scholarly journals Identification of p130Cas as a Mediator of Focal Adhesion Kinase–promoted Cell Migration

1998 ◽  
Vol 140 (1) ◽  
pp. 211-221 ◽  
Author(s):  
Leslie A. Cary ◽  
Dong Cho Han ◽  
Thomas R. Polte ◽  
Steven K. Hanks ◽  
Jun-Lin Guan
Keyword(s):  
Cell Migration ◽  
Binding Site ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Cho Cells ◽  
Mek Inhibitor ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Sh2 Domains ◽  
Promote Cell Migration

Previously we have demonstrated that focal adhesion kinase (FAK)-promoted migration on fibronectin (FN) by its overexpression in CHO cells is dependent on FAK autophosphorylation at Y397 and subsequent binding of Src to this site. In this report, we have examined the role of FAK association with Grb2 and p130Cas, two downstream events of the FAK/Src complex that could mediate integrin-stimulated activation of extracellular signal-regulated kinases (Erks). We show that a Y925F FAK mutant was able to promote cell migration as efficiently as FAK and that the transfected FAK demonstrated no detectable association with Grb2 in CHO cells. In contrast, cells expressing a FAK P712/715A mutant demonstrated a level of migration comparable to that of control cells. This mutation did not affect FAK kinase activity, autophosphorylation, or Src association but did significantly reduce p130Cas association with FAK. Furthermore, FAK expression in CHO cells increased tyrosine phosphorylation of p130Cas and its subsequent binding to several SH2 domains, which depended on both the p130Cas binding site and the Src binding site. However, we did not detect increased activation of Erks in cells expressing FAK, and the MEK inhibitor PD98059 did not decrease FAK-promoted cell migration. Finally, we show that coexpression of p130Cas further increased cell migration on FN and coexpression of the p130Cas SH3 domain alone functioned as a dominant negative mutant and decreased cell migration. Together, these results demonstrate that p130Cas, but not Grb2, is a mediator of FAK-promoted cell migration and suggest that FAK/ p130Cas complex targets downstream pathways other than Erks in mediating FAK-promoted cell migration.

Calcium-and integrin-binding protein regulates focal adhesion kinase activity during platelet spreading on immobilized fibrinogen

Blood ◽  
2003 ◽  
Vol 102 (10) ◽  
pp. 3629-3636 ◽  
Author(s):  
Meghna U. Naik ◽  
Ulhas P. Naik
Keyword(s):  
Cell Migration ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Cho Cells ◽  
Binding Protein ◽  
Chinese Hamster ◽  
Focal Adhesions ◽  
Dominant Negative ◽  
Focal Complex ◽  
Platelet Spreading

AbstractPlatelet spreading on the subendothelium in response to vascular injury is fundamental to the regulation of physiologic hemostasis. Previously, we have shown that, when bound to glycoprotein IIb (GPIIb), calcium- and integrin-binding protein (CIB) regulates platelet spreading on immobilized fibrinogen (Fg). In this study, we investigated the signaling events that occur downstream of CIB in the absence of signaling that occurs as a result of granular secretion. Using Chinese hamster ovary (CHO) cells as a model, we demonstrate that CIB induces cell migration. Immunofluorescence analysis of CIB localization indicates that endogenous CIB accumulates in areas of focal adhesions, and its overexpression up-regulates the formation of focal adhesion complexes compared with control cells. Immunoprecipitation analysis indicates that CIB associates with focal adhesion kinase (FAK), a key regulator in focal complex formation, and up-regulates its activity. Overexpression of dominant-negative FAK, FRNK, along with CIB in CHO cells completely inhibits CIB-induced cell migration. Further, confirmation of these data in the platelet system indicates that CIB and FAK associate throughout all stages of platelet spreading but only on Fg binding to GPIIb/IIIa. Taken together, our results suggest that CIB regulates platelet spreading through the regulation of FAK activation. (Blood. 2003;102: 3629-3636)

Induced focal adhesion kinase expression suppresses apoptosis by activating NF-κB signaling in intestinal epithelial cells

