Clinical significance of FAK expression in human neoplasia

Focal Adhesion Kinase is a 119-121 kDa nonreceptor protein kinase widely expressed in various tissues and cell types. Several studies showed that FAK plays an important role in integrin signaling. Once activated by integrin and non-integrin stimuli, it binds and activates several other molecules, such as Src, p130Cas, Grb2, PI3K and paxillin, thus promoting signaling transduction. In normal cells FAK activity is under constant regulation by mechanisms such as gene amplification, alternative splicing and action of phosphatases. On the contrary, in vitro studies showed that in transformed cells unopposed FAK signaling promoted cancer cells' malignant characteristics. FAK was held responsible for cancer cells' uninhibited proliferation, protection from apoptosis, invasion, migration, adhesion and spreading, as well as tumor angiogenesis. Several in vivo studies supported the above observations and further correlated FAK expression with various clinicopathological parameters of several types of human malignancies. The purpose of this article is a comprehensive review of the existing data on FAK expression and signaling and their clinical significance in human malignancy.     

Paxillin and Focal Adhesion Kinase (FAK) Regulate Cardiac Contractility in the Zebrafish Heart

An orchestrated interplay of adaptor and signaling proteins at mechano-sensitive sites is essential to maintain cardiac contractility and when defective leads to heart failure. We recently showed that Integrin-linked Kinase (ILK), ß-Parvin and PINCH form the IPP-complex to grant tuned Protein Kinase B (PKB) signaling in the heart. Loss of one of the IPP-complex components results in destabilization of the whole complex, defective PKB signaling and finally heart failure. Two components of IPP, ILK and ß-Parvin directly bind to Paxillin; however, the impact of this direct interaction on the maintenance of heart function is not known yet. Here, we show that targeted gene inactivation of Paxillin results in progressive decrease of cardiac contractility and heart failure in zebrafish without affecting IPP-complex stability and PKB phosphorylation. However, we found that Paxillin deficiency leads to the destabilization of its known binding partner Focal Adhesion Kinase (FAK) and vice versa resulting in degradation of Vinculin and thereby heart failure. Our findings highlight an essential role of Paxillin and FAK in controlling cardiac contractility via the recruitment of Vinculin to mechano-sensitive sites in cardiomyocytes.     

Regulation of CDC42 GTPase by proline-rich tyrosine kinase 2 interacting with PSGAP, a novel pleckstrin homology and Src homology 3 domain containing rhoGAP protein

Proline-rich tyrosine kinase 2 (PYK2), a tyrosine kinase structurally related to focal adhesion kinase (FAK), is implicated in regulating cytoskeletal organization. However, mechanisms by which PYK2 participates in and regulates cytoskeletal organization remain largely unknown. Here we report identification of PSGAP, a novel protein that interacts with PYK2 and FAK and contains multiple domains including a pleckstrin homology domain, a rhoGTPase-activating protein domain, and a Src homology 3 domain. PYK2 interacts with PSGAP Src homology 3 domain via the carboxyl-terminal proline-rich sequence. PSGAP is able to increase GTPase activity of CDC42 and RhoA in vitro and in vivo. Remarkably, PYK2, but not FAK, can activate CDC42 via inhibition of PSGAP-mediated GTP hydrolysis of CDC42. Moreover, PSGAP is localized at cell periphery in fibroblasts in a pleckstrin homology domain-dependent manner. Over expression of PSGAP in fibroblasts results in reorganization of cytoskeletal structures and changes of cellular morphology, which requires rhoGTPase-activating activity. Taken together, our results suggest that PSGAP is a signaling protein essential for PYK2 regulation of cytoskeletal organization via Rho family GTPases.     

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.     

