Differential regulation of components of the focal adhesion complex by heregulin: role of phosphatase SHP-2.

Heregulin (HRG) has been implicated in the progression of breast cancer cells to a malignant phenotype, a process that involves changes in cell motility and adhesion. Here we demonstrate that HRG differentially regulates the site-specific phosphorylation of the focal adhesion components focal adhesion kinase (FAK) and paxilin in a dose-dependent manner. HRG at suboptimal doses (0.01 and 0.1 nM) increased adhesion of cells to the substratum, induced phosphorylation of FAK at Tyr-577, -925, and induced formation of well-defined focal points in breast cancer cell line MCF-7. HRG at a dose of 1 nM, increased migratory potential of breast cancer cells, selectively dephosphorylated FAK at Tyr-577, -925, and paxillin at Tyr-31. Tyrosine phosphorylation of FAK at Tyr-397 remained unaffected by HRG stimulation. FAK associated with HER2 only in response to 0.01 nM HRG. In contrast, 1 nM HRG induced activation and increased association of tyrosine phosphatase SHP-2 with HER2 but decreased association of HER2 with FAK. Expression of dominant-negative SHP-2 blocked HRG-mediated dephosphorylation of FAK and paxillin, leading to persistent accumulation of mature focal points. Our results suggest that HRG differentially regulates signaling from focal adhesion complexes through selective phosphorylation and dephosphorylation and that tyrosine phosphatase SHP-2 has a role in the HRG signaling.     

Phosphospecific antibodies reveal focal adhesion kinase activation loop phosphorylation in nascent and mature focal adhesions and requirement for the autophosphorylation site.

Focal adhesion kinase (FAK) is a key signaling molecule regulating cellular responses to integrin-mediated adhesion. Integrin engagement promotes FAK phosphorylation at multiple sites to achieve full FAK activation. Phosphorylation of FAK Tyr-397 creates a binding site for Src-family kinases, and phosphorylation of FAK Tyr-576/Tyr-577 in the kinase domain activation loop enhances catalytic activity. Using novel phosphospecific antibody reagents, we show that FAK activation loop phosphorylation is significantly elevated in cells expressing activated Src and is an early event following cell adhesion to fibronectin. In both cases, this regulation is largely dependent on Tyr-397. We also show that the FAK activation loop tyrosines are required for maximal Tyr-397 phosphorylation. Finally, immunostaining analyses revealed that tyrosine-phosphorylated forms of FAK are present in both newly forming and mature focal adhesions. Our findings support a model for reciprocal activation of FAK and Src-family kinases and suggest that FAK/Src signaling may occur during both focal adhesion assembly and turnover.     

Induced Focal Adhesion Kinase (FAK) Expression in FAK-Null Cells Enhances Cell Spreading and Migration Requiring Both Auto- and Activation Loop Phosphorylation Sites and Inhibits Adhesion-Dependent Tyrosine Phosphorylation of Pyk2

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase involved in integrin-mediated control of cell behavior. Following cell adhesion to components of the extracellular matrix, FAK becomes phosphorylated at multiple sites, including tyrosines 397, 576, and 577. Tyr-397 is an autophosphorylation site that promotes interaction with c-Src or Fyn. Tyr-576 and Tyr-577 lie in the putative activation loop of the kinase domain, and FAK catalytic activity may be elevated through phosphorylation of these residues by associated Src family kinase. Recent studies have implicated FAK as a positive regulator of cell spreading and migration. To further study the mechanism of adhesion-induced FAK activation and the possible role and signaling requirements for FAK in cell spreading and migration, we utilized the tetracycline repression system to achieve inducible expression of either wild-type FAK or phosphorylation site mutants in fibroblasts derived from FAK-null mouse embryos. Using these Tet-FAK cells, we demonstrated that both the FAK autophosphorylation and activation loop sites are critical for maximum adhesion-induced FAK activation and FAK-enhanced cell spreading and migration responses. Negative effects on cell spreading and migration, as well as decreased phosphorylation of the substrate p130(Cas), were observed upon induced expression of the FAK autophosphorylation site mutant. These negative effects appear to result from an inhibition of integrin-mediated signaling by the FAK-related kinase Pyk2/CAKbeta/RAFTK/CadTK.     

