Concerted modulation of paxillin dynamics at focal adhesions by deleted in liver cancer-1 and focal adhesion kinase during early cell spreading

Cytoskeleton ◽  
2014 ◽  
Vol 71 (12) ◽  
pp. 677-694 ◽  
Author(s):  
Shelly Kaushik ◽  
Archna Ravi ◽  
Feroz M. Hameed ◽  
Boon Chuan Low
Keyword(s):  
Liver Cancer ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Cell Spreading

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 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 The Association of ASAP1, an ADP Ribosylation Factor-GTPase Activating Protein, with Focal Adhesion Kinase Contributes to the Process of Focal Adhesion Assembly

2002 ◽  
Vol 13 (6) ◽  
pp. 2147-2156 ◽  
Author(s):  
Yunhao Liu ◽  
Joost C. Loijens ◽  
Karen H. Martin ◽  
Andrei V. Karginov ◽  
J. Thomas Parsons
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Ph Domain ◽  
Wild Type ◽  
Gtpase Activating Protein ◽  
Adp Ribosylation ◽  
Transient Overexpression ◽  
Adp Ribosylation Factor

ASAP1 (ADP ribosylation factor [ARF]- GTPase-activating protein [GAP] containing SH3, ANK repeats, and PH domain) is a phospholipid-dependent ARF-GAP that binds to and is phosphorylated by pp60Src. Using affinity chromatography and yeast two-hybrid interaction screens, we identified ASAP1 as a major binding partner of protein tyrosine kinase focal adhesion kinase (FAK). GlutathioneS-transferase pull-down and coimmunoprecipitation assays showed the binding of ASAP1 to FAK is mediated by an interaction between the C-terminal SH3 domain of ASAP1 with the second proline-rich motif in the C-terminal region of FAK. Transient overexpression of wild-type ASAP1 significantly retarded the spreading of REF52 cells plated on fibronectin. In contrast, overexpression of a truncated variant of ASAP1 that failed to bind FAK or a catalytically inactive variant of ASAP1 lacking GAP activity resulted in a less pronounced inhibition of cell spreading. Transient overexpression of wild-type ASAP1 prevented the efficient organization of paxillin and FAK in focal adhesions during cell spreading, while failing to significantly alter vinculin localization and organization. We conclude from these studies that modulation of ARF activity by ASAP1 is important for the regulation of focal adhesion assembly and/or organization by influencing the mechanisms responsible for the recruitment and organization of selected focal adhesion proteins such as paxillin and FAK.

scholarly journals Cardiac fibroblasts require focal adhesion kinase for normal proliferation and migration

2009 ◽  
Vol 296 (3) ◽  
pp. H627-H638 ◽  
Author(s):  
Ana Maria Manso ◽  
Seok-Min Kang ◽  
Sergey V. Plotnikov ◽  
Ingo Thievessen ◽  
Jaewon Oh ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Adult Mouse ◽  
Cardiac Fibroblasts ◽  
Focal Adhesions ◽  
Protein Levels ◽  
Tyrosine Kinase 2 ◽  
A Cell ◽  
And Migration ◽  
Proliferation And Migration

Migration and proliferation of cardiac fibroblasts (CFs) play an important role in the myocardial remodeling process. While many factors have been identified that regulate CF growth and migration, less is known about the signaling mechanisms involved in these processes. Here, we utilized Cre-LoxP technology to obtain focal adhesion kinase (FAK)-deficient adult mouse CFs and studied how FAK functioned in modulating cell adhesion, proliferation, and migration of these cells. Treatment of FAKflox/flox CFs with Ad/Cre virus caused over 70% reduction of FAK protein levels within a cell population. FAK-deficient CFs showed no changes in focal adhesions, cell morphology, or protein expression levels of vinculin, talin, or paxillin; proline-rich tyrosine kinase 2 (Pyk2) expression and activity were increased. Knockdown of FAK protein in CFs increased PDGF-BB-induced proliferation, while it reduced PDGF-BB-induced migration. Adhesion to fibronectin was not altered. To distinguish between the function of FAK and Pyk2, FAK function was inhibited via adenoviral-mediated overexpression of the natural FAK inhibitor FAK-related nonkinase (FRNK). Ad/FRNK had no effect on Pyk2 expression, inhibited the PDGF-BB-induced migration, but did not change the PDGF-BB-induced proliferation. FAK deficiency had only modest effects on increasing PDGF-BB activation of p38 and JNK MAPKs, with no alteration in the ERK response vs. control cells. These results demonstrate that FAK is required for the PDGF-BB-induced migratory response of adult mouse CFs and suggest that FAK could play an essential role in the wound-healing response that occurs in numerous cardiac pathologies.

