References of "Plançon, Sébastien 50002869"
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See detailRegulation of Neutrophil Degranulation and Cytokine Secretion: A Novel Model Approach Based on Linear Fitting
Naegelen, Isabelle UL; Beaume, Nicolas UL; Plançon, Sébastien UL et al

in Journal of Immunology Research (2015), 2015

Neutrophils participate in the maintenance of host integrity by releasing various cytotoxic proteins during degranulation. Due to recent advances, a major role has been attributed to neutrophil-derived ... [more ▼]

Neutrophils participate in the maintenance of host integrity by releasing various cytotoxic proteins during degranulation. Due to recent advances, a major role has been attributed to neutrophil-derived cytokine secretion in the initiation, exacerbation, and resolution of inflammatory responses. Because the release of neutrophil-derived products orchestrates the action of other immune cells at the infection site and, thus, can contribute to the development of chronic inflammatory diseases, we aimed to investigate in more detail the spatiotemporal regulation of neutrophil-mediated release mechanisms of proinflammatory mediators. Purified human neutrophils were stimulated for different time points with lipopolysaccharide. Cells and supernatants were analyzed by flow cytometry techniques and used to establish secretion profiles of granules and cytokines. To analyze the link between cytokine release and degranulation time series, we propose an original strategy based on linear fitting, which may be used as a guideline, to (i) define the relationship of granule proteins and cytokines secreted to the inflammatory site and (ii) investigate the spatial regulation of neutrophil cytokine release. The model approach presented here aims to predict the correlation between neutrophil-derived cytokine secretion and degranulation and may easily be extrapolated to investigate the relationship between other types of time series of functional processes. [less ▲]

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See detailEffects of Endocrine Disruptor Compounds, Alone or in Combination, on Human Macrophage-Like THP-1 Cell Response
Couleau, Nicolas; Falla, Jairo; Beillerot, Adeline et al

in PLoS ONE (2015), 10(7), 0131428

The aim of the present study was to evaluate the immunological effects on human macrophages of four endocrine disruptor compounds (EDCs) using the differentiated human THP-1 cell line as a model. We ... [more ▼]

The aim of the present study was to evaluate the immunological effects on human macrophages of four endocrine disruptor compounds (EDCs) using the differentiated human THP-1 cell line as a model. We studied first the effects of these EDCs, including Bisphenol A (BPA), di-ethylhexyl-phthalate (DEHP), dibutyl phthalate (DBP) and 4-tert-octylphenol (4-OP), either alone or in combination, on cytokine secretion, and phagocytosis. We then determined whether or not these effects were mediated by estrogen receptors via MAPK pathways. It was found that all four EDCs studied reduced strongly the phagocytosis of the differentiated THP-1 cells and that several of these EDCs disturbed also TNF-α, IL-1 β and IL-8 cytokine secretions. Furthermore, relative to control treatment, decreased ERK 1/2 phosphorylation was always associated with EDCs treatments-either alone or in certain combinations (at 0.1 μM for each condition). Lastly, as treatments by an estrogen receptor antagonist suppressed the negative effects on ERK 1/2 phosphorylation observed in cells treated either alone with BPA, DEHP, 4-OP or with the combined treatment of BPA and DEHP, we suggested that estrogen receptor-dependent pathway is involved in mediating the effects of EDCs on human immune system. Altogether, these results advocate that EDCs can disturb human immune response at very low concentrations. [less ▲]

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See detailAn essential role of syntaxin 3 protein for granule exocytosis and secretion of IL-1α, IL-1β, IL-12b, and CCL4 from differentiated HL-60 cells
Naegelen, Isabelle UL; Plançon, Sébastien UL; Nicot, Nathalie et al

in Journal of Leukocyte Biology (2014), 97

Besides their roles in the killing of pathogens, neutrophils have the capacity to package a variety of cytokines into cytoplasmic granules for subsequent release upon inflammatory conditions. Because the ... [more ▼]