Differential regulation of cell motility and invasion by FAK

Cell migration and invasion are fundamental components of tumor cell metastasis. Increased focal adhesion kinase (FAK) expression and tyrosine phosphorylation are connected with elevated tumorigenesis. Null mutation of FAK results in embryonic lethality, and FAK-/- fibroblasts exhibit cell migration defects in culture. Here we show that viral Src (v-Src) transformation of FAK-/- cells promotes integrin-stimulated motility equal to stable FAK reexpression. However, FAK-/- v-Src cells were not invasive, and FAK reexpression, Tyr-397 phosphorylation, and FAK kinase activity were required for the generation of an invasive cell phenotype. Cell invasion was linked to transient FAK accumulation at lamellipodia, formation of a FAK-Src-p130Cas-Dock180 signaling complex, elevated Rac and c-Jun NH2-terminal kinase activation, and increased matrix metalloproteinase expression and activity. Our studies support a dual role for FAK in promoting cell motility and invasion through the activation of distinct signaling pathways.     

Hyperosmotic stress induces rapid focal adhesion kinase phosphorylation at tyrosines 397 and 577. Role of Src family kinases and Rho family GTPases

Hyperosmotic stress induced by treatment of Swiss 3T3 cells with the non-permeant solutes sucrose or sorbitol, rapidly and robustly stimulated endogenous focal adhesion kinase (FAK) phosphorylation at Tyr-397, the major autophosphorylation site, and at Tyr-577, within the kinase activation loop. Hyperosmotic stress-stimulated FAK phosphorylation at Tyr-397 occurred via a Src-independent pathway, whereas Tyr-577 phosphorylation was completely blocked by exposure to the Src family kinase inhibitor PP-2. Inhibition of p38 MAP kinase or phosphatidylinositol 3-kinases did not prevent FAK phosphorylation stimulated by hyperosmotic stress. Overexpression of N17 RhoA did not reduce hyperosmotic stress-mediated localization of phosphorylated FAK to focal contacts and treatment with the Rho-associated kinase inhibitor Y-27632 did not prevent FAK translocation and tyrosine phosphorylation in response to hyperosmotic stress. Overexpression of N17 Rac only slightly altered the hyperosmotic stress-mediated localization of phosphorylated FAK to focal contacts. In contrast, overexpression of the N17 mutant of Cdc42 disrupted hyperosmotic stress-stimulated FAK Tyr-397 localization to focal contacts. Additionally, treatment of cells with Clostridium difficile toxin B potently inhibited hyperosmotic stress-induced FAK tyrosine phosphorylation. Furthermore, FAK null fibroblasts compared with their FAK containing controls show markedly increased sensitivity, manifest by subsequent apoptosis, to sustained hyperosmotic stress. Our results indicate that FAK plays a fundamental role in protecting cells from hyperosmotic stress, and that the pathway(s) that mediates FAK autophosphorylation at Tyr-397 in response to osmotic stress can be distinguished from the pathways utilized by many other stimuli, including neuropeptides and bioactive lipids (Rho- and Rho-associated kinase-dependent), tyrosine kinase receptor agonists (phosphatidylinositol 3-kinase-dependent), and integrins (Src-dependent).     

Phosphorylated alpha-actinin and protein-tyrosine phosphatase 1B coregulate the disassembly of the focal adhesion kinase x Src complex and promote cell migration

The focal adhesion kinase (FAK) is a key regulator of cell migration. Phosphorylation at Tyr-397 activates FAK and creates a binding site for Src family kinases. FAK phosphorylates the cytoskeletal protein alpha-actinin at Tyr-12. Here we report that protein-tyrosine phosphatase 1B (PTP 1B) is an alpha-actinin phosphatase. PTP 1B-dependent dephosphorylation of alpha-actinin was seen in COS-7 cells and PTP 1B-null fibroblasts reconstituted with PTP 1B. Furthermore, we show that coexpression of wild-type alpha-actinin and PTP 1B causes dephosphorylation at Tyr-397 in FAK. No dephosphorylation was observed in cells coexpressing the alpha-actinin phosphorylation mutant Y12F and PTP 1B. Furthermore, the phosphorylation at four other sites in FAK was not altered by PTP 1B. In addition, we found that phosphorylated alpha-actinin bound to Src and reduced the binding of FAK to Src. The dephosphorylation at Tyr-397 in FAK triggered by wild-type alpha-actinin and PTP 1B caused a significant increase in cell migration. We propose that phosphorylated alpha-actinin disrupts the FAK x Src complex exposing Tyr-397 in FAK to PTP 1B. These findings uncover a novel feedback loop involving phosphorylated alpha-actinin and PTP 1B that regulates FAK x Src interaction and cell migration.     

