Focal Adhesion Targeting Domain Research Articles

Abstract LB431: Direct comparison of focal adhesion kinase (FAK) therapeutic modalities in cancer: Kinase, FAT, and PROTAC

Abstract Introduction: Focal Adhesion Kinase (FAK) is a multifunctional non-receptor tyrosine kinase and scaffolding protein that has emerged as a potential therapeutic target for the treatment of cancer due to its upregulation and role in cancer cell survival. However, it remains unknown which cancer types are most susceptible to FAK modulation or the best pharmacological strategy to target FAK. FAK is endogenously regulated by an alternative transcript termed FAK-related non-kinase (FRNK), which inhibits FAK through the Focal Adhesion Targeting (FAT) domain. Current FAK-kinase inhibitors that block the catalytic site have shown limited efficacy in multiple Phase I/II clinical trials. FAK-degrading PROTACs have also shown inadequate induction of antiproliferative effects despite successful protein degradation. However, we have shown promising results with our novel FAK-FAT inhibitors, which target the scaffolding function of FAK to induce anoikis and inhibit proliferation in cancer cells. A direct comparison of these different FAK modulators in various cancer types has yet to be performed, creating a significant gap in understanding the best approach for targeted FAK inhibition for the treatment of cancer. Methods: The Broad Institute’s Cancer Dependency Map (DepMap) was utilized to select a cancer cell line cohort based on dependency on FAK for survival. Four cancer types, including breast, brain, lung, and skin cancers were selected based on the correlation of FAK expression for survival and previous literature evidence for dependency on FAK. Three FAK-dependent and one FAK-independent cell lines per cancer type were selected for a total of 16 cancer cell lines. Genetic adenoviral constructs that block the FAK-FAT domain (Ad-FRNK) and knockdown FAK levels (shRNA) were utilized to investigate the role of FAK in cancer cell survival. These constructs were used as they recapitulate the pharmacological strategy of our FAK-FAT inhibitor UA-2012 and FAK degrading PROTACs, respectively. Results: Cell viability was significantly decreased in 10/13 FAK-dependent cell lines after transduction with either adFRNK or FAK targeting shRNAs, six of which showed a statically significant decrease in cell viability upon transduction with adFRNK compared to shRNAs. No cell lines showed a greater response to FAK shRNAs compared to adFRNK. We then evaluated the ability of the FAK kinase inhibitor Defactinib, FAK degrading PROTAC BI-3663, and our novel FAK-FAT domain inhibitor UA-2012 to induce cancer cell death. Overall, FAK-FAT domain inhibition with UA-2012 resulted in the most potent reduction in viability of FAK-dependent cells, particularly in skin and breast cancer cell lines. Treatment with Defactinib resulted in low IC50 values regardless of FAK, and PROTAC BI-3663 displayed minimal effects with high IC50 values often above 200 µM despite successful protein degradation. Conclusion: This data suggests that the FAK-FAT domain may be the major modulator of FAK-mediated survival in breast and skin cancer cells. Citation Format: Lauren Reyes, Ruchi Khatiwala, Timothy Marlowe. Direct comparison of focal adhesion kinase (FAK) therapeutic modalities in cancer: Kinase, FAT, and PROTAC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB431.

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.

Protein tyrosine phosphatase 1B targets focal adhesion kinase and paxillin in cell-matrix adhesions.

Protein tyrosine phosphatase 1B (PTP1B, also known as PTPN1) is an established regulator of cell-matrix adhesion and motility. However, the nature of substrate targets at adhesion sites remains to be validated. Here, we used bimolecular fluorescence complementation assays, in combination with a substrate trapping mutant of PTP1B, to directly examine whether relevant phosphotyrosines on paxillin and focal adhesion kinase (FAK, also known as PTK2) are substrates of the phosphatase in the context of cell-matrix adhesion sites. We found that the formation of catalytic complexes at cell-matrix adhesions requires intact tyrosine residues Y31 and Y118 on paxillin, and the localization of FAK at adhesion sites. Additionally, we found that PTP1B specifically targets Y925 on the focal adhesion targeting (FAT) domain of FAK at adhesion sites. Electrostatic analysis indicated that dephosphorylation of this residue promotes the closed conformation of the FAT 4-helix bundle and its interaction with paxillin at adhesion sites.

Evaluation of the Binding Kinetics of RHEB with mTORC1 by In-Cell and In Vitro Assays.

