Roles of focal adhesion kinase (FAK) in megakaryopoiesis and platelet function: studies using a megakaryocyte lineage–specific FAK knockout

Focal adhesion kinase (FAK) plays a key role in mediating signaling downstream of integrins and growth factor receptors. In this study, we determined the roles of FAK in vivo by generating a megakaryocyte lineage–specific FAK-null mouse (Pf4-Cre/FAK-floxed). Megakaryocyte and platelet FAK expression was ablated in Pf4-Cre/FAK-floxed mice without affecting expression of the FAK homologue PYK2, although PYK2 phosphorylation was increased in FAK−/− megakaryocytes in response to fibrinogen. Megakaryopoiesis is greatly enhanced in Pf4-Cre/FAK-floxed mice, with significant increases in megakaryocytic progenitors (CFU-MK), mature megakaryocytes, megakaryocyte ploidy, and moderate increases in resting platelet number and platelet recovery following a thrombocytopenic stress. Thrombopoietin (Tpo)–mediated activation of Lyn kinase, a negative regulator of megakaryopoiesis, is severely attenuated in FAK-null megakaryocytes compared with wild-type controls. In contrast, Tpo-mediated activation of positive megakaryopoiesis regulators such as ERK1/2 and AKT is increased in FAK-null megakaryocytes, providing a plausible explanation for the observed increases in megakaryopoiesis in these mice. In Pf4-Cre/FAK-floxed mice, rebleeding times are significantly increased, and FAK-null platelets exhibit diminished spreading on immobilized fibrinogen. These studies establish clear roles for FAK in megakaryocyte growth and platelet function, setting the stage for manipulation of this component of the Tpo signaling apparatus for therapeutic benefit.

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The Headpiece Domain of Dematin Regulates Cell Shape, Motility, and Wound Healing by Modulating RhoA Activation

Molecular and Cellular Biology ◽

10.1128/mcb.00237-08 ◽

2008 ◽

Vol 28 (15) ◽

pp. 4712-4718 ◽

Cited By ~ 10

Author(s):

Morvarid Mohseni ◽

Athar H. Chishti

Keyword(s):

Wound Healing ◽

Focal Adhesion Kinase ◽

Signaling Pathways ◽

Focal Adhesion ◽

Mouse Skin ◽

Fiber Formation ◽

Negative Regulator ◽

Rhoa Activation ◽

Primary Mouse

ABSTRACT RhoA is known to participate in cytoskeletal remodeling events through several signaling pathways, yet the precise mechanism of its activation remains unknown. Here, we provide the first evidence that dematin functions upstream of RhoA and regulates its activation. Primary mouse embryonic fibroblasts were generated from a dematin headpiece domain null (HPKO) mouse, and the visualization of the actin morphology revealed a time-dependent defect in stress fiber formation, membrane protrusions, cell motility, and cell adhesion. Rescue experiments using RNA interference and transfection assays revealed that the observed phenotypes are due to a null effect and not a gain of function in the mutant fibroblasts. In vivo wounding of adult HPKO mouse skin showed a decrease in wound healing (reepithelialization and granulation) compared to the wild-type control. Biochemical analysis of the HPKO fibroblasts revealed a sustained hyperphosphorylation of focal adhesion kinase (FAK) at tyrosine 397 as well as a twofold increase in RhoA activation. Inhibition of both RhoA and FAK signaling using C3 toxin and FRNK (focal adhesion kinase nonrelated kinase), respectively, revealed that dematin acts upstream of RhoA. Together, these results unveil a new function of dematin as a negative regulator of the RhoA activation pathway with physiological implications for normal and pathogenic signaling pathways.

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Protein Kinase C Phosphorylation of a γ-Protocadherin C-terminal Lipid Binding Domain Regulates Focal Adhesion Kinase Inhibition and Dendrite Arborization

Journal of Biological Chemistry ◽

10.1074/jbc.m115.642306 ◽

2015 ◽

Vol 290 (34) ◽

pp. 20674-20686 ◽

Cited By ~ 28

Author(s):

Austin B. Keeler ◽

Dietmar Schreiner ◽

Joshua A. Weiner

Keyword(s):

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Cortical Neurons ◽

Lipid Binding ◽

Binding Motif ◽

Wild Type ◽

Western Blots ◽

Phospholipid Binding ◽

C Terminus

The γ-protocadherins (γ-Pcdhs) are a family of 22 adhesion molecules with multiple critical developmental functions, including the proper formation of dendritic arbors by forebrain neurons. The γ-Pcdhs bind to and inhibit focal adhesion kinase (FAK) via a constant C-terminal cytoplasmic domain shared by all 22 proteins. In cortical neurons lacking the γ-Pcdhs, aberrantly high activity of FAK and of PKC disrupts dendrite arborization. Little is known, however, about how γ-Pcdh function is regulated by other factors. Here we show that PKC phosphorylates a serine residue situated within a phospholipid binding motif at the shared γ-Pcdh C terminus. Western blots using a novel phospho-specific antibody against this site suggest that a portion of γ-Pcdh proteins is phosphorylated in the cortex in vivo. We find that PKC phosphorylation disrupts both phospholipid binding and the γ-Pcdh inhibition of (but not binding to) FAK. Introduction of a non-phosphorylatable (S922A) γ-Pcdh construct into wild-type cortical neurons significantly increases dendrite arborization. This same S922A construct can also rescue dendrite arborization defects in γ-Pcdh null neurons cell autonomously. Consistent with these data, introduction of a phosphomimetic (S/D) γ-Pcdh construct or treatment with a PKC activator reduces dendrite arborization in wild-type cortical neurons. Together, these data identify a novel mechanism through which γ-Pcdh control of a signaling pathway important for dendrite arborization is regulated.