Induction of Apoptosis after Expression of PYK2, a Tyrosine Kinase Structurally Related to Focal Adhesion Kinase

Many cells (e.g., epithelial cells) require attachment to the extracellular matrix (ECM) to survive, a phenomenon known as anchorage-dependent cell survival. Disruption of the cell–ECM interactions mediated by the integrin receptors results in apoptosis. Focal adhesion kinase (FAK), a 125-kD protein tyrosine kinase activated by integrin engagement, appears to be involved in mediating cell attachment and survival. Proline-rich tyrosine kinase 2 (PYK2), also known as cellular adhesion kinase β (CAKβ) and related adhesion focal tyrosine kinase, is a second member of the FAK subfamily and is activated by an increase in intracellular calcium levels, or treatment with TNFα and UV light. However, the function of PYK2 remains largely unknown. In this study, we show that over-expression of PYK2, but not FAK, in rat and mouse fibroblasts leads to apoptotic cell death. Using a series of deletion mutants and chimeric fusion proteins of PYK2/FAK, we determined that the NH₂-terminal domain and tyrosine kinase activity of PYK2 were required for the efficient induction of apoptosis. Furthermore, the apoptosis mediated by PYK2 could be suppressed by over-expressing catalytically active v-Src, c-Src, phosphatidylinositol-3-kinase, or Akt/protein kinase B. In addition, it could also be suppressed by overexpressing an ICE or ICE-like proteinase inhibitor, crmA, but not Bcl2. Collectively, our results suggest that PYK2 and FAK, albeit highly homologous in primary structure, appear to have different functions; FAK is required for cell survival, whereas PYK2 induces apoptosis in fibroblasts.     

PYK2 expression and phosphorylation increases in pressure overload-induced left ventricular hypertrophy

Proline-rich tyrosine kinase 2 (PYK2) is a member of the focal adhesion kinase (FAK) family of nonreceptor protein tyrosine kinases. PYK2 has been implicated in linking G protein-coupled receptors to activation of mitogen-activated protein kinase cascades and cellular growth in a variety of cell types. To determine whether PYK2 expression and phosphorylation is altered in left ventricular (LV) myocardium undergoing LV hypertrophy (LVH) and heart failure in vivo, suprarenal abdominal aortic coarctation was performed in 160-g male Sprague-Dawley rats. Immunohistochemistry and Western blotting were performed on LV tissue 1, 8, and 24 wk after aortic banding. Aortic banding produced sustained hypertension and gradually developing LVH. PYK2 levels were increased 1.8 +/- 0.2-, 2.7 +/- 0.6-, and 2.0 +/- 0.2-fold in 1-, 8-, and 24-wk banded animals compared with their respective sham-operated controls. The increase in PYK2 expression was paralleled by an increase in PYK2 phosphorylation, both of which preceded the development of LVH. Immunohistochemistry revealed that enhanced PYK2 expression occurred predominantly in the cardiomyocyte population. Furthermore, there was a high degree of correlation (R = 0.75; P < 0.001) between the level of PYK2 and the degree of LVH in 24-wk sham and banded animals. In contrast, FAK levels and FAK phosphorylation were not increased before the development of LVH. However, there was a high degree of correlation (R = 0.68; P < 0.001) between the level of FAK and the degree of LVH in 24-wk sham and banded rats. There was also a significant increase in the ratio of phosphospecific anti-FAK to FAK at this time point. These data are consistent with a role for PYK2 in the induction of pressure overload-induced cardiomyocyte hypertrophy, and suggest that PYK2 and FAK have distinctly different roles in LVH progression.     

FAK Acts as a Suppressor of RTK-MAP Kinase Signalling in Drosophila melanogaster Epithelia and Human Cancer Cells

Receptor Tyrosine Kinases (RTKs) and Focal Adhesion Kinase (FAK) regulate multiple signalling pathways, including mitogen-activated protein (MAP) kinase pathway. FAK interacts with several RTKs but little is known about how FAK regulates their downstream signalling. Here we investigated how FAK regulates signalling resulting from the overexpression of the RTKs RET and EGFR. FAK suppressed RTKs signalling in Drosophila melanogaster epithelia by impairing MAPK pathway. This regulation was also observed in MDA-MB-231 human breast cancer cells, suggesting it is a conserved phenomenon in humans. Mechanistically, FAK reduced receptor recycling into the plasma membrane, which resulted in lower MAPK activation. Conversely, increasing the membrane pool of the receptor increased MAPK pathway signalling. FAK is widely considered as a therapeutic target in cancer biology; however, it also has tumour suppressor properties in some contexts. Therefore, the FAK-mediated negative regulation of RTK/MAPK signalling described here may have potential implications in the designing of therapy strategies for RTK-driven tumours.     

