Wild-type Focal Adhesion Kinase Research Articles

Abstract C065: Dissecting the role of CXCL16 downstream of FAK inhibition in the anti-tumor immune response against pancreatic cancer

Abstract Background: Unfortunately, the impact of immunotherapy in pancreatic cancer has, to date, been limited. Nonetheless, despite lack of success with checkpoint blockade alone, there is hope that combinations of agents may unlock the potential for immunotherapy success in pancreatic cancer. FAK (Focal Adhesion Kinase) is a non-receptor tyrosine kinase that is elevated in human Pancreatic Ductal Adenocarcinoma (PDAC) tissue and correlates with high levels of fibrosis and poor CD8 cytotoxic T cell infiltration. We, and others, have already demonstrated a role for FAK in promoting tumor evasion by inducing an immunosuppressive microenvironment, specifically by regulation of cytokines such as CCL5. This has led to trials of the FAK inhibitor (defactinib) in conjunction with immunotherapy. Here we set out to improve our understanding of the role of FAK in regulating chemokine/cytokine mediated signaling in PDAC with a view to identify clinically targetable pathways with potential therapeutic benefit to pancreatic cancer patients. Methods: We combined CRISPR, NanoString, Forward Phase Protein Arrays (FPPA) and ELISA with in vivo experiments using a transplantable KrasG12Dp53R172H mouse model of Pancreatic Cancer and validated findings using human patient-derived PDAC cell lines. Results: We used CRISPR-Cas9 gene editing to deplete FAK gene expression in Panc47 cells, a syngeneic cell line isolated from PDAC arising in fully back-crossed C57BL/6 KPC mice, and reconstituted wild-type FAK (FAK-WT) expression into a clonal Panc47 FAK null (FAK-/-) cell line. Subsequent NanoString and FPPA analyses of secreted cytokines in Panc47 FAK-WT and Panc47 FAK-/- cell lines identified CXCL16 as one of the most significantly increased chemokines upon FAK depletion. Similar results were observed when CRISPR mediated FAK depletion was performed in patient-derived PDAC cell lines. Previous work in our lab has shown that depletion of Signal Transducer and Activator of Transcription-3 (STAT3), a key mediator of immune evasion, in combination with FAK depletion in orthotopically implanted PDAC tumors significantly increases CD8 T-cell infiltration into murine tumors when compared with FAK-/- tumors alone. Here we identify that dual FAK/STAT3 depletion further elevates CXCL16 expression and show that CXCR6, the receptor for CXCL16, is associated with both murine and human CD8 T-cells. Conclusions: These findings identify a FAK-dependent CXCL16:CXCR6 paracrine signaling axis associated with elevated recruitment of CD8 T-cells into PDAC tumors. This requires the dual targeting of FAK and STAT3, identifying a novel therapeutic strategy that warrants further investigation. Citation Format: Jane L. Parry, Marta Canel, Alan Serrels. Dissecting the role of CXCL16 downstream of FAK inhibition in the anti-tumor immune response against pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C065.

