Serine Phosphorylation of Focal Adhesion Kinase in Interphase and Mitosis: A Possible Role in Modulating Binding to p130Cas

Focal adhesion kinase (FAK) is an important regulator of integrin signaling in adherent cells and accordingly its activity is significantly modulated during mitosis when cells detach from the extracellular matrix. During mitosis, FAK becomes heavily phosphorylated on serine residues concomitant with its inactivation and dephosphorylation on tyrosine. Little is known about the regulation of FAK activity by serine phosphorylation. In this report, we characterize two novel sites of serine phosphorylation within the C-terminal domain of FAK. Phosphorylation-specific antibodies directed to these sites and against two previously characterized sites of serine phosphorylation were used to study the regulated phosphorylation of FAK in unsynchronized and mitotic cells. Among the four major phosphorylation sites, designated pS1-pS4, phosphorylation of pS1 (Ser722) is unchanged in unsynchronized and mitotic cells. In contrast, pS3 and pS4 (Ser843 and Ser910) exhibit increased phosphorylation during mitosis. In vitro peptide binding experiments provide evidence that phosphorylation of pS1 (Ser722) may play a role in modulating FAK binding to the SH3 domain of the adapter protein p130Cas.

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cAMP- and cGMP-dependent protein kinase phosphorylation sites of the focal adhesion vasodilator-stimulated phosphoprotein (VASP) in vitro and in intact human platelets.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)36652-8 ◽

1994 ◽

Vol 269 (20) ◽

pp. 14509-14517 ◽

Cited By ~ 8

Author(s):

E. Butt ◽

K. Abel ◽

M. Krieger ◽

D. Palm ◽

V. Hoppe ◽

...

Keyword(s):

Protein Kinase ◽

Focal Adhesion ◽

Human Platelets ◽

Phosphorylation Sites ◽

Dependent Protein Kinase ◽

Cgmp Dependent Protein Kinase ◽

Dependent Protein ◽

Kinase Phosphorylation ◽

Camp And Cgmp

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Focal adhesion kinase (FAK) and mechanical stimulation negatively regulate the transition of airway smooth muscle tissues to a synthetic phenotype

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00299.2016 ◽

2016 ◽

Vol 311 (5) ◽

pp. L893-L902 ◽

Cited By ~ 10

Author(s):

Yidi Wu ◽

Youliang Huang ◽

Susan J. Gunst

Keyword(s):

Smooth Muscle ◽

Focal Adhesion Kinase ◽

Airway Smooth Muscle ◽

Focal Adhesion ◽

Inflammatory Responses ◽

Mechanical Load ◽

Akt Activation ◽

Lower Load ◽

Synthetic Phenotype

The effects of mechanical forces and focal adhesion kinase (FAK) in regulating the inflammatory responses of airway smooth muscle (ASM) tissues to stimulation with interleukin (IL)-13 were investigated. Canine tracheal tissues were subjected to different mechanical loads in vitro, and the effects of mechanical load on eotaxin secretion and inflammatory signaling pathways in response to IL-13 were determined. Eotaxin secretion by tissues in response to IL-13 was significantly inhibited in muscles maintained at a higher (+) load compared with those at a lower (−) load as assessed by ELISA, and Akt activation was also reduced in the higher (+) loaded tissues. Conversely the (+) mechanical load increased activation of the focal adhesion proteins FAK and paxillin in the tissues. The role of FAK in regulating the mechanosensitive responses was assessed by overexpressing FAK-related nonkinase in the tissues, by expressing the FAK kinase-dead mutant FAK Y397F, or by treating tissues with the FAK inhibitor PF-573228. FAK inactivation potentiated Akt activity and increased eotaxin secretion in response to IL-13. FAK inhibition also suppressed the mechanosensitivity of Akt activation and eotaxin secretion. In addition, FAK inactivation suppressed smooth muscle myosin heavy chain expression induced by the higher (+) mechanical load. The results demonstrate that the imposition of a higher mechanical load on airway smooth muscle stimulates FAK activation, which promotes the expression of the differentiated contractile phenotype and suppresses the synthetic phenotype and the inflammatory responses of the muscle tissue.

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Focal adhesion kinase and paxillin bind to peptides mimicking beta integrin cytoplasmic domains.

The Journal of Cell Biology ◽

10.1083/jcb.130.5.1181 ◽

1995 ◽

Vol 130 (5) ◽

pp. 1181-1187 ◽

Cited By ~ 404

Author(s):

M D Schaller ◽

C A Otey ◽

J D Hildebrand ◽

J T Parsons

Keyword(s):

