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James E Casanova - One of the best experts on this subject based on the ideXlab platform.
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activation of arf6 by arno stimulates epithelial cell migration through downstream activation of both rac1 and phospholipase d
Journal of Cell Biology, 2001Co-Authors: Lorraine C Santy, James E CasanovaAbstract:Migration of epithelial cells is essential for tissue morphogenesis, wound healing, and metastasis of epithelial tumors. Here we show that ARNO, a guanine nucleotide exchange factor for ADP-ribosylation factor (ARF) GTPases, induces Madin-Darby canine kidney epithelial cells to develop broad lamellipodia, to separate from neighboring cells, and to exhibit a dramatic increase in migratory behavior. This transition requires ARNO catalytic activity, which we show leads to enhanced activation of endogenous ARF6, but not ARF1, using a novel pulldown assay. We further demonstrate that expression of ARNO leads to increased activation of endogenous Rac1, and that Rac activation is required for ARNO-induced cell motility. Finally, ARNO-induced activation of ARF6 also results in increased activation of phospholipase D (PLD), and inhibition of PLD activity also inhibits motility. However, inhibition of PLD does not prevent activation of Rac. Together, these data suggest that ARF6 activation stimulates two distinct signaling pathways, one leading to Rac activation, the other to changes in membrane phospholipid composition, and that both pathways are required for cell motility.
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intra endosomal ph sensitive recruitment of the arf nucleotide exchange factor arno and arf6 from cytoplasm to proximal tubule endosomes
Journal of Biological Chemistry, 2001Co-Authors: Bruno Maranda, Patrick Vinay, James E Casanova, Dennis Brown, Sylvain G Bourgoin, D A Ausiello, Vladimir MarshanskyAbstract:Abstract Kidney proximal tubule epithelial cells have an extensive apical endocytotic apparatus that is critical for the reabsorption and degradation of proteins that traverse the glomerular filtration barrier and that is also involved in the extensive recycling of functionally important apical plasma membrane transporters. We show here that an Arf-nucleotide exchange factor, ARNO (ADP-ribosylation factor nucleotide site opener) as well as Arf6 and Arf1 small GTPases are located in the kidney proximal tubule receptor-mediated endocytosis pathway, and that ARNO and Arf6 recruitment from cytosol to endosomes is pH-dependent. In proximal tubules in situ, ARNO and Arf6 partially co-localized with the V-ATPase in apical endosomes in proximal tubules. Arf1 was localized both at the apical pole of proximal tubule epithelial cells, but also in the Golgi. By Western blot analysis ARNO, Arf6, and Arf1 were detected both in purified endosomes and in proximal tubule cytosol. A translocation assay showed that ATP-driven endosomal acidification triggered the recruitment of ARNO and Arf6 from proximal tubule cytosol to endosomal membranes. The translocation of both ARNO and Arf6 was reversed by V-type ATPase inhibitors and by uncouplers of endosomal intralumenal pH, and was correlated with the magnitude of intra-endosomal acidification. Our data suggest that V-type ATPase-dependent acidification stimulates the selective recruitment of ARNO and Arf6 to proximal tubule early endosomes. This mechanism may play an important role in the pH-dependent regulation of receptor-mediated endocytosis in proximal tubulesin situ.