Abstract 669: Nuclear FAK drives thyroid cancer growth and survival

Abstract Background: Late stage thyroid cancers characterized by metastasis and extranodal invasion have a poor prognosis compared to those with localized disease. However, there are limited therapeutic options and few biomarkers to indicate which patients will develop aggressive disease. Our lab has identified Focal Adhesion Kinase (FAK) as a key regulator of thyroid cancer growth, invasion, and metastasis. FAK is a non-receptor tyrosine kinase that is auto-phosphorylated at tyrosine 397 (Y397) in response to integrin or growth factor receptor signaling resulting in the activation of downstream signaling pathways. While FAK is predominantly localized at the plasma membrane, FAK has also been shown to accumulate in the nucleus via a nuclear localization sequence (NLS) to promote cell survival. We have found that FAK localizes to the nucleus in a subset of thyroid cancer patient tumors and that phosphorylated Y397 FAK (pY397 FAK) specifically accumulates in the nucleolus. The nucleolus plays a key role in cancer progression through the synthesis of ribosomal RNA (rRNA) and subsequent increase in ribosome biogenesis, protein synthesis, and tumor growth. It is unclear how pY397 FAK localizes to the nucleolus and what the function of pY397 FAK is in the nucleolus. Hypothesis: Nuclear FAK drives thyroid cancer growth and survival through the phosphorylation of nucleolar proteins involved in rRNA transcription. Results: To address the functional role of FAK in the nucleus, we excluded FAK from the nucleus by mutating its NLS and found that it significantly decreased anchorage independent growth compared to wild type (WT) FAK in our thyroid cancer cells (BCPAP and 8505C). Importantly, non-phosphorylatable FAK mutant (Y397F) and kinase dead FAK mutant also decreased growth indicating that nuclear and pY397 FAK are required for anchorage independent growth. Furthermore, we found that forcing FAK into the nucleus with an SV40 NLS resulted in increased FAK nucleolar accumulation which was eliminated when FAK is forced into the nucleus with Y397F FAK. These data indicate that pY397 FAK is required for FAK nucleolar accumulation. To investigate the role of pY397 FAK in the nucleolus, we performed BioID to identify novel protein-protein interactions for WT FAK and Y397F FAK. We found that pY397 FAK interacts with a network of nucleolar proteins including NPM1, TOP1, and DDX46. Interestingly, the majority of these nucleolar proteins are involved in transcription of pre-rRNA which is essential for protein synthesis and growth. Of note, Nucleophosmin 1 (NPM1) is an endoribonuclease that regulates pre-rRNA synthesis through the cleavage of the 46S rRNA transcript. We confirmed that pY397 FAK specifically interacts with NPM1 by Proximity Ligation Assay. Conclusion: These data indicate that pY397 FAK interacts with a network of nucleolar proteins involved in rRNA transcription and that nuclear pY397 FAK drives growth and survival in thyroid cancer. Citation Format: Meghan Kellett, Vibha Sharma, Brittelle Kessler, Sharon Sams, Molishree Joshi, Rebecca E. Schweppe. Nuclear FAK drives thyroid cancer growth and survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 669.

EB1 Restricts Breast Cancer Cell Invadopodia Formation and Matrix Proteolysis via FAK

Regulation of microtubule dynamics by plus-end tracking proteins (+TIPs) plays an essential role in cancer cell migration. However, the role of +TIPs in cancer cell invasion has been poorly addressed. Invadopodia, actin-rich protrusions specialized in extracellular matrix degradation, are essential for cancer cell invasion and metastasis, the leading cause of death in breast cancer. We, therefore, investigated the role of the End Binding protein, EB1, a major hub of the +TIP network, in invadopodia functions. EB1 silencing increased matrix degradation by breast cancer cells. This was recapitulated by depletion of two additional +TIPs and EB1 partners, APC and ACF7, but not by the knockdown of other +TIPs, such as CLASP1/2 or CLIP170. The knockdown of Focal Adhesion Kinase (FAK) was previously proposed to similarly promote invadopodia formation as a consequence of a switch of the Src kinase from focal adhesions to invadopodia. Interestingly, EB1-, APC-, or ACF7-depleted cells had decreased expression/activation of FAK. Remarkably, overexpression of wild type FAK, but not of FAK mutated to prevent Src recruitment, prevented the increased degradative activity induced by EB1 depletion. Overall, we propose that EB1 restricts invadopodia formation through the control of FAK and, consequently, the spatial regulation of Src activity.

A Synergistic Anticancer FAK and HDAC Inhibitor Combination Discovered by a Novel Chemical-Genetic High-Content Phenotypic Screen.