An endogenous inhibitor of focal adhesion kinase blocks Rac1/JNK but not Ras/ERK-dependent signaling in vascular smooth muscle cells

Humoral factors and extracellular matrix are critical co-regulators of smooth muscle cell (SMC) migration and proliferation. We reported previously that focal adhesion kinase (FAK)-related non-kinase (FRNK) is expressed selectively in SMC and can inhibit platelet-derived growth factor BB homodimer (PDGF-BB)-induced proliferation and migration of SMC by attenuating FAK activity. The goal of the current studies was to identify the mechanism by which FAK/FRNK regulates SMC growth and migration in response to diverse mitogenic signals. Transient overexpression of FRNK in SMC attenuated autophosphorylation of FAK at Tyr-397, reduced Src family-dependent tyrosine phosphorylation of FAK at Tyr-576, Tyr-577, and Tyr-881, and reduced phosphorylation of the FAK/Src substrates Cas and paxillin. However, FRNK expression did not alter the magnitude or dynamics of ERK activation induced by PDGF-BB or angiotensin II. Instead, FRNK expression markedly attenuated PDGF-BB-, angiotensin II-, and integrin-stimulated Rac1 activity and attenuates downstream signaling to JNK. Importantly, constitutively active Rac1 rescued the proliferation defects in FRNK expressing cells. Based on these observations, we hypothesize that FAK activation is required to integrate integrin signals with those from receptor tyrosine kinases and G protein-coupled receptors through downstream activation of Rac1 and that in SMC, FRNK may control proliferation and migration by buffering FAK-dependent Rac1 activation.     

Src family kinases are required for integrin-mediated but not for G protein-coupled receptor stimulation of focal adhesion kinase autophosphorylation at Tyr-397

Plating suspended Swiss 3T3 cells onto fibronectin-coated dishes promoted phosphorylation of endogenous focal adhesion kinase (FAK) at Tyr-397, the major autophosphorylation site, and at Tyr-577, located in the activation loop, as revealed by site-specific antibodies that recognize the phosphorylated form of these residues. Treatment with the selective Src family kinase inhibitor pyrazolopyrimidine 2 (PP-2) markedly reduced the phosphorylation of both Tyr-397 and Tyr-577 induced by fibronectin. Furthermore, fibronectin-mediated FAK phosphorylation at Tyr-397 was dramatically reduced in SYF cells (deficient in Src, Yes, and Fyn expression). Stimulation of Swiss 3T3 cells with bombesin also induced a rapid increase in the phosphorylation of endogenous FAK at Tyr-397. In contrast to the results obtained with fibronectin, PP-2 did not prevent FAK Tyr-397 phosphorylation stimulated by bombesin at a concentration (10 micrometer) that suppressed bombesin-induced FAK Tyr-577 phosphorylation. Similarly, PP-2 did not prevent Tyr-397 phosphorylation in Swiss 3T3 cells stimulated with other G protein-coupled receptor agonists including vasopressin, bradykinin, endothelin, and lysophosphatidic acid. Lysophosphatidic acid also induced FAK phosphorylation at Tyr-397 in SYF cells. Our results identify, for first time, the existence of Src-dependent and Src-independent pathways leading to FAK autophosphorylation at Tyr-397 stimulated by adhesion-dependent signals and G protein-coupled receptor agonists in the same cell.     

