Endogenous Focal Adhesion Kinase Research Articles

A Synergistic Anticancer FAK and HDAC Inhibitor Combination Discovered by a Novel Chemical-Genetic High-Content Phenotypic Screen.

We mutated the focal adhesion kinase (FAK) catalytic domain to inhibit binding of the chaperone Cdc37 and ATP, mimicking the actions of a FAK kinase inhibitor. We reexpressed mutant and wild-type FAK in squamous cell carcinoma (SCC) cells from which endogenous FAK had been deleted, genetically fixing one axis of a FAK inhibitor combination high-content phenotypic screen to discover drugs that may synergize with FAK inhibitors. Histone deacetylase (HDAC) inhibitors represented the major class of compounds that potently induced multiparametric phenotypic changes when FAK was rendered kinase-defective or inhibited pharmacologically in SCC cells. Combined FAK and HDAC inhibitors arrest proliferation and induce apoptosis in a subset of cancer cell lines in vitro and efficiently inhibit their growth as tumors in vivo Mechanistically, HDAC inhibitors potentiate inhibitor-induced FAK inactivation and impair FAK-associated nuclear YAP in sensitive cancer cell lines. Here, we report the discovery of a new, clinically actionable, synergistic combination between FAK and HDAC inhibitors.

Kisspeptin regulation of human decidual stromal cells motility via FAK-Src intracellular tyrosine kinases.

Can of Clinical Genetics, Maastricht University Medical Centre, Maastricht kisspeptin and its analogues regulate the motility of human decidual stromal cells and what intracellular signaling pathways are involved? Kisspeptin analogue-mediated cell motility in human decidual stromal cells via the focal adhesion kinase (FAK)-steroid receptor coactivator (Src) pathway suggesting that kisspeptin may modulate embryo implantation and decidual programming in human pregnancy. The extravillous trophoblast invades the maternal decidua during embryo implantation and placentation. The motile behavior and invasive potential of decidual stromal cells regulate embryo implantation and programming of human pregnancy. Human decidual stromal cells were isolated from healthy women undergoing elective termination of a normal pregnancy at 6- to 12-week gestation, after informed consent. Kisspeptin analogues were synthetic peptides. Cell motility was estimated by an invasion and migration assay. Immunoblot analysis was performed to investigate the expression of kisspeptin receptor and the effects of kisspeptin analogues on the phosphorylation of FAK and Src. Small interfering RNAs (siRNAs) were used to knock down the expression of kisspeptin receptor, FAK, Src, matrix metallo-proteinases (MMPs) 2 and 9, and extracellular signal-regulated protein kinase (ERK) 1/2. The kisspeptin receptor was expressed in human decidual stromal cells. Kisspeptin agonist decreased, but antagonist increased, cell motility. Kisspeptin agonist decreased the phosphorylation of FAK and Src tyrosine kinases, whereas antagonist increased it. These effects on phosphorylation were abolished by kisspeptin receptor siRNA. The activation of cell motility by kisspeptin analogues was suppressed by siRNA knockdown of endogenous FAK (decreased 66%), Src (decreased 60%), kisspeptin receptor (decreased 26%), MMP-2 (decreased 36%), MMP-9 (decreased 23%), and ERK 1/2 inhibitor (decreased 27%). Human decidual stromal cells were obtained from women having terminations after 6-12weeks of pregnancy and differences in timing could affect their properties. Kisspeptin acting within the endometrium has a potential modulatory role on embryo implantation and decidual programming of human pregnancy. This work was supported by grant NSC-104-2314-B-182A-146-MY2 (to H.-M.W.) from the Ministry of Science and Technology, Taiwan, and grants CMRPG3E0401 and CMRPG3E0402 (to H.-M.W.). This work was also supported by grants from the Canadian Institutes of Health Research to P.C.K.L. P.C.K.L. is the recipient of a Child & Family Research Institute Distinguished Investigator Award. The authors have no conflicts of interest to disclose. N/A.

