Focal Adhesion Kinase Tyrosine Research Articles

Role of FRNK tyrosine phosphorylation in vascular smooth muscle spreading and migration

Focal adhesion kinase (FAK) and its autonomously expressed, C-terminal inhibitor FAK-related non-kinase (FRNK), are important regulators of vascular smooth muscle cell (VSMC) spreading and migration. However, the mechanisms of FRNK-mediated inhibition of FAK-dependent signalling are not fully defined. The aim of this study was to determine the potential role of FRNK tyrosine phosphorylation in regulating these processes. Rat carotid arteries were balloon-injured and FAK and FRNK expression and phosphorylation were examined by immunocytochemistry, immunoprecipitation, and western blotting with total and phosphospecific antibodies. FAK and FRNK expression increased four- and nine-fold, respectively, in alpha-smooth muscle actin-positive VSMCs of injured arteries when compared with contralateral control arteries, and the upregulated FRNK was phosphorylated at residues Y168 and Y232. In A7r5 cells (an embryonic rat VSMC line), endogenously expressed FRNK was also phosphorylated at Y168 and Y232 under basal conditions, and Y168/Y232 phosphorylation increased in response to angiotensin II treatment. When overexpressed in A7r5 cells and adult rat aortic smooth muscle cells (RASM), wild-type (wt) GFP-tagged FRNK was also phosphorylated at residues Y168 and Y232, and GFP-wtFRNK inhibited cell spreading and migration. Mutation of GFP-FRNK at Y168 (GFP-Y168F-FRNK) abrogated FRNK-mediated inhibition of cell spreading and migration, but did not affect its localization in VSMC focal adhesions or its ability to inhibit FAK tyrosine phosphorylation. Phosphorylation of Y168 on FRNK may represent a novel mechanism by which FRNK inhibits cell spreading and migration in VSMCs.

PTEN and SHIP2 regulates PI3K/Akt pathway through focal adhesion kinase

Our laboratory has established a novel role of focal adhesion kinase (FAK) in vitro and in vivo, as a positive regulator of insulin signaling pathway. In vitro studies reported tyrosine dephosphorylation of FAK under insulin resistance in C2C12 skeletal muscle cells. A decrease in FAK tyrosine phosphorylation was also observed in skeletal muscle of insulin resistant Sprague–Dawley rats fed on high-fat-diet. Present study was undertaken to explore the cellular mechanism of FAK dephosphorylation under insulin resistance in C2C12 skeletal muscle cells. Here we report that PTEN and SHIP2, the phosphatases widely implicated as negative regulators of insulin signaling, to be responsible for dephosphorylation of FAK. Data propose that under insulin resistance upregulation of PTEN and SHIP2 act through changes in FAK phosphorylation to impair insulin signaling suggesting FAK to be a key mediator of PTEN and SHIP2 in the regulation of insulin signaling. Thus data elucidates a part of molecular mechanism of insulin resistance in skeletal muscle cells.

Up-Regulation of α5-Integrin by E-Cadherin Loss in Hypoxia and Its Key Role in the Migration of Extravillous Trophoblast Cells during Early Implantation

During early pregnancy, cytotrophoblast cells differentiate into extravillous trophoblast (EVT) cells and invade the uterine spiral arteries. This physiological process is essential for the development of maternal-fetal circulation. Because EVT cells are exposed to a low-oxygen environment during this process, we investigated the role of hypoxia in the mechanism that regulates the invasive behavior of EVT cells. Real-time PCR and immunofluorescent analysis were performed to investigate how hypoxia influences the expression of E-cadherin in villous explants cultures and in trophoblast-derived BeWo cells. We determined that hypoxia induced E-cadherin down-regulation through Snail up-regulation in villous explant cultures. The influence of E-cadherin loss was examined by analyzing the expression of alpha(5)-integrin and phosphorylated focal adhesion kinase (FAK) by Western blot and evaluating trophoblast invasion using a matrigel invasion assay. E-cadherin loss induced the up-regulation of alpha(5)-integrin, which leads to the tyrosine phosphorylation of FAK, resulting in an increase in the invasive activity of EVT cells. An alpha(5)-integrin neutralizing antibody inhibited the invasion of EVT cells by attenuating FAK tyrosine phosphorylation. Immunohistochemical analysis using clinical placental bed biopsies revealed that alpha(5)-integrin was up-regulated and FAK tyrosine phosphorylated (Try(861)) as EVT cells invade the uterine myometrium, whereas E-cadherin expression was down-regulated. These results suggest that alpha(5)-integrin up-regulation induced by E-cadherin loss under hypoxia has a crucial role in regulating the migration of EVT cells. This finding should help us reach a better understanding of the pathogenesis of critical gestational diseases, such as preeclampsia.