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the adp ribosylation factor nucleotide exchange factor arno promotes β arrestin release necessary for luteinizing hormone choriogonadotropin receptor desensitization
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Sutapa Mukherjee, Mariefrance Bader, Richard A Kahn, Scott R Frank, James E Casanova, Vsevolod V Gurevich, Jonathan C R Jones, Evelyn T Maizels, Krzysztof Palczewski, Klaus AktoriesAbstract:Desensitization of guanine nucleotide binding protein-coupled receptors is a ubiquitous phenomenon characterized by declining effector activity upon persistent agonist stimulation. The luteinizing hormone/choriogonadotropin receptor (LH/CGR) in ovarian follicles exhibits desensitization of effector adenylyl cyclase activity in response to the mid-cycle surge of LH. We have previously shown that uncoupling of the agonist-activated LH/CGR from the stimulatory G protein (Gs) is dependent on GTP and attributable to binding of β-arrestin present in adenylyl cyclase-rich follicular membrane fraction to the third intracellular (3i) loop of the receptor. Here, we report that LH/CGR-dependent desensitization is mimicked by ADP ribosylation factor nucleotide-binding site opener, a guanine nucleotide exchange factor of the small G proteins ADP ribosylation factors (Arfs) 1 and 6, and blocked by synthetic N-terminal Arf6 peptide, suggesting that the GTP-dependent step of LH/CGR desensitization is receptor-dependent Arf6 activation. Arf activation by GTP and ADP ribosylation factor nucelotide-binding site opener promotes the release of docked β-arrestin from the membrane, making β-arrestin available for LH/CGR; Arf6 but not Arf1 peptides block β-arrestin release from the membrane. Thus, LH/CGR appears to activate two membrane delimited signaling cascades via two types of G proteins: heterotrimeric Gs and small G protein Arf6. Arf6 activation releases docked β-arrestin necessary for receptor desensitization, providing a feedback mechanism for receptor self-regulation.
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arno is a guanine nucleotide exchange factor for adp ribosylation factor 6
Journal of Biological Chemistry, 1998Co-Authors: Scott R Frank, Sunil Upender, Steen H Hansen, James E CasanovaAbstract:ADP-ribosylation factors (ARFs) constitute a family of small monomeric GTPases. ARFs 1 and 3 function in the recruitment of coat proteins to membranes of the Golgi apparatus, whereas ARF6 is localized to the plasma membrane, where it appears to modulate both the assembly of the actin cytoskeleton and endocytosis. Like other GTPases, ARF activation is facilitated by specific guanine nucleotide exchange factors (GEFs). ARNO (ARF nucleotide-binding site opener) is a member of a growing family of ARF-GEFs that share a common, tripartite structure consisting of an N-terminal coiled-coil domain, a central domain with homology to the yeast protein Sec7p, and a C-terminal pleckstrin homology domain. Recently, ARNO and its close homologue cytohesin-1 were found to catalyze in vitro nucleotide exchange on ARF1 and ARF3, respectively, raising the possibility that these GEFs function in the Golgi. However, the actual function of these proteins may be determined in part by their ability to interact with specific ARFs and in part by their subcellular localization. We report here that in vitro ARNO can stimulate nucleotide exchange on both ARF1 and ARF6. Furthermore, based on subcellular fractionation and immunolocalization experiments, we find that ARNO is localized to the plasma membrane in mammalian cells rather than the Golgi. It is therefore likely that ARNO functions in plasma membrane events by modulating the activity of ARF6 in vivo. These findings are consistent with the previous observation that cytohesin-1 regulates the adhesiveness of alphaLbeta2 integrins at the plasma membrane of lymphocytes.
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arno is a guanine nucleotide exchange factor for adp ribosylation factor 6
Journal of Biological Chemistry, 1998Co-Authors: Scott R Frank, Sunil Upender, Steen H Hansen, James E CasanovaAbstract:ADP-ribosylation factors (ARFs) constitute a family of small monomeric GTPases. ARFs 1 and 3 function in the recruitment of coat proteins to membranes of the Golgi apparatus, whereas ARF6 is localized to the plasma membrane, where it appears to modulate both the assembly of the actin cytoskeleton and endocytosis. Like other GTPases, ARF activation is facilitated by specific guanine nucleotide exchange factors (GEFs). ARNO (ARF nucleotide-binding site opener) is a member of a growing family of ARF-GEFs that share a common, tripartite structure consisting of an N-terminal coiled-coil domain, a central domain with homology to the yeast protein Sec7p, and a C-terminal pleckstrin homology domain. Recently, ARNO and its close homologue cytohesin-1 were found to catalyze in vitronucleotide exchange on ARF1 and ARF3, respectively, raising the possibility that these GEFs function in the Golgi. However, the actual function of these proteins may be determined in part by their ability to interact with specific ARFs and in part by their subcellular localization. We report here that in vitro ARNO can stimulate nucleotide exchange on both ARF1 and ARF6. Furthermore, based on subcellular fractionation and immunolocalization experiments, we find that ARNO is localized to the plasma membrane in mammalian cells rather than the Golgi. It is therefore likely that ARNO functions in plasma membrane events by modulating the activity of ARF6 in vivo. These findings are consistent with the previous observation that cytohesin-1 regulates the adhesiveness of αLβ2integrins at the plasma membrane of lymphocytes.