Besides their roles in the killing of pathogens, neutrophils have the capacity to package a variety of cytokines into cytoplasmic granules for subsequent release upon inflammatory conditions. Because the rapid secretion of cytokines orchestrates the action of other immune cells at the infection site and thus, can contribute to the development and chronicity of inflammatory diseases, we aimed to determine the intracellular SNARE machinery responsible for the regulation of cytokine secretion and degranulation. From a constructed gene-expression network, we first selected relevant cytokines for functional validation by the CBA approach. We established a cytokine-secretion profile for human neutrophils and dHL-60 cells, underlining their similar ability to secrete a broad variety of cytokines within proinflammatory conditions mimicked by LPS stimulation. Secondly, after screening of SNARE genes by microarray experiments, we selected STX3 for further functional studies. With the use of a siRNA strategy, we show that STX3 is clearly required for the maximal release of IL-1α, IL-1β, IL-12b, and CCL4 without alteration of other cytokine secretion in dHL-60 cells. In addition, we demonstrate that STX3 is involved in MMP-9 exocytosis from gelatinase granules, where STX3 is partly localized. Our results suggest that the secretion of IL-1α, IL-1β, IL-12b, and CCL4 occurs during gelatinase degranulation, a process controlled by STX3. In summary, these findings provide first evidence that STX3 has an essential role in trafficking pathways of cytokines in neutrophil granulocytes. [less ▲]

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See detailNew Insights into the Regulation of Neutrophil NADPH Oxidase Activity in the Phagosome: A Focus on the Role of Lipid and Ca(2+) Signaling
Bréchard, Sabrina UL; Plançon, Sébastien UL; Tschirhart, Eric UL

in Antioxidants & Redox Signaling (2013), 18(6), 661-676

Significance: Reactive oxygen species, produced by the phagosomal NADPH oxidase of neutrophils, play a significant physiological role during normal defense. Their role is not only to kill invading ... [more ▼]

Significance: Reactive oxygen species, produced by the phagosomal NADPH oxidase of neutrophils, play a significant physiological role during normal defense. Their role is not only to kill invading pathogens, but also to act as modulators of global physiological functions of phagosomes. Given the importance of NADPH oxidase in the immune system, its activity has to be decisively controlled by distinctive mechanisms to ensure appropriate regulation at the phagosome. Recent Advances: Here, we describe the signal transduction pathways that regulate phagosomal NADPH oxidase in neutrophils, with an emphasis on the role of lipid metabolism and intracellular Ca(2+) mobilization. Critical Issues: The potential involvement of Ca(2+)-binding S100A8 and S100A9 proteins, known to interact with the plasma membrane NADPH oxidase, is also considered. Future Directions: Recent technical progress in advanced live imaging microscopy will permit to focus more accurately on phagosomal rather than plasma membrane NADPH oxidase regulation during neutrophil phagocytosis. [less ▲]

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See detailiPLA 2 , a novel determinant in Ca2+ - and phosphorylation-dependent S100A8/A9 regulated NOX2 activity
Schenten, Véronique UL; Bréchard, Sabrina UL; Plançon, Sébastien UL et al

in Biochimica et Biophysica Acta-Molecular Cell Research (2010), 1803(7), 840-847

The neutrophil NADPH oxidase (NOX2) is a key enzyme responsible for host defense against invading pathogens, via the production of reactive oxygen species. Dysfunction of NOX2 can contribute to ... [more ▼]

The neutrophil NADPH oxidase (NOX2) is a key enzyme responsible for host defense against invading pathogens, via the production of reactive oxygen species. Dysfunction of NOX2 can contribute to inflammatory processes, which could lead to the development of diseases such as atherosclerosis. In this paper, we characterize a pathway leading to NOX2 activation in which iPLA(2)-regulated p38 MAPK activity is a key regulator of S100A8/A9 translocation via S100A9 phosphorylation. Studies in cell-free or recombinant systems involved two Ca2+-binding proteins of the S100 family, namely S100A8 and S100A9, in NOX2 activation dependent on intracellular Ca2+ concentration ([Ca2+](i)) elevation. Using differentiated HL-60 cells as a model of neutrophils, we provide evidence that [Ca2+](i)-regulated S100A8/A9 translocation is mediated by an increase in [Ca2+](i) through intracellular Ca2+ store depletion. Moreover, we confirm that p38 MAPK induces S100A9 phosphorylation, a mandatory precondition for S100 translocation. Based on a pharmacological approach and an siRNA strategy, we identify iPLA(2) as a new molecular player aiding S100 translocation and NOX2 activity. Inhibition of p38 MAPK activity and S100A9 phosphorylation by bromoenol lactone, a selective inhibitor of iPLA(2), indicated that p38 MAPK-mediated S100A9 phosphorylation is dependent on iPLA(2). In conclusion, we have characterized a pathway leading to NOX2 activation in which iPLA(2)-regulated p38 MAPK activity is a key regulator of S100A8/A9 translocation via S100A9 phosphorylation. [less ▲]