We mutated the focal adhesion kinase (FAK) catalytic domain to inhibit binding of the chaperone Cdc37 and ATP, mimicking the actions of a FAK kinase inhibitor. We reexpressed mutant and wild-type FAK in squamous cell carcinoma (SCC) cells from which endogenous FAK had been deleted, genetically fixing one axis of a FAK inhibitor combination high-content phenotypic screen to discover drugs that may synergize with FAK inhibitors. Histone deacetylase (HDAC) inhibitors represented the major class of compounds that potently induced multiparametric phenotypic changes when FAK was rendered kinase-defective or inhibited pharmacologically in SCC cells. Combined FAK and HDAC inhibitors arrest proliferation and induce apoptosis in a subset of cancer cell lines in vitro and efficiently inhibit their growth as tumors in vivo Mechanistically, HDAC inhibitors potentiate inhibitor-induced FAK inactivation and impair FAK-associated nuclear YAP in sensitive cancer cell lines. Here, we report the discovery of a new, clinically actionable, synergistic combination between FAK and HDAC inhibitors.

Spatial arrangement of LD motif-interacting residues on focal adhesion targeting domain of Focal Adhesion Kinase determine domain-motif interaction affinity and specificity

BackgroundLeucine rich Aspartate motifs (LD motifs) are molecular recognition motifs on Paxillin that recognize LD-motif binding domains (LDBD) of a number of focal adhesion proteins in order to carry out downstream signaling and actin cytoskeleton remodeling. In this study, we identified structural features within LDBDs that influence their binding affinity with Paxillin LD motifs. MethodsVarious point mutants of focal adhesion targeting (FAT) domain of Focal Adhesion Kinase (FAK) were created by moving a key Lysine residue two and three helical turns in order to match the unique conformations as observed in LDBDs of two other focal adhesion proteins, Vinculin and CCM3. ResultsThis led to identify a mutant of FAT domain of FAK, named as FAT(NV) (Asn992 of FAT domain was replaced by Val), with remarkable high affinity for LD1 (Kd = 1.5 μM vs no-binding with wild type) and LD2 peptides (Kd = 7.2 μM vs 63 μM with wild type). Consistently, the focal adhesions of MCF7 cells expressing FAK(NV) were highly stable (turnover rate = 1.25 × 10−5 μm2/s) as compared to wild type FAK transfected cells (turnover rate = 1.5 × 10−3 μm2/s). ConclusionsWe observed that the relative disposition of key LD binding amino-acids at LDBD surface, hydrophobic burial of long Leucine side chains of LD-motifs and complementarity of charged surfaces are the key factors determining the binding affinities of LD motifs with LDBDs. General significanceOur study will help in protein engineering of FAT domain of FAK by modulating FAK-LD motif interactions which have implications in cellular focal adhesions and cell migration.

Intravascular Delivery of TAT‐conjugated Focal Adhesion Kinase Protects against Acute Lung Injury

Acute lung injury (ALI), a major cause of high mortality in all age groups, occurs because of endothelial barrier failure. But it lacks a cure. We showed that endothelial focal adhesion kinase (FAK) is barrier protective (PMID: 23033075), suggesting FAK may be a cure for ALI. To test this hypothesis, we purified wild type FAK (FAKWT) and FAK with YàF mutation at tyrosine 925 (FAKMUT), which is expressed in FAK's focal adhesion targeting domain. Then we non‐covalently conjugated the proteins with the cell‐permeable peptide TAT (HIV‐1 transactivator of transcription). We intranasally instilled LPS in anesthetized mice. After 24h, we intravenously infused TAT‐FAKp or TAT‐FAKpY925F. After 4 h, we gave intravenous Evans Blue‐albumin (EBA). After a further 4 h, we assayed ALI in terms of EBA concentration and leukocyte counts in the bronchoalveolar lavage (BAL). Also, by live confocal microscopy of isolated, blood‐perfused lungs, we assayed endothelial F‐actin in terms of the lung endothelial fluorescence of Rhodamin Phalloidin. LPS increased EBA and leukocytes two‐times above baseline (p<0.05 each). TAT‐FAKWT, but not TAT‐FAKpMUT inhibited both responses (p<0.05). Endothelial F‐actin was markedly higher after TAT‐FAKWT than after TAT‐FAKpMUT infusion (p<0.05). LPS‐induced mortality in 5 days was 80% for untreated or TAT‐FAKpMUT infused mice. However, TAT‐FAKpWT infusion decreased the mortality to 25% (p<0.05). Our findings indicate that purified FAK can be effectively given by intravenous injection. TAT‐FAKWT, given well after the initiation of LPS‐induced ALI, markedly increased survival. The protective effect was due to FAK's Y925 residue that is likely responsible for enhancing cortical F‐actin, thereby strengthening the endothelial barrier (PMID:23033075). We propose that intravenous delivery of TAT‐FAKpWT might be effective therapy for ALI (HL36024).Support or Funding InformationNIH: HL36024This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Interactions of the Protein-tyrosine Phosphatase-α with the Focal Adhesion Targeting Domain of Focal Adhesion Kinase Are Involved in Interleukin-1 Signaling in Fibroblasts