Julie G. Donaldson - One of the best experts on this subject based on the ideXlab platform.
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arf1 and arf6 promote ventral actin structures formed by acute activation of protein kinase c and src
Cytoskeleton, 2014Co-Authors: Juliane P Caviston, Lee Ann Cohen, Julie G. DonaldsonAbstract:Arf proteins regulate membrane traffic and organelle structure. Although Arf6 is known to initiate actin-based changes in cell surface architecture, Arf1 may also function at the plasma membrane. Here we show that acute activation of protein kinase C (PKC) induced by the phorbol ester PMA led to the formation of motile actin structures on the ventral surface of Beas-2b cells, a lung bronchial epithelial cell line. Ventral actin structures also formed in PMA-treated HeLa cells that had elevated levels of Arf activation. For both cell types, formation of the ventral actin structures was enhanced by expression of active forms of either Arf1 or Arf6 and by the expression of guanine nucleotide exchange factors that activate these Arfs. By contrast, formation of these structures was blocked by inhibitors of PKC and Src and required phosphatidylinositol 4, 5-bisphosphate, Rac, Arf6, and Arf1. Furthermore, expression of ASAP1, an Arf1 GTPase activating protein (GAP) was more effective at inhibiting the ventral actin structures than was ACAP1, an Arf6 GAP. This study adds to the expanding role for Arf1 in the periphery and identifies a requirement for Arf1, a "Golgi Arf," in the reorganization of the cortical actin cytoskeleton on ventral surfaces, against the substratum.
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Arf6 recruits the Rac GEF Kalirin to the plasma membrane facilitating Rac activation-5
2011Co-Authors: Tae Hyeon Koo, Betty A. Eipper, Julie G. DonaldsonAbstract:Copyright information:Taken from "Arf6 recruits the Rac GEF Kalirin to the plasma membrane facilitating Rac activation"http://www.biomedcentral.com/1471-2121/8/29BMC Cell Biology 2007;8():29-29.Published online 18 Jul 2007PMCID:PMC1939998.DH-PH motifs. (Also depicted, Sec14 domain, oval and SH2 domain, hexagon). B. Arf6, but not Arf1 or ARF5, binds to SR region of Kalirin. COS7 cells were transfected with C-terminally HA-tagged Arf isoforms, and cell extracts were incubated with GST-KalSR4-7 or with GST. Arfs were detected by immunoblotting with anti-HA antibody. The GST fusion proteins were visualized by Coomassie staining. C. Arf6-GDP co-immunoprecipitates with Kalirin5. FLAG-tagged Kalirin5 was co-expressed with HA-tagged Arf6, Arf6Q67L, Arf6T27N or with Arf1 in COS7 cells. Lysates were immunoprecipitated with FLAG antibody and then immunobloted with HA antibody to detect Arf proteins. D. Expression of EFA6 decreases the interaction between Arf6 and Kalirin5. FLAG-tagged EFA6 was co-expressed with Arf6 and HA-tagged Kalirin5 in COS7 cells and the cell lysates were immunoprecipitated with HA antibody and then immunobloted as indicated. E. Energy depletion increases the interaction between Arf6 and Kalirin5. COS7 cells co-transfected with HA-tagged Arf6 WT and FLAG-tagged Kalirin5 were incubated with 50 mM 2-deoxyglucose and 0.02 % sodium azide to deplete cellular ATP and GTP levels for 0, 15, 30, or 60 minutes prior to immunoprecipitation. For each panel, the graph summarizes data for 3 independent experiments (mean ± s.d.). Statistical comparison using one-way ANOVA. *p < 0.05 and **P < 0.01