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See detailSTIM1 but not STIM2 is an essential regulator of Ca2+ influx-mediated NADPH oxidase activity in neutrophil-like HL-60 cells
Bréchard, Sabrina UL; Plançon, Sébastien UL; Melchior, Chantal UL et al

in Biochemical Pharmacology (2009), 78(5), 504-513

Extracellular Ca2+ entry, primarily mediated through store-operated Ca2+ entry (SOCE), is known to be a critical event for NADPH oxidase (NOX2) regulation in neutrophils. While defective NOX2 activity has ... [more ▼]

Extracellular Ca2+ entry, primarily mediated through store-operated Ca2+ entry (SOCE), is known to be a critical event for NADPH oxidase (NOX2) regulation in neutrophils. While defective NOX2 activity has been linked to various inflammatory diseases, regulatory mechanisms that control Ca2+ influx-induced NOX2 activation are poorly understood in SOCE. The role of STIM1, a Ca2+ sensor that transduces the store depletion signal to the plasma membrane, seems well established and supported by numerous studies in non-phagocytic cells. Here, in neutrophil-like HL-60 cells we used a siRNA approach to delineate the effect of STIM1 knock-down on NOX2 activity regulated by Ca2+ influx. Because the function of the STIM1 homolog, STIM2, is still unclear, we determined the consequence of STIM2 knock-down on Ca2+ and NOX2. STIM1 and STIM2 knock-down was effective and isoform specific when assayed by real-time PCR and Western blotting. Consistent with a unique role of STIM1 in the regulation of SOCE, STIM1, but not STIM2, siRNA significantly decreased Ca2+ influx induced by fMLF or the SERCA pump inhibitor thapsigargin. A redistribution of STIM1, originally localized intracellularly, near the plasma membrane was observed by confocal microscopy upon stimulation by fMLF. Inhibition of STIM1-induced SOCE led to a marked decrease in NOX2 activity while STIM2 siRNA had no effect. Thus, our results provide evidence for a role of STIM1 protein in the control of Ca2+ influx in neutrophils excluding a STIM2 involvement in this process. It also places STIM1 as a key modulator of NOX2 activity with a potential interest for anti-inflammatory pharmacological development. [less ▲]

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See detailCell passaging rapidly affects expression, secretion and activity of MMP9 as well as mobility of HL60 leukemia cells
Bernard, Yohann UL; Plançon, Sébastien UL; Melchior, Chantal UL et al

in Journal of Cell and Animal Biology (2008), 2(9), 160-165

The HL60 cell line, derived from acute promyelocytic leukemia cells, can differentiate into neutrophil-like cell following DMSO treatment. Mobility of HL60, or DMSO-differentiated HL60 cells (≠HL60 ... [more ▼]

The HL60 cell line, derived from acute promyelocytic leukemia cells, can differentiate into neutrophil-like cell following DMSO treatment. Mobility of HL60, or DMSO-differentiated HL60 cells (≠HL60), requires surface expression of adhesion molecules and production of matrix metalloproteinases (MMPs). The aim of this study was to investigate in HL60 and ≠HL60 the effects of cell passaging (over 5 passages after delivery (P and P+5)) on i) surface expression of adhesion molecule CD11b, which is considered a neutrophil differentiation marker ii) MMP9 mRNA expression, protein release and zymographic activity and iii) cellular mobility. As expected, CD11b expression at both cell passages increased in ≠HL60 relative to undifferentiated HL60, but expression levels of this neutrophils marker did not change over 5 passages. MMP9 mRNA expression however, in basal conditions was increased in HL60 at P+5. At P+5 versus P, MMP9 protein levels, MMP9 zymographic activity and cellular mobility in HL60 and ≠HL60 were elevated. Stimulation by N-formyl-L-Methionyl-L-Leucyl-L-Phenylalanine had no effects on HL60, but raised MMP9 protein concentration and zymographic activity in ≠HL60. Since passage history is likely to also influence cellular functions other than MMP-related effects, it is important to carefully consider passage numbers when designing experiments [less ▲]

