Faculty Opinions recommendation of Targeting membrane-localized focal adhesion kinase to focal adhesions: roles of tyrosine phosphorylation and SRC family kinases.

2003 ◽  
Author(s):  
Mark Ginsberg
Keyword(s):  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Src Family Kinases

scholarly journals Autophosphorylation of Tyr397 and its phosphorylation by Src-family kinases are altered in focal-adhesion-kinase neuronal isoforms

2000 ◽  
Vol 348 (1) ◽  
pp. 119-128 ◽  
Author(s):  
Madeleine TOUTANT ◽  
Jeanne-Marie STUDLER ◽  
Ferran BURGAYA ◽  
Alicia COSTA ◽  
Pascal EZAN ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Fluorescent Protein ◽  
Focal Adhesions ◽  
Src Family Kinases ◽  
Critical Residue ◽  
Green Fluorescent ◽  
And Function

In brain, focal adhesion kinase (FAK) is regulated by neurotransmitters and has a higher molecular mass than in other tissues, due to alternative splicing. Two exons code for additional peptides of six and seven residues (‘boxes’ 6 and 7), located on either side of Tyr397, which increase its autophosphorylation. Using in situ hybridization and a monoclonal antibody (Mab77) which does not recognize FAK containing box 7, we show that, although mRNAs coding for boxes 6 and 7 have different patterns of expression in brain, FAK+6,7 is the main isoform in forebrain neurons. The various FAK isoforms fused to green fluorescent protein were all targeted to focal adhesions in non-neuronal cells. Phosphorylation-state-specific antibodies were used to study in detail the phosphorylation of Tyr397, a critical residue for the activation and function of FAK. The presence of boxes 6 and 7 increased autophosphorylation of Tyr397 independently and additively, whereas they had a weak effect on FAK kinase activity towards poly(Glu,Tyr). Src-family kinases were also able to phosphorylate Tyr397 in cells, but this phosphorylation was decreased in the presence of box 6 or 7, and abolished in the presence of both. Thus the additional exons characteristic of neuronal isoforms of FAK do not alter its targeting, but change dramatically the phosphorylation of Tyr397. They increase its autophosphorylation in vitro and in transfected COS-7 cells, whereas they prevent its phosphorylation when co-transfected with Src-family kinases.

scholarly journals Tyrosine phosphorylation of focal adhesion kinase (p125FAK): regulation by cAMP and thrombin in mesangial cells.

1996 ◽  
Vol 7 (3) ◽  
pp. 415-423
Author(s):  
D A Troyer ◽  
A Bouton ◽  
R Bedolla ◽  
R Padilla
Keyword(s):  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Actin Filaments ◽  
Mesangial Cells ◽  
Close Contact ◽  
Focal Adhesions ◽  
Cytochalasin D ◽  
Fiber Formation ◽  
Stress Fibers

Stress fibers, composed of actin filaments, converge upon and associate with a number of proteins, including focal adhesion kinase (p125FAK), and integrin receptors to form areas of close contact between cells and the extracellular matrix referred to as focal adhesions. Treatment of mesangial cells with cAMP-elevating agents causes a loss of focal adhesions, fragmentation of stress fibers, and decreased tyrosine phosphorylation of p125FAK. Thrombin reverses these effects of cAMP, and this model can be used to address some of the cellular mechanisms involved in regulating the loss and formation of focal adhesions. This study reports the effects of cAMP and thrombin on mesangial cell shape, distribution of actin, formation of stress fibers, and tyrosine phosphorylation of p125FAK. cAMP-treated cells display a condensed cell body with slender processes that traverse the area formerly covered by the cell. Addition of thrombin to these cells restores actin filaments (stress fibers) and increases tyrosine phosphorylation of p125FAK, and the cells resume a flattened morphology, even in the continued presence of cAMP-elevating agents. Peptides that mimic the tethered ligand portion of the thrombin receptor have the same effects on cell morphology and stress fiber formation as thrombin. In selected experiments, agents that disrupt either stress fibers (cytochalasin D) or microtubules (nocodazole; Sigma Chemical, St. Louis, MO) were used to examine the role of these cytoskeletal elements in thrombin-induced restoration of focal adhesions. Cytochalasin D blocked the ability of thrombin to restore focal adhesions and phosphorylate p125FAK. The effects of nocodazole, an agent that destabilizes microtubules (but which has no known receptor), are very similar to those of thrombin. The findings discussed in this study indicate that thrombin can modulate the formation of focal adhesions. The organization of stress fibers and microtubules is apparently intimately related to the phosphorylation of p125FAK and can be modulated by soluble receptor agonists such as thrombin or via altered polymerization of microtubules.