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Arf6 recruits the Rac GEF Kalirin to the plasma membrane facilitating Rac activation-0
2011Co-Authors: Tae Hyeon Koo, Betty A. Eipper, Julie G. DonaldsonAbstract:Copyright information:Taken from "Arf6 recruits the Rac GEF Kalirin to the plasma membrane facilitating Rac activation"http://www.biomedcentral.com/1471-2121/8/29BMC Cell Biology 2007;8():29-29.Published online 18 Jul 2007PMCID:PMC1939998.DH-PH motifs. (Also depicted, Sec14 domain, oval and SH2 domain, hexagon). B. Arf6, but not Arf1 or ARF5, binds to SR region of Kalirin. COS7 cells were transfected with C-terminally HA-tagged Arf isoforms, and cell extracts were incubated with GST-KalSR4-7 or with GST. Arfs were detected by immunoblotting with anti-HA antibody. The GST fusion proteins were visualized by Coomassie staining. C. Arf6-GDP co-immunoprecipitates with Kalirin5. FLAG-tagged Kalirin5 was co-expressed with HA-tagged Arf6, Arf6Q67L, Arf6T27N or with Arf1 in COS7 cells. Lysates were immunoprecipitated with FLAG antibody and then immunobloted with HA antibody to detect Arf proteins. D. Expression of EFA6 decreases the interaction between Arf6 and Kalirin5. FLAG-tagged EFA6 was co-expressed with Arf6 and HA-tagged Kalirin5 in COS7 cells and the cell lysates were immunoprecipitated with HA antibody and then immunobloted as indicated. E. Energy depletion increases the interaction between Arf6 and Kalirin5. COS7 cells co-transfected with HA-tagged Arf6 WT and FLAG-tagged Kalirin5 were incubated with 50 mM 2-deoxyglucose and 0.02 % sodium azide to deplete cellular ATP and GTP levels for 0, 15, 30, or 60 minutes prior to immunoprecipitation. For each panel, the graph summarizes data for 3 independent experiments (mean ± s.d.). Statistical comparison using one-way ANOVA. *p < 0.05 and **P < 0.01
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active arf6 recruits arno cytohesin gefs to the pm by binding their ph domains
Molecular Biology of the Cell, 2007Co-Authors: Lee Ann Cohen, Akira Honda, Fraser D Brown, Peter Varnai, Tamas Balla, Julie G. DonaldsonAbstract:ARNO is a soluble guanine nucleotide exchange factor (GEF) for the Arf family of GTPases. Although in biochemical assays ARNO prefers Arf1 over Arf6 as a substrate, its localization in cells at the plasma membrane (PM) suggests an interaction with Arf6. In this study, we found that ARNO activated Arf1 in HeLa and COS-7 cells resulting in the recruitment of Arf1 on to dynamic PM ruffles. By contrast, Arf6 was activated less by ARNO than EFA6, a canonical Arf6 GEF. Remarkably, Arf6 in its GTP-bound form recruited ARNO to the PM and the two proteins could be immunoprecipitated. ARNO binding to Arf6 was not mediated through the catalytic Sec7 domain, but via the pleckstrin homology (PH) domain. Active Arf6 also bound the PH domain of Grp1, another ARNO family member. This interaction was direct and required both inositol phospholipids and GTP. We propose a model of sequential Arf activation at the PM whereby Arf6-GTP recruits ARNO family GEFs for further activation of other Arf isoforms.
