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 Identification and Characterization of Rvs162/Rvs167-3, a Novel N-BAR Heterodimer in the Human Fungal Pathogen Candida albicans

2014 ◽  
Vol 14 (2) ◽  
pp. 182-193 ◽  
Author(s):  
Areti Gkourtsa ◽  
Janny van den Burg ◽  
Karin Strijbis ◽  
Teja Avula ◽  
Sietske Bijvoets ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Fluorescent Protein ◽  
Genetic Interaction ◽  
Phenotypic Analysis ◽  
Content Type ◽  
Associated Functions ◽  
Increased Sensitivity ◽  
Green Fluorescent ◽  
And Function

ABSTRACT Membrane reshaping resides at the core of many important cellular processes, and among its mediators are the BAR (Bin, Amphiphysin, Rvs) domain-containing proteins. We have explored the diversity and function of the Rvs BAR proteins in Candida albicans and identified a novel family member, Rvs167-3 (orf19.1861). We show that Rvs167-3 specifically interacts with Rvs162 to form a stable BAR heterodimer able to bind liposomes in vitro . A second, distinct heterodimer is formed by the canonical BAR proteins Rvs161 and Rvs167. Purified Rvs161/Rvs167 complex also binds liposomes, indicating that C. albicans expresses two functional BAR heterodimers. We used live-cell imaging to localize green fluorescent protein (GFP)-tagged Rvs167-3 and Rvs167 and show that both proteins concentrate in small cortical spots. However, while Rvs167 strictly colocalizes with the endocytic marker protein Abp1, we do not observe any colocalization of Rvs167-3 with sites of endocytosis marked by Abp1. Furthermore, the rvs167-3 Δ/Δ mutant is not defective in endocytosis and strains lacking Rvs167-3 or its partner Rvs162 do not display increased sensitivity to high salt concentrations or decreased cell wall integrity, phenotypes which have been observed for rvs167 Δ/Δ and rvs161 Δ/Δ strains and which are linked to endocytosis defects. Taken together, our results indicate different roles for the two BAR heterodimers in C. albicans : the canonical Rvs161/Rvs167 heterodimer functions in endocytosis, whereas the novel Rvs162/Rvs167-3 heterodimer seems not to be involved in this process. Nevertheless, despite their different roles, our phenotypic analysis revealed a genetic interaction between the two BAR heterodimers, suggesting that they may have related but distinct membrane-associated functions.

scholarly journals Akt Phosphorylation of Serine 21 on Pak1 Modulates Nck Binding and Cell Migration

2003 ◽  
Vol 23 (22) ◽  
pp. 8058-8069 ◽  
Author(s):  
Guo-Lei Zhou ◽  
Ya Zhuo ◽  
Charles C. King ◽  
Benjamin H. Fryer ◽  
Gary M. Bokoch ◽  
...  
Keyword(s):  
Cell Migration ◽  
Protein Kinases ◽  
Cellular Proliferation ◽  
Fluorescent Protein ◽  
Focal Adhesions ◽  
Adaptor Protein ◽  
Small Gtpases ◽  
Akt Phosphorylation ◽  
Green Fluorescent

ABSTRACT The p21-activated protein kinases (Paks) regulate cellular proliferation, differentiation, transformation, and survival through multiple downstream signals. Paks are activated directly by the small GTPases Rac and Cdc42 and several protein kinases including Akt and PDK-1. We found that Akt phosphorylated and modestly activated Pak1 in vitro. The major site phosphorylated by Akt on Pak1 mapped to serine 21, a site originally shown to be weakly autophosphorylated on Pak1 when Cdc42 or Rac activates it. A peptide derived from the region surrounding serine 21 was a substrate for Akt but not Pak1 in vitro, and Akt stimulated serine 21 phosphorylation on the full-length Pak1 much better than Rac did. The adaptor protein Nck binds Pak near serine 21, and its association is regulated by phosphorylation of this site. We found that either treatment of Pak1 in vitro with Akt or coexpression of constitutively active Akt with Pak1 reduced Nck binding to Pak1. In HeLa cells, green fluorescent protein-tagged Pak1 was concentrated at focal adhesions and was released when Akt was cotransfected. A peptide containing the Nck binding site of Pak1 fused to a portion of human immunodeficiency virus Tat to allow it to enter cells was used to test the functional importance of Nck/Pak binding in Akt-stimulated cell migration. This Tat-Nck peptide reduced Akt-stimulated cell migration. Together, these data suggest that Akt modulates the association of Pak with Nck to regulate cell migration.

scholarly journals Integrins on eggs: focal adhesion kinase is activated at fertilization, forms a complex with integrins, and is necessary for cortex formation and cell cycle initiation

