Recruitment Of Focal Adhesion Kinase Research Articles

Hydrogels with tunable mechanical plasticity regulate endothelial cell outgrowth in vasculogenesis and angiogenesis

The endothelial cell (EC) outgrowth in both vasculogenesis and angiogenesis starts with remodeling surrounding matrix and proceeds with the crosstalk between cells for the multicellular vasculature formation. The mechanical plasticity of matrix, defined as the ability to permanently deform by external traction, is pivotal in modulating cell behaviors. Nevertheless, the implications of matrix plasticity on cell-to-cell interactions during EC outgrowth, along with the molecular pathways involved, remain elusive. Here we develop a collagen-hyaluronic acid based hydrogel platform with tunable plasticity by using compositing strategy of dynamic and covalent networks. We show that although the increasing plasticity of the hydrogel facilitates the matrix remodeling by ECs, the largest tubular lumens and the longest invading distance unexpectedly appear in hydrogels with medium plasticity instead of the highest ones. We unravel that the high plasticity of the hydrogels promotes stable integrin cluster of ECs and recruitment of focal adhesion kinase with an overenhanced contractility which downregulates the vascular endothelial cadherin expression and destabilizes the adherens junctions between individual ECs. Our results, further validated with mathematical simulations and in vivo angiogenic tests, demonstrate that a balance of matrix plasticity facilitates both cell-matrix binding and cell-to-cell adherens, for promoting vascular assembly and invasion.

Substrate rigidity modulates traction forces and stoichiometry of cell-matrix adhesions.

In cell-matrix adhesions, integrin receptors and associated proteins provide a dynamic coupling of the extracellular matrix (ECM) to the cytoskeleton. This allows bidirectional transmission of forces between the ECM and the cytoskeleton, which tunes intracellular signaling cascades that control survival, proliferation, differentiation, and motility. The quantitative relationships between recruitment of distinct cell-matrix adhesion proteins and local cellular traction forces are not known. Here, we applied quantitative super-resolution microscopy to cell-matrix adhesions formed on fibronectin-stamped elastomeric pillars and developed an approach to relate the number of talin, vinculin, paxillin, and focal adhesion kinase (FAK) molecules to the local cellular traction force. We find that FAK recruitment does not show an association with traction-force application, whereas a ∼60 pN force increase is associated with the recruitment of one talin, two vinculin, and two paxillin molecules on a substrate with an effective stiffness of 47kPa. On a substrate with a fourfold lower effective stiffness, the stoichiometry of talin:vinculin:paxillin changes to 2:12:6 for the same ∼60 pN traction force. The relative change in force-related vinculin recruitment indicates a stiffness-dependent switch in vinculin function in cell-matrix adhesions. Our results reveal a substrate-stiffness-dependent modulation of the relationship between cellular traction-force and the molecular stoichiometry of cell-matrix adhesions.

Determining Integrin Molecular Tension for the Recruitment and the Activation of Focal Adhesion Kinase

Focal adhesion kinase (FAK), a member of protein tyrosine kinase family, is an important mediator of signals transduced by integrins (major mechanosensitive transmembrane receptors transmitting cellular forces at cell matrix interface). FAK regulates various cellular functions such as cell adhesion, proliferation, differentiation and spreading. Although the biochemical role of FAK in integrin signaling is relatively more understood, the biomechanical processes for FAK's recruitment and activation by integrins remains less clear. To study the mechanobiology of FAK, Our group adopted two molecular tension tools, tension gauge tether (TGT) and integrative tension sensor (ITS), both having a designable tension threshold, selectable in the range of 10-60 pN. TGT globally knocks down integrin tensions in live cells to a designed force level, while ITS visualizes integrin tension by fluorescence imaging with high resolution. With TGT, ITS and a FRET-based biosensor reporting FAK activation, we manipulated and mapped integrin tension in live cells, and studied the consequent FAK aggregation and activation. ITS and FAK live cell imaging revealed that integrin tension higher than 12 pN was produced after FAK aggregation, indicating that FAK recruitment to focal adhesions does not require integrin tension higher than 12 pN. TGT and FAK biosensor revealed that FAK was not activated on TGT surfaces with tension tolerance (Ttol) of 12, 23 and 33 pN, but on the surfaces with Ttol of 43 pN and 54 pN. These results suggest that FAK activation by integrin signaling requires integrin tension higher than 33 pN. Overall, our study revealed that FAK recruitment and FAK activation have distinct requirement for integrin tensions at separate force levels.