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Tyrosine Kinases ◽

Protein Tyrosine Kinase ◽

Cytoplasmic Domain ◽

Embryo Cell ◽

Vitro Assay ◽

Protein Tyrosine ◽

Signaling Complex

The integrins have recently been implicated in signal transduction. A likely mediator of integrin signaling is focal adhesion kinase (pp125FAK or FAK), a structurally distinct protein tyrosine kinase that becomes enzymatically activated upon engagement of integrins with their ligands. A second candidate signaling molecule is paxillin, a focal adhesion associated, cytoskeletal protein that coordinately becomes phosphorylated on tyrosine upon activation of pp125FAK. Paxillin physically complexes with two protein tyrosine kinases, pp60src and Csk (COOH-terminal src kinase), and the oncoprotein p47gag-crk, each of which could function as part of a paxillin signaling complex. Using an in vitro assay we have established that the cytoplasmic domain of the beta 1 integrin can bind to paxillin and pp125FAK from chicken embryo cell lysates. The NH2-terminal, noncatalytic domain of pp125FAK can bind directly to the cytoplasmic tail of beta 1 and recognizes integrin sequences distinct from those involved in binding to alpha-actinin. Paxillin binding is independent of pp125FAK binding despite the fact that both bind to the same region of beta 1. These results demonstrate that the cytoplasmic domain of the beta subunits of integrins contain binding sites for both signaling molecules and structural proteins suggesting that integrins can coordinate the generation of cytoplasmic signals in addition to their role in anchoring components of the cytoskeleton.

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Cytoskeletal changes induced by GRAF, the GTPase regulator associated with focal adhesion kinase, are mediated by Rho

Journal of Cell Science ◽

10.1242/jcs.112.2.231 ◽

1999 ◽

Vol 112 (2) ◽

pp. 231-242 ◽

Cited By ~ 3

Author(s):

J.M. Taylor ◽

M.M. Macklem ◽

J.T. Parsons

Keyword(s):

Focal Adhesion Kinase ◽

Pc12 Cells ◽

Focal Adhesion ◽

Fiber Formation ◽

Serum Starvation ◽

3T3 Cells ◽

Neurite Retraction ◽

Swiss 3T3

Graf, the GTPase regulator associated with focal adhesion kinase was previously shown to have GAP activity for Ρ A and Cdc42 in vitro (Hildebrand et al 1996 Mol. Cell Biol. 16: 3169–3178). In this study we sought to determine whether Graf acted at the level of Cdc42, Rho, or both in vivo and whether Graf was a signal terminator or transducer for these proteins. Microinjection of Graf cDNA into subconfluent Swiss 3T3 cells (in the presence of serum) has marked effects on cell shape and actin localization. Graf expression causes clearing of stress fibers followed by formation of long actin based filopodial-like extensions. Similar phenotypes were observed following injection of the Rho-inhibitor, C3 into these cells. The Graf response was dependent on GAP activity, since injection of Graf cDNA containing point mutations in the GAP domain (R236Q or N351V) which block enzymatic activity, does not confer this phenotype. Injection of Graf into Swiss 3T3 cells in which Rho has been down-regulated by serum starvation has no effect on cell morphology. Using this system, we demonstrate that Graf blocks sphingosine-1-phosphate (SPP) stimulated (Rho-mediated) stress fiber formation. Conversely, Graf expression does not inhibit bradykinin stimulated (Cdc42-mediated) filopodial extensions. These data indicate that Graf is a GAP for Rho in vivo. To further substantiate these results we examined the effect of Graf over-expression on Rho-mediated neurite retraction in nerve growth factor (NGF)-differentiated PC12 cells. In PC12 cells, which express relatively high levels of endogenous Graf, overexpression of Graf (but not Graf containing the R236Q mutation) enhances SPP-induced neurite retraction. These data indicate the possibility that Graf may be an effector for Rho in certain cell types.

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Focal adhesion kinase inhibitor TAE226 combined with Sorafenib slows down hepatocellular carcinoma by multiple epigenetics effects

10.21203/rs.3.rs-892488/v1 ◽

2021 ◽

Author(s):

Ilaria Romito ◽

Manuela Porru ◽

Maria Rita Braghini ◽

Luca Pompili ◽

Nadia Panera ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Kinase Inhibitor ◽

Malignant Tumours ◽

Nurd Complex ◽

Antitumor Effects ◽

Combination Regimens

Abstract Background Hepatocellular carcinoma (HCC) is one of the most common and lethal malignant tumours worldwide. Sorafenib (SOR) is one of the most effective single-drug systemic therapy against advanced HCC, but the identification of novel combination regimens for a continued improvement in overall survival is a big challenge. Recent studies highlighted the crucial role of focal adhesion kinase (FAK) in HCC growth. The aim of this study was to investigate the antitumor effects of three different FAK inhibitors, alone or in combination with SOR, using in vitro and in vivo models of HCC. Methods The effect of PND1186, PF431396, TAE226 on cell viability was compared to SOR. Among them TAE226, emerging as the most effective FAKi, was then tested alone or in combination with SOR using 2D/3D human HCC cell line cultures and HCC xenograft murine models. The mechanisms of action were assessed by gene/protein expression and imaging approaches, combined with high-throughput methods. Results TAE226 emerged as the more effective FAKi to be combined with SOR against HCC. Combined TAE226 and SOR treatment reduced HCC growth both in vitro and in vivo by affecting tumour-promoting gene expression and inducing epigenetic changes via dysregulation of the nuclear interactome of FAK. We characterized a novel nuclear functional interaction between FAK and the NuRD complex. TAE226-mediated FAK depletion and SOR-promoted MAPK down-modulation causing an increase of histone H3 lysine 27 acetylation, counteracting its trimethylation by decreasing the nuclear amount of HDAC1/2. Conclusions Altogether, our findings provide the first evidence that TAE226 combined with SOR efficiently reduce HCC growth in vitro and in vivo. Our data also highlight that deep analysis of FAK nuclear interactome may lead to the identification of new promising therapeutic approaches for HCC.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e10-10-0821 ◽

2011 ◽

Vol 22 (8) ◽

pp. 1409-1419 ◽

Cited By ~ 29

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.