Inhibiting focal adhesion kinase (FAK) blocks IL-4 induced VCAM-1 expression and eosinophil recruitment in vitro and in vivo.

Leukocyte recruitment plays a critical role during both normal inflammation and chronic inflammatory diseases, and ongoing studies endeavor to better understand the complexities of this process. Focal adhesion kinase (FAK) is well known for its role in cancer, yet it also has been shown to regulate aspects of neutrophil and B16 melanoma cell recruitment by rapidly influencing endothelial cell focal adhesion dynamics and junctional opening. Recently, we found that FAK related non-kinase (FRNK), a protein that is often used as a FAK dominant negative, blocked eosinophil transmigration by preventing the transcription of vascular cell adhesion molecule-1 (VCAM-1) and eotaxin-3 (CCL26). Surprisingly, the blocking occurred even in the absence of endogenous FAK. To better understand the role of FAK in leukocyte recruitment, we used a FAK-specific inhibitor (PF-573228) and determined the effect on IL-4 induced eosinophil recruitment in vitro and in vivo. PF-573228 prevented the expression of VCAM-1 and CCL26 expression in IL-4-stimulated human endothelial cells in vitro. As a result, eosinophil adhesion and transmigration were blocked. PF-572338 also prevented IL-4-induced VCAM-1 expression in vivo. Using brightfield intravital microscopy, we found that PF-573228 decreased leukocyte rolling flux, adhesion, and emigration. We specifically examined eosinophil recruitment in vivo by using an eosinophil-GFP reporter mouse and found PF-573228 attenuated eosinophil emigration. This study reveals that a FAK inhibitor influences inflammation through its action on eosinophil recruitment.

Oncogenic activation of FAK drives apoptosis suppression in a 3D-culture model of breast cancer initiation.

A key hallmark of cancer cells is the loss of positional control over growth and survival. Focal adhesion kinase (FAK) is a tyrosine kinase localised at sites of integrin-mediated cell adhesion to the extracellular matrix. FAK controls a number of adhesion-dependent cellular functions, including migration, proliferation and survival. Although FAK is overexpressed and activated in metastatic tumours, where it promotes invasion, it can also be elevated in cancers that have yet to become invasive. The contribution of FAK to the early stages of tumourigenesis is not known. We have examined the effect of activating FAK in non-transformed mammary epithelial cells (MECs) to understand its role in tumour initiation. In agreement with previous studies, we find FAK activation in 2D-culture promotes proliferation, migration, and epithelial-to-mesenchymal transition. However in 3D-cultures that better resemble normal tissue morphology, mammary cells largely respond to FAK activation via suppression of apoptosis, promoting aberrant acinar morphogenesis. This is an acquired function of FAK, because endogenous FAK signalling is not required for normal morphogenesis in 3D-culture or in vivo. Thus, FAK activation may facilitate tumour initiation by causing resistance to apoptosis. We suggest that aberrant FAK activation in breast epithelia is dependent upon the tissue context in which it occurs.

PIAS1-FAK Interaction Promotes the Survival and Progression of Non-Small Cell Lung Cancer