scholarly journals Identification of integrin-stimulated sites of serine phosphorylation in FRNK, the separately expressed C-terminal domain of focal adhesion kinase: a potential role for protein kinase A

1997 ◽  
Vol 324 (1) ◽  
pp. 141-149 ◽  
Author(s):  
Alan RICHARDSON ◽  
John D. SHANNON ◽  
Reid B. ADAMS ◽  
Michael D. SCHALLER ◽  
J. Thomas PARSONS
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Serine Phosphorylation ◽  
Early Event ◽  
Kinase A

Focal adhesion kinase (pp125FAK) is a protein tyrosine kinase that is localized to focal adhesions in many cell types and which undergoes tyrosine phosphorylation after integrin binding to extracellular matrix. In some cells the C-terminal non-catalytic domain of pp125FAK is expressed as a separate protein referred to as FRNK (FAK-related, non-kinase). We have previously shown that overexpression of FRNK inhibits tyrosine phosphorylation of pp125FAK and its substrates as well as inhibiting cell spreading on fibronectin. In this report we identify Ser148 and Ser151 as residues in FRNK that are phosphorylated after tyrosine phosphorylation of pp125FAK and in response to integrin binding to fibronectin. Tyrosine phosphorylation of pp125FAK appears to be an early event after integrin occupancy, and serine phosphorylation of FRNK occurs significantly later. Treatment of fibroblasts with a series of protein kinase A inhibitors delayed serine phosphorylation of FRNK as well as cell spreading on fibronectin and tyrosine phosphorylation of pp125FAK. However, these PKA inhibitors are unlikely to delay cell spreading simply by preventing serine phosphorylation of FRNK, as overexpression of FRNK containing mutations of Ser148 and Ser151 either singly or jointly to either alanine or glutamate residues did not significantly alter the ability of FRNK to act as an inhibitor of pp125FAK.

scholarly journals Complex Formation with Focal Adhesion Kinase: A Mechanism to Regulate Activity and Subcellular Localization of Src Kinases

1999 ◽  
Vol 10 (10) ◽  
pp. 3489-3505 ◽  
Author(s):  
Michael D. Schaller ◽  
Jeffrey D. Hildebrand ◽  
J. Thomas Parsons
Keyword(s):  
Subcellular Localization ◽  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Tyrosine Kinases ◽  
Focal Adhesions ◽  
Src Kinases ◽  
High Affinity Binding Site ◽  
Src Homology ◽  
Src Homology 2 Domain

Tyrosine phosphorylation of focal adhesion kinase (FAK) creates a high-affinity binding site for the src homology 2 domain of the Src family of tyrosine kinases. Assembly of a complex between FAK and Src kinases may serve to regulate the subcellular localization and the enzymatic activity of members of the Src family of kinases. We show that simultaneous overexpression of FAK and pp60c-srcor p59fynresults in the enhancement of the tyrosine phosphorylation of a limited number of cellular substrates, including paxillin. Under these conditions, tyrosine phosphorylation of paxillin is largely cell adhesion dependent. FAK mutants defective for Src binding or focal adhesion targeting fail to cooperate with pp60c-srcor p59fynto induce paxillin phosphorylation, whereas catalytically defective FAK mutants can direct paxillin phosphorylation. The negative regulatory site of pp60c-srcis hypophosphorylated when in complex with FAK, and coexpression with FAK leads to a redistribution of pp60c-srcfrom a diffuse cellular location to focal adhesions. A FAK mutant defective for Src binding does not effectively induce the translocation of pp60c-srcto focal adhesions. These results suggest that association with FAK can alter the localization of Src kinases and that FAK functions to direct phosphorylation of cellular substrates by recruitment of Src kinases.