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See detailCa2+-dependent regulation of NOX2 activity via MRP proteins in HL-60 granulocytes
Schenten, Véronique UL; Bréchard, Sabrina UL; Melchior, Chantal UL et al

in Calcium Binding Proteins (2008), 3(1), 25-30

Recently, two proteins of the S100 protein family, the myeloid-related calcium-binding proteins MRP-8 and MRP-14 have been implicated in the Ca2+-induced activation of the neutrophil NADPH oxidase (NOX2 ... [more ▼]

Recently, two proteins of the S100 protein family, the myeloid-related calcium-binding proteins MRP-8 and MRP-14 have been implicated in the Ca2+-induced activation of the neutrophil NADPH oxidase (NOX2) but the mechanism underlying this process remains unclear. In this study, the role of MRP-8/14 in the Ca2+-dependent regulation of NOX2 activity was characterized in neutrophil-like HL-60 cells using small interfering RNAs (siRNAs) to knock-down endogenous MRP-8 and/or MRP-14 expression. Real-time PCR and Western blot revealed that MRP-8 and MRP-14 expression was 20 times higher in dimethylsulfoxide-differentiated neutrophil-like HL-60 cells compared to quiescent HL-60 cells. Knock-down of MRP-8 and MRP-14 in differentiated HL-60 cells decreased protein levels by 30 and 45% respectively. The impact of the reduced MRP-8/14 protein expression on NOX2 activity was investigated by measuring fMLF-induced H2O2 production. In cells simultaneously transfected with MRP-8 and MRP14 siRNAs, H2O2 production was reduced by 50%, suggesting that both MRP-8 and MRP-14 are required for NOX2 activity; single knock-downs were inefficient. To elucidate the role of Ca2+ in MRP8/14, and consequently in NOX2 activation, siRNA-transfected cells were treated with the Ca2+ ionophore ionomycin prior to stimulation with PMA, a Ca2+-independent protein kinase C activator. PMA-induced H2O2 production was enhanced by ionomycin. This amplification of NOX2 activity was abolished by MRP8/14 knock-down, indicating that both MRP-8 and MRP-14 are necessary to regulate Ca2+-induced NOX2 activation. Taken together, our results suggest that the mechanism of MRPs activation is highly dependent on the increase of intracellular Ca2+ level for a full activation of NOX2. [less ▲]

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See detailStore-operated Ca2+ channels formed by TRPC1, TRPC6 and Orai1 and non-store-operated channels formed by TRPC3 are involved in the regulation of NADPH oxidase in HL-60 granulocytes
Bréchard, Sabrina UL; Melchior, Chantal UL; Plançon, Sébastien UL et al

in Cell Calcium (2008)

Ca(2+) influx has been shown to be essential for NADPH oxidase activity which is involved in the inflammatory process. Ca(2+) conditions underlying the oxidative response are clearly delineated. Here, we ... [more ▼]

Ca(2+) influx has been shown to be essential for NADPH oxidase activity which is involved in the inflammatory process. Ca(2+) conditions underlying the oxidative response are clearly delineated. Here, we show that store-operated Ca(2+) entry (SOCE) is required at the beginning of NADPH oxidase activation in response to fMLF (N-formyl-l-methionyl-l-leucyl-l-phenylalanine ) in neutrophil-like HL-60 cells. When extracellular Ca(2+) is initially removed, early addition of Ca(2+) after stimulation causes a complete restoration of Ca(2+) entry and H(2)O(2) production. Both Ca(2+) entry and H(2)O(2) production are decreased by purported SOCE blockers, 2-aminoethoxydiphenyl borane (2-APB) and SK&F 96365. Endogenously expressed TRPC (transient receptor potential canonical) homologues and Orai1 were investigated for their role in supporting store-operated Ca(2+) channels activity. TRPC1, TRPC6 and Orai1 knock-out by siRNA resulted in the inhibition of Ca(2+) influx and H(2)O(2) production in response to fMLF and thapsigargin while suppression of TRPC3 had no effect on thapsigargin induced-SOCE. 2-APB and SK&F 96365 were able to amplify the reduction of fMLF-stimulated Ca(2+) entry and H(2)O(2) production observed in cells transfected by TRPC3 siRNA. In summary, Ca(2+) influx in HL-60 cells relies on different membrane TRPC channels and Orai1 for allowing NADPH oxidase activation. TRPC3 primarily mediates SOCE-independent pathways and TRPC1, TRPC6 and Orai1 exclusively contribute to SOCE. [less ▲]