Hypoxia-inducible transgelin 2 selects epithelial-to-mesenchymal transition and γ-radiation-resistant subtypes by focal adhesion kinase-associated insulin-like growth factor 1 receptor activation in non-small-cell lung cancer cells.

Microenvironment, such as hypoxia common to cancer, plays a critical role in the epithelial‐to‐mesenchymal transition (EMT) program, which is a major route of cancer metastasis and confers γ‐radiation resistance to cells. Herein, we showed that transgelin 2 (TAGLN2), an actin‐binding protein, is significantly induced in hypoxic lung cancer cells and that Snail1 is simultaneously increased, which induces EMT by downregulating E‐cadherin expression. Forced TAGLN2 expression induced severe cell death; however, a small population of cells surviving after forced TAGLN2 overexpression showed γ‐radiation resistance, which might promote tumor relapse and recurrence. These surviving cells showed high metastatic activity with an increase of EMT markers including Snail1. In these cells, TAGLN2 activated the insulin‐like growth factor 1 receptor β (IGF1Rβ)/PI3K/AKT pathway by recruitment of focal adhesion kinase to the IGF1R signaling complex. Activation of the IGF1Rβ/PI3K/AKT pathway also induced inactivation of glycogen synthase kinase 3β (GSK3β), which is involved in Snail1 stabilization. Therefore, both the IGF1Rβ inhibitor (AG1024) and the PI3K inhibitor (LY294002) or AKT inactivation with MK2206 lower the cellular level of Snail1. Involvement of GSK3β was also confirmed by treatment with lithium chloride, the inducer of GSK3β phosphorylation, or MG132, the 26S proteasomal inhibitor, which also stabilized Snail1. In conclusion, the present study provides important evidence that hypoxia‐inducible TAGLN2 is involved in the selection of cancer cells with enhanced EMT properties to overcome the detrimental environment of cancer cells.

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.

The KDEL receptor signalling cascade targets focal adhesion kinase on focal adhesions and invadopodia.

Membrane trafficking via the Golgi-localised KDEL receptor activates signalling cascades that coordinate both trafficking and other cellular functions, including autophagy and extracellular matrix degradation. In this study, we provide evidence that membrane trafficking activates KDEL receptor and the Src family kinases at focal adhesions of HeLa cells, where this phosphorylates ADP-ribosylation factor GTPase-activating protein with SH3 domain, ankyrin repeat and PH domain (ASAP)1 and focal adhesion kinase (FAK). Previous studies have reported extracellular matrix degradation at focal adhesions. Here, matrix degradation was not seen at focal adhesions, although it occurred at invadopodia, where it was increased by KDEL receptor activation. This activation of KDEL receptor at invadopodia of A375 cells promoted recruitment and phosphorylation of FAK on tyrosines 397 and 861. From the functional standpoint, FAK overexpression inhibited steady-state and KDEL-receptor-stimulated extracellular matrix degradation, whereas overexpression of the FAK-Y397F mutant only inhibited KDEL-receptor-stimulated matrix degradation. Finally, we show that the Src and FAK activated downstream of KDEL receptor are part of parallel signalling pathways. In conclusion, membrane-traffic-generated signalling via KDEL receptor activates Src not only at the Golgi complex, but also at focal adhesions. By acting on Src and FAK, KDEL receptor increases invadopodia-mediated matrix degradation.

Involvement of integrin β1/FAK signaling in the analgesic effects induced by glial cell line-derived neurotrophic factor in neuropathic pain.

Treatment of neuropathic pain (NP) continues to be a clinical challenge and the underlying mechanisms of NP remain elusive. More evidence suggests that glial cell line-derived neurotrophic factor (GDNF) has potent anti-nociceptive effects on NP, but the underlying mechanisms are still largely unknown. Recent data have shown that integrin β1 plays an important part in NP induction, and that the activity of integrin β1 signaling is associated with the phosphorylation of the conserved threonines in the cytoplasmic domain and recruitment of focal adhesion kinase (FAK) to the integrin β1 tail and phosphorylation. We assessed the effect of GDNF on integrinβ1/FAK signaling in NP states. Immunostaining results showed that integrin β1 was mainly observed in the superficial dorsal horn in the spinal cord of rats, and was mostly expressed in intrinsic neurons. Expression of p-integrin β1 and the phosphorylation of integrin β1-associated FAK, but not integrin β1 itself, was up-regulated after chronic constriction injury (CCI), which could be reversed by GDNF, and the effect of GDNF on integrin β1/FAK signaling was inhibited by pre-treatment with RET function-blocking antibody (RET Ab). Moreover, pre-treatment with RET Ab could antagonize the effect of GDNF on inhibiting the NP induced by CCI. These data suggest that GDNF can regulate integrin β1 activity via a RET-related mechanism.