A FAK-p120RasGAP-p190RhoGAP complex regulates polarity in migrating cells

Directional motility is a complex process requiring the spatiotemporal integration of signals that regulate cytoskeletal changes, and the establishment of an anteroposterior or polarized cell axis. Focal adhesion kinase (FAK) promotes cell migration, but a molecular role for FAK in promoting cell polarity remains undefined. Here, using wound healing and Golgi-reorientation analyses, we show that fibroblast, endothelial and carcinoma polarity during cell migration requires FAK and is associated with a complex between FAK, p120RasGAP and p190RhoGAP (p190A), leading to p190A tyrosine phosphorylation. Fibronectin-integrin-mediated FAK activation and phosphorylation promote SH2-mediated binding of p120RasGAP to FAK and FAK-mediated p190A tyrosine phosphorylation. The association of p120RasGAP with FAK facilitates the formation of a FAK-p120RasGAP-p190A complex targeted to leading-edge focal adhesions by FAK. Knockdown of p120RasGAP, mutation of FAK Y397 or inhibition of FAK activity prevent the association of FAK with p190A and subsequent tyrosine phosphorylation of p190A, and result in the loss of cell polarity. Because reconstitution of FAK-null fibroblasts with FAK or a Pyk2-FAK chimera restore the normal decrease in RhoA GTP binding upon cell spreading on fibronectin, our studies support a model whereby FAK activity facilitates the recruitment and stabilization of a p120RasGAP-p190A complex at leading-edge focal adhesions connected to the transient inhibition of RhoA activity and the regulation of cell polarity.

Integrins and proximal signaling mechanisms in cardiovascular disease

Integrins are heterodimeric cell-surface molecules, which act as the principle mediators of molecular dialog between a cell and its extracellular matrix environment. In addition to their structural functions, integrins mediate signaling from the extracellular space into the cell through integrin-associated signaling and adaptor molecules such as FAK (focal adhesion kinase), ILK (integrin-linked kinase), PINCH (particularly interesting new cysteine-histidine rich protein) and Nck2 (non-catalytic (region of) tyrosine kinase adaptor protein-2). Via these molecules, integrin signaling tightly and cooperatively interacts with receptor tyrosine kinases (RTKs) signaling to regulate survival, proliferation and cell shape as well as polarity, adhesion, migration and differentiation. In the heart and blood vessels, the function and regulation of these molecules can be partially disturbed and thus contribute to cardiovascular diseases such as cardiac hypertrophy and atherosclerosis. In this review, we discuss the primary mechanisms of action and signaling of integrins in the cardiac and vascular system in normal and pathological states, as well as therapeutic strategies for targeting these systems (1).

Resistance of Tumor Cells to Cytolytic T Lymphocytes Involves Rho-GTPases and Focal Adhesion Kinase Activation

Tumor cells evade adaptive immunity by a variety of mechanisms, including selection of variants that are resistant to specific cytotoxic T lymphocyte (CTL) pressure. Recently, we have reported that the reorganization of the actin cytoskeleton can be used by tumor cells as a strategy to promote their resistance to CTL-mediated lysis. In this study, we further examined the functional features of a CTL-resistant tumor variant and investigated the relationship between cytoskeleton alteration, the acquisition of tumor resistance to CTL-induced cell death, Rho-GTPases, and focal adhesion kinase (FAK) pathways. Our data indicate that although the resistant cells do not display an increased migratory potential, an alteration of adhesion to the extracellular matrix was observed. When Rho-GTPases were activated in cells by the bacterial CNF1 (cytotoxic necrotizing factor 1), striking changes in the cell morphology, including actin cytoskeleton, focal adhesions, and membrane extensions, were observed. More importantly, such activation also resulted in a significant attenuation of resistance to CTL-induced cell death. Furthermore, we demonstrate that FAK signaling pathways were constitutively defective in the resistant cells. Silencing of FAK in the sensitive target cells resulted in the inhibition of immune synapse formation with specific CTLs and their subsequent lysis. Expression of the FAK mutant (Y397F) resulted in an inhibition of IGR-Heu cell adhesion and of their susceptibility to specific lysis. These results suggest that FAK activation plays a role in the control of tumor cell susceptibility to CTL-mediated lysis.