The sequence of genomic alterations acquired by cancer cells during tumor progression and metastasis is poorly understood. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that integrates cytoskeleton remodeling, mitogenic signaling and cell survival. FAK has previously been reported to undergo nuclear localization during cell migration, cell differentiation and apoptosis. However, the mechanism behind FAK nuclear accumulation and its contribution to tumor progression has remained elusive. We report that amplification of FAK and the SUMO E3 ligase PIAS1 gene loci frequently co-occur in non-small cell lung cancer (NSCLC) cells, and that both gene products are enriched in a subset of primary NSCLCs. We demonstrate that endogenous FAK and PIAS1 proteins interact in the cytoplasm and the cell nucleus of NSCLC cells. Ectopic expression of PIAS1 promotes proteolytic cleavage of the FAK C-terminus, focal adhesion maturation and FAK nuclear localization. Silencing of PIAS1 deregulates focal adhesion turnover, increases susceptibility to apoptosis in vitro and impairs tumor xenograft formation in vivo. Nuclear FAK in turn stimulates gene transcription favoring DNA repair, cell metabolism and cytoskeleton regulation. Consistently, ablation of FAK by CRISPR/Cas9 editing, results in basal DNA damage, susceptibility to ionizing radiation and impaired oxidative phosphorylation. Our findings provide insight into a mechanism regulating FAK cytoplasm-nuclear distribution and demonstrate that FAK activity in the nucleus promotes NSCLC survival and progression by increasing cell-ECM interaction and DNA repair regulation.

Generation of point-mutant FAK knockin mice.

Focal adhesion kinase is a non-receptor protein tyrosine kinase with signaling functions downstream of integrins and growth factor receptors. In addition to its role in adhesion, migration, and proliferation it also has non-kinase scaffolding functions in the nucleus. Focal adhesion kinase (FAK) activation involves the following: (1) ligand bound growth factors or clustered integrins activate FAK kinase domain; (2) FAK autophosphorylates tyrosine (Y) 397; (3) Src binds pY397 and phosphorylates FAK at various other sites including Y861; (4) downstream signaling of activated FAK elicits changes in cellular behavior. Although many studies have demonstrated roles for the kinase domain, Y397 and Y861 sites, in vitro much less is known about their functions in vivo. Here, we report the generation of a series of FAK-mutant knockin mice where mutant FAK, either kinase dead, non-phosphorylatable mutants Y397F and Y861F, or mutant Y397E-containing a phosphomimetic site that results in a constitutive active Y397, can be expressed in a Cre inducible fashion driven by the ROSA26 promoter. In future studies, intercrossing these mice with FAKflox/flox mice and inducible cre-expressing mice will enable the in vivo study of mutant FAK function in the absence of endogenous FAK in a spatially and temporally regulated fashion within the whole organism.

MicroRNA-543 suppresses endometrial cancer oncogenicity via targeting FAK and TWIST1 expression

Focal adhesion kinase (FAK) is a critical mediator of extracellular matrix signaling, cell survival, proliferation and motility. Twist homolog 1 (TWIST1) is a transcription factor and serves as a powerful oncogene. Increased FAK and TWIST1 expression are observed in a variety of solid human tumors and correlate with metastasis and poor survival. Here, we identify miR-543 as a direct regulator of FAK and TWIST1 expression in endometrial cancer. Forced expression of miR-543 in endometrial cancer cell lines decreases both endogenous FAK and TWIST1 mRNA and protein levels. Forced expression of miR-543 in aggressive endometrial cancer cell lines also impairs tumor cell monolayer proliferation, anchorage-independent growth, migration and invasion. Endogenous miR-543 expression is decreased in malignant versus normal endometrium tissue and the levels of miR-543 inversely correlate with mRNA levels of FAK and TWIST1. miR-543 expression is decreased in endometrial cancer and serves as a tumor suppressor by targeting FAK and TWIST1 expression.

Focal Adhesion Kinase Regulates Expression of Thioredoxin-interacting Protein (TXNIP) in Cancer Cells

Focal Adhesion Kinase (FAK) plays an important role in cancer cell survival. Previous microarray gene profiling study detected inverse regulation between expression of thioredoxin-interacting protein (TXNIP) and FAK, where down-regulation of FAK by siRNA in MCF-7 cells caused up-regulation of TXNIP mRNA level, and in contrast up-regulation of doxycyclin- induced FAK caused repression of TXNIP. In the present report, we show that overexpression of FAK in MCF-7 cells repressed TXNIP promoter activity. Treatment of MCF-7 cells with 1alpha, 25-dihydroxyvitamin D3 (1,25D) down-regulated endogenous FAK and up-regulated TXNIP protein level, and treatment with 5-FU decreased FAK protein expression and up-regulated TXNIP protein expression in 293 cells. Moreover, silencing of FAK with siRNA increased TXNIP protein expression, while overexpression of FAK inhibited TXNIP protein expression in 293 cells. In addition, treatment of DBTRG glioblastoma cells with FAK inhibitor Y15 increased TXNIP mRNA, decreased cancer cell viability and increased apoptosis. These results for the first time demonstrate FAK-regulated TXNIP expression which is important for apoptotic, survival and oxidative stress signaling pathways in cancer cells.