Oxidation of KCNB1 potassium channels triggers apoptotic integrin signaling in the brain

Oxidative modification of the voltage-gated potassium (K+) channel KCNB1 promotes apoptosis in the neurons of cortex and hippocampus through a signaling pathway mediated by Src tyrosine kinases. How oxidation of the channel is transduced into Src recruitment and activation, however, was not known. Here we show that the apoptotic signal originates from integrins, which form macromolecular complexes with KCNB1 channels. The initial stimulus is transduced to Fyn and possibly other Src family members by focal adhesion kinase (FAK). Thus KCNB1 and integrin alpha chain V (integrin-α5) coimmunoprecipitated in the mouse brain and these interactions were retained upon channel’s oxidation. Pharmacological inhibition of integrin signaling or FAK suppressed apoptosis induced by oxidation of KCNB1, as well as FAK and Src/Fyn activation. Most importantly, the activation of the integrin–FAK–Src/Fyn cascade was negligible in the presence of non-oxidizable C73A KCNB1 mutant channels, even though they normally interacted with integrin-α5. This leads us to conclude that the transition between the non-oxidized and oxidized state of KCNB1 activates integrin signaling. KCNB1 oxidation may favor integrin clustering, thereby facilitating the recruitment and activation of FAK and Src/Fyn kinases.

Thyroid Hormone Controls Breast Cancer Cell Movement via Integrin αV/β3/SRC/FAK/PI3-Kinases.

Thyroid hormones (TH) play a fundamental role in diverse processes, including cellular movement. Cell migration requires the integration of events that induce changes in cell structure towards the direction of migration. These actions are driven by actin remodeling and stabilized by the development of adhesion sites to extracellular matrix via transmembrane receptors linked to the actin cytoskeleton. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that promotes cell migration and invasion through the control of focal adhesion turnover. In this work, we demonstrate that the thyroid hormone triiodothyronine (T3) regulates actin remodeling and cell movement in breast cancer T-47D cells through the recruitment of FAK. T3 controls FAK phosphorylation and translocation at sites where focal adhesion complexes are assembled. This process is triggered via rapid signaling to integrin αV/β3, Src, phosphatidylinositol 3-OH kinase (PI3K), and FAK. In addition, we established a cellular model with different concentration of T3 levels: normal, absence, and excess in T-47D breast cancer cells. We found that the expression of Src, FAK, and PI3K remained at normal levels in the excess of T3 model, while it was significantly reduced in the absence model. In conclusion, these results suggest a novel role for T3 as an important modulator of cell migration, providing a starting point for the development of new therapeutic strategies for breast cancer treatment.

Synergistic Activation upon MET and ALK Coamplification Sustains Targeted Therapy in Sarcomatoid Carcinoma, a Deadly Subtype of Lung Cancer