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 Controls Aggressive Phenotype of Androgen-Independent Prostate Cancer

Overexpression of focal adhesion kinase (FAK) has been well correlated with tumor development and/or the maintenance of tumor phenotype. In addition, inappropriate activation of the extracellular regulated kinase (ERK) signaling pathway is common to many human cancers. In the present study, we investigated the interplay between FAK and ERK in androgen-independent prostate cancer cells (PC3 and DU145 cells). We observed that suppression of FAK expression using small interfering RNA-mediated knockdown decreased the clonogenic activity, whereas overexpression of FAK increased it. We also observed that detachment of PC3 and DU145 cells from their substrate induced tyrosine phosphorylation of FAK. ERK knockdown diminished FAK protein levels and tyrosine phosphorylation of FAK as well as FAK promoter-reporter activity. We also tested the effect of MEK inhibitors and small interfering RNA-mediated knockdown of ERK1 and/or ERK2 on cell proliferation, invasiveness, and growth in soft agar of PC3 and DU145 cells. Inhibition of ERK signaling grossly impaired clonogenicity as well as invasion through Matrigel. However, inhibition of ERK signaling resulted in only a modest inhibition of 3H-thymidine incorporation and no effect on overall viability of the cells or increased sensitivity to anoikis. Taken together, these data show, for the first time, a requirement for FAK in aggressive phenotype of prostate cancer cells; reveal interdependence of FAK and ERK1/2 for clonogenic and invasive activity of androgen-independent prostate cancer cells; suggest a role for ERK regulation of FAK in substrate-dependent survival; and show for the first time, in any cell type, the regulation of FAK expression by ERK signaling pathway.

δ-Opioid receptors activate ERK/MAP kinase via integrin-stimulated receptor tyrosine kinases

Integrin-mediated cell adherence to extracellular matrix proteins results in stimulation of ERK1/2 activity, a mechanism involving focal adhesion tyrosine kinases (pp125FAK, Pyk-2) and epidermal growth factor receptors (EGFRs). G protein-coupled receptors (GPCRs) may also mediate ERK1/2 activation in an integrin-dependent manner, the underlying signaling mechanism of which still remains unclear. Here we demonstrate that the δ-opioid receptor (DOR), a typical GPCR, stimulates ERK1/2 activity in HEK293 cells via integrin-mediated transactivation of EGFR function. Inhibition of integrin signaling by RGDT peptides, cytochalasin, and by keeping the cells in suspension culture both blocked [D-Ala2, D-Leu5]enkephalin (DADLE)- and etorphine-stimulated ERK1/2 activity. Integrin-dependent ERK1/2 activation does not involve FAK/Pyk-2, because over-expression of the FAK/Pyk-2 inhibitor SOCS-3 failed to attenuate DOR signaling. Exposure of the cells to the EGFR inhibitors AG1478 and BPIQ-I blocked DOR-mediated ERK1/2 activation. Because RGDT peptides also prevented DOR-mediated EGFR activation, the present findings indicate that in HEK293 cells DOR-stimulated ERK1/2 activity is mediated by integrin-stimulated EGFRs. Further studies with the phospholipase C (PLC) inhibitors U73122 and ET-18-OCH3 revealed that opioid-stimulated integrin activation is sensitive to PLC. In contrast, integrin-mediated transactivation of EGFR function appears to be dependent on PKC-δ, as indicated by studies with rottlerin and siRNA knock-down. A similar ERK1/2 signaling pathway was observed for NG108-15 cells, a neuronal cell line endogenously expressing the DOR. In these cells, the nerve growth factor TrkA receptor replaces the EGFR in connecting DOR-activated integrins to the Ras/Raf/ERK1/2 pathway. Together, these data describe an alternative ERK1/2 signaling pathway in which the DOR transactivates the growth factor receptor associated mitogen-activated protein kinase cascade in an integrin-dependent manner.