scholarly journals Contractility modulates cell adhesion strengthening through focal adhesion kinase and assembly of vinculin-containing focal adhesions

2010 ◽  
pp. n/a-n/a ◽  
Author(s):  
David W. Dumbauld ◽  
Heungsoo Shin ◽  
Nathan D. Gallant ◽  
Kristin E. Michael ◽  
Harish Radhakrishna ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Focal Adhesions

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 Calcium Rises Locally Trigger Focal Adhesion Disassembly and Enhance Residency of Focal Adhesion Kinase at Focal Adhesions

2004 ◽  
Vol 279 (27) ◽  
pp. 28715-28723 ◽  
Author(s):  
Grégory Giannone ◽  
Philippe Rondé ◽  
Mireille Gaire ◽  
Joël Beaudouin ◽  
Jacques Haiech ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Focal Adhesions

scholarly journals Direct association of pp125FAK with paxillin, the focal adhesion-targeting mechanism of pp125FAK.

1995 ◽  
Vol 182 (4) ◽  
pp. 1089-1099 ◽  
Author(s):  
K Tachibana ◽  
T Sato ◽  
N D'Avirro ◽  
C Morimoto
Keyword(s):  
Amino Acids ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Fusion Proteins ◽  
Focal Adhesions ◽  
Immunohistochemical Analysis ◽  
Binding Activity ◽  
Glutathione S Transferase ◽  
Cytoskeleton Protein ◽  
Direct Association

Focal adhesion kinase (pp125FAK) is localized to focal adhesions and tyrosine phosphorylated by the engagement of beta 1 integrins. However, it is unclear how pp125FAK is linked to integrin molecules. We demonstrate that pp125FAK is directly associated with paxillin, a 68-kD cytoskeleton protein. The COOH-terminal domain of pp125FAK spanning FAK residues 919-1042 is sufficient for paxillin binding and has vinculin-homologous amino acids, which are essential for paxillin binding. Microinjection and subsequent immunohistochemical analysis reveal that glutathione S-transferase-FAK fusion proteins, which bind to paxillin, localize to focal adhesions, whereas fusion proteins with no paxillin-binding activity do not localize to focal adhesions. These findings strongly suggest that pp125FAK is localized to focal adhesions by the direct association with paxillin.

Focal Adhesion Kinase Regulates Critical Functions in Hematopoiesis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1407-1407
Author(s):  
Sasidhar Vemula ◽  
Benjamin P. Abratigue ◽  
Premchand Gandra ◽  
John T. O’Malley ◽  
Ayek-Nati N. Ahyi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Extracellular Matrix ◽  
Growth Factor ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Egf Receptor ◽  
Bone Marrow Cells ◽  
Focal Adhesions ◽  
Growth Factor Receptors ◽  
Fold Reduction