Function of Focal Adhesion Kinase Scaffolding to Mediate Endophilin A2 Phosphorylation Promotes Epithelial-Mesenchymal Transition and Mammary Cancer Stem Cell Activities in Vivo

Tyrosine kinases have been shown to play critical roles in cancer development and progression, and their inhibitors hold the potential as effective targeted therapies for breast and other cancers. However, some of these kinases like focal adhesion kinase (FAK) also possess scaffolding functions in intracellular signaling, but such kinase-independent functions of FAK or other kinases have not been examined in cancer directly in vivo. Here, we report that disruption of the function of FAK scaffolding through its Pro-878/881 motif suppressed mammary tumor growth and metastasis in a well characterized murine model of human breast cancer. P878A/P881A mutation in the endogenous FAK gene decreased the expression of markers for epithelial-mesenchymal transition (EMT) and mammary cancer stem cell (MaCSC) activities in tumors derived from mutant mice. This mutation disrupted the function of FAK scaffolding to mediate endophilin A2 phosphorylation at Tyr-315 by Src, leading to the decreased surface expression of MT1-MMP, as observed previously in transformed fibroblasts in vitro. Inhibition of the downstream components of this FAK scaffolding function by Y315F endophilin A2 mutant or MT1-MMP knockdown reduced markers for EMT and MaCSC activities. Conversely, bypass of the scaffolding function using the phosphorylation mimic mutant Y315E endophilin A2 or endophilin A2 knockdown rescued the decreased markers for EMT and MaCSCs as well as surface expression of MT1-MMP in tumor cells harboring the P878A/P881A mutation. Together, these results identify a novel role of FAK scaffolding function in breast cancer, which could serve as a new target in combination with kinase inhibition for more effective treatment strategies.

Protein tyrosine kinase 6 protects cells from anoikis by directly phosphorylating focal adhesion kinase and activating AKT

Protein tyrosine kinase 6 (PTK6) is a non-receptor tyrosine kinase expressed in epithelial cancers. Disruption of Ptk6 decreases AOM-induced colon tumorigenesis in mice by preventing STAT3 activation. Relocalization of PTK6 in prostate cancers contributes to increased growth. Although not expressed in normal breast or ovary, PTK6 promotes anchorage-independent survival of breast and ovarian tumor cells. We identified several potential PTK6 substrates in the human SW620 colon cancer cell line using mass spectrometry, including FAK (focal adhesion kinase). We show that FAK is a direct substrate of PTK6 in vitro and in vivo. Expression of membrane targeted active PTK6 (Palm-PTK6-YF) induces constitutive activation of FAK and cell morphology changes, which are independent of SRC family kinases in Src−/−, Yes−/−, Fyn−/− (SYF) mouse embryonic fibroblasts (MEFs). Palm-PTK6-YF expressing SYF cells are transformed and overcome contact inhibition, form colonies in transformation assays, proliferate in suspension, and form tumors in a xenograft model. Expression of FAK and Palm-PTK6-YF in Fak−/− MEFs synergistically activates AKT and protects cells against anoikis. However, expression of Palm-PTK6-YF in Akt1/2−/− MEFs fails to protect cells from anoikis, indicating AKT is critical in PTK6 and FAK mediated survival signaling. In a conditional Pten knockout murine prostate cancer model, we identify prostate epithelial cells with enhanced activation of endogenous PTK6 and FAK at the plasma membrane. Knockdown of PTK6 in the PC3 human prostate cancer cell line disrupts FAK and AKT activation and promotes anoikis, which can be rescued by exogenous expression of FAK. Our data reveal important roles for a PTK6-FAK-AKT signaling axis in promoting anchorage-independent cell survival.