IntroductionGenetic alterations suitable for targeted therapy are poorly known issues in pulmonary sarcomatoid carcinoma (PSC), an uncommon and life-threatening family of non–small cell lung cancers. MethodsNinety-eight PSCs were assessed for MNNG HOS Transforming gene (MET) and anaplastic lymphoma receptor tyrosine kinase gene (ALK) status by fluorescence in situ hybridization (FISH) and for relevant protein expression by immunohistochemical analysis, also taking advantage of phosphorylated (p-) antibodies. Moreover, levels of ALK and MET mRNA were also determined by real-time polymerase chain reaction and Western blot analysis for downstream activation pathways involving p-MET, p–protein kinase B, p–mitogen-activated protein kinase, p-SRC proto-oncogene tyrosine-protein kinase, and p–focal adhesion kinase (p-FAK). ResultsMET amplification was detected by FISH in 25 of 98 PSCs (25.6%) and ALK amplification (but not the relevant rearrangement) was found in 16 of 98 (16.3%), with all ALK-amplified tumors also showing MET amplification (p < 0.0001). Nine PSCs, however, showed MET amplification without any ALK gene alteration. ALK protein expression was always lacking, whereas MET and p-MET were confined to the relevant amplified tumors only. Increased levels of ALK and MET mRNA were detectable in tumors with no direct relationship between mRNA content, protein expression, or alterations detected by FISH. Western blot assays showed complete activation of downstream signal pathways up to p-SRC proto-oncogene tyrosine-protein kinase, and p–focal adhesion kinase recruitment in MET and ALK–coamplified tumors only, whereas isolated MET amplification, MET and ALK borderline amplification (5%–10% of tumor cells with ≥15 copies of the relevant gene), or negative tumors showing eusomy or chromosome polysomy were confined to p–mitogen-activated protein kinase, p–protein kinase B, and/or p-MET activation. Multivariate survival analysis pushed a higher percentage of MET altered cells or a higher value of MET copy gain per cell to marginally emerge for overall survival (p = 0.140) and disease-free survival (p = 0.060), respectively. ConclusionsALK and MET seemed to act as synergistic, nonrandom coactivators of downstream signal when coamplified in a subset of patients with PSC, thus likely suggesting a combined mechanism of oncogene addiction. These alterations could be a suitable target for therapy based on specific inhibitors.

A novel role for RhoA GTPase in the regulation of airway smooth muscle contraction.

Recent studies have demonstrated a novel molecular mechanism for the regulation of airway smooth muscle (ASM) contraction by RhoA GTPase. In ASM tissues, both myosin light chain (MLC) phosphorylation and actin polymerization are required for active tension generation. RhoA inactivation dramatically suppresses agonist-induced tension development and completely inhibits agonist-induced actin polymerization, but only slightly reduces MLC phosphorylation. The inhibition of MLC phosphatase does not reverse the effects of RhoA inactivation on contraction or actin polymerization. Thus, RhoA regulates ASM contraction through its effects on actin polymerization rather than MLC phosphorylation. Contractile stimulation of ASM induces the recruitment and assembly of paxillin, vinculin, and focal adhesion kinase (FAK) into membrane adhesion complexes (adhesomes) that regulate actin polymerization by catalyzing the activation of cdc42 GTPase by the G-protein-coupled receptor kinase-interacting target (GIT) - p21-activated kinase (PAK) - PAK-interacting exchange factor (PIX) complex. Cdc42 is a necessary and specific activator of the actin filament nucleation activator, N-WASp. The recruitment and activation of paxillin, vinculin, and FAK is prevented by RhoA inactivation, thus preventing cdc42 and N-WASp activation. We conclude that RhoA regulates ASM contraction by catalyzing the assembly and activation of membrane adhesome signaling modules that regulate actin polymerization, and that the RhoA-mediated assembly of adhesome complexes is a fundamental step in the signal transduction process in response to a contractile agonist.

The Chlamydia Effector TarP Mimics the Mammalian Leucine-Aspartic Acid Motif of Paxillin to Subvert the Focal Adhesion Kinase during Invasion

Host cell signal transduction pathways are often targets of bacterial pathogens, especially during the process of invasion when robust actin remodeling is required. We demonstrate that the host cell focal adhesion kinase (FAK) was necessary for the invasion by the obligate intracellular pathogen Chlamydia caviae. Bacterial adhesion triggered the transient recruitment of FAK to the plasma membrane to mediate a Cdc42- and Arp2/3-dependent actin assembly. FAK recruitment was via binding to a domain within the virulence factor TarP that mimicked the LD2 motif of the FAK binding partner paxillin. Importantly, bacterial two-hybrid and quantitative imaging assays revealed a similar level of interaction between paxillin-LD2 and TarP-LD. The conserved leucine residues within the L(D/E)XLLXXL motif were essential to the recruitment of FAK, Cdc42, p34(Arc), and actin to the plasma membrane. In the absence of FAK, TarP-LD-mediated F-actin assembly was reduced, highlighting the functional relevance of this interaction. Together, the data indicate that a prokaryotic version of the paxillin LD2 domain targets the FAK signaling pathway, with TarP representing the first example of an LD-containing Type III virulence effector.

