Src-Mediated Phosphorylation of Focal Adhesion Kinase Couples Actin and Adhesion Dynamics to Survival Signaling

ABSTRACT Integrin-associated focal adhesions not only provide adhesive links between cellular actin and extracellular matrix but also are sites of signal transmission into the cell interior. Many cell responses signal through focal adhesion kinase (FAK), often by integrin-induced autophosphorylation of FAK or phosphorylation by Src family kinases. Here, we used an interfering FAK mutant (4-9F-FAK) to show that Src-dependent FAK phosphorylation is required for focal adhesion turnover and cell migration, by controlling assembly of a calpain 2/FAK/Src/p42ERK complex, calpain activation, and proteolysis of FAK. Expression of 4-9F-FAK in FAK-deficient fibroblasts also disrupts F-actin assembly associated with normal adhesion and spreading. In addition, we found that FAK's ability to regulate both assembly and disassembly of the actin and adhesion networks may be linked to regulation of the protease calpain. Surprisingly, we also found that the same interfering 4-9F-FAK mutant protein causes apoptosis of serum-deprived, transformed cells and suppresses anchorage-independent growth. These data show that Src-mediated phosphorylation of FAK acts as a pivotal regulator of both actin and adhesion dynamics and survival signaling, which, in turn, control apparently distinct processes such as cell migration and anchorage-independent growth. This also highlights that dynamic regulation of actin and adhesions (which include the integrin matrix receptors) is critical to signaling output and biological responses.

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Focal adhesion kinase confers pro-migratory and anti-apoptotic properties and is a potential therapeutic target in Ewing sarcoma

10.1101/604207 ◽

2019 ◽

Author(s):

Konrad Steinestel ◽

Esther-Pia Jansen ◽

Marcel Trautmann ◽

Uta Dirksen ◽

Jan Rehkämper ◽

...

Keyword(s):

Cell Migration ◽

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Ewing Sarcoma ◽

Chorioallantoic Membrane ◽

Focal Adhesions ◽

Childhood And Adolescence ◽

Tissue Samples

ABSTRACTOncogenesis of Ewing sarcoma (EwS), the second most common malignant bone tumor of childhood and adolescence, is dependent on the expression of chimeric EWSR1-ETS fusion oncogenes, most often EWSR1-FLI1 (E/F).E/F expression leads to dysregulation of focal adhesions (FAs) enhancing the migratory capacity of EwS cells. Here we show that, in EwS cell lines and tissue samples, focal adhesion kinase (FAK) is expressed and phosphorylated at Y397 in an E/F-dependent way involving Ezrin. Employing different EwS cell as in vitro models, we found that key malignant properties of E/F are mediated via substrate-independent autophosphorylation of FAK on Y397. This phosphorylation results in enhanced FA formation, Rho-dependent cell migration, and impaired caspase-3-mediated apoptosis in vitro. Conversely, treatment with the FAK inhibitor Y15 enhanced caspase-mediated apoptosis and EwS cell migration, independent from the respective EWSR1-ETS fusion type, mimicking an anoikis-like phenotype. Our findings were confirmed in vivo using an avian chorioallantoic membrane (CAM) model. Our results provide a first rationale for the therapeutic use of FAK inhibitors to impair metastatic dissemination of EwS.

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Focal adhesion kinase suppresses Rho activity to promote focal adhesion turnover

Journal of Cell Science ◽

10.1242/jcs.113.20.3673 ◽

2000 ◽

Vol 113 (20) ◽

pp. 3673-3678 ◽

Cited By ~ 27

Author(s):

X.D. Ren ◽

W.B. Kiosses ◽

D.J. Sieg ◽

C.A. Otey ◽

D.D. Schlaepfer ◽

...

Keyword(s):

Cell Migration ◽

Cell Adhesion ◽

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Focal Adhesions ◽

Small Gtpase ◽

Embryonic Lethality ◽

Extracellular Matrices ◽

Constitutive Activation ◽

Focal Adhesion Turnover

Focal adhesion kinase (FAK) is activated and localized at focal adhesions upon cell adhesion to extracellular matrices. Cells lacking FAK show increased focal adhesion number and decreased cell migration, functions that are regulated by the small GTPase Rho. We now report that fibroblasts from FAK-/- mice failed to transiently inhibit Rho activity when plated on fibronectin. Re-expression of FAK restored normal Rho regulation. Turnover of focal adhesions correlated inversely with Rho activity. The presence or absence of FAK was mimicked by inhibiting or activating Rho, respectively. These data suggest that loss of FAK resulting in constitutive activation of Rho and inhibition of focal adhesion turnover can account for deficiencies in cell migration and embryonic lethality of the FAK knockout.