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See detailA new functional role of the fibrinogen RGD motif as the molecular switch that selectively triggers integrin alphaIIbbeta3-dependent RhoA activation during cell spreading
Salsmann, Alexandre UL; Schaffner-Reckinger, Elisabeth UL; Kabile, Fabrice UL et al

in Journal of Biological Chemistry (2005), 280(39), 33610-33619

A number of RGD-type integrins rely on a synergistic site in addition to the canonical RGD site for ligand binding and signaling, although it is still unclear whether these two recognition sites function ... [more ▼]

A number of RGD-type integrins rely on a synergistic site in addition to the canonical RGD site for ligand binding and signaling, although it is still unclear whether these two recognition sites function independently, synergistically, or competitively. Experimental evidence has suggested that fibrinogen binding to the RGD-type integrin alphaIIbbeta3 occurs exclusively through the synergistic gamma(400-411) sequence, thus questioning the functional role of the RGD recognition site. Here we have investigated the respective role of the fibrinogen gamma(400-411) sequence and the RGD motif in the molecular events leading to ligand-induced alphaIIbbeta3-dependent Chinese hamster ovary (CHO) cell or platelet spreading, by using intact fibrinogen and well characterized plasmin-generated fibrinogen fragments containing either the RGD motif (fragment C) or the gamma(400-411) sequence (fragment D), and CHO cells expressing resting wild type (alphaIIbbeta3wt), constitutively active (alphaIIbbeta3T562N), or non-functional (alphaIIbbeta3D119Y) receptors. Our data provide evidence that the gamma(400-411) site by itself is able to initiate alphaIIbbeta3 clustering and recruitment of intracellular proteins to early focal complexes, mediating cell attachment, FAK phosphorylation, and Rac1 activation, while the RGD motif subsequently acts as a molecular switch on the beta3 subunit to trigger cell spreading. More importantly, we show that the premier functional role of the RGD site is not to reinforce cell attachment but, rather, to imprint a conformational change on the beta3 subunit leading to maximal RhoA activation and actin cytoskeleton organization in CHO cells as well as in platelets. Finally, alphaIIbbeta3-dependent RhoA stimulation and cell spreading, but not cell attachment, are Src-dependent and phosphoinositide 3-kinase-independent and are inhibited by the Src antagonist PP2. [less ▲]

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See detailA fluorescence cell biology approach to map the second integrin-binding site of talin to a 130-amino acid sequence within the rod domain
Tremuth, L. A; Kreis, Stephanie UL; Melchior, C. A et al

in Journal of Biological Chemistry (2004), 279(21), 22258-22266

The cytoskeletal protein talin, which provides a direct link between integrins and actin filaments, has been shown to contain two distinct binding sites for integrin β subunits. Here, we report the ... [more ▼]

The cytoskeletal protein talin, which provides a direct link between integrins and actin filaments, has been shown to contain two distinct binding sites for integrin β subunits. Here, we report the precise delimitation and a first functional analysis of the talin rod domain integrin-binding site. Partially overlapping cDNAs covering the entire human talin gene were transiently expressed as DsRed fusion proteins in Chinese hamster ovary cells expressing α IIbβ 3, linked to green fluorescent protein (GFP). Two-color fluorescence analysis of the transfected cells, spread on fibrinogen, revealed distinct subcellular staining patterns including focal adhesion, actin filament, and granular labeling for different talin fragments. The rod domain fragment G (residues 1984-2344), devoid of any known actin- or vinculin-binding sites, colocalized with β 3-GFP in focal adhesions. Direct in vitro interaction of fragment G with native platelet integrin α IIbβ 3 or with the recombinant wild type, but not the Y747A mutant β 3 cytoplasmic tail, linked to glutathione S-transferase, was demonstrated by surface plasmon resonance analysis and pull-down assays, respectively. Here, we demonstrate for the first time the in vivo relevance of this interaction by fluorescence resonance energy transfer between β 3-GFP and DsRed-talin fragment G. Further in vitro pull-down studies allowed us to map out the integrin-binding site within fragment G to a stretch of 130 residues (fragment J, residues 1984-2113) that also localized to focal adhesions. Finally, we show by a cell biology approach that this integrin- binding site within the talin rod domain is important for β 3-cytoskeletal interactions but does not participate in α IIbβ 3 activation. [less ▲]