Physics Meets Molecules

See related article, pages 538–545 Arteries and veins are permanently exposed to hemodynamic forces because of the pulsatile nature of blood pressure and flow. Hence, the endothelium is constantly detecting different biomechanical forces, cyclic stretch and shear stress in particular,1–2 and converts the latter stimuli into intra- and extracellular signals. Endothelial cells thereby modulate multiple of physiological and pathophysiological processes: production of growth-promoting and growth-inhibiting hormones, enzymes, cytokines, etc; mediation of inflammatory responses through the expression of chemotactic and adhesion molecules on the endothelial surface; modulation of hemostasis and thrombosis via secretion of procoagulant, anticoagulant, and fibrinolytic agents; and the regulation of vascular smooth muscle cell contraction through the release of vasodilators and vasoconstrictors.3–5 This being the case, the equilibrium between physiological levels of blood flow (shear stress) and the endothelium is tightly counterbalanced. Thereby, the lumen radius of an artery is the most important denominator, which signifies that the smaller the lumen the higher the shear stress. However, once physiological shear forces are reduced, several pathological conditions may arise: proatherogenic and/or prothrombotic states and hence atherosclerosis and/or thrombosis.6–7 Inversely, high levels of shear forces play a key role in adaptive phases of arteriogenesis (collateral artery growth), the most clinically relevant mechanism of vascular development.8 In case of an arterial stenosis, these arterial/arteriolar anastomoses are the only anastomosis to the low-pressure territory and perfuse the periphery with nutrient blood flow. Elevation of shear stress and concomitant cyclic stretch are currently discussed to be the strongest inducers of arteriogenesis. In a rabbit model of femoral artery ligation, Schaper and colleagues induced an abrupt increase in shear stress by means of an arterial–venous shunt (pressure drop across collateral arteries) and accelerated the speed of arteriogenesis, surpassing the conductance values of the occluded artery when shear …

OipA plays a role in Helicobacter pylori-induced focal adhesion kinase activation and cytoskeletal re-organization

The initial signalling events leading to Helicobacter pylori infection associated changes in motility, cytoskeletal reorganization and elongation of gastric epithelial cells remain poorly understood. Because focal adhesion kinase (FAK) is known to play important roles in regulating actin cytoskeletal organization and cell motility we examined the effect of H. pylori in gastric epithelial cells co-cultured with H. pylori or its isogenic cag pathogenicity island (PAI) or oipA mutants. H. pylori induced FAK phosphorylation at distinct tyrosine residues in a dose- and time-dependent manner. Autophosphorylation of FAK Y397 was followed by phosphorylation of Src Y418 and resulted in phosphorylation of the five remaining FAK tyrosine sites. Phosphorylated FAK and Src activated Erk and induced actin stress fibre formation. FAK knock-down by FAK-siRNA inhibited H. pylori-mediated Erk phosphorylation and abolished stress fibre formation. Infection with oipA mutants reduced phosphorylation of Y397, Y576, Y577, Y861 and Y925, inhibited stress fibre formation and altered cell morphology. cag PAI mutants reduced phosphorylation of only FAK Y407 and had less effect on stress fibre formation than oipA mutants. We propose that activation of FAK and Src are responsible for H. pylori-induced induction of signalling pathways resulting in the changes in cell phenotype important for pathogenesis.

Cleavage of focal adhesion kinase is an early marker and modulator of oxidative stress-induced apoptosis

Focal adhesion kinase (FAK) is a signaling molecule associated with cell survival. Previously, we showed that thimerosal, a reactive oxygen species (ROS) generator, can acutely induce FAK tyrosine phosphorylation (within minutes) and chronically induce apoptosis (within days) by redox modulation in HeLa S cells. In the present study, we report that a prolonged oxidative stress by thimerosal induces a remarkable cleavage of FAK, which is accompanied with apoptosis. In fact, the kinetics of FAK cleavage has a good correlation with and actually preceding the apoptosis that was independent of anoikis. The effects were almost completely blocked by the pretreatment with either N-acetyl- l-cysteine (ROS scavenger) or Z-VAD-FMK (pan-caspase inhibitor), suggesting ROS-induced caspase activation as a key mechanism. They could be also reproduced by hydrogen peroxide alone, which appeared to be responsible for thimerosal-mediated oxidative stress-induced apoptosis. Additionally, the down regulation of FAK with antisense oligonucleotide dramatically augmented thimerosal-induced apoptosis. We could observe similar results using human corneal epithelial cells. Taken together, our results show that FAK is a critical cellular target of caspases during oxidative stress (particularly by hydrogen peroxide), resulting in the acceleration of subsequent apoptosis regardless of the anchorage status of cells. From the present results, it is more likely that not cell detachment but the proteolytic cleavage (or inhibition) of FAK is a key modulator as well as a promising indicator of apoptosis in epithelial cells under oxidative stress.

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.

