates pHi and tumor progression (Webb et al., 2011).Also in the early 1990s, FAK was the first PTK shown to localize to focal adhesions and to be activated by integrins (Parsons, 2003). FAK and the closely related proline-rich tyrosine kinase 2 (Pyk2) share a common domain structure of an N-terminal FERM domain and an ∼40 amino acid linker domain containing an autophosphorylation site (Y397 in FAK) that serves as a Src homology 2 (SH2) binding site for Src-family PTKs. The FAK linker is followed by a central kinase domain, a scaffolding region containing two proline-rich motifs that are SH3 domain binding sites, and a C-terminal focal adhesion–targeting (FAT) domain (Fig. 1 A). It is the FAK FAT domain that binds to integrin-associated proteins (paxillin and talin) and facilitates FAK phosphorylation at Y397 via protein clustering (Toutant et al., 2002). However, the importance of the results of Choi et al. (2013) lies in the role of the FAK FERM domain in the intramolecular regulation of FAK Y397 autophosphorylation.Open in a separate windowFigure 1.Overview of FAK structure and activation. (A) FAK schematic. Depicted is the FAK N-terminal FERM domain comprised of three lobes (F1, F2, and F3), a linker domain, central kinase domain, and a C-terminal focal adhesion–targeting (FAT) domain. Shown are histidine (H) residue 58 and tyrosine (Y) residues 194, 397, 576/577, and 925, and proline-rich domains (Pro-1, Pro-2, and Pro-3) that are sites for SH3 domain binding. FERM plus linker (F+L), FERM-linker-kinase (F+L+K), and linker-kinase (L+K) are constructs used by Choi et al. (2013). (B) Model of growth factor–stimulated FAK activation. Upon binding of the FAK FERM F2 lobe to c-Met receptor and/or phosphatidylinositol 4,5-P₂ (PIP2) lipid, FAK undergoes conformational changes and Y194 phosphorylation, leading to FAK Y397 autophosphorylation in the linker region. Src binding to and phosphorylation of FAK within the kinase domain leads to full FAK activation. HGF, hepatocyte growth factor.FERM domains are typically comprised of three lobes (F1, F2, and F3) grouped in a cloverleaf-like structure (Frame et al., 2010). In an inactive conformation, the FAK FERM F2 lobe binds to and blocks the FAK kinase domain active site. The FAK FERM F1 lobe binds to and sequesters FAK Y397 in the linker region (Fig. 1 B). Point mutations of FAK in the F1 lobe, F2 lobe, or within the kinase domain can weaken these inhibitory intramolecular binding interactions and result in elevated FAK Y397 phosphorylation (Lietha et al., 2007). It has been hypothesized that the normal sequence of events for FAK activation starts with the binding of some “activating” factor to the FAK F2 lobe that would trigger FERM lobe displacement and allow FAK cis or trans auto-phosphorylation of Y397 (Fig. 1 B). Subsequent full FAK activation occurs via SH2 domain binding of Src to phosphorylated Y397, resulting in Src-mediated phosphorylation within the FAK kinase domain at Y576 and Y577 to promote catalytic activation and phosphorylation within the FAT domain at Y925 to promote Grb2 SH2 binding (Schaller, 2010). Recently, growth factor receptor phosphorylation of FAK at Y194 within the FERM F2 lobe was shown to promote FAK activation (Fig. 1 B; Chen et al., 2011). Conformational changes triggered by FAK kinase activity also regulate FAK FERM–mediated binding to targets such as VE-cadherin (Chen et al., 2012). Outside of FAK phosphorylation at Y194, and a potential role for lipid binding to FAK FERM (Cai et al., 2008), additional “initiators” of FERM conformational changes remain undefined.Choi et al. (2013) provide new and important insights in FERM-mediated FAK activation by changes in pHi. By using a chimeric paxillin construct fused to the pH biosensor pHluorin and to mCherry, Choi et al. (2013) showed that pHi increases within peripheral adhesions in mouse embryo fibroblasts (MEFs) spreading on fibronectin (FN) at 30 to 50 min after plating. Stable NHE-1 knockdown resulted in the lowering of pHi and the inhibition of FAK but not Src activation at 30 and 60 min during MEF spreading on FN. Interestingly, NHE-1 knockdown or pharmacological NHE-1 inhibition resulted in MEFs with a rounded morphology and an increased number of small focal adhesions. This adhesion and spreading phenotype is similar to that of FAK-null MEFs (Sieg et al., 1999).To determine if there was a connection between increased pHi and FAK activation, Choi et al. (2013) performed recombinant FAK in vitro phosphorylation assays. The FAK FERM-linker-kinase (F+L+K) fragment but not the linker-kinase (L+K) (Fig. 1 A) exhibited increased Y397 phosphorylation as a function of pH (pH 7.5 > pH 6.5). Intriguingly, regulation of FAK Y397 phosphorylation by pH was dependent upon the presence of the FAK FERM domain, which was shown by using the FERM-linker (F+L) as a substrate in trans (Fig. 1 A). These results support the hypothesis that exposure of the FAK linker region for phosphorylation is pH dependent (Fig. 2). Changes in amino acid protonation can be considered a post-translational modification, as ionic interactions contribute to secondary and tertiary protein structure (Schönichen et al., 2013). The pK_a of histidine is ∼6.5, and variations from this value depend upon the local protein environment. The challenge is to determine which sites functionally serve as pH sensors in vivo.Open in a separate windowFigure 2.Simplified model of FAK activation via histidine 58 (H58) deprotonation. The FAK FERM F1 lobe sequesters FAK Y397 in the linker region keeping FAK in an inactive and closed conformation. Integrin engagement at focal adhesions results in transient and local increases of pHi through NHE-1 activity. Changes in pHi result in H58 deprotonation within the FERM F1 lobe, leading to FAK conformational changes that expose the FAK linker region and enabling FAK Y397 autophosphorylation. Src binding to and phosphorylation of FAK within the kinase domain leads to full FAK activation.The FAK FERM domain contains seven histidine residues, three of which (H41, H58, and H75) are within the FERM F1 lobe. Mutation of these sites individually to alanine revealed that H58A mutation selectively enhanced F+L and F+L+K Y397 phosphorylation at pH 6.5. This approach allowed Choi et al. (2013) to conclude that H58 deprotonation confers some type of change within the F+L region to facilitate Y397 autophosphorylation (Fig. 2). Using the crystal structure of FAK F+L+K as a template, molecular dynamic simulations with H58 (neutral or positively charged) revealed conformational differences within residues of the linker region around Y397 despite the lack of direct binding to H58. Although Choi et al. (2013) did not identify key partner electrostatic interactions that contribute to conformational changes upon H58 deprotonation, recent studies by (Ritt et al. (2013) proposed that E466 within the FAK kinase domain may be important for this regulation within full-length FAK. However, this does not explain results from Choi et al. (2013) for the pH dependence of Y397 phosphorylation within F+L.Lastly, full-length H58A FAK exhibited elevated Y397 phosphorylation upon re-expression in FAK-null MEFs in combination with NHE-1 shRNA knockdown. H58A FAK promoted spreading and adhesion changes in both control and NHE-1 shRNA FAK-null MEFs, whereas wild-type FAK did not rescue FAK-null phenotypes in the absence of NHE-1 expression. Choi et al. (2013) note that pH-regulated FAK Y397 phosphorylation required combined integrin stimulation and elevated pH to activate FAK. In tumor cells, NHE-1 inhibition prevents elevated FAK Y397 phosphorylation and recent studies show that FAK can phosphorylate cortactin to promote adhesion turnover (Tomar et al., 2012). Cortactin tyrosine phosphorylation facilitates the recruitment of NHE-1 to tumor cell invadopodia (Magalhaes et al., 2011), leading to the pH-dependent release of actin-depolymerizing factor cofilin from cortactin (Frantz et al., 2008). As FAK activity promotes ovarian and breast tumor metastasis (Walsh et al., 2010; Ward et al., 2013), it is possible that FAK may serve as a pH-dependent sensor to initiate cell spreading.In the control of cell motility, NHE-1 is postulated to create pH nanodomains at focal adhesions to control protein–protein interactions (Ludwig et al., 2013). A simplistic model is that integrin clustering facilitates rapid FAK recruitment to focal adhesion where increases in pH trigger FERM conformational changes, release of the FAK linker region, and allow for FAK Y397 phosphorylation in cis or trans (Fig. 2). Adhesion turnover is increased at alkaline pHi, consistent with leading edge cell spreading and extension. At nascent adhesions FAK recruits talin (Lawson et al., 2012), and at alkaline pH FAK signaling activity may be enhanced over talin binding to filamentous actin needed for adhesion maturation (Srivastava et al., 2008). However, as pHi falls, pH sensor residues within the talin rod domain confer enhanced actin binding and this may be part of a signaling switch to promote a cycle of focal adhesion maturation. Additionally, pHi changes may alter phosphorylation site specificity by Src within the FAK FAT domain (Cable et al., 2012). Thus, pHi can affect FAK activity and FAK phosphorylation. Moreover, the FAK-related Pyk2 PTK is activated by acidic pHi within cells of the kidney (Li et al., 2004). Although the molecular mechanism is not known as to how Pyk2 is regulated by acidity, clearly there is much more to discover about the role of pHi changes at adhesions and invadopodia and how this may alter PTK activation in the control of cell movement and invasion.     