The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) is activated by the small G-protein, Ras homolog enriched in brain (RHEB–GTPase). On lysosome, RHEB activates mTORC1 by binding the domains of N-heat, M-heat, and the focal adhesion targeting (FAT) domain, which allosterically regulates ATP binding in the active site for further phosphorylation. The crucial role of RHEB in regulating growth and survival through mTORC1 makes it a targetable site for anti-cancer therapeutics. However, the binding kinetics of RHEB to mTORC1 is still unknown at the molecular level. Therefore, we studied the kinetics by in vitro and in-cell protein–protein interaction (PPI) assays. To this end, we used the split-luciferase system (NanoBiT®) for in-cell studies and prepared proteins for the in vitro measurements. Consequently, we demonstrated that RHEB binds to the whole mTOR both in the presence or absence of GTPγS, with five-fold weaker affinity in the presence of GTPγS. In addition, RHEB bound to the truncated mTOR fragments of N-heat domain (∆N, aa 60–167) or M-heat domain (∆M, aa 967–1023) with the same affinity in the absence of GTP. The reconstructed binding site of RHEB, ∆N-FAT-M, however, bound to RHEB with the same affinity as ∆N-M, indicating that the FAT domain (∆FAT, aa 1240–1360) is dispensable for RHEB binding. Furthermore, RHEB bound to the truncated kinase domain (∆ATP, aa 2148–2300) with higher affinity than to ∆N-FAT-M. In conclusion, RHEB engages two different binding sites of mTOR, ∆N-FAT-M and ∆ATP, with higher affinity for ∆ATP, which likely regulates the kinase activity of mTOR through multiple different biding modes.

Inhibiting FAK-Paxillin Interaction Reduces Migration and Invadopodia-Mediated Matrix Degradation in Metastatic Melanoma Cells.

Simple SummaryThe focal adhesion kinase (FAK) is over-expressed in a variety of human tumors and is involved in many aspects of the metastatic process. This has led to the development of small inhibitors of FAK kinase function which are currently evaluated in clinical trials. We demonstrate here that this class of inhibitors, while decreasing melanoma cell migration, increases invadopodia activity in metastatic melanoma cells. Searching for an alternative strategy to inhibit the oncogenic activity of FAK, we show that inhibiting FAK scaffolding function using a small peptide altering FAK–paxillin interactions reduces both migration and invadopodia-mediated matrix degradation in metastatic melanoma cells.The nonreceptor tyrosine kinase FAK is a promising target for solid tumor treatment because it promotes invasion, tumor progression, and drug resistance when overexpressed. Investigating the role of FAK in human melanoma cells, we found that both in situ and metastatic melanoma cells strongly express FAK, where it controls tumor cells’ invasiveness by regulating focal adhesion-mediated cell motility. Inhibiting FAK in human metastatic melanoma cells with either siRNA or a small inhibitor targeting the kinase domain impaired migration but led to increased invadopodia formation and extracellular matrix degradation. Using FAK mutated at Y397, we found that this unexpected increase in invadopodia activity is due to the lack of phosphorylation at this residue. To preserve FAK–Src interaction while inhibiting pro-migratory functions of FAK, we found that altering FAK–paxillin interaction, with either FAK mutation in the focal adhesion targeting (FAT) domain or a competitive inhibitor peptide mimicking paxillin LD domains drastically reduces cell migration and matrix degradation by preserving FAK activity in the cytoplasm. In conclusion, our data show that targeting FAK–paxillin interactions could be a potential therapeutic strategy to prevent metastasis formation, and molecules targeting this interface could be alternative to inhibitors of FAK kinase activity which display unexpected effects.

Recognition of LD motifs by the focal adhesion targeting domains of focal adhesion kinase and proline-rich tyrosine kinase 2-beta: Insights from molecular dynamics simulations.