A Novel Protein Encoded by Kaposi’s Sarcoma-Associated Herpesvirus Open Reading Frame 36 Inhibits Cell Spreading and Focal Adhesion Kinase Activation

Objective: Kaposi’s sarcoma-associated herpesvirus (KSHV) is a γ-herpesvirus implicated in the development of Kaposi’s sarcoma, primary effusion lymphoma and some forms of multicentric Castleman’s disease. The KSHV open reading frame (ORF) 36 encodes a viral serine/threonine protein kinase. The aim of this study was to characterize the cellular function of the ORF36 protein. Methods: The expression kinetics of the ORF36 protein and its localization were determined. The wild-type ORF36 and its mutant proteins were subjected to in vitro kinase assay. Cell morphology change by ORF36 protein was studied. The focal adhesion kinase (FAK) tyrosine phosphorylation and cleavage were determined when ORF36 was expressed. Results: ORF36 protein expressed in the late phase during the KSHV reactivation. The C-terminal domain of ORF36 protein was important for kinase activity. Moreover, the ORF36 protein altered cell morphology to a round shape, similar to the phenotype of FAK-deficient cells. The kinase activity of ORF36 protein was required for the inhibition of cell spreading. Interestingly, ORF36 protein colocalized with FAK, suppressed its tyrosine phosphorylation and promoted FAK cleavage. Conclusion: Our results collectively demonstrate that the KSHV ORF36 protein is a viral protein kinase that inhibits cell spreading and FAK activation.

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.

Functional Characterization of Src-interacting Na/K-ATPase Using RNA Interference Assay

We have shown that the Na/K-ATPase and Src form a signaling receptor complex. Here we determined how alterations in the amount and properties of the Na/K-ATPase affect basal Src activity and ouabain-induced signal transduction. Several alpha1 subunit knockdown cell lines were generated by transfecting LLC-PK1 cells with a vector expressing alpha1-specific small interference RNA. Although the alpha1 knockdown resulted in significant decreases in Na/K-ATPase activity, it increased the basal Src activity and tyrosine phosphorylation of focal adhesion kinase, a Src effector. Concomitantly it also abolished ouabain-induced activation of Src and ERK1/2. When the knockdown cells were rescued by a rat alpha1, both Na/K-ATPase activity and the basal Src activity were restored. In addition, ouabain was able to stimulate Src and ERK1/2 in the rescued cells at a much higher concentration, consistent with the established differences in ouabain sensitivity between pig and rat alpha1. Finally both fluorescence resonance energy transfer analysis and co-immunoprecipitation assay indicated that the pumping-null rat alpha1 (D371E) mutant could also bind Src. Expression of this mutant restored the basal Src activity and focal adhesion kinase tyrosine phosphorylation. Taken together, the new findings suggest that LLC-PK1 cells contain a pool of Src-interacting Na/K-ATPase that not only regulates Src activity but also serves as a receptor for ouabain to activate protein kinases.

Integrin-induced Tyrosine Phosphorylation of Protein-tyrosine Phosphatase-α Is Required for Cytoskeletal Reorganization and Cell Migration

Protein-tyrosine phosphatase-alpha (PTPalpha) activates Src family kinases (SFKs) to promote the integrin-stimulated early autophosphorylation of focal adhesion kinase (FAK). We report here that integrin stimulation induces tyrosine phosphorylation of PTPalpha. PTPalpha was dephosphorylated upon fibroblast detachment from the substratum and rephosphorylated when cells were plated on the integrin ligand fibronectin. alpha PTP phosphorylation occurred at Tyr789 and required SFKs (Src or Fyn/Yes), FAK, and an intact cytoskeleton. It also required active PTPalpha or constitutively active Src. These observations indicate that PTPalpha activates SFKs and that the subsequently activated SFK.FAK tyrosine kinase complex in turn phosphorylates PTPalpha. Reintroduction of wild-type PTPalpha or unphosphorylatable PTPalpha(Y789F) (but not inactive PTPalpha) into PTPalpha-null fibroblasts restored defective integrin-induced SFK activation, FAK phosphorylation, and paxillin phosphorylation. PTPalpha(Y789F) and inactive PTPalpha could not rescue delayed actin stress fiber assembly and focal adhesion formation or defective cell migration. This study distinguishes two roles of PTPalpha in integrin signaling: an early role as an activator of SFKs and FAK with no requirement for PTPalpha phosphorylation and a later downstream role in cytoskeleton-associated events for which PTPalpha phosphorylation at Tyr789 is essential.

