Regulation and activation of focal adhesion kinase and paxillin during the adhesion, proliferation, and differentiation of prostatic epithelial cells in vitro and in vivo

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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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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A Potential Role of Progestin-Induced Laminin-5/α6-Integrin Signaling in the Formation of Side Branches in the Mammary Gland

Endocrinology ◽

10.1210/en.2012-1518 ◽

2012 ◽

Vol 153 (10) ◽

pp. 4990-5001 ◽

Cited By ~ 7

Author(s):

Gabriele Meyer ◽

Jeffrey Leipprandt ◽

Jianwei Xie ◽

Mark D. Aupperlee ◽

Sandra Z. Haslam

Keyword(s):

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Nuclear Factor Κb ◽

Inhibitory Effect ◽

Collagen Gels ◽

Paracrine Factor ◽

Laminin 5 ◽

Adult Mice

Abstract Mammary organoids from adult mice produce tubules, analogous to mammary ducts in vivo, in response to hepatocyte growth factor (HGF) when cultured in collagen gels. The combination of HGF plus progestin (R5020) causes reduced tubule number and length. We hypothesized that the inhibitory effect on tubulogenesis was due to progestin-mediated alteration of HGF/c-Met signaling. Using molecular inhibitors and short hairpin RNA, it was determined that HGF activation of Ras-related C3 botulinum toxin substrate (Rac1) was required for the formation of cytoplasmic extensions, the first step of tubulogenesis, and that Rac1 activity was Src kinase (Src) and focal adhesion kinase (FAK) dependent. The highly novel finding was that R5020 reduced tubulogenesis by up-regulating and increasing extracellular laminin and α6-integrin ligation to reduce activation of the Src, focal adhesion kinase, and Rac1 pathway. Receptor activator of nuclear factor-κB ligand, another progesterone-induced paracrine factor, did not replicate this effect of R5020. The inhibitory effect of R5020 on tubulogenesis was likely mediated through progesterone receptor (PR) isoform A (PRA), because PRA is the predominant PR isoform expressed in the organoids, and the progestin-induced effect was prevented by the PR antagonist RU486. These results provide a plausible mechanism that explains progestin/PRA-mediated blunting of HGF-induced tubulogenesis in vitro and is proposed to be relevant to progesterone/PRA-induced side-branching in vivo during pregnancy.

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Direct interaction of v-Src with the focal adhesion kinase mediated by the Src SH2 domain.

Molecular Biology of the Cell ◽

10.1091/mbc.5.4.413 ◽

1994 ◽

Vol 5 (4) ◽

pp. 413-421 ◽

Cited By ~ 223

Author(s):

Z Xing ◽

H C Chen ◽

J K Nowlen ◽

S J Taylor ◽

D Shalloway ◽

...

Keyword(s):

Signal Transduction ◽

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Intracellular Signaling ◽

Direct Interaction ◽

Sh2 Domain ◽

Sh2 Domains ◽

Intracellular Signaling Molecules

The recently described focal adhesion kinase (FAK) has been implicated in signal transduction pathways initiated by cell adhesion receptor integrins and by neuropeptide growth factors. To examine the mechanisms by which FAK relays signals from the membrane to the cell interior, we carried out a series of experiments to detect potential FAK interactions with proteins containing Src homology 2 (SH2) domains that are important intracellular signaling molecules. Using v-Src-transformed NIH3T3 cells, we showed that FAK was present in the immune-complex precipitated by anti-Src antibody, suggesting potential interaction of FAK with v-Src in vivo. We also showed potentially direct interaction of FAK with v-Src in vivo using the yeast two-hybrid system. Using recombinant FAK expressed in insect cells and bacterial fusion proteins containing Src SH2 domains, we showed direct binding of FAK to the Src SH2 domain but not to the SH3 domain in vitro. A kinase-defective mutant of FAK, which is not autophosphorylated, did not interact with the Src SH2 domain under the same conditions, suggesting the involvement of the FAK autophosphorylation sites. Treatment of FAK with a protein-tyrosine phosphatase decreased its binding to the Src SH2 domain, whereas autophosphorylation in vitro increased its binding. These results confirm the importance of FAK autophosphorylation sites in its interaction with SH2 domain-containing proteins. Taken together, these results suggest that FAK may mediate signal transduction events initiated on the cell surface by kinase activation and autophosphorylation that result in its binding to other key intracellular signaling molecules.

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Src Inhibitors Pyrazolo[3,4-d]pyrimidines, Si306 and Pro-Si306, Inhibit Focal Adhesion Kinase and Suppress Human Glioblastoma Invasion In Vitro and In Vivo

Cancers ◽

10.3390/cancers12061570 ◽

2020 ◽

Vol 12 (6) ◽

pp. 1570 ◽

Cited By ~ 1

Author(s):

Marija Nešović ◽

Aleksandra Divac Rankov ◽

Ana Podolski-Renić ◽

Igor Nikolić ◽

Goran Tasić ◽

...

Keyword(s):

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Growth Factor Receptor ◽

Similar Efficacy ◽

Src Tyrosine Kinase ◽

Downstream Signaling ◽

Egfr Expression ◽

Human Gbm

Glioblastoma (GBM), as the most aggressive brain tumor, displays a high expression of Src tyrosine kinase, which is involved in the survival, migration, and invasiveness of tumor cells. Thus, Src emerged as a potential target for GBM therapy. The effects of Src inhibitors pyrazolo[3,4-d]pyrimidines, Si306 and its prodrug pro-Si306 were investigated in human GBM cell lines (U87 and U87-TxR) and three primary GBM cell cultures. Primary GBM cells were more resistant to Si306 and pro-Si306 according to the 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. However, the ability of all GBM cells to degrade the extracellular matrix was considerably compromised after Si306 and pro-Si306 applications. Besides reducing the phosphorylation of Src and its downstream signaling pathway components, both compounds decreased the phosphorylated form of focal adhesion kinase (FAK) and epidermal growth factor receptor (EGFR) expression, showing the potential to suppress the aggressiveness of GBM. In vivo, Si306 and pro-Si306 displayed an anti-invasive effect against U87 xenografts in the zebrafish embryo model. Considering that Si306 and pro-Si306 are able to cross the blood–brain barrier and suppress the spread of GBM cells, we anticipate their clinical testing in the near future. Moreover, the prodrug showed similar efficacy to the drug, implying the rationality of its use in clinical settings.

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