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Regulation of Focal Adhesion Kinase by Its Amino-Terminal Domain through an Autoinhibitory Interaction

Molecular and Cellular Biology ◽

10.1128/mcb.23.22.8030-8041.2003 ◽

2003 ◽

Vol 23 (22) ◽

pp. 8030-8041 ◽

Cited By ~ 119

Author(s):

Lee Ann Cooper ◽

Tang-Long Shen ◽

Jun-Lin Guan

Keyword(s):

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Mammalian Cells ◽

Cell Cycle Progression ◽

Kinase Domain ◽

Negative Regulator ◽

Amino Terminus ◽

Amino Terminal

ABSTRACT We have investigated a role for the amino-terminal FERM-like domain of the focal adhesion kinase (FAK) as a negative regulator of its own activity and phosphorylation state. Deletion of the first 375 amino acids from the amino terminus of FAK increases its catalytic activity in vitro, its phosphorylation when expressed in mammalian cells, and the phosphorylation of a FAK substrate, paxillin. Deletion mutants are phosphorylated in suspension, suggesting that they are no longer regulated by adhesion. The amino terminus of FAK can interact with the kinase domain of FAK in vitro and in vivo, suggesting that it might act as an autoinhibitor of FAK activity. The amino terminus of FAK can act in trans to inhibit FAK phosphorylation when expressed in mammalian cells or to directly inhibit FAK activity in vitro. Expression of the amino terminus of FAK inhibits cell cycle progression in CHO cells, consistent with its inhibition of FAK phosphorylation and function in trans. A glutathione S-transferase fusion protein containing the cytoplasmic tail of the β1 integrin stimulates FAK activity in vitro, suggesting that FAK could be regulated by molecular interactions with the amino terminus. Based on these and previous data, we propose a working model for activation of FAK in cell adhesion.

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GSK3β is a negative regulator of platelet function and thrombosis

Blood ◽

10.1182/blood-2007-09-111518 ◽

2008 ◽

Vol 111 (7) ◽

pp. 3522-3530 ◽

Cited By ~ 64

Author(s):

Dongjun Li ◽

Shelley August ◽

Donna S. Woulfe

Keyword(s):

Platelet Function ◽

Glycogen Synthase ◽

Human Platelets ◽

Dense Granule ◽

Negative Regulator ◽

Wild Type ◽

Single Allele ◽

Induced Aggregation

Abstract Glycogen synthase kinase (GSK)3β is a ser-thr kinase that is phosphorylated by the kinase Akt. Although Akt has been shown to regulate platelet function and arterial thrombosis, its effectors in platelets remain unknown. We show here that agonist-dependent phosphorylation of GSK3β in platelets is Akt dependent. To determine whether GSK3β regulates platelet function, platelets from mice lacking a single allele of GSK3β were compared with those of wild-type (WT) controls. GSK3β+/− platelets demonstrated enhanced agonist-dependent aggregation, dense granule secretion, and fibrinogen binding, compared with WT platelets. Treatment of human platelets with GSK3 inhibitors renders them more sensitive to agonist-induced aggregation, suggesting that GSK3 suppresses platelet function in vitro. Finally, the effect of GSK3β on platelet function in vivo was evaluated using 2 thrombosis models in mice. In the first, 80% of GSK3β+/− mice (n = 10) formed stable occlusive thrombi after ferric chloride carotid artery injury, whereas the majority of wild-type mice (67%) formed no thrombi (n = 15). In a disseminated thrombosis model, deletion of a single allele of GSK3β in mice conferred enhanced sensitivity to thrombotic insult. Taken together, these results suggest that GSK3β acts as a negative regulator of platelet function in vitro and in vivo.

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Poldip2 controls leukocyte infiltration into the ischemic brain by regulating focal adhesion kinase-mediated VCAM-1 induction

Scientific Reports ◽

10.1038/s41598-021-84987-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Lori N. Eidson ◽

Qingzeng Gao ◽

Hongyan Qu ◽

Daniel S. Kikuchi ◽

Ana Carolina P. Campos ◽

...