Abstract 185: The Impact of Cyclic Strain on Focal Adhesion Signaling and miRNA Profile in Vascular Smooth Muscle Cells

Vascular smooth muscle cells (VSCM) are major contributors to the regulation of vascular tone through their contractile flexibility. Along with other stimulus, they are exposed to mechanical cyclic strain. This is induced by changes in arterial blood pressure waveforms, generated from the left ventricle. It is thought such stimulus can be a major regulator of phenotypic modulation in smooth muscle cells. These altered states of VSMC fate can have a direct influence on cell function, including proliferation, apoptosis, migration and adhesion. This is considered to play a critical role in the pathogenesis of cardiovascular disease. We have shown that cyclic strain, an important physiological stimulus, has a compounding influence on smooth muscle cell function via mechanotransduction. Increased strain has shown to increase SMC adhesion, proliferation and migration. We hypothesise that the effect of mechanical cyclic strain is transduced through the cell via integrin rich focal adhesions, which can alter the miRNA signature of vascular smooth muscle cells and ultimately play a pivotal role in cell function. The Focal Adhesion Kinase (FAK) protein and its endogenous negative regulator FAK Related Non Kinase (FRNK) are known to play a commanding role in integrin signaling. We propose that down regulation of FAK by either siRNA technology or over expression of FRNK will negate the effect of pathological mechanical strain on cell function. We also predict regulation of expression on individual miRNA, through use of Pre-Mir and Anti-Mir technology could yield a similar effect. In the current study we exposed primary derived human aortic smooth muscle cells, in an in vitro model, to cyclic strain to assess its influence on focal adhesions, integrin and miRNA expression. We have concluded thus far that increased cyclic strain shows increased cell migration, adhesion and proliferation. It also alters the miRNA signature as we have shown with a panel of mechano-sensitive miRNA. Coinciding with this we have also demonstrated the impact on cellular function from knockdown of miRNA biogenesis molecules such as Drosha, Dicer, Exportin 5 and Argonaute2 through use of siRNA.

Germline deletion of FAK-related non-kinase delays post-natal cardiomyocyte mitotic arrest

The cardiomyocyte phenotypic switch from a proliferative to terminally differentiated state impacts normal heart development and pathologic myocardial remodeling, yet the signaling mechanisms that regulate this vital process are incompletely understood. Studies from our lab and others indicate that focal adhesion kinase (FAK) is a critical regulator of cardiac growth and remodeling and we found that expression of the endogenous FAK inhibitor, FAK-related non kinase (FRNK) coincided with postnatal cardiomyocyte arrest. Mis-expression of FRNK in the embryonic heart led to pre-term lethality associated with reduced cardiomyocyte proliferation and led us to speculate that the postnatal FRNK surge might be required to promote quiescence in this growth promoting environment. Herein, we provide strong evidence that endogenous FRNK contributes to post-mitotic arrest. Depletion of FRNK promoted DNA synthesis in post-natal day (P) 10 hearts accompanied by a transient increase in DNA content and multi-nucleation by P14, indicative of DNA replication without cell division. Interestingly, a reduction in tri- and tetra-nucleated cardiomyocytes, concomitant with an increase in bi-nucleated cells by P21, indicated the possibility that FRNK-depleted cardiomyocytes underwent eventual cytokinesis. In support of this conclusion, Aurora B-labeled central spindles (a hallmark of cytokinesis) were observed in tetra-nucleated P20 FRNK(-/-) but not wt cardiomyocytes, while no evidence of apoptosis was observed. Moreover, hearts from FRNK null mice developed ventricular enlargement that persisted until young adulthood which resulted from myocyte expansion rather than myocyte hypertrophy or interstitial growth. These data indicate that endogenous FRNK serves an important role in limiting DNA synthesis and regulating the un-coupling between DNA synthesis and cytokinesis in the post-natal myocardium.