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.     

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.     

Unexpected effects of FERM domain mutations on catalytic activity of Jak3: structural implication for Janus kinases.

Janus kinases comprise carboxyterminal kinase, pseudokinase, SH2-like, and N-terminal FERM domains. We identified three patient-derived mutations in the FERM domain of Jak3 and investigated the functional consequences of these mutations. These mutations inhibited receptor binding and also abrogated kinase activity, suggesting interactions between the FERM and kinase domains. In fact, the domains were found to physically associate, and coexpression of the FERM domain enhanced activity of the isolated kinase domain. Conversely, staurosporine, which alters kinase domain structure, disrupted receptor binding, even though the catalytic activity of Jak3 is dispensable for receptor binding. Thus, the Jak FERM domain appears to have two critical functions: receptor interaction and maintenance of kinase integrity.     

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.     

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.     

Ligand-mediated Structural Dynamics of a Mammalian Pancreatic KATP Channel

Regulation of pancreatic K_ATP channels involves orchestrated interactions of their subunits, Kir6.2 and SUR1, and ligands. Previously we reported K_ATP channel cryo-EM structures in the presence and absence of pharmacological inhibitors and ATP, focusing on the mechanisms by which inhibitors act as pharmacological chaperones of K_ATP channels (Martin et al., 2019). Here we analyzed the same cryo-EM datasets with a focus on channel conformational dynamics to elucidate structural correlates pertinent to ligand interactions and channel gating. We found pharmacological inhibitors and ATP enrich a channel conformation in which the Kir6.2 cytoplasmic domain is closely associated with the transmembrane domain, while depleting one where the Kir6.2 cytoplasmic domain is extended away into the cytoplasm. This conformational change remodels a network of intra- and inter-subunit interactions as well as the ATP and PIP₂ binding pockets. The structures resolved key contacts between the distal N-terminus of Kir6.2 and SUR1′s ABC module involving residues implicated in channel function and showed a SUR1 residue, K134, participates in PIP₂ binding. Molecular dynamics simulations revealed two Kir6.2 residues, K39 and R54, that mediate both ATP and PIP₂ binding, suggesting a mechanism for competitive gating by ATP and PIP₂.     

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.     

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.