FAK promotes recruitment of talin to nascent adhesions to control cell motility

Cell migration is a dynamic process that involves the continuous formation, maturation, and turnover of matrix-cell adhesion sites. New (nascent) adhesions form at the protruding cell edge in a tension-independent manner and are comprised of integrin receptors, signaling, and cytoskeletal-associated proteins. Integrins recruit focal adhesion kinase (FAK) and the cytoskeletal protein talin to nascent adhesions. Canonical models support a role for talin in mediating FAK localization and activation at adhesions. Here, alternatively, we show that FAK promotes talin recruitment to nascent adhesions occurring independently of talin binding to β1 integrins. The direct binding site for talin on FAK was identified, and a point mutation in FAK (E1015A) prevented talin association and talin localization to nascent adhesions but did not alter integrin-mediated FAK recruitment and activation at adhesions. Moreover, FAK E1015A inhibited cell motility and proteolytic talin cleavage needed for efficient adhesion dynamics. These results support an alternative linkage for FAK-talin interactions within nascent adhesions essential for the control of cell migration.

FAK promotes recruitment of talin to nascent adhesions to control cell motility

Cell migration is a dynamic process that involves the continuous formation, maturation, and turnover of matrix-cell adhesion sites. New (nascent) adhesions form at the protruding cell edge in a tension-independent manner and are comprised of integrin receptors, signaling, and cytoskeletal-associated proteins. Integrins recruit focal adhesion kinase (FAK) and the cytoskeletal protein talin to nascent adhesions. Canonical models support a role for talin in mediating FAK localization and activation at adhesions. Here, alternatively, we show that FAK promotes talin recruitment to nascent adhesions occurring independently of talin binding to β1 integrins. The direct binding site for talin on FAK was identified, and a point mutation in FAK (E1015A) prevented talin association and talin localization to nascent adhesions but did not alter integrin-mediated FAK recruitment and activation at adhesions. Moreover, FAK E1015A inhibited cell motility and proteolytic talin cleavage needed for efficient adhesion dynamics. These results support an alternative linkage for FAK-talin interactions within nascent adhesions essential for the control of cell migration.

Essential diurnal Rac1 activation during retinal phagocytosis requires αvβ5 integrin but not tyrosine kinases focal adhesion kinase or Mer tyrosine kinase

Diurnal phagocytosis of shed photoreceptor outer-segment particles by retinal pigment epithelial (RPE) cells belongs to a group of conserved clearance mechanisms employing αv integrins upstream of tyrosine kinases and Rho GTPases. In this study, we tested the interdependence of the tyrosine kinases focal adhesion kinase (FAK) and Mer tyrosine kinase (MerTK) and Rho GTPases during engulfment. RPE cells activated and redistributed Rac1, but not RhoA or Cdc42, during phagocytosis. Toxin B, overexpression of dominant-negative Rac1, or decreasing Rac1 expression prevented particle engulfment. Fluorescence microscopy showed that Rac1 inhibition had no obvious effect on F-actin arrangement in resting RPE but prevented recruitment of F-actin to surface-bound phagocytic particles. Quantification of active GTP-Rac1 in wild-type and mutant RPE in culture and in vivo revealed that Rac1 activation during phagocytosis requires αvβ5 integrin and its ligand milk fat globule EGF factor-8 (MFG-E8) but not the receptor tyrosine kinase MerTK. Abolishing tyrosine kinase signaling downstream of αvβ5 toward MerTK by inhibiting FAK specifically or tyrosine kinases generally neither prevented Rac1 activation nor F-actin recruitment during phagocytosis. Likewise, inhibiting Rac1 had no effect on FAK or MerTK activation. We conclude that MerTK activation via FAK and F-actin recruitment via Rac1 both require MFG-E8-ligated αvβ5 integrin. Both pathways are independently activated and required for clearance phagocytosis.

Estrogen receptor- promotes endothelial cell motility through focal adhesion kinase

Sex steroids play a key role in cell movement and tissue organization. Cell migration requires the integration of events that induce changes in cell structure such as protrusion, polarization and traction toward the direction of migration. These actions are driven by actin remodeling and are stabilized by the development of adhesion sites to extracellular matrix via transmembrane receptors linked to the actin cytoskeleton. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that facilitates cell migration via the control of the turnover of focal adhesion complexes. In this work, we demonstrated that 17β-estradiol (E(2)) regulates actin remodeling and cell movement in human umbilical vein endothelial cells through the recruitment of FAK. E(2) induces phosphorylation of FAK and its translocation toward membrane sites where focal adhesion complexes are assembled. This process is triggered via a Gα/Gβ protein-dependent, rapid extra-nuclear signaling of estrogen receptor-α (ERα) that interacts in a multiprotein complex with c-Src, phosphatidylinositol 3-OH kinase and FAK. Phosphorylation of FAK is fundamental for its activation, translocation to the plasmatic membrane and the subsequent formation of focal adhesion complexes. In conclusion, we found that ERα enhances endothelial cell motility through the dynamic control of actin arrangement and the formation of focal adhesion complexes. The identification of these processes broadens the understanding of the actions of estrogens on endothelial cells and could be relevant in physiological or pathological settings.