Growth factors stimulate kidney proximal tubule cell migration independent of augmented tyrosine phosphorylation of focal adhesion kinase

Migration of human proximal tubule cells (HKC-5) was stimulated by epidermal growth factor (EGF), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1). Integrin signaling via phosphorylation of focal adhesion kinase (FAK) appears to play a central role in cell migration. Once stimulated, FAK undergoes autophosphorylation at tyrosine (Y) 397, followed by phosphorylation of several sites including Y576/Y577 which increases FAK's kinase activity, as well as at Y407, Y861, and Y925. EGF, HGF, and IGF-1 stimulate FAK phosphorylation in various cells. We showed that endothelin stimulated phosphorylation of Y397 in fibroblasts but not HKC-5 cells. After EGF stimulation, HKC-5 cells showed no change in tyrosine phosphorylation at FAK Y397, 407, 576, 861, or 925. Similarly, HGF and IGF-1 did not stimulate the phosphorylation of FAK Y397 in HKC-5 cells. Further, after inhibition of FAK expression by siRNA, cell migration was similar to cells treated with non-target siRNA and responded to EGF with increased migration. Thus, in proximal tubule cells, stimulation of cell migration by growth factors was independent of augmented FAK tyrosine phosphorylation.     

PTP alpha regulates integrin-stimulated FAK autophosphorylation and cytoskeletal rearrangement in cell spreading and migration

We investigated the molecular and cellular actions of receptor protein tyrosine phosphatase (PTP) alpha in integrin signaling using immortalized fibroblasts derived from wild-type and PTP alpha-deficient mouse embryos. Defects in PTP alpha-/- migration in a wound healing assay were associated with altered cell shape and focal adhesion kinase (FAK) phosphorylation. The reduced haptotaxis to fibronectin (FN) of PTP alpha-/- cells was increased by expression of active (but not inactive) PTP alpha. Integrin-mediated formation of src-FAK and fyn-FAK complexes was reduced or abolished in PTP alpha-/- cells on FN, concomitant with markedly reduced phosphorylation of FAK at Tyr397. Reintroduction of active (but not inactive) PTP alpha restored FAK Tyr-397 phosphorylation. FN-induced cytoskeletal rearrangement was retarded in PTP alpha-/- cells, with delayed filamentous actin stress fiber assembly and focal adhesion formation. This mimicked the effects of treating wild-type fibroblasts with the src family protein tyrosine kinase (Src-PTK) inhibitor PP2. These results, together with the reduced src/fyn tyrosine kinase activity in PTP alpha-/- fibroblasts (Ponniah et al., 1999; Su et al., 1999), suggest that PTP alpha functions in integrin signaling and cell migration as an Src-PTK activator. Our paper establishes that PTP alpha is required for early integrin-proximal events, acting upstream of FAK to affect the timely and efficient phosphorylation of FAK Tyr-397.     

Tyrosine phosphorylation of focal adhesion kinase at sites in the catalytic domain regulates kinase activity: a role for Src family kinases.

Focal adhesion kinase (FAK) is a widely expressed nonreceptor protein-tyrosine kinase implicated in integrin-mediated signal transduction pathways and in the process of oncogenic transformation by v-Src. Elevation of FAK's phosphotyrosine content, following both cell adhesion to extracellular matrix substrata and cell transformation by Rous sarcoma virus, correlates directly with an increased kinase activity. To help elucidate the role of FAK phosphorylation in signal transduction events, we used a tryptic phosphopeptide mapping approach to identify tyrosine sites of phosphorylation responsive to both cell adhesion and Src transformation. We have identified four tyrosines, 397, 407, 576, and 577, which are phosphorylated in mouse BALB/3T3 fibroblasts in an adhesion-dependent manner. Tyrosine 397 has been previously recognized as the major site of FAK autophosphorylation. Phosphorylation of tyrosines 407, 576, and 577, which are previously unrecognized sites, is significantly elevated in the presence of c-Src in vitro and v-Src in vivo. Tyrosines 576 and 577 lie within catalytic subdomain VIII--a region recognized as a target for phosphorylation-mediated regulation of protein kinase activity. We found that maximal kinase activity of FAK immune complexes requires phosphorylation of both tyrosines 576 and 577. Our results indicate that phosphorylation of FAK by Src (or other Src family kinases) is an important step in the formation of an active signaling complex.     