Reduced Expression of Focal Adhesion Kinase Disrupts Insulin Action in Skeletal Muscle Cells

Integrins mediate interactions between cells and extracellular matrix proteins that modulate growth factor signaling. Focal adhesion kinase (FAK) is a key multifunctional integrin pathway protein. We recently reported that disruption of FAK impairs insulin-mediated glycogen synthesis in hepatocytes. To test the hypothesis that FAK regulates skeletal muscle insulin action, we reduced FAK expression in L6 myotubes using FAK antisense. In untransfected myotubes, insulin stimulated both FAK tyrosine phosphorylation and kinase activity. Cells treated with antisense FAK showed 78 and 53% reductions in FAK mRNA and FAK protein, respectively, whereas insulin receptor substrate 1/2 and paxillin abundance were unaffected. Insulin-stimulated U-(14)C-glucose incorporation into glycogen was abolished by FAK antisense, and 2-deoxy-glucose uptake and glucose transporter 4 (GLUT4) translocation were both markedly attenuated. Antisense FAK did not alter GLUT1 or GLUT3 protein abundance. Immunofluorescence staining showed decreased FAK Tyr(397) phosphorylation and reduced actin stress fibers. Thus, in skeletal myotubes, FAK regulates the insulin-mediated cytoskeletal rearrangement essential for normal glucose transport and glycogen synthesis. Integrin signaling may play an important regulatory role in muscle insulin action.

Integrin α2-mediated ERK and Calpain Activation Play a Critical Role in Cell Adhesion and Motility via Focal Adhesion Kinase Signaling: IDENTIFICATION OF A NOVEL SIGNALING PATHWAY

Higher levels of focal adhesion kinase (FAK) are expressed in colon metastatic carcinomas. However, the signaling pathways and their mechanisms that control cell adhesion and motility, important components of cancer metastasis, are not well understood. We sought to identify the integrin-mediated mechanism of FAK cleavage and downstream signaling as well as its role in motility in human colon cancer GEO cells. Our results demonstrate that phosphorylated FAK (tyrosine 397) is cleaved at distinct sites by integrin signaling when cells attach to collagen IV. Specific blocking antibodies (clone P1E6) to integrin alpha2 inhibited FAK activation and cell motility (micromotion). Ectopic expression of the FAK C-terminal domain FRNK attenuated FAK and ERK phosphorylation and micromotion. Calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal blocked FAK cleavage, cell adhesion, and micromotion. Antisense approaches established an important role for mu-calpain in cell motility. Expression of wild type mu-calpain increased cell micromotion, whereas its point mutant reversed the effect. Further, cytochalasin D inhibited FAK phosphorylation and cleavage, cell adhesion, locomotion, and ERK phosphorylation, thus showing FAK activation downstream of actin assembly. We also found a pivotal role for FAK Tyr(861) phosphorylation in cell motility and ERK activation. Our results reveal a novel functional connection between integrin alpha2 engagement, FAK, ERK, and mu-calpain activation in cell motility and a direct link between FAK cleavage and enhanced cell motility. The data suggest that blocking the integrin alpha2/FAK/ERK/mu-calpain pathway may be an important strategy to reduce cancer progression.

Antianoikis Effect of Nuclear Factor-κB through Up-regulated Expression of Osteoprotegerin, BCL-2, and IAP-1

Epithelial cells undergo a form of apoptosis termed anoikis when they lose extracellular attachments. We evaluated the role of transcription factor NF-kappaB in the regulation of anoikis susceptibility of intestinal epithelial cells. Culture of rat intestinal epithelial cells in suspension induced NF-kappaB activation, which blocked the anoikis of those cells, as assessed by internucleosomal DNA fragmentation and caspase-3 cleavage. Activation of NF-kappaB after the loss of extracellular attachments required focal adhesion kinase tyrosine 397 phosphorylation. This triggered a signaling cascade through phosphatidylinositol 3-kinase and AKT, to induce DNA binding of the RelA/p65 NF-kappaB polypeptide. NF-kappaB activated in this manner induced the up-regulated expression of a distinct program of genes that included osteoprotegerin, BCL-2, and IAP-1 (inhibitor of apoptosis protein-1). Chromatin immunoprecipitation experiments revealed that NF-kappaB directly regulated the promoters of these 3 genes. Knock-down of the expression of osteoprotegerin, BCL-2, or inhibitor of apoptosis protein-1 by RNA interference showed that these factors inhibit anoikis, and genetic reconstitution of their expression alone or in combination restored normal levels of anoikis to NF-kappaB-inactive intestinal epithelial cells. Together, these findings have identified the molecular components of a previously unrecognized antianoikis pathway in intestinal epithelial cells.