Periostin mediates vascular smooth muscle cell migration through the integrins ανβ3 and ανβ5 and focal adhesion kinase (FAK) pathway

Smooth muscle cell (SMC) migration involves interactions of integrin receptors with extracellular matrix (ECM) and is an important process of neointimal formation in atherosclerosis and restenosis after vascular interventions. Previous studies have shown that periostin (PN), a novel ECM protein, is upregulated in rat carotid artery after balloon injury, and growth factor-stimulated expression of PN promotes SMC migration in vitro. Here, we address the mechanism by which PN–integrin interaction mediates SMC migration in vitro. Aortic SMCs isolated from PN null mice exhibited a significantly reduced ability to migrate and proliferate in vitro. Endogenous PN protein was absent and very low in the culture medium from the primary cultures of PN−/− and wildtype SMCs, respectively. In both types of SMCs, adenovirus-mediated overexpression of HA-tagged PN to a similar extent, which induced a robust cell migration concomitantly with an increase in β3-integrin expression and phosphorylation of FAK (Tyr397). Furthermore, in cultured human SMCs, specific integrin blocking antibodies showed that interactions of PN-ανβ3 and PN-ανβ5, but not PN-β1 integrins, are required for SMC migration. Inhibition of FAK signaling by overexpression of an endogenous FAK inhibitor termed FRNK (FAK-related nonkinase) significantly attenuated FAK (Tyr397) phosphorylation and the SMC migration induced by PN. These results reveal a mechanism whereby PN mediates vascular SMC migration through an interaction with alphaV-integrins (mainly ανβ3) and subsequent activation of FAK pathway.

FAK alters invadopodia and focal adhesion composition and dynamics to regulate breast cancer invasion

Focal adhesion kinase (FAK) is important for breast cancer progression and invasion and is necessary for the dynamic turnover of focal adhesions. However, it has not been determined whether FAK also regulates the dynamics of invasive adhesions formed in cancer cells known as invadopodia. In this study, we report that endogenous FAK functions upstream of cellular Src (c-Src) as a negative regulator of invadopodia formation and dynamics in breast cancer cells. We show that depletion of FAK induces the formation of active invadopodia but impairs invasive cell migration. FAK-deficient MTLn3 breast cancer cells display enhanced assembly and dynamics of invadopodia that are rescued by expression of wild-type FAK but not by FAK that cannot be phosphorylated at tyrosine 397. Moreover, our findings demonstrate that FAK depletion switches phosphotyrosine-containing proteins from focal adhesions to invadopodia through the temporal and spatial regulation of c-Src activity. Collectively, our findings provide novel insight into the interplay between FAK and Src to promote invasion.

Focal Adhesion Kinase as a RhoA-activable Signaling Scaffold Mediating Akt Activation and Cardiomyocyte Protection

