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.

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 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.

scholarly journals Identification of Tyr-397 as the primary site of tyrosine phosphorylation and pp60src association in the focal adhesion kinase, pp125FAK.

1995 ◽  
Vol 15 (5) ◽  
pp. 2819-2827 ◽  
Author(s):  
B L Eide ◽  
C W Turck ◽  
J A Escobedo
Keyword(s):  
Extracellular Matrix ◽  
Cell Adhesion ◽  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Consensus Sequence ◽  
Sh2 Domain ◽  
Protein Tyrosine ◽  
Adhesion Plaques

A number of cellular processes, such as proliferation, differentiation, and transformation, are regulated by cell-extracellular matrix interactions. Previous studies have identified a novel tyrosine kinase, the focal adhesion kinase p125FAK, as a component of cell adhesion plaques. p125FAK was identified as a 125-kDa tyrosine-phosphorylated protein in cells transformed by the v-src oncogene. p125FAK is an intracellular protein composed of three domains: a central domain with homology to protein tyrosine kinases, flanked by two noncatalytic domains of 400 amino acids which bear no significant homology to previously cloned proteins. p125FAK is believed to play an important regulatory role in cell adhesion because it localizes to cell adhesion plaques and because its phosphorylation on tyrosine residues is regulated by binding of cell surface integrins to the extracellular matrix. Recent studies have shown that Src, through its SH2 domain, stably associates with pp125FAK and that this association prevents dephosphorylation of pp125FAK in vitro by protein tyrosine phosphatases. In this report, we identify Tyr-397 as the primary in vivo and in vitro site of p125FAK tyrosine phosphorylation and association with Src. Substituting phenylalanine for tyrosine at position 397 significantly reduces p125FAK tyrosine phosphorylation and association with Src but does not abolish p125FAK kinase activity. In addition, p125FAK kinase is able to trans-phosphorylate Tyr-397 in vitro in a kinase-deficient p125FAK variant. Phosphorylation of Tyr-397 provides a site [Y(P)AEI] that fits the consensus sequence for the binding of Src.

scholarly journals Impaired Integrin-mediated Adhesion and Signaling in Fibroblasts Expressing a Dominant-negative Mutant PTP1B

1998 ◽  
Vol 143 (3) ◽  
pp. 861-873 ◽  
Author(s):  
Carlos O. Arregui ◽  
Janne Balsamo ◽  
Jack Lilien
Keyword(s):  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Tyrosine Phosphatase ◽  
Focal Adhesions ◽  
Dominant Negative ◽  
Stress Fibers ◽  
Dominant Negative Mutant ◽  
Β1 Integrin ◽  
Wild Type ◽  
In Cells

To investigate the role of nonreceptor protein tyrosine phosphatase 1B (PTP1B) in β1-integrin– mediated adhesion and signaling, we transfected mouse L cells with normal and catalytically inactive forms of the phosphatase. Parental cells and cells expressing the wild-type or mutant PTP1B were assayed for (a) adhesion, (b) spreading, (c) presence of focal adhesions and stress fibers, and (d) tyrosine phosphorylation. Parental cells and cells expressing wild-type PTP1B show similar morphology, are able to attach and spread on fibronectin, and form focal adhesions and stress fibers. In contrast, cells expressing the inactive PTP1B have a spindle-shaped morphology, reduced adhesion and spreading on fibronectin, and almost a complete absence of focal adhesions and stress fibers. Attachment to fibronectin induces tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin in parental cells and cells transfected with the wild-type PTP1B, while in cells transfected with the mutant PTP1B, such induction is not observed. Additionally, in cells expressing the mutant PTP1B, tyrosine phosphorylation of Src is enhanced and activity is reduced. Lysophosphatidic acid temporarily reverses the effects of the mutant PTP1B, suggesting the existence of a signaling pathway triggering focal adhesion assembly that bypasses the need for active PTP1B. PTP1B coimmunoprecipitates with β1-integrin from nonionic detergent extracts and colocalizes with vinculin and the ends of actin stress fibers in focal adhesions. Our data suggest that PTP1B is a critical regulatory component of integrin signaling pathways, which is essential for adhesion, spreading, and formation of focal adhesions.

scholarly journals Focal Adhesion Kinase Is a Substrate and Downstream Effector of SHP-2 Complexed with Helicobacter pylori CagA

2006 ◽  
Vol 26 (1) ◽  
pp. 261-276 ◽  
Author(s):  
Ryouhei Tsutsumi ◽  
Atsushi Takahashi ◽  
Takeshi Azuma ◽  
Hideaki Higashi ◽  
Masanori Hatakeyama
Keyword(s):  
Helicobacter Pylori ◽  
Epithelial Cells ◽  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Cell Shape ◽  
Dominant Negative ◽  
Gastric Epithelial Cells ◽  
Dependent Inhibition ◽  
H Pylori

ABSTRACT Infection with cagA-positive Helicobacter pylori (H. pylori) is associated with atrophic gastritis, peptic ulcer, and gastric adenocarcinoma. The cagA gene product CagA is translocated from H. pylori into gastric epithelial cells and undergoes tyrosine phosphorylation by Src family kinases (SFKs). Tyrosine-phosphorylated CagA binds and activates SHP-2 phosphatase and the C-terminal Src kinase (Csk) while inducing an elongated cell shape termed the “hummingbird phenotype.” Here we show that CagA reduces the level of focal adhesion kinase (FAK) tyrosine phosphorylation in gastric epithelial cells. The decrease in phosphorylated FAK is due to SHP-2-mediated dephosphorylation of FAK at the activating phosphorylation sites, not due to Csk-dependent inhibition of SFKs, which phosphorylate FAK. Coexpression of constitutively active FAK with CagA inhibits induction of the hummingbird phenotype, whereas expression of dominant-negative FAK elicits an elongated cell shape characteristic of the hummingbird phenotype. These results indicate that inhibition of FAK by SHP-2 plays a crucial role in the morphogenetic activity of CagA. Impaired cell adhesion and increased motility by CagA may be involved in the development of gastric lesions associated with cagA-positive H. pylori infection.