Abstract Focal adhesion kinase (FAK) initially identified as a unique cytoplasmic tyrosine kinase involved in focal adhesions, has been studied extensively in fibroblasts. In these cells, FAK has been shown to play an essential role in bridging signals between integrin and growth factor receptors such as the PDGF and the EGF receptor. In fibroblasts, FAK localizes to regions of the cell that attach to the extracellular matrix and coordinates signals from integrins, cytokines, growth factor receptors, and oncogenes. In some tumors, FAK is over-expressed or constitutively activated, which correlates with increased motility, invasiveness, and proliferation. More recently, expression of FAK in acute myeloid leukemia was associated with enhanced blast migration, increased cellularity, and poor prognosis. However, virtually nothing is known about FAKs role in normal hematopoiesis. FAK is expressed in blood cells, including in bone marrow derived KIT+, Gr-1+, Mac-1+, CD4+, CD8+ and B220+ cells. To determine how loss of FAK affects hematopoiesis, we have generated a mouse model with hematopoietic restricted deletion of FAK. We deleted FAK in bone marrow cells by crossing the FAK-flox mice to Mx.Cre+ expressing mice and by treating Mx.cre+FAK+/+ and Mx.cre+FAKflox/flox mice with poly (I)-poly(C) and then analyzing mice 1 month after the last injection. After one month of poly(I)-poly(C) induction, the progeny failed to express detectable levels of FAK in bone marrow, spleen as well as in bone marrow derived macrophages as determined by PCR and western blotting. Evaluation of peripheral blood counts in control and FAK deleted mice revealed modest but significant differences in different lineages (WBC k/μl: FAK; 14 vs. FAK−/−; 10.3, n=7, *p<0.05, LY k/μl: FAK; 10.48 vs. FAK−/−; 7.26, n=7, *p<0.005, RBC k/μl: FAK; 9.76 X106 vs. FAK−/−;8.58 X106 n=7 *p<0.003, PLT k/μl: FAK; 644 vs. FAK−/−; 434 n=7 *p<0.007). Since macrophages express abundant levels of FAK and are rapidly recruited in large numbers to sites of infection, we initially examined the role of FAK in macrophages by creating a well studied model of aseptic thioglycolate-induced peritonitis. Our results demonstrate a ∼1.5 fold reduction in the migration of macrophages to the peritoneal cavity of FAK−/− mice compared to controls (n=5, FAK; 1.8 X 106 vs. FAK−/−; 1.213 X106, *p<0.03). The reduction in recruitment of FAK−/− macrophages was observed in spite of comparable levels of F4/80 expression (WT; 92.98% vs. FAK−/−; 94.55%) as well as integrin (α4β1 & α5β1) expression (WT; 68% & 83.79% vs. FAK−/−; 60.39% & 83.17%, respectively) between WT and FAK−/− macrophages. Further analysis of FAK−/− macrophages revealed a significant decrease in extracellular matrix/integrin directed migration of these cells in response to M-CSF on fibronectin (40% reduction), laminin (55% reduction) and collagen (60% reduction) (n=3, *p<0.004) coated plates as well as a decrease in migration in a wound healing assay (n=3, *p<0.003). The reduction in migration of FAK−/− macrophages was associated with a significant decrease in adhesion on fibronectin (63%), laminin (52%) and collagen (56%) as well as spreading (n=3, *p<0.03). Taken together, our results provide a critical physiologic role for FAK in regulating several adhesive and migratory functions in cells of myeloid lineage. Additional functions of FAK in other hematopoietic lineages will be discussed.

scholarly journals Focal adhesion kinase protein regulates Wnt3a gene expression to control cell fate specification in the developing neural plate

2011 ◽  
Vol 22 (13) ◽  
pp. 2409-2421 ◽  
Author(s):  
Yuri Fonar ◽  
Yoni E. Gutkovich ◽  
Heather Root ◽  
Anastasia Malyarova ◽  
Emil Aamar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Wnt Signaling ◽  
Focal Adhesion Kinase ◽  
Cell Fate ◽  
Focal Adhesion ◽  
Control Cell ◽  
Focal Adhesions ◽  
Neural Plate ◽  
Cell Fate Specification ◽  
Fate Specification

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase protein localized to regions called focal adhesions, which are contact points between cells and the extracellular matrix. FAK protein acts as a scaffold to transfer adhesion-dependent and growth factor signals into the cell. Increased FAK expression is linked to aggressive metastatic and invasive tumors. However, little is known about its normal embryonic function. FAK protein knockdown during early Xenopus laevis development anteriorizes the embryo. Morphant embryos express increased levels of anterior neural markers, with reciprocally reduced posterior neural marker expression. Posterior neural plate folding and convergence-extension is also inhibited. This anteriorized phenotype resembles that of embryos knocked down zygotically for canonical Wnt signaling. FAK and Wnt3a genes are both expressed in the neural plate, and Wnt3a expression is FAK dependent. Ectopic Wnt expression rescues this FAK morphant anteriorized phenotype. Wnt3a thus acts downstream of FAK to balance anterior–posterior cell fate specification in the developing neural plate. Wnt3a gene expression is also FAK dependent in human breast cancer cells, suggesting that this FAK–Wnt linkage is highly conserved. This unique observation connects the FAK- and Wnt-signaling pathways, both of which act to promote cancer when aberrantly activated in mammalian cells.

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