RhoA a small G-protein that has an established role in cell growth and in regulation of the actin cytoskeleton. Far less is known about whether RhoA can modulate cell fate. We previously reported that sustained RhoA activation induces cardiomyocyte apoptosis (Del Re, D. P., Miyamoto, S., and Brown, J. H. (2007) J. Biol. Chem. 282, 8069-8078). Here we demonstrate that less chronic RhoA activation affords a survival advantage, protecting cardiomyocytes from apoptotic insult induced by either hydrogen peroxide treatment or glucose deprivation. Under conditions where RhoA is protective, we observe Rho kinase-dependent cytoskeletal rearrangement and activation of focal adhesion kinase (FAK). Activation of endogenous cardiomyocyte FAK leads to its increased association with the p85 regulatory subunit of phosphatidylinositol-3-kinase (PI3K) and to concomitant activation of Akt. Treatment of isolated perfused hearts with sphingosine 1-phosphate recapitulates this response. The pathway by which RhoA mediates cardiomyocyte Akt activation is demonstrated to require Rho kinase, FAK and PI3K, but not Src, based on studies with pharmacological inhibitors (Y-27632, LY294002, PF271 and PP2) and inhibitory protein expression (FAK-related nonkinase). Inhibition of RhoA-mediated Akt activation at any of these steps, including inhibition of FAK, prevents RhoA from protecting cardiomyocytes against apoptotic insult. We further demonstrate that stretch of cardiomyocytes, which activates endogenous RhoA, induces the aforementioned signaling pathway, providing a physiologic context in which RhoA-mediated FAK phosphorylation can activate PI3K and Akt. We suggest that RhoA-mediated effects on the cardiomyocyte cytoskeleton provide a novel mechanism for protection from apoptosis.

A novel focal adhesion kinase from Marsupenaeus japonicus and its response to WSSV infection

Focal adhesion kinase (FAK) is a cytoplasmic protein-tyrosine kinase involved in integrin-mediated signal transduction which regulates multiple cell functions in mammalian cells. In contrast to the well document of FAK in mammalian cells, the properties of FAK in crustacean have not been reported yet and even none of their gene or protein sequences is known to date. Here, we report for the first time the cloning of FAK from Marsupenaeus japonicus (designated as MjFAK) and the identification of its involvement in the virus infection and host defense. Sequence analysis displayed that MjFAK shared strong similarity to FAK family protein-tyrosine kinase, including conserved tyrosine phosphorylation sites, PTK domain and FAT domain. Immunofluorescence staining analysis showed that MjFAK was located prominently at the cell periphery and partly in cytoplasm and nucleus. Notably, considerable high content of MjFAK and MjFAK (pY399) were found in shrimp, which differs greatly from the low level of endogenous FAK and pFAK in the mammalian cells. It implies that pMjFAK may play a significant role in shrimp. Moreover, pMjFAK increased at the early infection stage, and the hemocyte adhesion activity of fibronectin also increased significantly accompanying with its phosphorylation. These results suggest that pMjFAK may not only promote the WSSV infection, but also participate in the defense mechanism via the enhancement of the immune-cell adhesion. Our data provide a clue to recognize the FAK-mediated signaling connection in the control of immunity and virus infection in crustaceans, which will be helpful to shrimp viral disease control.

Focal adhesion kinase regulates insulin resistance in skeletal muscle

On the basis of our previous studies, we investigated the possible role of focal adhesion kinase (FAK) in the development of insulin resistance in skeletal muscle, a major organ responsible for insulin-stimulated glucose uptake. Insulin-resistant C2C12 skeletal muscle cells were transfected with FAK wild-type or FAK mutant plasmids, knocked down using small interfering RNA (siRNA), and their effects on the levels and activities of insulin-signalling molecules and on glucose uptake were determined. A significant decrease in tyrosine phosphorylation of FAK in insulin-resistant C2C12 cells was observed. A similar decrease was observed in skeletal muscle obtained from insulin-resistant Sprague-Dawley rats fed a high-fat diet. Increased levels of FAK in insulin-resistant C2C12 skeletal muscle cells increased insulin sensitivity and glucose uptake. These effects were reversed by an increase in the level of kinase activity mutant FAK or suppression of endogenous FAK by siRNA. FAK was also found to interact downstream with insulin receptor substrate-1, phosphatidylinositol 3-kinase and protein kinase C and glycogen synthase kinase 3beta, leading to translocation of glucose transporter 4 and resulting in the regulation of glucose uptake. The present study provides strong evidence that the modulation of FAK level regulates the insulin sensitivity of skeletal muscle cells. The results demonstrate a direct role of FAK in insulin-resistant skeletal muscle cells for the first time.