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somatostatin receptors signal through efa6a arf6 to activate phospholipase d in clonal β cells
Journal of Biological Chemistry, 2007Co-Authors: Justin Adam Grodnitzky, Julie G. Donaldson, Nasser Syed, Michael J Kimber, Tim A Day, Walter H HsuAbstract:Abstract Somatostatin (SS) is a peptide hormone that inhibits insulin secretion in β-cells by activating its Gi/o-coupled receptors. Our previous work indicated that a βγ-dimer of Gi/o coupled to SS receptors can activate phospholipase D1 (PLD1) (Cheng, H., Grodnitzky, J. A., Yibchok-anun, S., Ding, J., and Hsu, W. H. (2005) Mol. Pharmacol. 67, 2162–2172). The aim of the present study was to elucidate the mechanisms underlying SS-induced PLD activation. We demonstrated the presence of ADP-ribosylation factor Arf1 and Arf6 in clonal β-cells, HIT-T15. We also determined that the activation of PLD1 was mediated through Arf6. Overexpression of dominant-negative (dn) Arf6 mutant, Arf6(T27N), and suppression of mRNA levels using siRNA, both abolished SS-induced PLD activation, while overexpression of wild type Arf6 further enhanced this PLD activation. In contrast, overexpression of dn-Arf1 mutant Arf1(T31N) or dn-ARF5 mutant ARF5(T31N) failed to reduce SS-induced PLD activation. These findings suggested that Arf6, but not Arf1 or ARF5, mediates the effect of SS. We further determined the involvement of the Arf6 guanine nucleotide exchange factor (GEF) EFA6A, a GEF previously thought to be found predominantly in the brain, in the activation of PLD1 in HIT-T15 cells. Using Northern and Western blot analyses, both mRNA and protein of EFA6A were found in these cells. Overexpression of dn-EFA6A mutant, EFA6A(E242K), and suppression of mRNA levels using siRNA, both abolished SS-induced PLD activation, whereas overexpression of dn-EFA6B mutant, EFA6B(E651K), failed to reduce SS-induced PLD activation. In addition, overexpression of dn-ARNO mutant, ARNO(E156K), another GEF of Arf6, had no effect on SS-induced activation of PLD. Taken together, these results suggest that SS signals through EFA6A to activate Arf6-PLD cascade.
Scott R Frank - One of the best experts on this subject based on the ideXlab platform.
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the adp ribosylation factor nucleotide exchange factor arno promotes β arrestin release necessary for luteinizing hormone choriogonadotropin receptor desensitization
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Sutapa Mukherjee, Mariefrance Bader, Richard A Kahn, Scott R Frank, James E Casanova, Vsevolod V Gurevich, Jonathan C R Jones, Evelyn T Maizels, Krzysztof Palczewski, Klaus AktoriesAbstract:Desensitization of guanine nucleotide binding protein-coupled receptors is a ubiquitous phenomenon characterized by declining effector activity upon persistent agonist stimulation. The luteinizing hormone/choriogonadotropin receptor (LH/CGR) in ovarian follicles exhibits desensitization of effector adenylyl cyclase activity in response to the mid-cycle surge of LH. We have previously shown that uncoupling of the agonist-activated LH/CGR from the stimulatory G protein (Gs) is dependent on GTP and attributable to binding of β-arrestin present in adenylyl cyclase-rich follicular membrane fraction to the third intracellular (3i) loop of the receptor. Here, we report that LH/CGR-dependent desensitization is mimicked by ADP ribosylation factor nucleotide-binding site opener, a guanine nucleotide exchange factor of the small G proteins ADP ribosylation factors (Arfs) 1 and 6, and blocked by synthetic N-terminal Arf6 peptide, suggesting that the GTP-dependent step of LH/CGR desensitization is receptor-dependent Arf6 activation. Arf activation by GTP and ADP ribosylation factor nucelotide-binding site opener promotes the release of docked β-arrestin from the membrane, making β-arrestin available for LH/CGR; Arf6 but not Arf1 peptides block β-arrestin release from the membrane. Thus, LH/CGR appears to activate two membrane delimited signaling cascades via two types of G proteins: heterotrimeric Gs and small G protein Arf6. Arf6 activation releases docked β-arrestin necessary for receptor desensitization, providing a feedback mechanism for receptor self-regulation.
