Focal Adhesion Kinase Autophosphorylation Research Articles

SPP1 promotes tumor progression in esophageal carcinoma by activating focal adhesion pathway.

Recurrence and metastasis are the major obstacles affecting the therapeutic efficacy and clinical outcomes for patients with esophageal carcinoma (ESCA). Secreted phosphoprotein 1 (SPP1) is considered as a hub gene in ESCA and is negatively associated with disease-free survival (DFS) in ESCA. However, the exact roles and underlying mechanisms remain elusive. This study aims to examine the roles of SPP1 on ESCA, and elucidate the potential mechanisms. Bioinformatics were used to analyze the expression of SPP1 in ESCA tissues, and its relations with clinicopathological characteristics and clinical prognosis in patients with ESCA based on The Cancer Genome Atlas (TCGA) dataset. Loss-of-function was conducted to examine the roles of SPP1 on malignant behaviors of ESCA cells by cell counting kit-8 (CCK8), plate clone, wound healing, and transwell assays. Gene set enrichment analysis (GSEA) was conducted to screen the pathways associated with SPP1 in ESCA. Then, the enriched pathway and the underlying mechanism were elucidated by western blotting, cell adhesion, and cell spreading assays. Lastly, Y15 [a specific inhibitor of focal adhesion kinase (FAK)] was used to examine its potential to inhibit tumor growth in ESCA cells. SPP1 was upregulated in ESCA tissues compared to the adjacent nontumorous tissues, which was closely associated with clinical stage, lymph node metastasis, histological subtype, and p53 mutation. A high expression of SPP1 indicated a poor clinical prognosis in patients with ESCA. The knockdown of SPP1 inhibited cell proliferative, migratory, and invasive capacities in ESCA cells. GSEA indicated that the focal adhesion pathway was closely related with SPP1 in ESCA. Further studies confirmed that the knockdown of SPP1 suppressed cell adhesion ability and reduced the expression of p-FAK and p-Erk in ESCA cells. In addition, Y15 inhibited FAK autophosphorylation and dramatically inhibited cell proliferation, migration, and invasion in ESCA cells. SPP1 promotes tumor progression in ESCA by activating FAK/Erk pathway, and FAK is a potential therapeutic target to overcome tumor recurrence and metastasis of ESCA.

Abstract 2098: A Novel Mechanism Of Focal Adhesion Kinase Activation By Integrins And Its Role In Outside-in Signaling In Platelets

Ligand binding to integrin αIIbβ3 mediates platelet adhesion/aggregation and transmits outside-in signaling, leading to platelet spreading, granule secretion, and significant expansion of platelet thrombi resulting in thrombosis. Focal Adhesion Kinase (FAK) has been recognized in many cells to play an important role during integrin outside-in signaling. Whereas FAK activation downstream of integrins is characterized by autophosphorylation of FAK at Y 397 followed by Y 576/7 phosphorylation, it is not clear how FAK phosphorylation and activation is regulated and how FAK play roles in outside-in signaling in platelets. Here we show that reactive oxygen species (ROS) plays a key role in integrin-dependent stimulation of FAK phosphorylation and activation and ROS-stimulated FAK activation is downstream of the Gα13-integrin interaction, NOX2 activation, and the Src signaling pathway. Furthermore, FAK, following ROS-dependent activation, is important for the integrin outside-in signaling-mediated activation of Syk and immunoreceptor tyrosine-based activation motif (ITAM) signaling pathway. We further show that FAK is important in amplification of platelet activation and aggregation as well as in vivo thrombosis. Thus, we have identified a novel mechanism of FAK activation and a key role of FAK in integrin outside-in signaling and integrin-dependent ITAM pathway activation, leading to amplification of platelet activation, aggregation, and thrombosis.