Interleukin-1 (IL-1) signaling in fibroblasts is mediated through focal adhesions, organelles that are enriched with adaptor and cytoskeletal proteins that regulate signal transduction. We examined interactions of the focal adhesion kinase (FAK) with protein-tyrosine phosphatase-α (PTP-α) in IL-1 signaling. In wild type and FAK knock-out mouse embryonic fibroblasts, we found by immunoblotting, immunoprecipitation, immunostaining, and gene silencing that FAK is required for IL-1-mediated sequestration of PTPα to focal adhesions. Immunoprecipitation and pulldown assays of purified proteins demonstrated a direct interaction between FAK and PTPα, which was dependent on the FAT domain of FAK and by an intact membrane-proximal phosphatase domain of PTPα. Recruitment of PTPα to focal adhesions, IL-1-induced Ca(2+) release from the endoplasmic reticulum, ERK activation, and IL-6, MMP-3, and MMP-9 expression were all blocked in FAK knock-out fibroblasts. These processes were restored in FAK knock-out cells transfected with wild type FAK, FAT domain, and FRNK. Our data indicate that IL-1-induced signaling through focal adhesions involves interactions between the FAT domain of FAK and PTPα.

Focal Adhesion Kinase Plays a Role in Osteoblast Mechanotransduction In Vitro but Does Not Affect Load-Induced Bone Formation In Vivo

A healthy skeleton relies on bone's ability to respond to external mechanical forces. The molecular mechanisms by which bone cells sense and convert mechanical stimuli into biochemical signals, a process known as mechanotransduction, are unclear. Focal adhesions play a critical role in cell survival, migration and sensing physical force. Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase that controls focal adhesion dynamics and can mediate reparative bone formation in vivo and osteoblast mechanotransduction in vitro. Based on these data, we hypothesized that FAK plays a role in load-induced bone formation. To test this hypothesis, we performed in vitro fluid flow experiments and in vivo bone loading studies in FAK−/− clonal lines and conditional FAK knockout mice, respectively. FAK−/− osteoblasts showed an ablated prostaglandin E2 (PGE2) response to fluid flow shear. This effect was reversed with the re-expression of wild-type FAK. Re-expression of FAK containing site-specific mutations at Tyr-397 and Tyr-925 phosphorylation sites did not rescue the phenotype, suggesting that these sites are important in osteoblast mechanotransduction. Interestingly, mice in which FAK was conditionally deleted in osteoblasts and osteocytes did not exhibit altered load-induced periosteal bone formation. Together these data suggest that although FAK is important in mechanically-induced signaling in osteoblasts in vitro, it is not required for an adaptive response in vivo, possibly due to a compensatory mechanism that does not exist in the cell culture system.

Serine-910 phosphorylation of focal adhesion kinase is critical for sarcomere reorganization in cardiomyocyte hypertrophy