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Fibronectin-stimulated signaling from a focal adhesion kinase-c-Src complex: involvement of the Grb2, p130cas, and Nck adaptor proteins.

Molecular and Cellular Biology ◽

10.1128/mcb.17.3.1702 ◽

1997 ◽

Vol 17 (3) ◽

pp. 1702-1713 ◽

Cited By ~ 320

Author(s):

D D Schlaepfer ◽

M A Broome ◽

T Hunter

Keyword(s):

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Intracellular Signaling ◽

Protein Tyrosine Kinase ◽

Adaptor Protein ◽

Adaptor Proteins ◽

Sh2 Domain ◽

Mitogen Activated Protein Kinase

The focal adhesion kinase (FAK), a protein-tyrosine kinase (PTK), associates with integrin receptors and is activated by cell binding to extracellular matrix proteins, such as fibronectin (FN). FAK autophosphorylation at Tyr-397 promotes Src homology 2 (SH2) domain binding of Src family PTKs, and c-Src phosphorylation of FAK at Tyr-925 creates an SH2 binding site for the Grb2 SH2-SH3 adaptor protein. FN-stimulated Grb2 binding to FAK may facilitate intracellular signaling to targets such as ERK2-mitogen-activated protein kinase. We examined FN-stimulated signaling to ERK2 and found that ERK2 activation was reduced 10-fold in Src- fibroblasts, compared to that of Src- fibroblasts stably reexpressing wild-type c-Src. FN-stimulated FAK phosphotyrosine (P.Tyr) and Grb2 binding to FAK were reduced, whereas the tyrosine phosphorylation of another signaling protein, p130cas, was not detected in the Src- cells. Stable expression of residues 1 to 298 of Src (Src 1-298, which encompass the SH3 and SH2 domains of c-Src) in the Src- cells blocked Grb2 binding to FAK; but surprisingly, Src 1-298 expression also resulted in elevated p130cas P.Tyr levels and a two- to threefold increase in FN-stimulated ERK2 activity compared to levels in Src- cells. Src 1-298 bound to both FAK and p130cas and promoted FAK association with p130cas in vivo. FAK was observed to phosphorylate p130cas in vitro and could thus phosphorylate p130cas upon FN stimulation of the Src 1-298-expressing cells. FAK-induced phosphorylation of p130cas in the Src 1-298 cells promoted the SH2 domain-dependent binding of the Nck adaptor protein to p130cas, which may facilitate signaling to ERK2. These results show that there are additional FN-stimulated pathways to ERK2 that do not involve Grb2 binding to FAK.

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Regulation of Focal Adhesion Kinase by a Novel Protein Inhibitor FIP200

Molecular Biology of the Cell ◽

10.1091/mbc.e02-05-0295 ◽

2002 ◽

Vol 13 (9) ◽

pp. 3178-3191 ◽

Cited By ~ 78

Author(s):

Smita Abbi ◽

Hiroki Ueda ◽

Chuanhai Zheng ◽

Lee Ann Cooper ◽

Jihe Zhao ◽

...

Keyword(s):

Cell Cycle ◽

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Cell Cycle Progression ◽

Protein Inhibitor ◽

Cellular Functions ◽

Cycle Progression ◽

Novel Protein

Focal adhesion kinase (FAK) is a major mediator of integrin signaling pathways. The mechanisms of regulation of FAK activity and its associated cellular functions are not very well understood. Here, we present data suggesting that a novel protein FIP200 functions as an inhibitor for FAK. We show the association of endogenous FIP200 with FAK, which is decreased upon integrin-mediated cell adhesion concomitant with FAK activation. In vitro- and in vivo-binding studies indicate that FIP200 interacts with FAK through multiple domains directly. FIP200 bound to the kinase domain of FAK inhibited its kinase activity in vitro and its autophosphorylation in vivo. Overexpression of FIP200 or its segments inhibited cell spreading, cell migration, and cell cycle progression, which correlated with their inhibition of FAK activity in vivo. The inhibition of these cellular functions by FIP200 could be rescued by coexpression of FAK. Last, we show that disruption of the functional interaction between endogenous FIP200 with FAK leads to increased FAK phosphorylation and partial restoration of cell cycle progression in cells plated on poly-l-lysine, providing further support for FIP200 as a negative regulator of FAK. Together, these results identify FIP200 as a novel protein inhibitor for FAK.

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

Biochemical Journal ◽

10.1042/bj3480119 ◽

2000 ◽

Vol 348 (1) ◽

pp. 119-128 ◽

Cited By ~ 16

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.

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