Abstract 2560: Post-translational modification of focal adhesion kinase (FAK) protein increases in breast cancer cells upon exposure to conditioned medium derived from in vitro and in vivo adipocytes

Abstract Under obese conditions, dysregulation of the normal secretion of adipokines from fat cells has been shown to drive cancer progression. However, the role adipokine dysregulation plays in driving metastasis is still unclear. Recent studies have reported the activation of metastasis-associated cell signaling pathways. One such pathway involves Focal Adhesion Kinase (FAK), a non-receptor cytoplasmic protein that acts as a signal transducer between receptor activation by adipokines and downstream effector proteins that control the biological phenomenon of metastasis. Nevertheless, the association between obesity-induced changes in adipocyte secretory factors and the subsequent increase in the transactivation of FAK through phosphorylation is unknown. To elucidate the potential relationship of adipocyte-secreted factors towards activating FAK, breast cancer cells with low (MCF7) and high (MDA-MB-231) metastatic potential were exposed to in vitro and in vivo, murine adipocyte-derived, conditioned medium. For the in vitro adipocyte model, 3T3-L1 MBX cells differentiated to obesity, with fatty acids supplementation, were used. For the in vivo model, adipocyte-secreted factors from organ-cultured visceral fat harvested from C3H/HeJ female mice fed an either high fat (46%) or control (11%) diets, were collected in the medium. Adipocyte secretions from the in vitro and in vivo adipocyte samples were determined using a mouse adipokine proteomic profiler array and ELISA kit. Under obese conditions, both the 3T3-L1 MBX cells and C3H/HeJ visceral fat were found to have significant changes in their adipokine secretions in comparison to controls. Results showed that the breast cancer cells got increased FAK autophosphorylation when exposed to either in vitro or in vivo-derived obese adipocyte-conditioned medium in comparison to the untreated breast cancer cells. Citation Format: Noshin Mubtasim, Caleb Boren, Benjamin Barr, Lauren Gollahon. Post-translational modification of focal adhesion kinase (FAK) protein increases in breast cancer cells upon exposure to conditioned medium derived from in vitro and in vivo adipocytes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2560.

The Sall2 transcription factor promotes cell migration regulating focal adhesion turnover and integrin β1 expression.

SALL2/Sall2 is a transcription factor associated with development, neuronal differentiation, and cancer. Interestingly, SALL2/Sall2 deficiency leads to failure of the optic fissure closure and neurite outgrowth, suggesting a positive role for SALL2/Sall2 in cell migration. However, in some cancer cells, SALL2 deficiency is associated with increased cell migration. To further investigate the role of Sall2 in the cell migration process, we used immortalized Sall2 knockout (Sall2 -/- ) and Sall2 wild-type (Sall2 +/+ ) mouse embryonic fibroblasts (iMEFs). Our results indicated that Sall2 positively regulates cell migration, promoting cell detachment and focal adhesions turnover. Sall2 deficiency decreased cell motility and altered focal adhesion dynamics. Accordingly, restoring Sall2 expression in the Sall2 -/- iMEFs by using a doxycycline-inducible Tet-On system recovered cell migratory capabilities and focal adhesion dynamics. In addition, Sall2 promoted the autophosphorylation of Focal Adhesion Kinase (FAK) at Y397 and increased integrin β1 mRNA and its protein expression at the cell surface. We demonstrated that SALL2 increases ITGB1 promoter activity and binds to conserved SALL2-binding sites at the proximal region of the ITGB1 promoter, validated by ChIP experiments. Furthermore, the overexpression of integrin β1 or its blockade generates a cell migration phenotype similar to that of Sall2 +/+ or Sall2 -/- cells, respectively. Altogether, our data showed that Sall2 promotes cell migration by modulating focal adhesion dynamics, and this phenotype is associated with SALL2/Sall2-transcriptional regulation of integrin β1 expression and FAK autophosphorylation. Since deregulation of cell migration promotes congenital abnormalities, tumor formation, and spread to other tissues, our findings suggest that the SALL2/Sall2-integrin β1 axis could be relevant for those processes.

ZINC40099027 promotes monolayer circular defect closure by a novel pathway involving cytosolic activation of focal adhesion kinase and downstream paxillin and ERK1/2.