Tyrosine-phosphorylated focal adhesion kinase (FAK) is required for the hypertrophic response of cardiomyocytes to growth factors and mechanical load, but the role of FAK serine phosphorylation in this process is unknown. The aims of the present study were to characterize FAK serine phosphorylation in cultured neonatal rat ventricular myocytes (NRVM), analyse its functional significance during hypertrophic signalling, and examine its potential role in the pathogenesis of human dilated cardiomyopathy (DCM). Endothelin-1 (ET-1) and other hypertrophic factors induced a time- and dose-dependent increase in FAK-S910 phosphorylation. ET-1-induced FAK-S910 phosphorylation required ET(A)R-dependent activation of PKCδ and Src via parallel Raf-1 → MEK1/2 → ERK1/2 and MEK5 → ERK5 signalling pathways. Replication-deficient adenoviruses expressing wild-type (WT) FAK and a non-phosphorylatable, S910A-FAK mutant were then used to examine the functional significance of FAK-S910 phosphorylation. Unlike WT-FAK, S910A-FAK increased the half-life of GFP-tagged paxillin within costameres (as determined by total internal reflection fluorescence microscopy and fluorescence recovery after photobleaching) and increased the steady-state FAK-paxillin interaction (as determined by co-immunoprecipitation and western blotting). These alterations resulted in reduced NRVM sarcomere reorganization and cell spreading. Finally, we found that FAK was serine-phosphorylated at multiple sites in non-failing, human left ventricular tissue. FAK-S910 phosphorylation and ERK5 expression were dramatically reduced in patients undergoing heart transplantation for end-stage DCM. FAK undergoes S910 phosphorylation via PKCδ and Src-dependent pathways that are important for cell spreading and sarcomere reorganization. Reduced FAK-S910 phosphorylation may contribute to sarcomere disorganization in DCM.

Phosphorylation of Focal Adhesion Kinase at Tyr397 in Gastric Carcinomas and its Clinical Significance

Focal adhesion kinase (FAK) has been implicated in tumorigenesis in various cancers; however, it remains unclear how FAK participates in tumor malignancy in vivo. This study seeks to understand the role of FAK activation in gastric cancer progression. Using immunohistochemical staining and Western blotting, we found that pY397 FAK, an autophosphorylation site on FAK activation, was abundant in the cancerous tissues of 21 of 59 patients with gastric carcinomas. We attempted to correlate clinicopathological parameters, including histological types, TNM staging, and cancer recurrence, with the expression of FAK and pY397 FAK in cancerous tissues. Intriguingly, patients with higher levels of pY397 FAK displayed higher incidences of gastric cancer recurrence after surgery and poor 5-year recurrence-free survival. Furthermore, multivariate analyses showed that pY397 FAK was an independent predictor of gastric cancer recurrence. As a result, expression of pY397 FAK is a significant prognostic factor for the recurrence of gastric cancer. Additionally, in vitro studies showed that overexpression of Y397F, a dominant-negative mutant of FAK, in AGS human gastric carcinoma cells impaired cell migration, invasion, and proliferation compared with cells overexpressing wild-type FAK. Thus, activation of FAK through autophosphorylation at Tyr397 leads to the progression of gastric carcinomas by promoting cell migration, invasion, and proliferation. Collectively, our results have provided valuable insights for the development of novel diagnoses and therapeutic targets for gastric cancer treatments.

Focal Adhesion Kinase, a Downstream Mediator of Raf-1 Signaling, Suppresses Cellular Adhesion, Migration, and Neuroendocrine Markers in BON Carcinoid Cells

We have recently reported that activation of the Raf-1/mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase 1/2 (MEK1/2)/ERK1/2 signaling cascade in gastrointestinal carcinoid cell line (BON) alters cellular morphology and neuroendocrine phenotype. The mechanisms by which Raf-1 mediates these changes in carcinoid cells are unclear. Here, we report that activation of the Raf-1 signaling cascade in BON cells induced the expression of focal adhesion kinase (FAK) protein, suppressed the production of neuroendocrine markers, and resulted in significant decreases in cellular adhesion and migration. Importantly, inactivation of MEK1/2 by 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene or abolition of FAK induction in Raf-1-activated BON cells by targeted siRNA led to reversal of the Raf-1-mediated reduction in neuroendocrine markers and cellular adhesion and migration. Phosphorylation site-specific antibodies detected the phosphorylated FAK(Tyr407), but not FAK(Tyr397), in these Raf-1-activated cells, indicating that FAK(Tyr407) may be associated with changes in the neuroendocrine phenotype. Overexpression of constitutively active FAK plasmids (wild-type FAK or FAK(Tyr397) mutant) into BON cells reduced neuroendocrine markers, whereas the FAK(Tyr407) mutant plasmid did not show any decrease in the levels of neuroendocrine markers, indicating that phosphorylation of FAK at the Tyr(407) residue may be important for these effects. Our results showed for the first time that FAK is an essential downstream effector of the Raf-1/MEK1/2/ERK1/2 signaling cascade and negatively regulated the neuroendocrine and metastatic phenotype in BON cells.