The focal adhesion kinase (FAK) and the proline-rich tyrosine kinase 2-beta (PYK2) are implicated in cancer progression and metastasis and represent promising biomarkers and targets for cancer therapy. FAK and PYK2 are recruited to focal adhesions (FAs) via interactions between their FA targeting (FAT) domains and conserved segments (LD motifs) on the proteins Paxillin, Leupaxin, and Hic-5. A promising new approach for the inhibition of FAK and PYK2 targets interactions of the FAK domains with proteins that promote localization at FAs. Advances toward this goal include the development of surface plasmon resonance, heteronuclear single quantum coherence nuclear magnetic resonance (HSQC-NMR) and fluorescence polarization assays for the identification of fragments or compounds interfering with the FAK-Paxillin interaction. We have recently validated this strategy, showing that Paxillin mimicking polypeptides with 2 to 3 LD motifs displace FAK from FAs and block kinase-dependent and independent functions of FAK, including downstream integrin signaling and FA localization of the protein p130Cas. In the present work we study by all-atom molecular dynamics simulations the recognition of peptides with the Paxillin and Leupaxin LD motifs by the FAK-FAT and PYK2-FAT domains. Our simulations and free-energy analysis interpret experimental data on binding of Paxillin and Leupaxin LD motifs at FAK-FAT and PYK2-FAT binding sites, and assess the roles of consensus LD regions and flanking residues. Our results can assist in the design of effective inhibitory peptides of the FAK-FAT: Paxillin and PYK2-FAT:Leupaxin complexes and the construction of pharmacophore models for the discovery of potential small-molecule inhibitors of the FAK-FAT and PYK2-FAT focal adhesion based functions.

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.

Development of a Fragment-Based Screening Assay for the Focal Adhesion Targeting Domain Using SPR and NMR.

The Focal Adhesion Targeting (FAT) domain of Focal Adhesion Kinase (FAK) is a promising drug target since FAK is overexpressed in many malignancies and promotes cancer cell metastasis. The FAT domain serves as a scaffolding protein, and its interaction with the protein paxillin localizes FAK to focal adhesions. Various studies have highlighted the importance of FAT-paxillin binding in tumor growth, cell invasion, and metastasis. Targeting this interaction through high-throughput screening (HTS) provides a challenge due to the large and complex binding interface. In this report, we describe a novel approach to targeting FAT through fragment-based drug discovery (FBDD). We developed two fragment-based screening assays—a primary SPR assay and a secondary heteronuclear single quantum coherence nuclear magnetic resonance (HSQC-NMR) assay. For SPR, we designed an AviTag construct, optimized SPR buffer conditions, and created mutant controls. For NMR, resonance backbone assignments of the human FAT domain were obtained for the HSQC assay. A 189-compound fragment library from Enamine was screened through our primary SPR assay to demonstrate the feasibility of a FAT-FBDD pipeline, with 19 initial hit compounds. A final total of 11 validated hits were identified after secondary screening on NMR. This screening pipeline is the first FBDD screen of the FAT domain reported and represents a valid method for further drug discovery efforts on this difficult target.

Development of a High-Throughput Fluorescence Polarization Assay to Detect Inhibitors of the FAK-Paxillin Interaction.

Focal adhesion kinase (FAK) is a promising cancer drug target due to its massive overexpression in multiple solid tumors and its critical role in the integration of signals that control proliferation, invasion, apoptosis, and metastasis. Previous FAK drug discovery and high-throughput screening have exclusively focused on the identification of inhibitors that target the kinase domain of FAK. Because FAK is both a kinase and scaffolding protein, the development of novel screening assays that detect inhibitors of FAK protein-protein interactions remains a critical need. In this report, we describe the development of a high-throughput fluorescence polarization (FP) screening assay that measures the interactions between FAK and paxillin, a focal adhesion-associated protein. We designed a tetramethylrhodamine (TAMRA)-tagged paxillin peptide based on the paxillin LD2 motif that binds to the focal adhesion targeting (FAT) domain with significant dynamic range, specificity, variability, stability, and a Z'-factor suitable for high-throughput screening. In addition, we performed a pilot screen of 1593 compounds using this FP assay, showing its feasibility for high-throughput drug screening. Finally, we identified three compounds that show dose-dependent competition of FAT-paxillin binding. This assay represents the first described high-throughput screening assay for FAK scaffold inhibitors and can accelerate drug discovery efforts for this promising drug target.

The dPix-Git complex is essential to coordinate epithelial morphogenesis and regulate myosin during Drosophila egg chamber development.

How biochemical and mechanical information are integrated during tissue development is a central question in morphogenesis. In many biological systems, the PIX-GIT complex localises to focal adhesions and integrates both physical and chemical information. We used Drosophila melanogaster egg chamber formation to study the function of PIX and GIT orthologues (dPix and Git, respectively), and discovered a central role for this complex in controlling myosin activity and epithelial monolayering. We found that Git’s focal adhesion targeting domain mediates basal localisation of this complex to filament structures and the leading edge of migrating cells. In the absence of dpix and git, tissue disruption is driven by contractile forces, as reduction of myosin activators restores egg production and morphology. Further, dpix and git mutant eggs closely phenocopy defects previously reported in pak mutant epithelia. Together, these results indicate that the dPix-Git complex controls egg chamber morphogenesis by controlling myosin contractility and Pak kinase downstream of focal adhesions.