Activation of Endogenous FAK via Expression of Its Amino Terminal Domain in Xenopus Embryos

Background

The Focal Adhesion Kinase is a well studied tyrosine kinase involved in a wide number of cellular processes including cell adhesion and migration. It has also been shown to play important roles during embryonic development and targeted disruption of the FAK gene in mice results in embryonic lethality by day 8.5.

Principal Findings

Here we examined the pattern of phosphorylation of FAK during Xenopus development and found that FAK is phosphorylated on all major tyrosine residues examined from early blastula stages well before any morphogenetic movements take place. We go on to show that FRNK fails to act as a dominant negative in the context of the early embryo and that the FERM domain has a major role in determining FAK’s localization at the plasma membrane. Finally, we show that autonomous expression of the FERM domain leads to the activation of endogenous FAK in a tyrosine 397 dependent fashion.

Conclusions

Overall, our data suggest an important role for the FERM domain in the activation of FAK and indicate that integrin signalling plays a limited role in the in vivo activation of FAK at least during the early stages of development.     

Focal adhesion kinase regulates cell-cell contact formation in epithelial cells via modulation of Rho

Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase that plays a key role in cellular processes such as cell adhesion, migration, proliferation and survival. Recent studies have also implicated FAK in the regulation of cell-cell adhesion. Here, evidence is presented showing that siRNA-mediated suppression of FAK levels in NBT-II cells and expression of dominant negative mutants of FAK caused loss of epithelial cell morphology and inhibited the formation of cell-cell adhesions. Rac and Rho have been implicated in the regulation of cell-cell adhesions and can be regulated by FAK signaling. Expression of active Rac or Rho in NBT-II cells disrupted formation of cell-cell contacts, thus promoting a phenotype similar to FAK-depleted cells. The loss of intercellular contacts in FAK-depleted cells is prevented upon expression of a dominant negative Rho mutant, but not a dominant negative Rac mutant. Inhibition of FAK decreased tyrosine phosphorylation of p190RhoGAP and elevated the level of GTP-bound Rho. This suggests that FAK regulates cell-cell contact formation by regulation of Rho.     

Focal Adhesion Kinase contributes to insulin-induced actin reorganization into a mesh harboring Glucose transporter-4 in insulin resistant skeletal muscle cells

Background  

Focal Adhesion Kinase (FAK) is recently reported to regulate insulin resistance by regulating glucose uptake in C2C12 skeletal muscle cells. However, the underlying mechanism for FAK-mediated glucose transporter-4 translocation (Glut-4), responsible for glucose uptake, remains unknown. Recently actin remodeling was reported to be essential for Glut-4 translocation. Therefore, we investigated whether FAK contributes to insulin-induced actin remodeling and harbor Glut-4 for glucose transport and whether downregulation of FAK affects the remodeling and causes insulin resistance.     

Src/FAK-mediated regulation of E-cadherin as a mechanism for controlling collective cell movement: Insights from in vivo imaging

Recent advances in confocal and multi-photon microscopy, together with fluorescent probe development, have enabled cancer biology studies to go beyond the culture dish and interrogate cancer-associated processes in the complex in vivo environment. Regulation of the tumor suppressor protein E-cadherin plays an important role in cancer development and progression, and may contribute to the decision between ‘single cell’ and ‘collective invasion’ in vivo. Mounting evidence from in vitro and in vivo experiments places the two nonreceptor protein tyrosine kinases Src and Focal Adhesion Kinase at the heart of E-cadherin regulation and the crosstalk between integrins and cadherins. Here we discuss recent insights, attained using high-resolution fluorescent in vivo imaging, into the regulation of E-cadherin and collective invasion. We focus on the regulatory crosstalk between the Src/FAK signaling axis and E-cadherin in vivo.Key words: Src, FAK, E-cadherin, EMT, fluorescent in vivo imaging     

PYK2 is overexpressed in chronic lymphocytic leukaemia: A potential new therapeutic target