ZINC40099027 (ZN27) is a specific focal adhesion kinase (FAK) activator that promotes murine mucosal wound closure after ischemic or NSAID-induced injury. Diverse motogenic pathways involve FAK, but the direct consequences of pure FAK activation have not been studied, and how ZN27-induced FAK activation stimulates wound closure remained unclear. We investigated signaling and focal adhesion (FA) turnover after FAK activation by ZN27 in Caco-2 cells, confirming key results in CCD841 cells. ZN27 increased Caco-2 FAK-Y-397, FAK-Y-576/7, paxillin-Y-118, and ERK 1/2 phosphorylation and decreased FAK-Y-925 phosphorylation, without altering FAK-Y-861, p38, Jnk, or Akt phosphorylation. ZN27 increased FAK-paxillin interaction while decreasing FAK-Grb2 association. ZN27 increased membrane-associated FAK-Y-397 and FAK-Y-576/7 phosphorylation and paxillin-Y-118 and ERK 1/2 phosphorylation but decreased FAK-Y-925 phosphorylation without altering Src or Grb2. Moreover, ZN27 increased the fluorescence intensity of GFP-FAK and pFAK-Y397 in FAs and increased the total number of FAs but reduced their size in GFP-FAK-transfected Caco-2 cells, consistent with increased FA turnover. In contrast, FAK-Y397F transfection prevented ZN27 effects on FAK size and number and FAK and pFAK fluorescent intensity in FAs. We confirmed the proposed FAK/paxillin/ERK pathway using PP2 and U0126 to block Src and MEK1/2 in Caco-2 and CCD841 cells. These results suggest that ZN27 promotes intestinal epithelial monolayer defect closure by stimulating autophosphorylation of FAK in the cytosol, distinct from classical models of FAK activation in the FA. Phosphorylated FAK translocates to the membrane, where its downstream substrates paxillin and ERK are phosphorylated, leading to FA turnover and human intestinal epithelial cell migration.

Discovery of novel chloropyramine-cinnamic acid hybrids as potential FAK inhibitors for intervention of metastatic triple-negative breast cancer

To search for novel focal adhesion kinase (FAK) inhibitors for intervention of metastatic triple-negative breast cancer (TNBC), a series of hybrids 7a-s from chloropyramine and cinnamic acid analogs were designed, synthesized and biologically evaluated. The most active compound 7d could potently inhibit the proliferation, invasion and migration of TNBC cells in vitro. The docking analysis of 7d was performed to elucidate its possible binding modes to focal adhesion targeting (FAT) domain of FAK scaffold. Further mechanism studies indicated the ability of 7d in disrupting Y925 autophosphorylation of FAK, reducing formation of focal adhesions (FAs) and stress fibers (SFs) as well as inducing apoptosis of TNBC cells. Together, 7d is a novel FAK inhibitor to inhibit the essential nonkinase scaffolding function of FAK via binding FAT domain and may be worth studying further for intervention of TNBC.

Design, synthesis, and biological evaluation of novel covalent inhibitors targeting focal adhesion kinase

Forty-one new focal adhesion kinase (FAK) covalent inhibitors were designed and synthesized based on FAK inhibitor TAE226. Compound 11w displayed the highest inhibition of FAK with an IC50 value of 35 nM and exhibited potent anticancer activity against Hela, HCT116 and MDA-MB-231 cell lines with IC50 values of 0.41, 0.01 and 0.11 μM respectively, compared to TAE226 (2.68, 0.64 and 4.19 μM respectively). 11w also inhibited the clone formation and migration of HCT-116 cells and stimulated cell cycle arrest in the G2/M phase, inducing tumor cell apoptosis. Compound 11w formed a covalent bond with the Cys427 residue of FAK in a docking model, inhibiting the autophosphorylation of FAK and downstream proteins in a dose-dependent manner. Moreover, 11w showed adequate oral bioavailability of 21.02%. A 74.20% inhibition of tumor growth in the HCT116 xenograft model was also observed. These data indicate that 11w is a promising covalent inhibitor of FAK.