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

Blood ◽

10.1182/blood-2003-05-1703 ◽

2003 ◽

Vol 102 (10) ◽

pp. 3629-3636 ◽

Cited By ~ 43

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)

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Rab40/Cullin5 complex regulates EPLIN and actin cytoskeleton dynamics during cell migration and invasion

10.1101/2021.04.01.438077 ◽

2021 ◽

Author(s):

Erik S Linklater ◽

Emily Duncan ◽

Ke Jun Han ◽

Algirdas Kaupinis ◽

Mindaugas Valius ◽

...

Keyword(s):

Cell Migration ◽

Actin Cytoskeleton ◽

Focal Adhesion ◽

Focal Adhesions ◽

Leading Edge ◽

Stress Fibers ◽

Actin Dynamics ◽

Migration And Invasion ◽

Matrix Remodeling ◽

Cell Migration And Invasion

Rab40b is a SOCS box containing protein that regulates the secretion of MMPs to facilitate extracellular matrix remodeling during cell migration. Here we show that Rab40b interacts with Cullin5 via the Rab40b SOCS domain. We demonstrate that loss of Rab40b/Cullin5 binding decreases cell motility and invasive potential, and show that defective cell migration and invasion stem from alteration to the actin cytoskeleton, leading to decreased invadopodia formation, decreased actin dynamics at the leading edge, and an increase in stress fibers. We also show that these stress fibers anchor at less dynamic, more stable focal adhesions. Mechanistically, changes in the cytoskeleton and focal adhesion dynamics are mediated in part by EPLIN, which we demonstrate to be a binding partner of Rab40b and a target for Rab40b/Cullin5 dependent localized ubiquitylation and degradation. Thus, we propose a model where the Rab40b/Cullin5 dependent ubiquitylation regulates EPLIN localization to promote cell migration and invasion by altering focal adhesion and cytoskeletal dynamics.

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Myocilin, a glaucoma-associated protein, promotes cell migration through activation of integrin-focal adhesion kinase-serine/threonine kinase signaling pathway

Journal of Cellular Physiology ◽

10.1002/jcp.22701 ◽

2011 ◽

Vol 226 (12) ◽

pp. 3392-3402 ◽

Cited By ~ 17

Author(s):

Heung Sun Kwon ◽

Stanislav I. Tomarev

Keyword(s):

Cell Migration ◽

Focal Adhesion Kinase ◽

Signaling Pathway ◽

Focal Adhesion ◽

Kinase Signaling ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Kinase Signaling Pathway

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Indomethacin Delays Gastric Restitution: Association with the Inhibition of Focal Adhesion Kinase and Tensin Phosphorylation and Reduced Actin Stress Fibers

Experimental Biology and Medicine ◽

10.1177/153537020222700607 ◽

2002 ◽

Vol 227 (6) ◽

pp. 412-424 ◽

Cited By ~ 20

Author(s):

Imre L. Szabó ◽

Rama Pai ◽

Michael K. Jones ◽

George R. Ehring ◽

Hirofumi Kawanaka ◽

...

Keyword(s):

Cell Migration ◽

Focal Adhesion ◽

Focal Adhesions ◽

Stress Fiber ◽

Fiber Formation ◽

Stress Fibers ◽

Actin Stress Fiber ◽

Stress Fiber Formation ◽

Actin Stress Fiber Formation

Repair of superficial gastric mucosal injury is accomplished by the process of restitution—migration of epithelial cells to restore continuity of the mucosal surface. Actin filaments, focal adhesions, and focal adhesion kinase (FAK) play crucial roles in cell motility essential for restitution. We studied whether epidermal growth factor (EGF) and/or indomethacin (IND) affect cell migration, actin stress fiber formation, and/or phosphorylation of FAK and tensin in wounded gastric monolayers. Human gastric epithelial monolayers (MKN 28 cells) were wounded and treated with either vehicle or 0.5 mM IND for 16 hr followed by EGF. EGF treatment significantly stimulated cell migration and actin stress fiber formation, and increased FAK localization to focal adhesions, and phosphorylation of FAK and tensin, whereas IND inhibited all these at the baseline and EGF-stimulated conditions. IND-induced inhibition of FAK phosphorylation preceded changes in actin polymerization, indicating that actin depolymerization might be the consequence of decreased FAK activity. In in vivo experiments, rats received either vehicle or IND (5 mg/kg i.g.), and 3 min later, they received water or 5% hypertonic NaCl; gastric mucosa was obtained at 1, 4, and 8 hr after injury. Four and 8 hr after hypertonic injury, FAK phosphorylation was induced in gastric mucosa compared with controls. IND pretreatment significantly delayed epithelial restitution in vivo, and reduced FAK phosphorylation and recruitment to adhesion points, as well as actin stress fiber formation in migrating surface epithelial cells. Our study indicates that FAK, tensin, and actin stress fibers are likely mediators of EGF-stimulated cell migration in wounded human gastric monolayers and potential targets for IND-induced inhibition of restitution.

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Cardiac fibroblasts require focal adhesion kinase for normal proliferation and migration

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00444.2008 ◽

2009 ◽

Vol 296 (3) ◽

pp. H627-H638 ◽

Cited By ~ 17

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.

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