2013 ◽  
Vol 24 (21) ◽  
pp. 3472-3481 ◽  
Author(s):  
D. Chan ◽  
C. J. Thomas ◽  
V. J. Taylor ◽  
R. D. Burke
Keyword(s):  
Cell Cycle ◽  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Fluorescent Protein ◽  
Cyclin E ◽  
Cortical Actin ◽  
Complex Forms ◽  
Green Fluorescent ◽  
Adhesion Complex ◽  
Egg Cortex

We investigate the proposal that integrins and focal adhesion kinase (FAK) form a complex that has structural and signaling functions in eggs. FAK protein is present in eggs and is phosphorylated at fertilization. pY397FAK localizes to the membrane 30 min after fertilization, which correlates with the expression of βC integrins and egg cortex development. The βC integrin and pY397FAK coimmunoprecipitate from egg cortex lysates. PF573 228 and Y11, inhibitors of FAK, interfere with pronuclear fusion and reduce the abundance of pY397FAK and cortical actin without affecting microvillar actin. Cyclin E normally accumulates in the nucleus 15 min after fertilization, then returns to background levels. PF573 228– or Y11-treated eggs accumulate cyclin E in the nucleus; however, levels remain high. In addition, PF573 228 interferes with the accumulation of pERK1/2 in the nucleus and in eggs initiating mitosis. Injection of eggs with a fusion protein consisting of the focal adhesion–targeting domain of FAK fused to green fluorescent protein interferes with cortex formation and produces abnormal nuclei. These data indicate that an integrin–FAK adhesion complex forms at the egg surface that functions in formation of actin arrays in the egg cortex and provides signaling inputs for cell cycle initiation.

Hic-5 promotes endothelial cell migration to lysophosphatidic acid

2007 ◽  
Vol 293 (1) ◽  
pp. H193-H203 ◽  
Author(s):  
C. Avraamides ◽  
M. E. Bromberg ◽  
J. P. Gaughan ◽  
S. M. Thomas ◽  
A. Y. Tsygankov ◽  
...  
Keyword(s):  
Cell Migration ◽  
Endothelial Cell ◽  
Focal Adhesion ◽  
Lysophosphatidic Acid ◽  
Fluorescent Protein ◽  
Focal Adhesions ◽  
Endothelial Cell Migration ◽  
Erk Phosphorylation ◽  
Green Fluorescent

Endothelial cell migration is critical for proper blood vessel development. Signals from growth factors and matrix proteins are integrated through focal adhesion proteins to alter cell migration. Hydrogen peroxide-inducible clone 5 (Hic-5), a paxillin family member, is enriched in the focal adhesions in bovine pulmonary artery endothelial (BPAE) cells, which migrate to lysophosphatidic acid (LPA) on denatured collagen. In this study, we investigate the role of Hic-5 in LPA-stimulated endothelial cell migration. LPA recruits Hic-5 to the focal adhesions and to the pseudopodia in BPAE cells plated on collagen, suggesting that recruitment of Hic-5 to focal adhesions is associated with endothelial cell migration. Knockdown of endogenous Hic-5 significantly decreases migration toward LPA, confirming involvement of Hic-5 in migration. To address the role of Hic-5 in endothelial cell migration, we exogenously expressed wild-type (WT) Hic-5 and green fluorescent protein Hic-5 C369A/C372A (LIM3 mutant) constructs in BPAE cells. WT Hic-5 expression increases chemotaxis of BPAE cells to LPA, whereas migration toward LPA of the green fluorescent protein Hic-5 C369A/C372A-expressing cells is similar to that shown in vector control cells. Additionally, ERK phosphorylation is enhanced in the presence of LPA in WT Hic-5 cells. A pharmacological inhibitor of MEK activity inhibits LPA-stimulated WT Hic-5 cell migration and ERK phosphorylation, suggesting Hic-5 enhances migration via MEK activation of ERK. Together, these studies indicate that Hic-5, a focal adhesion protein in endothelial cells, is recruited to the pseudopodia in the presence of LPA and enhances migration.

scholarly journals Paxillin, a tyrosine phosphorylated focal adhesion-associated protein binds to the carboxyl terminal domain of focal adhesion kinase.