The Netrin-1-Neogenin-1 signaling axis controls neuroblastoma cell migration via integrin-β1 and focal adhesion kinase activation

ABSTRACT Neuroblastoma is a highly metastatic tumor that emerges from neural crest cell progenitors. Focal Adhesion Kinase (FAK) is a regulator of cell migration that binds to the receptor Neogenin-1 and is upregulated in neuroblastoma. Here, we show that Netrin-1 ligand binding to Neogenin-1 leads to FAK autophosphorylation and integrin β1 activation in a FAK dependent manner, thus promoting neuroblastoma cell migration. Moreover, Neogenin-1, which was detected in all tumor stages and was required for neuroblastoma cell migration, was found in a complex with integrin β1, FAK, and Netrin-1. Importantly, Neogenin-1 promoted neuroblastoma metastases in an immunodeficient mouse model. Taken together, these data show that Neogenin-1 is a metastasis-promoting protein that associates with FAK, activates integrin β1 and promotes neuroblastoma cell migration.

Design, Synthesis, and Biological Evaluation of Covalent Inhibitors of Focal Adhesion Kinase (FAK) against Human Malignant Glioblastoma

Human malignant glioblastoma (GBM) is a highly invasive and lethal brain tumor. Targeting of integrin downstream signaling mediators in GBM such as focal adhesion kinase (FAK) seems reasonable and recently demonstrated promising results in early clinical studies. Herein, we report the structure-guided development of a series of covalent inhibitors of FAK. These new compounds displayed highly potent inhibitory potency against FAK enzymatic activity with IC50 values in the nanomolar range. Several inhibitors retarded tumor cell growth as assessed by a cell viability assay in multiple human glioblastoma cell lines. They also significantly reduced the rate of U-87 cell migration and delayed the cell cycle progression by stopping cells in the G2/M phase. Furthermore, these inhibitors showed a potent decrease of autophosphorylation of FAK in glioblastoma cells and its downstream effectors Akt and Erk as well as nuclear factor-κB. These data demonstrated that these inhibitors may have the potential to offer a promising new targeted therapy for human glioblastomas.

Structural basis of Focal Adhesion Kinase activation on lipid membranes.

Focal adhesion kinase (FAK) is a key component of the membrane proximal signaling layer in focal adhesion complexes, regulating important cellular processes, including cell migration, proliferation, and survival. In the cytosol, FAK adopts an autoinhibited state but is activated upon recruitment into focal adhesions, yet how this occurs or what induces structural changes is unknown. Here, we employ cryo-electron microscopy to reveal how FAK associates with lipid membranes and how membrane interactions unlock FAK autoinhibition to promote activation. Intriguingly, initial binding of FAK to the membrane causes steric clashes that release the kinase domain from autoinhibition, allowing it to undergo a large conformational change and interact itself with the membrane in an orientation that places the active site toward the membrane. In this conformation, the autophosphorylation site is exposed and multiple interfaces align to promote FAK oligomerization on the membrane. We show that interfaces responsible for initial dimerization and membrane attachment are essential for FAK autophosphorylation and resulting cellular activity including cancer cell invasion, while stable FAK oligomerization appears to be needed for optimal cancer cell proliferation in an anchorage-independent manner. Together, our data provide structural details of a key membrane bound state of FAK that is primed for efficient autophosphorylation and activation, hence revealing the critical event in integrin mediated FAK activation and signaling at focal adhesions.

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.

Focal adhesion kinase confers pro-migratory and antiapoptotic properties and is a potential therapeutic target in Ewing sarcoma.

Oncogenesis of Ewing sarcoma (EwS), the second most common malignant bone tumor of childhood and adolescence, is dependent on the expression of chimeric EWSR1‐ETS fusion oncogenes, most often EWSR1‐FLI1 (E/F). E/F expression leads to dysregulation of focal adhesions (FAs) enhancing the migratory capacity of EwS cells. Here, we show that, in EwS cell lines and tissue samples, focal adhesion kinase (FAK) is expressed and phosphorylated at Y397 in an E/F‐dependent way involving Ezrin. Employing different EwS cell lines as in vitro models, we found that key malignant properties of E/F are mediated via substrate‐independent autophosphorylation of FAK on Y397. This phosphorylation results in enhanced FA formation, Rho‐dependent cell migration, and impaired caspase‐3‐mediated apoptosis in vitro. Conversely, treatment with the FAK inhibitor 15 (1,2,4,5‐benzenetetraamine tetrahydrochloride (Y15) enhanced caspase‐mediated apoptosis and EwS cell migration, independent from the respective EWSR1‐ETS fusion type, mimicking an anoikis‐like phenotype and paralleling the effects of FAK siRNA knockdown. Our findings were confirmed in vivo using an avian chorioallantoic membrane model and provide a first rationale for the therapeutic use of FAK inhibitors to impair metastatic dissemination of EwS.