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See detailGreen fluorescent protein (GFP) tagged to the cytoplasmic tail of alphaIIb or beta3 allows the expression of a fully functional integrin alphaIIb(beta3): effect of beta3GFP on alphaIIb(beta3) ligand binding.
Plançon, Sébastien UL; Morel-Kopp, M. C.; Schaffner-Reckinger, Elisabeth UL et al

in The Biochemical journal (2001), 357(Pt 2), 529-36

Using green fluorescent protein (GFP) as an autofluorescent tag, we report the first successful visualization of a beta3 integrin in a living cell. GFP fused in frame to the cytoplasmic tail of either ... [more ▼]

Using green fluorescent protein (GFP) as an autofluorescent tag, we report the first successful visualization of a beta3 integrin in a living cell. GFP fused in frame to the cytoplasmic tail of either alphaIIb or beta3 allowed normal expression, heterodimerization, processing and surface exposure of alphaIIbGFPbeta3 and alphaIIb(beta3)GFP receptors in Chinese hamster ovary (CHO) cells. Direct microscopic observation of the autofluorescent cells in suspension following antibody-induced alphaIIb(beta3) capping revealed an intense autofluorescent cap corresponding to unlabelled immunoclustered GFP-tagged alphaIIb(beta3). GFP-tagged alphaIIbbeta3 receptors mediated fibrinogen-dependent cell adhesion, were readily detectable in focal adhesions of unstained living cells and triggered p125(FAK) tyrosine phosphorylation similar to wild-type alphaIIb(beta3) (where FAK corresponds to focal adhesion kinase). However, GFP tagged to beta3, but not to alphaIIb, induced spontaneous CHO cell aggregation in the presence of soluble fibrinogen, as well as binding of the fibrinogen mimetic monoclonal antibody PAC1 in the absence of alphaIIb(beta3) receptor activation. Time-lapse imaging of living transfectants revealed a characteristic redistribution of GFP-tagged alphaIIb(beta3) during the early stages of cell attachment and spreading, starting with alphaIIb(beta3) clustering at the rim of the cell contact area, that gradually overlapped with the boundary of the attached cell, and, with the onset of cell spreading, to a reorganization of alphaIIb(beta3) in focal adhesions. Taken together, our results demonstrate that (1) fusion of GFP to the cytoplasmic tail of either alphaIIb or beta3 integrin subunits allows normal cell surface expression of a functional receptor, and (2) structural modification of the beta3 integrin cytoplasmic tail, rather than the alphaIIb subunit, plays a major role in alphaIIb(beta3) affinity modulation. With the successful direct visualization of functional alphaIIb(beta3) receptors in living cells, the generation of autofluorescent integrins in transgenic animals will become possible, allowing new approaches to study the dynamics of integrin functions. [less ▲]

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See detailDistinct involvement of beta3 integrin cytoplasmic domain tyrosine residues 747 and 759 in integrin-mediated cytoskeletal assembly and phosphotyrosine signaling.
Schaffner-Reckinger, Elisabeth UL; Gouon, V.; Melchior, Chantal UL et al

in The Journal of biological chemistry (1998), 273(20), 12623-32

We have investigated the structural requirements of the beta3 integrin subunit cytoplasmic domain necessary for tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin during alphav beta3 ... [more ▼]

We have investigated the structural requirements of the beta3 integrin subunit cytoplasmic domain necessary for tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin during alphav beta3-mediated cell spreading. Using CHO cells transfected with various beta3 mutants, we demonstrate a close correlation between alphav beta3-mediated cell spreading and tyrosine phosphorylation of FAK and paxillin, and highlight a distinct involvement of the NPLY747 and NITY759 motifs in these signaling processes. Deletion of the NITY759 motif alone was sufficient to completely prevent alphav beta3-dependent focal contact formation, cell spreading, and FAK/paxillin phosphorylation. The single Y759A substitution induced a strong inhibitory phenotype, while the more conservative, but still phosphorylation-defective, Y759F mutation restored wild type receptor function. Alanine substitution of the highly conserved Tyr747 completely abolished alphav beta3-dependent formation of focal adhesion plaques, cell spreading, and FAK/paxillin phosphorylation, whereas a Y747F substitution only partially restored these events. As none of these mutations affected receptor-ligand interaction, our results suggest that the structural integrity of the NITY759 motif, rather than the phosphorylation status of Tyr759 is important for beta3-mediated cytoskeleton reorganization and tyrosine phosphorylation of FAK and paxillin, while the presence of Tyr at residue 747 within the NPLY747 motif is required for optimal beta3 post-ligand binding events. [less ▲]

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