Chronic Lymphocytic Leukaemia (CLL) is the most common adult B-cell leukaemia and despite improvement in patients' outcome, following the use of targeted therapies, it remains incurable. CLL supportive microenvironment plays a key role in both CLL progression and drug resistance through signals that can be sensed by the main components of the focal adhesion complex, such as FAK and PYK2 kinases. Dysregulations of both kinases have been observed in several metastatic cancers, but their role in haematological malignancies is still poorly defined. We characterized FAK and PYK2 expression and observed that PYK2 expression is higher in leukaemic B cells and its overexpression significantly correlates with their malignant transformation. When targeting both FAK and PYK2 with the specific inhibitor defactinib, we observed a dose–response effect on CLL cells viability and survival. In vivo treatment of a CLL mouse model showed a decrease of the leukaemic clone in all the lymphoid organs along with a significant reduction of macrophages and of the spleen weight and size. Our results first define a possible prognostic value for PYK2 in CLL, and show that both FAK and PYK2 might become putative targets for both CLL and its microenvironment (e.g. macrophages), thus paving the way to an innovative therapeutic strategy.     

Identification of sequences required for the efficient localization of the focal adhesion kinase, pp125FAK, to cellular focal adhesions

The integrin family of heterodimeric cell surface receptors play critical roles in multiple biological processes by mediating cellular adhesion to the extracellular matrix (ECM). Adhesion triggers intracellular signaling cascades, including tyrosine phosphorylation and elevation of [Ca2+]i. The Focal Adhesion Kinase (FAK or pp125FAK), a protein tyrosine kinase that colocalizes with integrins in cellular focal adhesions, is a prime candidate for a mediator of integrin signaling events. Here we report an analysis of the domain structure of FAK in which we have identified a contiguous stretch of 159 amino acids within the COOH terminus essential for correct subcellular localization. When placed in the context of an unrelated cytosolic protein, this Focal Adhesion Targeting (FAT) sequence functions to efficiently mediate the focal adhesion localization of this fusion protein. Furthermore, this analysis suggests that pp125FAK cannot be activated oncogenically by mutation. This result could be explained if pp125FK either exhibits a narrow substrate specificity or is diametrically opposed by cellular phosphatases or other cellular processes.     

Fibronectin induces macrophage migration through a SFK-FAK/CSF-1R pathway

Integrins, following binding to proteins of the extracellular matrix (ECM) including collagen, laminin and fibronectin (FN), are able to transduce molecular signals inside the cells and to regulate several biological functions such as migration, proliferation and differentiation. Besides activation of adaptor molecules and kinases, integrins transactivate Receptor Tyrosine Kinases (RTK). In particular, adhesion to the ECM may promote RTK activation in the absence of growth factors. The Colony-Stimulating Factor-1 Receptor (CSF-1R) is a RTK that supports the survival, proliferation, and motility of monocytes/macrophages, which are essential components of innate immunity and cancer development. Macrophage interaction with FN is recognized as an important aspect of host defense and wound repair. The aim of the present study was to investigate on a possible cross-talk between FN-elicited signals and CSF-1R in macrophages. FN induced migration in BAC1.2F5 and J774 murine macrophage cell lines and in human primary macrophages. Adhesion to FN determined phosphorylation of the Focal Adhesion Kinase (FAK) and Src Family Kinases (SFK) and activation of the SFK/FAK complex, as witnessed by paxillin phosphorylation. SFK activity was necessary for FAK activation and macrophage migration. Moreover, FN-induced migration was dependent on FAK in either murine macrophage cell lines or human primary macrophages. FN also induced FAK-dependent/ligand-independent CSF-1R phosphorylation, as well as the interaction between CSF-1R and β1. CSF-1R activity was necessary for FN-induced macrophage migration. Indeed, genetic or pharmacological inhibition of CSF-1R prevented FN-induced macrophage migration. Our results identified a new SFK-FAK/CSF-1R signaling pathway that mediates FN-induced migration of macrophages.     