Estrogen Receptor-α Promotes Breast Cancer Cell Motility and Invasion via Focal Adhesion Kinase and N-WASP

The ability of cancer cells to move and invade the surrounding environment is the basis of local and distant metastasis. Cancer cell movement requires dynamic remodeling of the cytoskeleton and cell membrane and is controlled by multiprotein complexes including focal adhesion kinase (FAK) or the Neural Wiskott-Aldrich Syndrome Protein (N-WASP). We show that 17β-estradiol induces phosphorylation of FAK and its translocation toward membrane sites where focal adhesion complexes are assembled. This process is triggered via a Gα/Gβ protein-dependent, rapid extranuclear signaling of estrogen receptor α interacts in a multiprotein complex with c-Src, phosphatidylinositol 3-OH kinase, and FAK. Within this complex FAK autophosphorylation ensues, and activated FAK recruits the small GTPase cdc42, which, in turn, triggers N-WASP phosphorylation. This results in the translocation of Arp2/3 complexes at sites where membrane structures related to cell movement are formed. Recruitment of FAK and N-WASP is necessary for cell migration and invasion induced by 17β-estradiol in breast cancer cells. Our findings identify an original mechanism through which estrogen promotes breast cancer cell motility and invasion. This information helps to understand the effects of estrogen on breast cancer metastasis and may provide new targets for therapeutic interventions.

X-linked inhibitor of apoptosis protein controls α5-integrin-mediated cell adhesion and migration

The association of integrins with caveolin-1 regulates cell adhesion. However, the vascular ramifications of this association remain to be clearly determined. We recently reported that the X chromosome-linked inhibitor of apoptosis protein (XIAP)-caveolin-1 interaction is critical to endothelial cell survival. Thus, we hypothesized that XIAP performs a crucial function in integrin/caveolin-1-mediated endothelial cell survival. In this study, we demonstrated that XIAP is recruited into the alpha(5)-integrin complex via caveolin-1 binding and mediates cell adhesion. We also determined that XIAP is critical to shear stress-stimulated ERK activation in an alpha(5)-integrin-dependent manner but is not important to VEGF-induced ERK activation. This differential activation of ERK is partly attributable to unique localizations of the receptors. Furthermore, we confirmed that XIAP is an essential molecule in the efficient recruitment of focal adhesion kinase (FAK) into the alpha(5)-integrin-associated complex. This alpha(5)-integrin-caveolin-1-XIAP-FAK multicomplex regulates endothelial cell migration via a mechanism that involves shear-dependent ERK activation. Together, our results indicate that XIAP stabilizes the alpha(5)-integrin-associated focal adhesion complex, thereby further regulating endothelial cell adhesion and migration. The findings of this study provide us with greater insight into the molecular mechanisms underlying the control of vascular function by integrins.

Focal Adhesion Kinase-Related Proline-Rich Tyrosine Kinase 2 and Focal Adhesion Kinase Are Co-Overexpressed in Early-Stage and Invasive ErbB-2-Positive Breast Cancer and Cooperate for Breast Cancer Cell Tumorigenesis and Invasiveness