Focal adhesion kinase‐dependent regulation of adhesive forces involves vinculin recruitment to focal adhesions

FAK (focal adhesion kinase), an essential non-receptor tyrosine kinase, plays pivotal roles in migratory responses, adhesive signalling and mechanotransduction. FAK-dependent regulation of cell migration involves focal adhesion turnover dynamics as well as actin cytoskeleton polymerization and lamellipodia protrusion. Whereas roles for FAK in migratory and mechanosensing responses have been established, the contribution of FAK to the generation of adhesive forces is not well understood. Using FAK-null cells expressing wild-type and mutant FAK under an inducible tetracycline promoter, we analysed the role of FAK in the generation of steady-state adhesive forces using micropatterned substrates and a hydrodynamic adhesion assay. FAK expression reduced steady-state strength by 30% compared with FAK-null cells. FAK expression reduced VCL (vinculin) localization to focal adhesions by 35% independently of changes in integrin binding and localization of talin and paxillin. RNAi (RNA interference) knock-down of VCL abrogated the FAK-dependent differences in adhesive forces. FAK-dependent changes in VCL localization and adhesive forces were confirmed in human primary fibroblasts with FAK knocked down by RNAi. The autophosphorylation Tyr-397 and kinase domain Tyr-576/Tyr-577 sites were differentially required for FAK-mediated adhesive responses. We demonstrate that FAK reduces steady-state adhesion strength by modulating VCL recruitment to focal adhesions. These findings provide insights into the role of FAK in mechanical interactions between a cell and the extracellular matrix.

FAK alters invadopodia and focal adhesion composition and dynamics to regulate breast cancer invasion

Focal adhesion kinase (FAK) is important for breast cancer progression and invasion and is necessary for the dynamic turnover of focal adhesions. However, it has not been determined whether FAK also regulates the dynamics of invasive adhesions formed in cancer cells known as invadopodia. In this study, we report that endogenous FAK functions upstream of cellular Src (c-Src) as a negative regulator of invadopodia formation and dynamics in breast cancer cells. We show that depletion of FAK induces the formation of active invadopodia but impairs invasive cell migration. FAK-deficient MTLn3 breast cancer cells display enhanced assembly and dynamics of invadopodia that are rescued by expression of wild-type FAK but not by FAK that cannot be phosphorylated at tyrosine 397. Moreover, our findings demonstrate that FAK depletion switches phosphotyrosine-containing proteins from focal adhesions to invadopodia through the temporal and spatial regulation of c-Src activity. Collectively, our findings provide novel insight into the interplay between FAK and Src to promote invasion.

Role of Focal Adhesion Kinase Ser-732 Phosphorylation in Centrosome Function during Mitosis

Focal adhesion kinase (FAK) is the major cytoplasmic tyrosine kinase in focal adhesions and a critical mediator of integrin signaling in a variety of cells, including endothelial cells (ECs). Here we describe a new function for FAK in the regulation of centrosome functions in a Ser-732 phosphorylation-dependent manner during mitosis. Deletion of FAK in primary ECs causes increases in centrosome numbers, multipolar and disorganized spindles, and unaligned chromosomes during mitosis. Re-expression of wild-type FAK, but not S732A mutant, rescued these mitotic defects, suggesting a role for Ser-732 phosphorylation in the regulation of centrosomal functions. Consistent with this possibility, Ser-732-phosphorylated FAK was found to co-localize in centrosomes in mitotic cells. FAK also associated with cytoplasmic dynein in a Ser-732 phosphorylation-dependent manner. Further analysis in FAK-null primary ECs showed that S732A mutant could rescue EC migration but not proliferation or tubulogenesis in vitro. Last, we showed that deletion of FAK in ECs reduced tumor angiogenesis in vivo, which could be restored by re-expression of wild-type FAK but not S732A mutant. Together, these studies demonstrated a novel role for Ser-732 phosphorylation of FAK in the regulation of centrosome function during mitosis, which may contribute to EC proliferation and angiogenesis.