Keyword(s):

Endothelial Cells ◽

Cerebral Ischemia ◽

Focal Adhesion Kinase ◽

Adhesion Molecules ◽

Focal Adhesion ◽

Vascular Inflammation ◽

Leukocyte Recruitment ◽

Ischemic Brain ◽

Inflammatory Monocytes

AbstractStroke is a multiphasic process involving a direct ischemic brain injury which is then exacerbated by the influx of immune cells into the brain tissue. Activation of brain endothelial cells leads to the expression of adhesion molecules such vascular cell adhesion molecule 1 (VCAM-1) on endothelial cells, further increasing leukocyte recruitment. Polymerase δ-interacting protein 2 (Poldip2) promotes brain vascular inflammation and leukocyte recruitment via unknown mechanisms. This study aimed to define the role of Poldip2 in mediating vascular inflammation and leukocyte recruitment following cerebral ischemia. Cerebral ischemia was induced in Poldip2+/+ and Poldip2+/− mice and brains were isolated and processed for flow cytometry or RT-PCR. Cultured rat brain microvascular endothelial cells were used to investigate the effect of Poldip2 depletion on focal adhesion kinase (FAK)-mediated VCAM-1 induction. Poldip2 depletion in vivo attenuated the infiltration of myeloid cells, inflammatory monocytes/macrophages and decreased the induction of adhesion molecules. Focusing on VCAM-1, we demonstrated mechanistically that FAK activation was a critical intermediary in Poldip2-mediated VCAM-1 induction. In conclusion, Poldip2 is an important mediator of endothelial dysfunction and leukocyte recruitment. Thus, Poldip2 could be a therapeutic target to improve morbidity following ischemic stroke.

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In Vivo Role of Focal Adhesion Kinase in Regulating Pancreatic -Cell Mass and Function Through Insulin Signaling, Actin Dynamics, and Granule Trafficking

Diabetes ◽

10.2337/db11-1344 ◽

2012 ◽

Vol 61 (7) ◽

pp. 1708-1718 ◽

Cited By ~ 42

Author(s):

E. P. Cai ◽

M. Casimir ◽

S. A. Schroer ◽

C. T. Luk ◽

S. Y. Shi ◽

...

Keyword(s):

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Insulin Signaling ◽

Cell Mass ◽

Actin Dynamics ◽

Pancreatic Cell ◽

And Function

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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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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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Abstract 15445: The Sirtuin SIRT3 Blocks Doxorubicin-induced Cardiac Hypertrophic Response of Mice

Circulation ◽

10.1161/circ.130.suppl_2.15445 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Vinodkumar Pillai ◽

Sadhana Samant ◽

Nagalingam Sundaresan ◽

Gene Kim ◽

Mahesh P Gupta

Keyword(s):

Negative Regulator ◽

Neonatal Rat ◽

Whole Body ◽

Wild Type ◽

Electron Microscopic ◽

Specific Expression ◽

Rat Cardiomyocytes ◽

Fluorescent Intensity ◽

Hypertrophic Response

Background and objective: Doxorubicin is a chemotherapeutic drug widely used to treat variety of cancers. One of the serious side effects of doxorubicin is its toxicity to the heart. Previously, we have shown that overexpression of SIRT3 blocks the hypertrophic response of the heart to agonist treatments. This study was undertaken to investigate whether SIRT3 can also attenuate the doxorubicin-induced cardiac hypertrophic response in mice. Methods and results: Neonatal rat cardiomyocytes overexpressed with SIRT3 and treated with doxorubicin (10μM) showed 28% reduced mean fluorescent intensity for CM-H 2 DCFDA dye, compared to mock infected control cells treated with doxorubicin, thus suggesting that SIRT3 was capable of blocking doxorubicin-induced ROS synthesis in cardiomyocytes. To examine the cardioprotective effects of SIRT3 in doxorubicin-induced cardiotoxicity in vivo ; we used a cumulative dose of 15mg/kg of doxorubicin for two different time points. One group of mice was treated intraperitoneally with 5mg/kg doxorubicin or an equal volume of saline every two weeks for a total of three doses. Transgenic mice having cardiac specific expression of SIRT3 (SIRT3-Tg) showed 33% reduced HW/BW ratio compared to control mice. Echocardiographic evaluation of hearts showed significantly reduced fractional shortening in control mice, compared to SIRT3-Tg mice (24.6 vs 34.7 %, P<0.05). SIRT3-Tg mice also showed significantly reduced fetal gene expression for ANF, βMHC and collagen-1 as determined by RT-PCR. Masson’s trichrome staining showed significantly reduced fibrosis in doxorubicin treated SIRT3-Tg mice compared to its control. Furthermore, electron microscopic analysis showed preserved mitochondrial and sarcomeres structures in doxorubicin treated SIRT3-Tg hearts, whereas in wild-type hearts these structures were highly disorganized. Second group of mice that received 15mg/kg dose for two weeks also showed similar results. Contrary to this, whole body SIRT3 knockout mice showed exacerbated cardiac hypertrophic response compared to wild-type mice in response to doxorubicin treatment. Conclusion: These results demonstrated that SIRT3 is an endogenous negative regulator of doxorubicin-induced cardiac hypertrophic response.

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