2006 ◽  
Vol 290 (5) ◽  
pp. C1310-C1320 ◽  
Author(s):  
Huifang M. Zhang ◽  
Kaspar M. Keledjian ◽  
Jaladanki N. Rao ◽  
Tongtong Zou ◽  
Lan Liu ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Intestinal Epithelial Cells ◽  
Ectopic Expression ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Intestinal Epithelial ◽  
Tnf Α ◽  
Induced Apoptosis

Focal adhesion kinase (FAK) integrates various extracellular and intracellular signals and is implicated in a variety of biological functions, but its exact role and downstream targeting signals in the regulation of apoptosis in intestinal epithelial cells (IECs) remains unclear. The current study tested the hypothesis that FAK has an antiapoptotic role in the IEC-6 cell line by altering NF-κB signaling. Induced FAK expression by stable transfection with the wild-type (WT)-FAK gene increased FAK phosphorylation, which was associated with an increase in NF-κB activity. These stable WT-FAK-transfected IECs also exhibited increased resistance to apoptosis when they were exposed to TNF-α plus cycloheximide (TNF-α/CHX). Specific inhibition of NF-κB by the recombinant adenoviral vector containing the IκBα superrepressor prevented increased resistance to apoptosis in WT-FAK-transfected cells. In contrast, inactivation of FAK by ectopic expression of dominant-negative mutant of FAK (DNM-FAK) inhibited NF-κB activity and increased the sensitivity to TNF-α/CHX-induced apoptosis. Furthermore, induced expression of endogenous FAK by depletion of cellular polyamines increased NF-κB activity and resulted in increased resistance to TNF-α/CHX-induced apoptosis, both of which were prevented by overexpression of DNM-FAK. These results indicate that increased expression of FAK suppresses TNF-α/CHX-induced apoptosis, at least partially, through the activation of NF-κB signaling in IECs.

scholarly journals Insulin-induced tyrosine dephosphorylation of paxillin and focal adhesion kinase requires active phosphotyrosine phosphatase 1D

1996 ◽  
Vol 318 (2) ◽  
pp. 609-614 ◽  
Author(s):  
D. Margriet OUWENS ◽  
Harald M. M. MIKKERS ◽  
Gerard C. M. van der ZON ◽  
Matthias STEIN-GERLACH ◽  
Axel ULLRICH ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Insulin Receptor ◽  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Dominant Negative ◽  
Matrix Proteins ◽  
Dominant Negative Mutant ◽  
Phosphotyrosine Phosphatase ◽  
Insulin Receptor Substrates

Insulin stimulation of fibroblasts rapidly induces the tyrosine dephosphorylation of proteins of 68 kDa and 125 kDa, in addition to the tyrosine phosphorylation of the insulin receptor β-chain, insulin receptor substrates 1 and 2, and Shc. Using specific antibodies, the 68 kDa and 125 kDa proteins were identified as paxillin and focal adhesion kinase (pp125FAK) respectively. We have examined whether dephosphorylation of paxillin and pp125FAK requires interaction of the cells with the extracellular matrix. For this, cells were grown on poly(l-lysine) plates, and the tyrosine phosphorylation of pp125FAK and paxillin was increased by addition of lysophosphatidic acid. Under these conditions, insulin still induced the complete dephosphorylation of pp125FAK and paxillin, indicating that this process can occur independently of the interaction of integrins with extracellular matrix proteins. We also studied whether dephosphorylation of pp125FAK and paxillin results from the action of a phosphotyrosine phosphatase. It was found that phenylarsine oxide, a phosphotyrosine phosphatase inhibitor, prevented the insulin-induced dephosphorylation of pp125FAK and paxillin. Furthermore, this insulin-induced dephosphorylation was also impaired in cells expressing a dominant-negative mutant of phosphotyrosine phosphatase 1D (PTP 1D). Thus we have identified paxillin as a target for dephosphorylation by insulin. In addition, we have obtained evidence that the insulin-mediated dephosphorylation of paxillin and pp125FAK requires active PTP 1D.

scholarly journals v-Crk Activates the Phosphoinositide 3-Kinase/AKT Pathway by Utilizing Focal Adhesion Kinase and H-Ras