scholarly journals The Cytoplasmic Tyrosines of Integrin Subunit β1 Are Involved in Focal Adhesion Kinase Activation

2000 ◽  
Vol 20 (15) ◽  
pp. 5758-5765 ◽  
Author(s):  
Krister Wennerberg ◽  
Annika Armulik ◽  
Takao Sakai ◽  
Marjam Karlsson ◽  
Reinhard Fässler ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Phosphorylation Site ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Integrin Subunit ◽  
Wild Type ◽  
Directed Cell Migration ◽  
Dependent Cell

ABSTRACT We have previously shown that mutation of the two tyrosines in the cytoplasmic domain of integrin subunit β1 (Y783 and Y795) to phenylalanines markedly reduces the capability of β1A integrins to mediate directed cell migration. In this study, β1-dependent cell spreading was found to be delayed in GD25 cells expressing β1AY783/795F compared to that in wild-type GD25-β1A. Focal adhesion kinase (FAK) tyrosine phosphorylation and activation were severely impaired in response to β1-dependent adhesion in GD25-β1AY783/795F cells compared to that in wild-type GD25-β1A or mutants in which only a single tyrosine was altered (β1AY783F or β1AY795F). Phosphorylation site-specific antibodies selective for FAK phosphotyrosine 397 indicated that the defect in FAK phosphorylation via β1AY783/795F lies at the level of the initial autophosphorylation step. Indeed, β1A-dependent tyrosine phosphorylation of tensin and paxillin was lost in the β1AY783/795F cells, consistent with the impairment in FAK activation. In contrast, p130CAS overall tyrosine phosphorylation was unaffected by the β1 mutations. Despite the defect in β1-mediated FAK activation, FAK was still localized to focal adhesions. Taken together, the phenotype of the GD25-β1AY783/795F cells resembles, but is distinct from, the phenotype observed in FAK-null cells. These observations argue that tyrosines 783 and 795 within the cytoplasmic tail of integrin subunit β1A are critical mediators of FAK activation and cell spreading in GD25 cells.

scholarly journals Inhibition of focal adhesion kinase (FAK) signaling in focal adhesions decreases cell motility and proliferation.

1996 ◽  
Vol 7 (8) ◽  
pp. 1209-1224 ◽  
Author(s):  
A P Gilmore ◽  
L H Romer
Keyword(s):  
Focal Adhesion Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Umbilical Vein ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Kinase Domain ◽  
Glutathione S Transferase ◽  
Umbilical Vein Endothelial Cells ◽  
And Function

It has been proposed that the focal adhesion kinase (FAK) mediates focal adhesion formation through tyrosine phosphorylation during cell adhesion. We investigated the role of FAK in focal adhesion structure and function. Loading cells with a glutathione-S-transferase fusion protein (GST-Cterm) containing the FAK focal adhesion targeting sequence, but not the kinase domain, decreased the association of endogenous FAK with focal adhesions. This displacement of endogenous FAK in both BALB/c 3T3 cells and human umbilical vein endothelial cells loaded with GST-Cterm decreased focal adhesion phosphotyrosine content. Neither cell type, however, exhibited a reduction in focal adhesions after GST-Cterm loading. These results indicate that FAK mediates adhesion-associated tyrosine phosphorylation, but not the formation of focal adhesions. We then examined the effect of inhibiting FAK function on other adhesion-dependent cell behavior. Cells microinjected with GST-Cterm exhibited decreased migration. In addition, cells injected with GST-Cterm had decreased DNA synthesis compared with control-injected or noninjected cells. These findings suggest that FAK functions in the regulation of cell migration and cell proliferation.

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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