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.

Increased expression of FAK isoforms as potential cancer biomarkers in ovarian cancer.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is expressed in most human cell types (example: Epithelial cells, fibroblasts and endothelial), it serves a key role in the control of cell survival, proliferation and motility. The abnormal expression of FAK has been associated with poor prognosis in cancer, including ovarian cancer. However, although FAK isoforms with specific molecular and functional properties have been characterized, there are a limited number of published studies that examine FAK isoforms in ovarian cancer. The aim of the present study was to analyze the expression level of FAK and its isoforms in ovarian cancer. The expression of FAK kinase and focal adhesion targeting (FAT) domains was determined with immunohistochemistry in healthy ovary, and serous and mucinous cystadenoma, borderline tumor and carcinoma samples. Additionally, the expression of FAK and its isoforms were investigated in three ovarian cancer-derived cell lines with western blotting and reverse transcription-semi-quantitative polymerase chain reaction. An increased expression of FAK kinase domain was determined in serous tumor samples and was associated with advancement of the lesion. FAK kinase domain expression was moderate-to-low in mucinous tumor samples. The expression of the FAK FAT domain in tumor samples was reduced, compared with healthy ovary samples; however, the FAT domain was localized to the cellular nucleus. Expression of alternative transcripts FAK°, FAK28,6 and FAK28 was determined in all three cell lines investigated. In conclusion, FAK kinase and FAT domains are differentially expressed among ovarian tumor types. These results indicated the presence of at least two isoforms of FAK (FAK and the putative FAK-related non-kinase) in tumor tissue, which is supported by the cells producing at least three FAK alternative transcripts. These results may support the use of FAK and its isoforms as biomarkers for ovarian cancer.

In Silico Screening to Identify Inhibitors of Growth Factor Receptor 2-Focal Adhesion Kinase Interaction for Therapeutic Treatment of Pathological Cardiac Hypertrophy.

The focal adhesion kinase-growth factor receptor 2 (FAK-Grb2) protein-protein interaction is implicated in pathogenesis of stress-induced cardiac hypertrophy. The focal adhesion targeting (FAT) domain of FAK unfolds to form a structural intermediate that interacts with a multibinding hot spot in the SH2 domain of Grb2. Disruption of the Grb2-FAT interaction is a therapeutic strategy for prevention of pathological cardiac hypertrophy. A pharmacophore was generated on the basis of structural and electrostatic properties of FAT bound to FAK using the Forge tool (Cresset). This pharmacophore was used as a query for Blaze server (Cresset) to screen a selectively enriched chemical library of 4,32,508 small molecules. The compounds selected were further filtered by hierarchical flexible docking approach using AutoDock v4. From the favorably docked compounds, five were selected on the basis of good adsorption, distribution, metabolism, excretion, and toxicity (ADMET) properties using SwissADME, MedChem Designer v.3, and MOLINSPIRATION. Stability of the binding mode of the inhibitors was further confirmed by molecular dynamic simulation study with AMBER v15 for a simulation time of 50 ns in aqueous environment. PM2307 was identified as the best inhibitor in terms of pharmacophoric features, dock score, and in silico ADMET analysis. The calculated binding affinity of PM2307 was better than that of the FAT-Grb2 complex as well as a previously reported small molecule inhibitor. PM2307 is also a quinolyl derivative sharing a similar scaffold with ofloxacin drugs, asserting its drug-like properties. Thus, it was proposed as a lead compound for development of drugs for pathological cardiac hypertrophy.

The binding of DCC-P3 motif and FAK-FAT domain mediates the initial step of netrin-1/DCC signaling for axon attraction

Netrin-1 plays a key role in axon guidance through binding to its receptor, Deleted in Colorectal Cancer (DCC). The initial step of signaling inside the cell after netrin-1/DCC ligation is the binding of DCC cytoplasmic P3 motif to focal adhesion targeting (FAT) domain of focal adhesion kinase (FAK). Here we report the crystal structure of P3/FAT complex. The helical P3 peptide interacts with a helix-swapped FAT dimer in a 2:2 ratio. Dimeric FAT binding is P3-specific and stabilized by a calcium ion. Biochemical studies showed that DCC-P3 motif and calcium ion could facilitate FAT dimerization in solution. Axon guidance assays confirm that the DCC/FAK complex is essential for netrin-1-induced chemoattraction. We propose that netrin-1/DCC engagement creates a small cluster of P3/FAT for FAK recruitment close to the cell membrane, which exerts a concerted effect with PIP2 for FAK signaling. We also compare P3/FAT binding with paxillin/FAT binding and discuss their distinct recognition specificity on a common FAT domain for axon attraction versus integrin signaling, respectively.