2002 ◽  
Vol 22 (20) ◽  
pp. 7015-7023 ◽  
Author(s):  
Tsuyoshi Akagi ◽  
Kazutaka Murata ◽  
Tomoyuki Shishido ◽  
Hidesaburo Hanafusa
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Critical Role ◽  
Sh3 Domain ◽  
Sh2 Domain ◽  
Dominant Negative ◽  
Regulatory Subunit ◽  
Akt Pathway ◽  
Dominant Negative Mutant ◽  
Phosphoinositide 3 Kinase

ABSTRACT v-Crk, an oncogene product of avian sarcoma virus CT10, efficiently transforms chicken embryo fibroblasts (CEF). We have recently reported that constitutive activation of the phosphoinositide 3-kinase (PI3K)/AKT pathway plays a critical role in the v-Crk-induced transformation of CEF. In the present study we investigated the molecular mechanism by which v-Crk activates the PI3K/AKT pathway. First, we found that v-Crk promotes the association of the p85 regulatory subunit of PI3K with focal adhesion kinase (FAK) by inducing the phosphorylation of the Y397 residue in FAK. This FAK phosphorylation needs activation of the Src family tyrosine kinase(s) for which the v-Crk SH2 domain is responsible. v-Crk was unable to activate the PI3K/AKT pathway in FAK-null cells, indicating the functional importance of FAK. In addition, we found that H-Ras is also required for the activation of the PI3K/AKT pathway. The v-Crk-induced activation of AKT was greatly enhanced by the overexpression of H-Ras or its guanine nucleotide exchange factor mSOS, which binds to the v-Crk SH3 domain, whereas a dominant-negative mutant of H-Ras almost completely suppressed this activation. Furthermore, we showed that v-Crk stimulates the interaction of H-Ras with the Ras binding domain in the PI3K p110 catalytic subunit. Our data indicated that the v-Crk-induced activation of PI3K/AKT pathway was cooperatively achieved by two distinct interactions. One is the interaction of p85 with tyrosine-phosphorylated FAK promoted by the v-Crk SH2 domain, and another is the interaction of p110 with H-Ras dictated by the v-Crk SH3 domain.

Stimulation of cell migration by overexpression of focal adhesion kinase and its association with Src and Fyn

1996 ◽  
Vol 109 (7) ◽  
pp. 1787-1794 ◽  
Author(s):  
L.A. Cary ◽  
J.F. Chang ◽  
J.L. Guan
Keyword(s):  
Cell Migration ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Cho Cells ◽  
Critical Role ◽  
Cellular Interactions ◽  
Cellular Functions ◽  
And Migration ◽  
Biochemical Signals

Cellular interactions with the extracellular matrix proteins play important roles in a variety of biological processes. Recent studies suggest that integrin-mediated cell-matrix interaction can transduce biochemical signals across the plasma membrane to regulate cellular functions such as proliferation, differentiation and migration. These studies have implicated a critical role of focal adhesion kinase (FAK) in integrin-mediated signal transduction pathways. We report here that overexpression of FAK in CHO cells increased their migration on fibronectin. A mutation of the major autophosphorylation site Y397 in FAK abolished its ability to stimulate cell migration, while phosphorylation of Y397 in a kinase-defective FAK by endogenous FAK led to increased migration. We also find that the wild-type and the kinase-defective FAK were associated with Src and Fyn in CHO cells whereas the F397 mutant was not. These results directly demonstrate a functional role for FAK in integrin signaling leading to cell migration. They also provide evidence for the functional significance of FAK/Src complex formation in vivo.

Phosphatidylinositol 3-kinase mediates integrin-dependent NF-(κ)B and MAPK activation through separate signaling pathways

2001 ◽  
Vol 114 (8) ◽  
pp. 1579-1589 ◽  
Author(s):  
M. Reyes-Reyes ◽  
N. Mora ◽  
A. Zentella ◽  
C. Rosales
Keyword(s):  
Signaling Pathways ◽  
Mapk Pathway ◽  
Mek Inhibitor ◽  
Small Gtpase ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Dependent Manner ◽  
Monocytic Leukemia ◽  
Monocytic Cells ◽  
Mapk Activation