scholarly journals Clustering of cell surface (beta)1,4-galactosyltransferase I induces transient tyrosine phosphorylation of focal adhesion kinase and loss of stress fibers

2000 ◽  
Vol 113 (2) ◽  
pp. 237-245 ◽  
Author(s):  
M.J. Wassler ◽  
B.D. Shur
Keyword(s):  
Extracellular Matrix ◽  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Basal Lamina ◽  
Stress Fibers ◽  
Intracellular Signal ◽  
Signal Cascades ◽  
Galactosyltransferase I ◽  
Actin Stress Fibers

It is well appreciated that clustering of receptors for the extracellular matrix, most notably the integrins, elicits intracellular signal cascades. One of the first indications that integrin-dependent signaling has occurred is by the activation of focal adhesion kinase (FAK). Another, although less well understood, receptor for the extracellular matrix is (beta)1, 4-galactosyltransferase I (GalT). GalT participates during lamellipodia formation and cell migration by recognizing terminal N-acetylglucosamine residues on basal lamina glycosides. In this study, we investigated whether GalT is also capable of eliciting intracellular signal cascades, specifically FAK activation, in response to ligand binding and/or aggregation. 3T3 fibroblasts were treated with two different reagents capable of aggregating GalT, either antibodies raised against recombinant GalT or multivalent polymers of N-acetylglucosamine, and the effects on tyrosine phosphorylation were analyzed. Both reagents induced an initial tyrosine phosphorylation (1-2 minutes) and subsequent dephosphorylation (5-10 minutes) of proteins with molecular mass 67 and 125 kDa. These proteins were identified as paxillin and FAK, respectively, by immunoprecipitation with anti-paxillin and anti-FAK antibodies. Preimmune IgG, anti-GalT Fab fragments, irrelevant polymers and monomeric N-acetylglucosamine had no effect. The ability of GalT aggregation to induce transient tyrosine phosphorylation was dependent upon cell density. In addition, FAK dephosphorylation was found to be sensitive to the phosphatase inhibitor, sodium pervanadate. Similar to the integrins, GalT requires association with the cytoskeleton in order to function as a matrix receptor. To determine if the transient tyrosine phosphorylation of FAK was dependent upon GalT binding to the cytoskeleton, stably transfected fibroblasts expressing different amounts of GalT were treated with polymeric N-acetylglucosamine. Cells expressing increased levels of GalT associated with the cytoskeleton showed increased levels of FAK tyrosine phosphorylation and prolonged dephosphorylation, relative to control cells. In contrast, cells in which a dominant negative form of GalT prevents association with the cytoskeleton showed no or weak response to polymeric N-acetylglucosamine. Concomitant with the GalT-stimulated dephosphorylation of FAK, cells treated with anti-GalT antibodies or polymeric N-acetylglucosamine showed a loss of actin stress fibers and focal adhesions. Pervanadate treatment inhibited the GalT-dependent loss of actin stress fibers. To confirm the requirement of GalT in transient FAK phosphorylation and stress fiber reorganization in this system, we created cells homozygous null for the GalT isoform that functions as a matrix receptor. These cells were incapable of phosphorylating FAK in response to GalT agonists and, interestingly, showed a lack of lamellar stress fibers when cultured on basal lamina matrices. These data suggest that GalT function as a basal lamina receptor involves transient activation of FAK and an associated reorganization of stress fibers.

scholarly journals Paxillin Binding Is Not the Sole Determinant of Focal Adhesion Localization or Dominant-Negative Activity of Focal Adhesion Kinase/Focal Adhesion Kinase-related Nonkinase

2000 ◽  
Vol 11 (9) ◽  
pp. 3247-3263 ◽  
Author(s):  
Marion A. Cooley ◽  
Jill M. Broome ◽  
Christoph Ohngemach ◽  
Lewis H. Romer ◽  
Michael D. Schaller
Keyword(s):  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Mutational Analysis ◽  
Focal Adhesions ◽  
Binding Activity ◽  
Dominant Negative ◽  
Dependent Inhibition ◽  
Dominant Negative Activity ◽  
Carboxy Terminal

The carboxy-terminal 150 residues of the focal adhesion kinase (FAK) comprise the focal adhesion-targeting sequence, which is responsible for its subcellular localization. The mechanism of focal adhesion targeting has not been fully elucidated. We describe a mutational analysis of the focal adhesion-targeting sequence of FAK to further examine the mechanism of focal adhesion targeting and explore additional functions encoded by the carboxy-terminus of FAK. The results demonstrate that paxillin binding is dispensable for focal adhesion targeting of FAK. Cell adhesion-dependent tyrosine phosphorylation strictly correlated with the ability of mutants to target to focal adhesions. Focal adhesion targeting was also a requirement for maximal FAK-dependent tyrosine phosphorylation of paxillin and FAK-related nonkinase (FRNK)–dependent inhibition of endogenous FAK function. However, there were additional requirements for these latter functions because we identified mutants that target to focal adhesions, yet are defective for the induction of paxillin phosphorylation or the dominant-negative function of FRNK. Furthermore, the paxillin-binding activity of FRNK mutants did not correlate with their ability to inhibit FAK, suggesting that FRNK has other targets in addition to paxillin.

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