Requirement of focal adhesion kinase in branching tubulogenesis

We previously demonstrated that α3β1 integrins are essential to hepatocyte growth factor (HGF)-independent branching tubulogenesis in Mardin-Darby Canine Kidney (MDCK) cells. However, the involvement of integrin downstream signaling molecules remains unclear. In the present study, we successfully isolated cell lines possessing different tubulogenic potentials from the MDCK cells; cyst clones (CA4, CA6) forming cystic structures when cultured in 0.3% type I collagen gel and mass clones (M610, M611, M612) forming aggregated masses. Cyst clones maintained cystic structure in 0.1% collagen gel, whereas mass clones spontaneously developed into tubules. Both clones exhibited various morphologies when cultured on a dish: cyst clones formed aggregated islands, while mass clones were more scattered and exhibited higher migration capacity. Among several focal adhesion machinery proteins examined, only the expression and phosphorylation level of focal adhesion kinase (FAK) in mass clones was higher than in cyst clones, while other proteins showed no obvious differences. However, overexpression of wild type FAK in CA6 cells did not facilitate branching tubule formation in 0.1% collagen gel. Targeted decrease in the expression level of FAK in M610 cells with the application of antisense cDNA resulted in a marked reduction of branching tubule formation in 0.1% collagen gel and showed a down-regulation of fibronectin assembly, which is known to promote tubulogenesis. In contrast, overexpression of wild type FAK in CA6 cells had no effect on fibronectin assembly. Taken together, our data demonstrates that FAK is required, but not sufficient for HGF-independent branching tubulogenesis in MDCK cells.

Focal adhesion kinase regulates laminin‐induced oligodendroglial process outgrowth

In the central nervous system (CNS), myelination of axons occurs when oligodendrocyte progenitor cells undergo terminal differentiation, and initiate process formation and axonal ensheathment. Although Fyn, a member of the Src-family kinases (SFKs), plays an important role in this differentiation process, the substrates of Fyn in oligodendrocytes are largely unknown. Using mass spectrometric analysis, we identified focal adhesion kinase (FAK) as a tyrosine-phosphorylated protein in the rat-derived CG4 oligodendrocyte cell line. Tyrosine phosphorylation of FAK was enhanced during differentiation of CG4 cells in a Fyn-dependent manner. In addition, phosphorylation of FAK was stimulated by laminin, one of the ligands for integrin. Knockdown of FAK expression in CG4 cells suppressed process outgrowth on laminin. Rac1 and Cdc42 activities, which are required for oligodendrocyte process formation, were down-regulated in FAK-knockdown cells. Expression of wild-type (WT) FAK in FAK-knockdown CG4 cells restored outgrowth of processes, but the Y397F mutant lacking the autophosphorylation site did not. These results suggest that FAK/Fyn-mediated activation of Rac1 and Cdc42 is critical for laminin-induced outgrowth of oligodendrocyte processes.

Targeting of FAK Ser910 by ERK5 and PP1δ in non-stimulated and phorbol ester-stimulated cells