Integrin-mediated signals play an important but poorly understood role in regulating many leukocyte functions. In monocytes and monocytic leukemia cells, (β)1 integrin-mediated adhesion results in a strong induction of immediate-early genes that are important in inflammation. To investigate the signaling pathways from integrins in monocytic cells, THP-1 cells were stimulated via (β)1 integrins by binding to fibronectin and by crosslinking the integrins with specific monoclonal antibodies. The involvement of MAPK and PI 3-K on nuclear factor (κ)B (NF-(κ)B) activation was then analyzed. We found that integrins activated both NF-(κ)B and MAPK in a PI 3-K-dependent manner, as wortmannin and LY294002 blocked these responses. However, the specific MEK inhibitor PD98059 did not prevent integrin-mediated NF-(κ)B activation. In contrast, a dominant negative mutant of Rac completely prevented NF-(κ)B activation, but it did not affect MAPK activation. These results indicate that integrin signaling to NF-(κ)B is not mediated by the MAPK pathway, but rather by the small GTPase Rac. In addition, a dominant negative form of Ρ augmented NF-(κ)B activation and blocked MAPK activation, implying that these two pathways are in competition with each other. These data suggest that integrins activate different signaling pathways in monocytic cells. One uses PI 3-K and Rac to activate NF-(κ)B, while the other uses PI 3-K, MEK, and MAPK to activate other nuclear factors, such as Elk-1.

scholarly journals PKCθ signaling is required for myoblast fusion by regulating the expression of caveolin-3 and β1D integrin upstream focal adhesion kinase

2011 ◽  
Vol 22 (8) ◽  
pp. 1409-1419 ◽  
Author(s):  
Luca Madaro ◽  
Valeria Marrocco ◽  
Piera Fiore ◽  
Paola Aulino ◽  
Piera Smeriglio ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Muscle Regeneration ◽  
Myoblast Fusion ◽  
Dominant Negative Mutant ◽  
Postnatal Life ◽  
Mutant Form ◽  
In Vitro Differentiation ◽  
Freeze Injury

Fusion of mononucleated myoblasts to form multinucleated myofibers is an essential phase of skeletal myogenesis, which occurs during muscle development as well as during postnatal life for muscle growth, turnover, and regeneration. Many cell adhesion proteins, including integrins, have been shown to be important for myoblast fusion in vertebrates, and recently focal adhesion kinase (FAK), has been proposed as a key mediator of myoblast fusion. Here we focused on the possible role of PKCθ, the PKC isoform predominantly expressed in skeletal muscle, in myoblast fusion. We found that the expression of PKCθ is strongly up-regulated following freeze injury–induced muscle regeneration, as well as during in vitro differentiation of satellite cells (SCs; the muscle stem cells). Using both PKCθ knockout and muscle-specific PKCθ dominant-negative mutant mouse models, we observed delayed body and muscle fiber growth during the first weeks of postnatal life, when compared with wild-type (WT) mice. We also found that myofiber formation, during muscle regeneration after freeze injury, was markedly impaired in PKCθ mutant mice, as compared with WT. This phenotype was associated with reduced expression of the myogenic differentiation program executor, myogenin, but not with that of the SC marker Pax7. Indeed in vitro differentiation of primary muscle-derived SCs from PKCθ mutants resulted in the formation of thinner myotubes with reduced numbers of myonuclei and reduced fusion rate, when compared with WT cells. These effects were associated to reduced expression of the profusion genes caveolin-3 and β1D integrin and to reduced activation/phosphorylation of their up-stream regulator FAK. Indeed the exogenous expression of a constitutively active mutant form of PKCθ in muscle cells induced FAK phosphorylation. Moreover pharmacologically mediated full inhibition of FAK activity led to similar fusion defects in both WT and PKCθ-null myoblasts. We thus propose that PKCθ signaling regulates myoblast fusion by regulating, at least in part, FAK activity, essential for profusion gene expression.

New concepts regarding focal adhesion kinase promotion of cell migration and proliferation

2006 ◽  
Vol 99 (1) ◽  
pp. 35-52 ◽  
Author(s):  
Braden D. Cox ◽  
Meera Natarajan ◽  
Michelle R. Stettner ◽  
Candece L. Gladson
Keyword(s):  
Cell Migration ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
New Concepts ◽  
Cell Migration And Proliferation
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