Lonomia obliqua bristle extract modulates Rac1 activation, membrane dynamics and cell adhesion properties

Lonomia obliqua is a caterpillar of potential therapeutic interest whose venom is able to induce severe blood leakage and modulate leukocyte migration. Since both phenotypes are associated with changes in cytoskeleton dynamics and cell adhesion properties, the aim of this study was to analyze the effects of Lonomia obliqua bristle extract (LOBE) in cell adhesion and migration signaling. Proteomic analysis revealed that epithelial cells (CHO-K1) exposed to LOBE (30 μg/mL, 30 min) exhibited changes in levels of actin regulatory proteins, including RhoGTPases. These changes correlated with an increase in the activity of the RhoGTPase family member Rac as measured by Förster resonance energy transfer (FRET). When plated in migration promoting conditions, CHO-K1 cells exposed to LOBE (10 μg/mL) showed an increase in membrane ruffling after short (30 min) period of incubation that was accompanied by changes in the distribution of the adhesion markers paxillin, vinculin and an increase of focal adhesion kinase autophosphorylation levels (Y397), suggesting changes in cell-extracellular matrix (ECM) adhesion properties and signaling. These data suggest that LOBE possesses bioactive molecules that are capable to modulated cell migration signaling, cytoskeletal dynamics and cell-ECM properties of several cell types.

FAK auto-phosphorylation site tyrosine 397 is required for development but dispensable for normal skin homeostasis.

Focal adhesion kinase (FAK) is an intensely studied non-receptor tyrosine kinase with roles in cancer and other common human diseases. Despite the large interest in FAK, the in vivo contribution of FAK auto-phosphorylation site tyrosine (Y) 397 to FAK function is incompletely understood. To study FAK Y397 in vivo we analyzed mice with ‘non-phosphorylatable’ Y-to-phenylalanine (F) and ‘phospho-mimicking’ Y-to-glutamate (E) mutations in the germline. We found that FAK Y397F mice die early during embryogenesis with abnormal angiogenesis like FAK kinase-dead mice. When Y397 is mutated to a glutamate mice survive beyond mid-gestation like mice where Y397 is lost by deletion of FAK exon 15. In culture, defects in proliferation, invasion and gene expression were more severe with the FAK Y397F than with the FAK Y397E mutation despite the inability of FAK Y397E to bind SRC. Conditional expression of FAK Y397F or Y397E in unchallenged avascular epidermis, however, resulted in no appreciable phenotype. We conclude that FAK Y397 is required for the highly dynamic tissue remodeling during development but dispensable for normal homeostasis of avascular epidermis. In contrast to the Y397F mutation, FAK Y397E retains sufficient biological activity to allow for development beyond mid-gestation.

Efficacy of the highly selective focal adhesion kinase inhibitor BI 853520 in adenocarcinoma xenograft models is linked to a mesenchymal tumor phenotype

Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, has attracted interest as a target for pharmacological intervention in malignant diseases. Here, we describe BI 853520, a novel ATP-competitive inhibitor distinguished by high potency and selectivity. In vitro, the compound inhibits FAK autophosphorylation in PC-3 prostate carcinoma cells with an IC50 of 1 nmol/L and blocks anchorage-independent proliferation of PC-3 cells with an EC50 of 3 nmol/L, whereas cells grown in conventional surface culture are 1000-fold less sensitive. In mice, the compound shows long half-life, high volume of distribution and high oral bioavailability; oral dosing of immunodeficient mice bearing subcutaneous PC-3 prostate adenocarcinoma xenografts resulted in rapid, long-lasting repression of FAK autophosphorylation in tumor tissue. Daily oral administration of BI 853520 to nude mice at doses of 50 mg/kg was well tolerated for prolonged periods of time. In a diverse panel of 16 subcutaneous adenocarcinoma xenograft models in nude mice, drug treatment resulted in a broad spectrum of outcomes, ranging from group median tumor growth inhibition values >100% and tumor regression in subsets of animals to complete lack of sensitivity. Biomarker analysis indicated that high sensitivity is linked to a mesenchymal tumor phenotype, initially defined by loss of E-cadherin expression and subsequently substantiated by gene set enrichment analysis. Further, we obtained microRNA expression profiles for 13 models and observed that hsa-miR-200c-3p expression is strongly correlated with efficacy (R2 = 0.889). BI 853520 is undergoing evaluation in early clinical trials.