Preferential phosphorylation of focal adhesion kinase tyrosine 861 is critical for mediating an anti-apoptotic response to hyperosmotic stress

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

Role of protein-tyrosine phosphatase SHP2 in focal adhesion kinase down-regulation during neutrophil cathepsin G-induced cardiomyocytes anoikis

Inflammatory cells and their proteases contribute to tissue reparation at site of inflammation. Although beneficial at early stages, excessive inflammatory reaction leads to cell death and tissue damage. Cathepsin G (Cat.G), a neutrophil-derived serine protease, has been shown to induce neonatal rat cardiomyocyte detachment and apoptosis by anoikis through caspase-3 dependent pathway. However the early mechanisms that trigger Cat.G-induced caspase-3 activation are not known. This study identifies focal adhesion kinase (FAK) tyrosine dephosphorylation as an early mechanism that regulates Cat.G-induced anoikis in cardiomyocytes. Both FAK tyrosine phosphorylation at Tyr-397 and kinase activity decrease rapidly upon Cat.G treatment and was associated with a decrease of FAK association with adapter and cytoskeletal proteins, p130(Cas) and paxillin, respectively. FAK-decreased tyrosine phosphorylation is required for Cat.G-induced myocyte anoikis as concurrent expression of phosphorylation-deficient FAK mutated at Tyr-397 or pretreatment with a protein-tyrosine phosphatase (PTP) inhibitor, pervanadate, blocks Cat.G-induced FAK tyrosine dephosphorylation, caspase-3 activation and DNA fragmentation. Analysis of PTPs activation shows that Cat.G treatment induces an increase of SHP2 and PTEN phosphorylation; however, only SHP2 forms a complex with FAK in response to Cat.G. Expression of dominant negative SHP2 mutant markedly attenuates FAK tyrosine dephosphorylation induced by Cat.G and protects myocytes to undergo apoptosis. In contrast, increased SHP2 expression exacerbates Cat.G-induced FAK tyrosine dephosphorylation and myocyte apoptosis. Taken together, these results show that Cat.G induces SHP2 activation that leads to FAK tyrosine dephosphorylation and promotes cardiomyocyte anoikis.     

CB1 cannabinoid receptors promote maximal FAK catalytic activity by stimulating cooperative signaling between receptor tyrosine kinases and integrins in neuronal cells

Tyrosine phosphorylation (Tyr-P) of focal adhesion kinase (FAK) regulates FAK activation. Phosphorylated FAK Tyr 397 binds Src family kinases (Src), which in turn directly phosphorylate FAK Tyr 576/577 to produce maximal FAK enzymatic activity. CB₁ cannabinoid receptors (CB₁) are abundantly expressed in the nervous system and influence FAK activation by presently unknown mechanisms. The current investigation determined that CB₁-stimulated maximal FAK catalytic activity is mediated by G_i/o proteins in N18TG2 neuronal cells, and that G_12/13 regulation of Rac1 and RhoA occurs concomitantly. Immunoblotting analyses using antibodies against FAK phospho-Tyr 397 and phospho-Tyr 576/577 demonstrated that the time-course of CB₁-stimulated FAK 576/577 Tyr-P occurred in three phases: Phase I (0–2 min) maximal Tyr-P, Phase II (5–20 min) rapid decline in Tyr-P, and Phase III (> 20 min) plateau in Tyr-P at submaximal levels. In contrast, FAK 397 Tyr-P was monophasic and significantly lower in magnitude. FAK 397 Tyr-P and Phase I FAK 576/577 Tyr-P involved protein tyrosine phosphatase (PTP1B and Shp1/Shp2)-mediated Src activation, Protein Kinase A (PKA) inhibition, and integrin activation. Phase I maximal FAK 576/577 Tyr-P also required cooperative signaling between receptor tyrosine kinases (RTKs) and integrins. The integrin antagonist RGDS peptide, Flk-1 vascular endothelial growth factor receptor (VEGFR) antagonist SU5416, and epidermal growth factor receptor (EGFR) antagonist AG 1478 blocked Phase I FAK 576/577 Tyr-P. CB₁ agonists failed to stimulate FAK Tyr-P in the absence of integrin activation upon suspension in serum-free culture media. In contrast, cells grown on the integrin ligands fibronectin and laminin displayed increased FAK 576/577 Tyr-P that was augmented by CB₁ agonists and blocked by the Src inhibitor PP2 and Flk-1 VEGFR antagonist SU5416. Taken together, these studies have identified a complex integrative pathway utilized by CB₁ to stimulate maximal FAK 576/577 Tyr-P in neuronal cells.     