1995 ◽  
Vol 6 (6) ◽  
pp. 637-647 ◽  
Author(s):  
J D Hildebrand ◽  
M D Schaller ◽  
J T Parsons
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Tyrosine Kinases ◽  
Focal Adhesions ◽  
Stable Complex ◽  
Suspension Cells ◽  
Terminal Domain ◽  
Carboxy Terminal

Focal adhesion kinase (pp125FAK or FAK) and paxillin colocalize with integrins in structures called focal adhesions. pp125FAK plays an important role in the transmission of integrin-induced cytoplasmic signals. Paxillin has also been implicated in cell signaling by virtue of its association with the protein tyrosine kinases pp60src and Csk (C-terminal Src kinase) as well as with the adapter/oncoprotein p47gag-crk. In this report we show that endogenous pp125FAK and paxillin form a stable complex both in vivo and in vitro and that this interaction is direct, requiring only pp125FAK and paxillin. The paxillin binding site on pp125FAK has been localized to the carboxy-terminal 148 residues of pp125FAK, but appears to be distinct from the previously identified focal adhesion-targeting sequence also present in the carboxy-terminal domain of pp125FAK. The interaction of paxillin and pp125FAK is independent of the adhesion of cells to the extracellular matrix, as the association can be detected in suspension cells as well as those attached to fibronectin.

scholarly journals Neuronal migration is mediated by inositol hexakisphosphate kinase 1 via α-actinin and focal adhesion kinase

2017 ◽  
Vol 114 (8) ◽  
pp. 2036-2041 ◽  
Author(s):  
Chenglai Fu ◽  
Jing Xu ◽  
Weiwei Cheng ◽  
Tomas Rojas ◽  
Alfred C. Chin ◽  
...  
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Neuronal Migration ◽  
Intracellular Localization ◽  
Focal Adhesions ◽  
Fiber Formation ◽  
Inositol Hexakisphosphate ◽  
Stress Fiber Formation ◽  
And Function ◽  
Pharmacologic Inhibition

Inositol hexakisphosphate kinase 1 (IP6K1), which generates 5-diphosphoinositol pentakisphosphate (5-IP7), physiologically mediates numerous functions. We report thatIP6K1deletion leads to brain malformation and abnormalities of neuronal migration. IP6K1 physiologically associates with α-actinin and localizes to focal adhesions.IP6K1deletion disrupts α-actinin’s intracellular localization and function. TheIP6K1deleted cells display substantial decreases of stress fiber formation and impaired cell migration and spreading. Regulation of α-actinin by IP6K1 requires its kinase activity. Deletion ofIP6K1abolishes α-actinin tyrosine phosphorylation, which is known to be regulated by focal adhesion kinase (FAK). FAK phosphorylation is substantially decreased inIP6K1deleted cells. 5-IP7, a product of IP6K1, promotes FAK autophosphorylation. Pharmacologic inhibition of IP6K by TNP [N2-(m-Trifluorobenzyl), N6-(p-nitrobenzyl)purine] recapitulates the phenotype ofIP6K1deletion. These findings establish that IP6K1 physiologically regulates neuronal migration by binding to α-actinin and influencing phosphorylation of both FAK and α-actinin through its product 5-IP7.

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

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.

scholarly journals A New Focal Adhesion Protein That Interacts with Integrin-Linked Kinase and Regulates Cell Adhesion and Spreading

2001 ◽  
Vol 153 (3) ◽  
pp. 585-598 ◽  
Author(s):  
Yizeng Tu ◽  
Yao Huang ◽  
Yongjun Zhang ◽  
Yun Hua ◽  
Chuanyue Wu
Keyword(s):  
Cell Adhesion ◽  
Focal Adhesion ◽  
Fluorescent Protein ◽  
Cytoskeleton Organization ◽  
Terminal Domain ◽  
Integrin Linked Kinase ◽  
Ch2 Domain ◽  
Green Fluorescent

Integrin-linked kinase (ILK) is a multidomain focal adhesion (FA) protein that functions as an important regulator of integrin-mediated processes. We report here the identification and characterization of a new calponin homology (CH) domain-containing ILK-binding protein (CH-ILKBP). CH-ILKBP is widely expressed and highly conserved among different organisms from nematodes to human. CH-ILKBP interacts with ILK in vitro and in vivo, and the ILK COOH-terminal domain and the CH-ILKBP CH2 domain mediate the interaction. CH-ILKBP, ILK, and PINCH, a FA protein that binds the NH2-terminal domain of ILK, form a complex in cells. Using multiple approaches (epitope-tagged CH-ILKBP, monoclonal anti–CH-ILKBP antibodies, and green fluorescent protein–CH-ILKBP), we demonstrate that CH-ILKBP localizes to FAs and associates with the cytoskeleton. Deletion of the ILK-binding CH2 domain abolished the ability of CH-ILKBP to localize to FAs. Furthermore, the CH2 domain alone is sufficient for FA targeting, and a point mutation that inhibits the ILK-binding impaired the FA localization of CH-ILKBP. Thus, the CH2 domain, through its interaction with ILK, mediates the FA localization of CH-ILKBP. Finally, we show that overexpression of the ILK-binding CH2 fragment or the ILK-binding defective point mutant inhibited cell adhesion and spreading. These findings reveal a novel CH-ILKBP–ILK–PINCH complex and provide important evidence for a crucial role of this complex in the regulation of cell adhesion and cytoskeleton organization.

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