Abstract B03: The EWS/FLI oncogene exerts pro-migratory and anti-apoptotic effects via dysregulation of focal adhesion kinase (FAK) signaling in Ewing sarcoma

Abstract Ewing sarcoma (ES), an aggressive bone tumor of childhood and adolescence, is one of the most genetically normal tumors. The oncogenic potential is derived from chimeric fusions of EWS and ETS family transcription factors, with EWS-FLI1 rearrangements in 85% of cases. (Micro-) metastatic dissemination is the major threat for patients, and earlier studies have shown that cytoskeletal rearrangements upon EWS/FLI oncoprotein expression contribute to a migratory phenotype in ES cells. Using a patient-derived ES cell line with a stable EWS/FLI knockdown (A673-iEF), we show here that key tumorigenic effects of EWS/FLI are mediated via autophosphorylation of FAK on tyrosine 397, resulting in enhanced formation of focal adhesions and actin stress fibers as well as tumor cell migration, but impaired caspase-3-mediated anoikis (detachment-induced cell death) in vitro. This effect was not limited to A673 cells, but could also be observed in TC-32 and CADO-ES1 ES cells. Application of the FAK inhibitor 1, 2, 4, 5-Benzenetetraamine tetrahydrochloride (Y15) impaired cell migration and enhanced caspase-mediated apoptosis. Taken together, our results show that key oncogenic effects of EWS rearrangements are mediated via FAK autophosphorylation in ES and provide a rationale for the use of FAK inhibitors to impair metastatic dissemination of ES. Citation Format: Konrad Steinestel, Marcel Trautmann, Jan Rehkaemper, Eva Wardelmann, Uta Dirksen, Wolfgang Hartmann. The EWS/FLI oncogene exerts pro-migratory and anti-apoptotic effects via dysregulation of focal adhesion kinase (FAK) signaling in Ewing sarcoma [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr B03.

Regulation of Focal Adhesion Kinase through a Direct Interaction with an Endogenous Inhibitor.

Focal adhesion kinase (FAK) plays a key role in integrin and growth factor signaling pathways. FAK-related non-kinase (FRNK) is an endogenous inhibitor of FAK that shares its primary structure with the C-terminal third of FAK. FAK S910 phosphorylation is known to regulate FAK protein-protein interactions, but the role of the equivalent site on FRNK (S217) is unknown. Here we determined that S217 is highly phosphorylated by ERK in cultured rat aortic smooth muscle cells. Blocking phosphorylation by mutation (S217A) greatly increased FRNK inhibitory potency, resulting in strong inhibition of FAK autophosphorylation at Y397 and induction of smooth muscle cell apoptosis. FRNK has been proposed to compete for FAK anchoring sites in focal adhesions, but we did not detect displacement of FAK by WT-FRNK or superinhibitory S217A-FRNK. Instead, we found FRNK interacted directly with FAK, and this interaction is markedly strengthened for the superinhibitory S217A-FRNK. The results suggest that FRNK limits growth and survival signaling pathways by binding directly to FAK in an inhibitory complex, and this inhibition is relieved by phosphorylation of FRNK at S217.