Role of Src-specific phosphorylation site on focal adhesion kinase for senescence-associated apoptosis resistance

A decreased apoptotic response toward noxious stress is an issuing characteristic of the aging phenotype. Hydrogen peroxide or staurosporine induced apoptosis readily in young cells but not in senescent cells. We showed that focal adhesion kinase (FAK) expression and its phosphorylation at Tyr³⁹⁷, autophosphorylation site for focal adhesion formation, and Tyr⁵⁷⁷, Src-dependent phosphorylation site, were both increased in senescent cells. Moreover, FAK was inactivated proteolytically by apoptotic stimuli in young cells, but not in senescent cells. In addition, senescent cells whose FAK expression was downregulated by siRNA showed the increased level of apoptosis by staurosporine treatment via caspase-3 activation but not by hydrogen peroxide treatment. Interestingly dephosphorylation at Tyr⁵⁷⁷ of FAK by PP2 treatment, Src-family kinase inhibitor, induced the apoptosis by staurosporine in senescent cells but dephosphorylation at Tyr³⁹⁷ by downregulation of caveolin-1 was not affected. These data suggest that FAK might differently regulate apoptosis and focal adhesion formation through site-specific tyrosine phosphorylation in senescent cells. Both authors contributed equally to this work.     

Expression of GRP and its receptor in well-differentiated colon cancer cells correlates with the presence of focal adhesion kinase phosphorylated at tyrosines 397 and 407.

Gastrin-releasing peptide (GRP) and its receptor (GRP-R) are not normally expressed by epithelial cells lining the colon but are aberrantly expressed in cancer, where they act as morphogens and regulate tumor cell differentiation. Studies of colon cancer formation in mice genetically incapable of synthesizing GRP-R suggested that this receptor's morphogenic properties were mediated via focal adhesion kinase (FAK). We therefore set out to determine the presence of both total and phosphorylated forms of FAK in human colon cancer specimens as a function of tumor cell differentiation and GRP/GRP-R co-expression. Ten colon cancers containing 25 regions of distinct differentiation were randomly selected from our GI Cancer Tumor Bank. All specimens were immunohistochemically probed using antibodies recognizing GRP, GRP-R, total FAK, and FAK specifically phosphorylated at tyrosine (Y) 397, 407, 576, 577, 861, and 925. Antibody-specific chromogen was determined by quantitative immunohistochemistry (IHC) for each region of defined differentiation. Here we confirm that GRP/GRP-R co-expression is a function of differentiation, with highest levels observed in well-differentiated tumor cells. We also show that the amount of total FAK and of FAK phosphorylated at Y397 and Y407 tightly correlates with differentiation and with the amount of GRP/GRP-R co-expression. These findings are consistent with GRP/GRP-R acting as a morphogen by activating FAK, and suggest that this occurs via phosphorylation of this enzyme at two specific tyrosine residues.     

A truncated FAK lacking the FERM domain displays high catalytic activity but retains responsiveness to adhesion-mediated signals

In order to determine the role of the FERM domain in the regulation of FAK phosphorylation at Tyr-397, the major autophosphorylation site, we generated a truncated FAK lacking a region of the N-terminus corresponding to amino acids 1-384 (FAKDelta384). FAKDelta384 showed a striking increase in phosphorylation, as compared with wild type FAK, in lysates of either HEK 293 or FAK-/- cells. Interestingly, the truncated form of FAK lacking the N-terminal domain retains responsiveness to integrin-mediated signals, as judged by its dephosphorylation by holding cells in suspension and by the recovery of the phosphorylation when replating the cells on fibronectin. We propose a model in which removal of FERM-mediated auto-inhibition is important to increase FAK catalytic activity but the translocation and clustering of this enzyme at the focal adhesions is required for maximal phosphorylation at Tyr-397.     