Structural and functional insights into the interaction between the Cas family scaffolding protein p130Cas and the focal adhesion-associated protein paxillin

The Cas family scaffolding protein p130Cas is a Src substrate localized in focal adhesions (FAs) and functions in integrin signaling to promote cell motility, invasion, proliferation, and survival. p130Cas targeting to FAs is essential for its tyrosine phosphorylation and downstream signaling. Although the N-terminal SH3 domain is important for p130Cas localization, it has also been reported that the C-terminal region is involved in p130Cas FA targeting. The C-terminal region of p130Cas or Cas family homology domain (CCHD) has been reported to adopt a structure similar to that of the focal adhesion kinase C-terminal focal adhesion-targeting domain. The mechanism by which the CCHD promotes FA targeting of p130Cas, however, remains unclear. In this study, using a calorimetry approach, we identified the first LD motif (LD1) of the FA-associated protein paxillin as the binding partner of the p130Cas CCHD (in a 1:1 stoichiometry with a Kd ∼4.2 μm) and elucidated the structure of the p130Cas CCHD in complex with the paxillin LD1 motif by X-ray crystallography. Of note, a comparison of the CCHD/LD1 complex with a previously solved structure of CCHD in complex with the SH2-containing protein NSP3 revealed that LD1 had almost identical positioning of key hydrophobic and acidic residues relative to NSP3. Because paxillin is one of the key scaffold molecules in FAs, we propose that the interaction between the p130Cas CCHD and the LD1 motif of paxillin plays an important role in p130Cas FA targeting.

Targeted Molecular Dynamics to determine Focal Adhesion Targeting Domain Folding Intermediates

Targeted Molecular Dynamics to determine Focal Adhesion Targeting Domain Folding Intermediates

Abstract P5-05-02: NEDD9 promotes breast cancer metastasis by regulating mitochondrial functions

Abstract NEDD9 has been characterized as a metastasis-promoting gene in various cancer cells including breast. We previously reported that NEDD9 promotes malignant phenotypes of breast cancer cells through distinct and non-overlapped domains. For example, the FAT (Focal Adhesion Targeting) domain of NEDD9 promotes cancer cell growth, while the SH-domain facilitates cell migration. These results suggest that NEDD9 promotes tumor metastasis by enhancing dissemination and growth in the tumor-host microenvironments through distinct and non-overlapped domains. Thus, targeting functions of NEDD9 is a promising approach for breast cancer therapies. In order to further characterize NEDD9-mediated breast cancer growth, we performed yeast-two hybrid (Y2H) screening to identify proteins that associate with the FAT domain of NEDD9. Using the FAT domain constructed in pGBKKT7 (Clonetech, CA) as a bait to screen library of human fibroblast (Clonetech, CA), we identified several proteins that associate with the domain. They are small GTPases (i.e. RAB11a and ARF4), cytoskeletal proteins (i.e. Nexilin), and cytosolic proteins (i.e. HAX-1). Among of these potential partner proteins, we focused on the interaction between NEDD9 and HAX-1 in breast cancer cells. Co-immunoprecipitation assays confirm the molecular complex of NEDD9-HAX-1 in both SK-Br3 and SUM149 cells. Importantly, p130cas, which harbors similar domain structures with NEDD9, was not precipitated with NEDD9, suggesting a specific interaction between NEDD9 and HAX-1. Given the fact of NEDD9 as a key metastasis promoting gene, these results suggest that NEDD9-HAX-1 plays a key role breast cancer metastasis by facilitating growth in microenvironments. While the biological function are not clear at present, previous studies demonstrated that HAX-1 localizes in mitochondria in breast cancer cells, Indeed, we demonstrated that NEDD9 was found in both cytosol and mitochondria fractions in malignant breast cancer cell MDA-MB-231, but not non-metastatic HCC38. These results suggest the presence of NEDD9-HAX1 complex in mitochondria and this complex may facilitate breast cancer metastasis. In addition to HAX-1, several mitochondrial proteins such as EFG1, DCTN6, and MMADHC were found in the Y2H screening system as described above. These results suggest that NEDD9 facilitates breast cancer metastasis through regulating multiple pathways including signaling pathways and mitochondrial functions, thus serving as a promising therapeutic target for cancer patients including breast. The view expressed in this article are those of the author and do not reflect the official policy of the Department of Defense, or U.S.Government. Citation Format: Iida J, Dorchak J, Slavik J, Clancy R, Cutler ML, Shriver CD. NEDD9 promotes breast cancer metastasis by regulating mitochondrial functions [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-05-02.