FAK phosphorylation plays a central role in thrombin-induced RPE cell migration

The migration of retinal pigment epithelial (RPE) cells is an important step in various pathologic conditions including subretinal neovascularization (SRN), proliferative vitreoretinopathy (PVR) and, importantly, as a consequence of retinal surgery. Therefore, the elucidation of the mechanisms underlying RPE trans-differentiation and migration is essential for devising effective treatments aimed to the prevention of these disorders. A common event in these pathologies is the alteration of the blood-retina barrier (BRB), which allows the interaction of RPE cells with thrombin, a pro-inflammatory protease contained in serum. Our previous work has demonstrated that thrombin induces RPE cell cytoskeletal remodeling and migration, hallmark processes in the development of PVR; however, the molecular mechanisms involved are still unclear. Cell migration requires the disassembly of focal adhesions induced by Focal Adhesion Kinase (FAK) phosphorylation, together with the formation of actin stress fibers. The aim of the present work was to identify thrombin-activated signaling pathways leading to FAK phosphorylation and to determine FAK participation in thrombin-induced RPE cell migration. Results demonstrate that the activation of PAR1 by thrombin induces FAK autophosphorylation at Y397 and the subsequent phosphorylation of Y576/577 within the activation loop. FAK phosphorylation was shown to be under the control of c/nPKC and PI3K/PKC-ζ, as well as by Rho/ROCK, since the inhibition of these pathways prevented thrombin-induced FAK phosphorylation and the consequent disassembly of focal adhesions, in parallel to FAK-dependent actin stress fiber formation and RPE cell migration. These findings demonstrate, for the first time, that thrombin stimulation of RPE cell transformation and migration are regulated by FAK tyrosine phosphorylation. Thus, targeting FAK phosphorylation may provide a strategical basis for PVR treatment.

Structural Insights How PIP2 Imposes Preferred Binding Orientations of FAK at Lipid Membranes.

Focal adhesion kinase (FAK) is a multidomain protein (FERM-kinase-FAT) with important signaling functions in the regulation of cell-substrate adhesions. Its inactive, autoinhibited form is recruited from the cytoplasm to the plasma membrane, where it becomes activated at PIP2 enriched regions. To elucidate the molecular basis of activation, we performed a systematic screening of binding orientations of FAK's autoinhibited FERM-kinase complex, as well as of the dissociated FERM and kinase domains alone, on model plasma membranes using coarse-grained scFix MARTINI simulations, partially corroborated by atomistic MD simulations. The proteins adopted many more different orientations than previously thought. The presence of PIP2 tuned and narrowed the complex map of competing interfacial orientations. The dissociated FERM domain most frequently interacted with the membrane through its autoinhibitory interface rather than with the "basic patch" residues. These findings suggest a PIP2-dependent activation mechanism in which membrane binding of the dissociated FERM domain competes with the rebinding of the kinase domain. This competition could promote FAK autophosphorylation on Y397 and subsequent Src binding. The orientation of peripheral proteins at membranes is crucial to understand cell adhesion processes and is furthermore required to exploit steered molecular dynamics to predict how tensile forces might switch their active states.

Neuronal migration is mediated by inositol hexakisphosphate kinase 1 via α-actinin and focal adhesion kinase.

Inositol hexakisphosphate kinase 1 (IP6K1), which generates 5-diphosphoinositol pentakisphosphate (5-IP7), physiologically mediates numerous functions. We report that IP6K1 deletion leads to brain malformation and abnormalities of neuronal migration. IP6K1 physiologically associates with α-actinin and localizes to focal adhesions. IP6K1 deletion disrupts α-actinin's intracellular localization and function. The IP6K1 deleted cells display substantial decreases of stress fiber formation and impaired cell migration and spreading. Regulation of α-actinin by IP6K1 requires its kinase activity. Deletion of IP6K1 abolishes α-actinin tyrosine phosphorylation, which is known to be regulated by focal adhesion kinase (FAK). FAK phosphorylation is substantially decreased in IP6K1 deleted cells. 5-IP7, a product of IP6K1, promotes FAK autophosphorylation. Pharmacologic inhibition of IP6K by TNP [N2-(m-Trifluorobenzyl), N6-(p-nitrobenzyl)purine] recapitulates the phenotype of IP6K1 deletion. These findings establish that IP6K1 physiologically regulates neuronal migration by binding to α-actinin and influencing phosphorylation of both FAK and α-actinin through its product 5-IP7.