Protein kinase Cε-dependent MARCKS phosphorylation in neonatal and adult rat ventricular myocytes

The myristoylated, alanine-rich protein kinase C substrate (MARCKS) is a cytoskeletal protein implicated in the regulation of cell spreading, stress fiber formation, and focal adhesion assembly in nonmuscle cells. However, its precise role in cardiomyocyte growth, and its PKC-dependent regulation have not been fully explored. In this report, we show that MARCKS is expressed and phosphorylated under basal conditions in cultured neonatal and adult rat ventricular myocytes (NRVM and ARVM, respectively). The PKC activators phenylephrine, angiotensin II, and endothelin-1 (ET) further increased MARCKS phosphorylation, with ET inducing the greatest response. To determine which PKC isoenzyme was responsible for agonist-induced MARCKS phosphorylation, NRVM and ARVM were infected with replication-defective adenoviruses (Adv) encoding wildtype (wt) and constitutively active (ca) mutants of PKCε, PKCδ, and PKCα. Only PKCε increased phosphorylated MARCKS (pMARCKS). In contrast, Adv-mediated overexpression of a dominant-negative (dn) mutant of PKCε reduced basal and ET-stimulated pMARCKS. dnPKCε overexpression also prevented ET-induced, apparent co-localization of pMARCKS with f-actin staining structures. Adv-mediated overexpression of GFP-tagged, wtMARCKS (wtMARCKS-GFP) increased phosphorylation of focal adhesion kinase (FAK) and also increased NRVM surface area. In contrast, overexpression of a GFP-tagged, non-phosphorylatable (np) MARCKS mutant (npMARCKS-GFP) decreased basal and ET-induced endogenous MARCKS and FAK phosphorylation, and blocked the ET-induced increase in NRVM surface area. We conclude that MARCKS is expressed in cardiomyocytes, is phosphorylated by PKCε, and participates in the regulation of FAK phosphorylation and cell spreading.

Activation of FAK is necessary for the osteogenic differentiation of human mesenchymal stem cells on laminin‐5

Human mesenchymal stem cell (hMSC) differentiation into osteoblasts and the signaling events involved are poorly understood. We recently established that contact with specific extracellular matrix (ECM) proteins, in particular laminin-5, is sufficient to induce an osteogenic phenotype in hMSC through an extracellular signal-related kinase (ERK)-dependent pathway. Activation of ERK 1/2 by laminin-5 induces phosphorylation of the runx2/cbfa-1 transcription factor that controls osteogenic gene expression. We hypothesized that focal adhesion kinase (FAK) mediated signaling pathways supply a link between cell surface integrin-ECM binding and activation of ERK 1/2, and that laminin-5 promotes its osteogenic effects through this pathway. To test this hypothesis, we plated hMSC on a laminin-5 matrix in the presence or absence of FAK-specific small inhibitory RNAs (siRNA), and assayed for phosphorylation of runx2/cbfa-1 as well as expression of established osteogenic differentiation markers (bone sialoprotein, osteocalcin, alkaline phosphatase, calcium deposition, and mineral:matrix ratio). We found that siRNA treatment reduced total endogenous FAK protein by approximately 40%, and reduced FAK phosphorylation on Y397 by approximately 33% in cells plated on laminin-5 for 30 min. SiRNA treated cells exhibited a decrease in ERK 1/2 phosphorylation after 1 h, and reduced serine/threonine phosphorylation of Runx2/Cbfa-1 after 8 days. Finally, FAK inhibition blocked osteogenic differentiation of hMSC, as assessed by lowered expression of osteogenic genes (RT-PCR), decreased alkaline phosphatase activity, greatly reduced calcium deposition, and a lower mineral:matrix ratio after 28 days in culture. These results establish FAK as an important mediator of laminin-5-induced osteogenic differentiation of hMSC.

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.