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arno is a guanine nucleotide exchange factor for adp ribosylation factor 6
Journal of Biological Chemistry, 1998Co-Authors: Scott R Frank, Sunil Upender, Steen H Hansen, James E CasanovaAbstract:ADP-ribosylation factors (ARFs) constitute a family of small monomeric GTPases. ARFs 1 and 3 function in the recruitment of coat proteins to membranes of the Golgi apparatus, whereas ARF6 is localized to the plasma membrane, where it appears to modulate both the assembly of the actin cytoskeleton and endocytosis. Like other GTPases, ARF activation is facilitated by specific guanine nucleotide exchange factors (GEFs). ARNO (ARF nucleotide-binding site opener) is a member of a growing family of ARF-GEFs that share a common, tripartite structure consisting of an N-terminal coiled-coil domain, a central domain with homology to the yeast protein Sec7p, and a C-terminal pleckstrin homology domain. Recently, ARNO and its close homologue cytohesin-1 were found to catalyze in vitro nucleotide exchange on ARF1 and ARF3, respectively, raising the possibility that these GEFs function in the Golgi. However, the actual function of these proteins may be determined in part by their ability to interact with specific ARFs and in part by their subcellular localization. We report here that in vitro ARNO can stimulate nucleotide exchange on both ARF1 and ARF6. Furthermore, based on subcellular fractionation and immunolocalization experiments, we find that ARNO is localized to the plasma membrane in mammalian cells rather than the Golgi. It is therefore likely that ARNO functions in plasma membrane events by modulating the activity of ARF6 in vivo. These findings are consistent with the previous observation that cytohesin-1 regulates the adhesiveness of alphaLbeta2 integrins at the plasma membrane of lymphocytes.
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arno is a guanine nucleotide exchange factor for adp ribosylation factor 6
Journal of Biological Chemistry, 1998Co-Authors: Scott R Frank, Sunil Upender, Steen H Hansen, James E CasanovaAbstract:ADP-ribosylation factors (ARFs) constitute a family of small monomeric GTPases. ARFs 1 and 3 function in the recruitment of coat proteins to membranes of the Golgi apparatus, whereas ARF6 is localized to the plasma membrane, where it appears to modulate both the assembly of the actin cytoskeleton and endocytosis. Like other GTPases, ARF activation is facilitated by specific guanine nucleotide exchange factors (GEFs). ARNO (ARF nucleotide-binding site opener) is a member of a growing family of ARF-GEFs that share a common, tripartite structure consisting of an N-terminal coiled-coil domain, a central domain with homology to the yeast protein Sec7p, and a C-terminal pleckstrin homology domain. Recently, ARNO and its close homologue cytohesin-1 were found to catalyze in vitronucleotide exchange on ARF1 and ARF3, respectively, raising the possibility that these GEFs function in the Golgi. However, the actual function of these proteins may be determined in part by their ability to interact with specific ARFs and in part by their subcellular localization. We report here that in vitro ARNO can stimulate nucleotide exchange on both ARF1 and ARF6. Furthermore, based on subcellular fractionation and immunolocalization experiments, we find that ARNO is localized to the plasma membrane in mammalian cells rather than the Golgi. It is therefore likely that ARNO functions in plasma membrane events by modulating the activity of ARF6 in vivo. These findings are consistent with the previous observation that cytohesin-1 regulates the adhesiveness of αLβ2integrins at the plasma membrane of lymphocytes.
Vassilis Koronakis - One of the best experts on this subject based on the ideXlab platform.