Ser910 of FAK (focal adhesion kinase) was phosphorylated in fibroblasts treated with the phorbol ester PMA and dephosphorylated by PP1d (protein phosphatase 1d), as indicated by shRNA (small-hairpin RNA) gene silencing. Ser910 of FAK was reported previously to be an ERK (extracellular-signal-regulated kinase) 1/2 target in cells treated with phorbol esters. In contrast, various approaches, including the use of the MEK (mitogen-activated protein kinase/ERK kinase) inhibitors UO126 and CI-1040 to inhibit ERK1/2 pointed to the involvement of ERK5. This hypothesis was confirmed by: (i) shRNA ERK5 gene silencing, which resulted in complete pSer910 loss in non-stimulated and PMA-stimulated cells; (ii) direct phosphorylation of recombinant FAK by ERK5; and (iii) ERK5 activation by PMA. PMA stimulation and ERK5 silencing in MDA-MB 231 and MDA-MB 361 breast cancer cells indicated Ser910 targeting by ERK5 also in these cells. Given the proximity of Ser910 to the FAT (focal adhesion targeting) regulatory domain of FAK, cell proliferation and morphology were investigated in FAK-/- cells expressing S910A mutant FAK. The cell growth rate decreased and exposure to PMA induced peculiar morphological changes in cells expressing S910A, with respect to wild-type FAK, suggesting a role for Ser910 in these processes. The present study indicates, for the first time, the phosphorylation of Ser910 of FAK by ERK5 and its dephosphorylation by PP1d, and suggested a role for Ser910 in the control of cell shape and proliferation.

Tumor Necrosis Factor-α Stimulates Focal Adhesion Kinase Activity Required for Mitogen-activated Kinase-associated Interleukin 6 Expression

Tumor Necrosis Factor-α Stimulates Focal Adhesion Kinase Activity Required for Mitogen-activated Kinase-associated Interleukin 6 Expression

Focal Adhesion Kinase Is Negatively Regulated by Phosphorylation at Tyrosine 407

Focal adhesion kinase (FAK) mediates signal transduction in response to multiple extracellular inputs via tyrosine phosphorylation at specific residues. Although several tyrosine phosphorylation events have been linked to FAK activation and downstream signal transduction, the function of FAK phosphorylation at Tyr(407) was previously unknown. Here, we show for the first time that phosphorylation of FAK Tyr(407) increases during serum starvation, contact inhibition, and cell cycle arrest, all conditions under which activating FAK Tyr(397) phosphorylation decreases. Transfection of NIH3T3 cells with a phosphorylation-mimicking FAK 407E mutant decreased autophosphorylation at Tyr(397) and inhibited both FAK kinase activity in vitro and FAK-mediated functions such as cell adhesion, spreading, proliferation, and migration. The opposite effects were observed in cells transfected with nonphosphorylatable mutant FAK 407F. Taken together, these data suggest the novel concept that FAK Tyr(407) phosphorylation negatively regulates the enzymatic and biological activities of FAK.

Preferential Phosphorylation of Focal Adhesion Kinase Tyrosine 861 Is Critical for Mediating an Anti-apoptotic Response to Hyperosmotic Stress

The results presented here demonstrate that focal adhesion kinase (FAK) Tyr-861 is the predominant tyrosine phosphorylation site stimulated by hyperosmotic stress in a variety of cell types, including epithelial cell lines (ileum-derived IEC-18, colon-derived Caco2, and stomach-derived NCI-N87), FAK null fibroblasts re-expressing FAK, and Src family kinase triple null fibroblasts (SYF cells) in which c-Src has been restored (YF cells). We show that hyperosmotic stress-stimulated FAK phosphorylation in epithelial cells is inhibited by Src family kinase inhibitors PP2 and SU6656 and that it does not occur in SYF cells. Unexpectedly, hyperosmotic stress-induced phosphorylation of FAK at Tyr-397, Tyr-576, and most dramatically at Tyr-861 was completely insensitive to the F-actin-disrupting agents, latrunculin A and cytochalasin D. Finally, we show that in FAK null cells exposed to hyperosmotic stress or growth factor withdrawal, re-expression of wild type FAK restored cell survival, whereas re-expression of FAK mutated from tyrosine to phenylalanine at position 861 (FAKY861F) did not. Our results indicate that FAK Tyr-861 phosphorylation is required for mammalian cell survival of hyperosmotic stress. Furthermore, the results suggest that FAK is an upstream regulator (rather than downstream effector) of F-actin reorganization in response to hyperosmotic stress. We propose that FAK/c-Src bipartite enzyme is a sensor of cytoplasmic shrinkage, and that the phosphorylation on FAK Tyr-861 by Src and subsequent reorganization of F-actin can initiate an anti-apoptotic signaling pathway that protects cells from hyperosmotic stress.