Early cancer cell migration and invasion of neighboring tissues are mediated by multiple events, including activation of focal adhesion signaling. Key regulators include the focal adhesion kinase (FAK) and FAK-related proline-rich tyrosine kinase 2 (Pyk2), whose distinct functions in cancer progression remain unclear. Here, we compared Pyk2 and FAK expression in breast cancer and their effects on ErbB-2-induced tumorigenesis and the potential therapeutic utility of targeting Pyk2 compared with FAK in preclinical models of breast cancer. Pyk2 is overexpressed in tissues from early and advanced breast cancers and overexpressed with both FAK and epidermal growth factor receptor-2 (ErbB-2) in a subset of breast cancer cases. Down-regulation of Pyk2 in ErbB-2-positive, FAK-proficient, and FAK-deficient cells reduced cell proliferation, which correlated with reduced mitogen-activated protein kinase (MAPK) activity. In contrast, Pyk2 silencing had little impact on cell migration and invasion. In vivo, Pyk2 down-regulation reduced primary tumor growth induced by a metastatic variant of ErbB-2-positive MDA 231 breast cancer cells but had little effect on lung metastases in contrast to FAK down-regulation. Dual reduction of Pyk2 and FAK expression resulted in strong inhibition of both primary tumor growth and lung metastases. Together, these data support the cooperative function of Pyk2 and FAK in breast cancer progression and suggest that dual inhibition of FAK and Pyk2 is an efficient therapeutic approach for targeting invasive breast cancer.

Focal Adhesion Kinase Regulates Critical Functions in Hematopoiesis.

Focal adhesion kinase (FAK) initially identified as a unique cytoplasmic tyrosine kinase involved in focal adhesions, has been studied extensively in fibroblasts. In these cells, FAK has been shown to play an essential role in bridging signals between integrin and growth factor receptors such as the PDGF and the EGF receptor. In fibroblasts, FAK localizes to regions of the cell that attach to the extracellular matrix and coordinates signals from integrins, cytokines, growth factor receptors, and oncogenes. In some tumors, FAK is over-expressed or constitutively activated, which correlates with increased motility, invasiveness, and proliferation. More recently, expression of FAK in acute myeloid leukemia was associated with enhanced blast migration, increased cellularity, and poor prognosis. However, virtually nothing is known about FAKs role in normal hematopoiesis. FAK is expressed in blood cells, including in bone marrow derived KIT+, Gr-1+, Mac-1+, CD4+, CD8+ and B220+ cells. To determine how loss of FAK affects hematopoiesis, we have generated a mouse model with hematopoietic restricted deletion of FAK. We deleted FAK in bone marrow cells by crossing the FAK-flox mice to Mx.Cre+ expressing mice and by treating Mx.cre+FAK+/+ and Mx.cre+FAKflox/flox mice with poly (I)-poly(C) and then analyzing mice 1 month after the last injection. After one month of poly(I)-poly(C) induction, the progeny failed to express detectable levels of FAK in bone marrow, spleen as well as in bone marrow derived macrophages as determined by PCR and western blotting. Evaluation of peripheral blood counts in control and FAK deleted mice revealed modest but significant differences in different lineages (WBC k/μl: FAK; 14 vs. FAK−/−; 10.3, n=7, *p<0.05, LY k/μl: FAK; 10.48 vs. FAK−/−; 7.26, n=7, *p<0.005, RBC k/μl: FAK; 9.76 X106 vs. FAK−/−;8.58 X106 n=7 *p<0.003, PLT k/μl: FAK; 644 vs. FAK−/−; 434 n=7 *p<0.007). Since macrophages express abundant levels of FAK and are rapidly recruited in large numbers to sites of infection, we initially examined the role of FAK in macrophages by creating a well studied model of aseptic thioglycolate-induced peritonitis. Our results demonstrate a ∼1.5 fold reduction in the migration of macrophages to the peritoneal cavity of FAK−/− mice compared to controls (n=5, FAK; 1.8 X 106 vs. FAK−/−; 1.213 X106, *p<0.03). The reduction in recruitment of FAK−/− macrophages was observed in spite of comparable levels of F4/80 expression (WT; 92.98% vs. FAK−/−; 94.55%) as well as integrin (α4β1 & α5β1) expression (WT; 68% & 83.79% vs. FAK−/−; 60.39% & 83.17%, respectively) between WT and FAK−/− macrophages. Further analysis of FAK−/− macrophages revealed a significant decrease in extracellular matrix/integrin directed migration of these cells in response to M-CSF on fibronectin (40% reduction), laminin (55% reduction) and collagen (60% reduction) (n=3, *p<0.004) coated plates as well as a decrease in migration in a wound healing assay (n=3, *p<0.003). The reduction in migration of FAK−/− macrophages was associated with a significant decrease in adhesion on fibronectin (63%), laminin (52%) and collagen (56%) as well as spreading (n=3, *p<0.03). Taken together, our results provide a critical physiologic role for FAK in regulating several adhesive and migratory functions in cells of myeloid lineage. Additional functions of FAK in other hematopoietic lineages will be discussed.