The focal adhesion targeting domain of p130Cas confers a mechanosensing function.

Members of the Cas family of focal adhesion proteins contain a highly conserved C-terminal focal adhesion targeting (FAT) domain. To determine the role of the FAT domain in these proteins, we compared wild-type exogenous NEDD9 with a hybrid construct in which the NEDD9 FAT domain had been exchanged for the p130Cas (also known as BCAR1) FAT domain. Fluorescence recovery after photobleaching (FRAP) revealed significantly slowed exchange of the fusion protein at focal adhesions and significantly slower two-dimensional migration. No differences were detected in cell stiffness as measured using atomic force microscopy (AFM) and in cell adhesion forces measured with a magnetic tweezer device. Thus, the slowed migration was not due to changes in cell stiffness or adhesion strength. Analysis of cell migration on surfaces of increasing rigidity revealed a striking reduction of cell motility in cells expressing the p130Cas FAT domain. The p130Cas FAT domain induced rigidity-dependent phosphorylation of tyrosine residues within NEDD9. This in turn reduced post-translational cleavage of NEDD9, which we show inhibits NEDD9-induced migration. Collectively, our data therefore suggest that the p130Cas FAT domain uniquely confers a mechanosensing function.

FAK Forms a Complex with MEF2 to Couple Biomechanical Signaling to Transcription in Cardiomyocytes

Focal adhesion kinase (FAK) has emerged as a mediator of mechanotransduction in cardiomyocytes, regulating gene expression during hypertrophic remodeling. However, how FAK signaling is relayed onward to the nucleus is unclear. Here, we show that FAK interacts with and regulates myocyte enhancer factor 2 (MEF2), a master cardiac transcriptional regulator. In cardiomyocytes exposed to biomechanical stimulation, FAK accumulates in the nucleus, binds to and upregulates the transcriptional activity of MEF2 through an interaction with the FAK focal adhesion targeting (FAT) domain. In the crystal structure (2.9Å resolution), FAT binds to a stably folded groove in the MEF2 dimer, known to interact with regulatory cofactors. FAK cooperates with MEF2 to enhance the expression of Jun in cardiomyocytes, an important component of hypertrophic response to mechanical stress. These findings underscore a connection between the mechanotransduction involving FAK and transcriptional regulation by MEF2, with potential relevance to the pathogenesis of cardiac disease.

Focal Adhesion Kinase Directly Interacts with TSC2 Through Its FAT Domain and Regulates Cell Proliferation in Cashmere Goat Fetal Fibroblasts.

Focal adhesion kinase (FAK) is a cytoplasmic nonreceptor tyrosine kinase that senses a variety of extracellular signals, such as growth factors and integrins, to control the process of cell proliferation and metabolism. We cloned three goat FAK transcript variants (KM655805, KM658268, and KM658269) that encode 1052, 1006, and 962 amino-acid residue proteins. Bioinformatics analysis indicated that the putative FAK protein contains an FERM domain, a PTK domain, two Proline-rich regions, and a focal adhesion-targeting (FAT) domain. All the three transcript variants of FAK were detected in seven different goat tissues, and variant 1 had the most accumulation whereas variant 2 and variant 3 had lower accumulation. Treatment of goat fetal fibroblasts (GFbs) with a specific FAK inhibitor, TAE226, inhibited cell proliferation (p < 0.05) and induced damage to the cell morphology in a dose- and time-dependent manner. Further research demonstrated that FAK directly interacted with TSC2 (Tuberous sclerosis 2) tuberin domain through its C-terminus, which contains the complete FAT domain. In conclusion, our results indicated that FAK may be widely expressed in Cashmere goat tissues and its products participate in the mammalian target of rapamycin signaling pathway and cell proliferation through a direct interaction with TSC2 in GFBs.