The translocation of focal adhesion kinase in brain synaptosomes is regulated by phosphorylation and actin assembly

Focal adhesion kinase (FAK) and the related proline-rich tyrosine kinase 2 (PYK2) are non-receptor protein tyrosine kinases that transduce extracellular signals through the activation of Src family kinases and are highly enriched in neurones. To further elucidate the regulation of FAK and PYK2 in nervous tissue, we investigated their distribution in brain subcellular fractions and analysed their translocation between membrane and cytosolic compartments. We have found that FAK and PYK2 are present in a small membrane-associated pool and a larger cytosolic pool in various neuronal compartments including nerve terminals. In intact nerve terminals, inhibition of Src kinases inhibited the membrane association of FAK, but not of PYK2, whereas tyrosine phosphatase inhibition sharply increased the membrane association of both FAK and PYK2. Disruption of the actin cytoskeleton was followed by a decrease in the membrane-associated pool of FAK, but not of PYK2. For both kinases, a significant correlation was found between autophosphorylation and membrane association. The data indicate that FAK and PYK2 are present in nerve terminals and that the membrane association of FAK is regulated by both phosphorylation and actin assembly, whereas that of PKY2 is primarily dependent on its phosphorylation state.     

Mechanism of Focal Adhesion Kinase Mechanosensing

Mechanosensing at focal adhesions regulates vital cellular processes. Here, we present results from molecular dynamics (MD) and mechano-biochemical network simulations that suggest a direct role of Focal Adhesion Kinase (FAK) as a mechano-sensor. Tensile forces, propagating from the membrane through the PIP₂ binding site of the FERM domain and from the cytoskeleton-anchored FAT domain, activate FAK by unlocking its central phosphorylation site (Tyr576/577) from the autoinhibitory FERM domain. Varying loading rates, pulling directions, and membrane PIP₂ concentrations corroborate the specific opening of the FERM-kinase domain interface, due to its remarkably lower mechanical stability compared to the individual alpha-helical domains and the PIP₂-FERM link. Analyzing downstream signaling networks provides further evidence for an intrinsic mechano-signaling role of FAK in broadcasting force signals through Ras to the nucleus. This distinguishes FAK from hitherto identified focal adhesion mechano-responsive molecules, allowing a new interpretation of cell stretching experiments.     

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 (K_d = 1.5 μM vs no-binding with wild type) and LD2 peptides (K_d = 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.     

The tyrosine phosphatase HD-PTP: A novel player in endothelial migration

Endothelial migration, pivotal step of angiogenesis, is tightly tuned by tyrosine phosphorylation of different substrates, which results from the coordinated action of tyrosine kinases and phosphatases. Here we report that the tyrosine phosphatase HD-PTP has a role in modulating endothelial motility. Indeed, we found that endothelial cells downregulating HD-PTP after transfection with siRNA acquire a scattered and spindle-shaped phenotype and migrate more than controls. We also show that HD-PTP binds Focal Adhesion Kinase (FAK), a crucial regulator of cell migration. This interaction is strongly inhibited by treatment with basic Fibroblast Growth Factor, an angiogenic factor which stimulates endothelial cell migration. In cells downregulating HD-PTP, FAK is hyperphosphorylated on tyrosine residues and localizes in the focal adhesions, at the leading edge of the cell.We suggest that HD-PTP contributes to the regulation of endothelial motility by modulating the tyrosine phosphorylation of FAK.     

Dibutyryl cyclic AMP-induced process formation in astrocytes is associated with a decrease in tyrosine phosphorylation of focal adhesion kinase and paxillin.

Focal adhesion kinase (FAK or pp125FAK) is a cytosolic protein tyrosine kinase which plays an important role in integrin-mediated signal transduction. Adhesion of cells to the substratum correlates with an increase in tyrosine phosphorylation of FAK as well as an associated protein, paxillin. In this report we show that the tyrosine phosphorylation of FAK and paxillin are decreased during dibutyryl cyclic AMP-induced (dB-cAMP) process formation in astrocytes. When astrocytes in suspension are treated with dB-cAMP, no alteration in morphology or tyrosine phosphorylation is observed, suggesting that both phenomena are linked and adhesion dependent. Furthermore, genistein, a tyrosine kinase inhibitor, can induce process formation in such cells, underscoring the significance of protein tyrosine kinases in maintaining the morphology of adherent cells. Finally, endothelin-1, a vasopeptide which is known to inhibit process formation in astrocytes, inhibited the tyrosine dephosphorylation of proteins associated with dB-cAMP treatment. These results suggest that the formation of asymmetric processes in astrocytes results from a coordinated set of alterations in the actin cytoskeleton as well as the adhesion of the cell to the substratum. Modification of the properties of such molecules is required for process formation and the dynamic modulation of astrocytic morphology in vitro and in vivo.