Alpha-actinin-1 phosphorylation modulates pressure-induced colon cancer cell adhesion through regulation of focal adhesion kinase-Src interaction

Physical forces including pressure, strain, and shear can be converted into intracellular signals that regulate diverse aspects of cell biology. Exposure to increased extracellular pressure stimulates colon cancer cell adhesion by a beta(1)-integrin-dependent mechanism that requires an intact cytoskeleton and activation of focal adhesion kinase (FAK) and Src. alpha-Actinin facilitates focal adhesion formation and physically links integrin-associated focal adhesion complexes with the cytoskeleton. We therefore hypothesized that alpha-actinin may be necessary for the mechanical response pathway that mediates pressure-stimulated cell adhesion. We reduced alpha-actinin-1 and alpha-actinin-4 expression with isoform-specific small interfering (si)RNA. Silencing of alpha-actinin-1, but not alpha-actinin-4, blocked pressure-stimulated cell adhesion in human SW620, HT-29, and Caco-2 colon cancer cell lines. Cell exposure to increased extracellular pressure stimulated alpha-actinin-1 tyrosine phosphorylation and alpha-actinin-1 interaction with FAK and/or Src, and enhanced FAK phosphorylation at residues Y397 and Y576. The requirement for alpha-actinin-1 phosphorylation in the pressure response was investigated by expressing the alpha-actinin-1 tyrosine phosphorylation mutant Y12F in the colon cancer cells. Expression of Y12F blocked pressure-mediated adhesion and inhibited the pressure-induced association of alpha-actinin-1 with FAK and Src, as well as FAK activation. Furthermore, siRNA-mediated reduction of alpha-actinin-1 eliminated the pressure-induced association of alpha-actinin-1 and Src with beta(1)-integrin receptor, as well as FAK-Src complex formation. These results suggest that alpha-actinin-1 phosphorylation at Y12 plays a crucial role in pressure-activated cell adhesion and mechanotransduction by facilitating Src recruitment to beta(1)-integrin, and consequently the association of FAK with Src, to enhance FAK phosphorylation.     

JNK Pathway-associated Phosphatase Dephosphorylates Focal Adhesion Kinase and Suppresses Cell Migration

JNK pathway-associated phosphatase (JKAP, also named DUSP22) is expressed in various tissues, indicating that JKAP may have an important biological function. We showed that JKAP localized in the actin filament-enriched region. Expression of JKAP reduced cell migration, whereas a JKAP mutant lacking catalytic activity promoted cell motility. JKAP efficiently removed tyrosine phosphorylation of several proteins. We have identified focal adhesion kinase (FAK) as a substrate of JKAP. Overexpression of JKAP, but not JKAP mutant lacking catalytic activity, decreased FAK phosphorylation at tyrosines 397, 576, and 577 in H1299 cells. Consistent with these results, decreasing JKAP expression by RNA interference promoted cell migration and Src-induced FAK phosphorylation. Taken together, this study identified a new role for JKAP in the modulation of FAK phosphorylation and cell motility.     

Transient forebrain ischemia effects FAK-coupled signaling in gerbil hippocampus

Focal adhesion kinase (FAK) is thought to play a major role in transducing extracellular matrix (ECM)-derived survival signals into cells. The function of FAK is linked to its autophosphorylation at Tyr-397 and then recruitment of several effector molecules. Thus, modulation of FAK activity may affect several intracellular signaling pathways and may participate in a variety of pathological settings. In the present study, we investigated the effect of short-term 5 min forebrain ischemia on levels and Tyr-397 phosphorylation of focal adhesion kinase and the interaction of this enzyme with Src protein tyrosine kinase and adapter protein p130Cas, involved in FAK-mediated signaling pathway in gerbil hippocampus. The total amount of focal adhesion kinase as well as its Tyr-397 phosphorylation declined substantially between 24 and 48 h after the insult, particularly in CA1 region of hippocampus. Concomitantly, a decreased amount of FAK/Src kinase complex has been observed. These data indicate that inhibition of FAK/Src-coupled signaling pathway may participate in the ischemia-induced neuronal degeneration in gerbil hippocampus. The temporal profile of FAK down-regulation in CA1 area coincides with metalloproteinases (MMPs) activation. These results suggest that extracellular proteolysis might belong to the mechanisms which govern the FAK-coupled pathway in ischemic hippocampus.