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The Arf GTPase-Activating Protein Family Is Exploited by Salmonella enterica Serovar Typhimurium To Invade Nonphagocytic Host Cells
2016Co-Authors: Anthony C Davidson, Daniel Humphreys, Peter J Hume, Andrew B. E. Brooks, Vassilis KoronakisAbstract:ABSTRACT To establish intracellular infections, Salmonella bacteria trigger host cell membrane ruffling and invasion by sub-verting cellular Arf guanine nucleotide exchange factors (GEFs) that activate Arf1 and Arf6 GTPases by promoting GTP binding. A family of cellular Arf GTPase-activating proteins (GAPs) can downregulate Arf signaling by stimulating GTP hydrolysis, but whether they do this during infection is unknown. Here, we uncovered a remarkable role for distinct Arf GAP family members in Salmonella invasion. The Arf6 GAPs ACAP1 and ADAP1 and the Arf1 GAP ASAP1 localized at Salmonella-induced ruffles, which was not the case for the plasmamembrane-localized Arf6 GAPs ARAP3 and GIT1 or the Golgi-associated Arf1 GAP1. Sur-prisingly, we found that loss of ACAP1, ADAP1, or ASAP1 impaired Salmonella invasion, revealing that GAPs cannot be consid-ered mere terminators of cytoskeleton remodeling. Salmonella invasion was restored in Arf GAP-depleted cells by expressing fast-cycling Arf derivatives, demonstrating that Arf GTP/GDP cycles facilitate Salmonella invasion. Consistent with this view, both constitutively active and dominant-negative Arf derivatives that cannot undergo GTP/GDP cycles inhibited invasion. Fur-thermore, we demonstrated that Arf GEFs and GAPs colocalize at invading Salmonella and collaborate to drive Arf1-dependent pathogen invasion. This study revealed that Salmonella bacteria exploit a remarkable interplay between Arf GEFs and GAPs to direct cycles of Arf GTPase activation and inactivation. These cycles drive Salmonella cytoskeleton remodeling and enable intra-cellular infections. IMPORTANCE To initiate infections, the Salmonella bacterial pathogen remodels the mammalian actin cytoskeleton and invade
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the arf gtpase activating protein family is exploited by salmonella enterica serovar typhimurium to invade nonphagocytic host cells
Mbio, 2015Co-Authors: Anthony C Davidson, Daniel Humphreys, Peter J Hume, Andrew B. E. Brooks, Vassilis KoronakisAbstract:ABSTRACT To establish intracellular infections, Salmonella bacteria trigger host cell membrane ruffling and invasion by subverting cellular Arf guanine nucleotide exchange factors (GEFs) that activate Arf1 and Arf6 GTPases by promoting GTP binding. A family of cellular Arf GTPase-activating proteins (GAPs) can downregulate Arf signaling by stimulating GTP hydrolysis, but whether they do this during infection is unknown. Here, we uncovered a remarkable role for distinct Arf GAP family members in Salmonella invasion. The Arf6 GAPs ACAP1 and ADAP1 and the Arf1 GAP ASAP1 localized at Salmonella-induced ruffles, which was not the case for the plasma membrane-localized Arf6 GAPs ARAP3 and GIT1 or the Golgi-associated Arf1 GAP1. Surprisingly, we found that loss of ACAP1, ADAP1, or ASAP1 impaired Salmonella invasion, revealing that GAPs cannot be considered mere terminators of cytoskeleton remodeling. Salmonella invasion was restored in Arf GAP-depleted cells by expressing fast-cycling Arf derivatives, demonstrating that Arf GTP/GDP cycles facilitate Salmonella invasion. Consistent with this view, both constitutively active and dominant-negative Arf derivatives that cannot undergo GTP/GDP cycles inhibited invasion. Furthermore, we demonstrated that Arf GEFs and GAPs colocalize at invading Salmonella and collaborate to drive Arf1-dependent pathogen invasion. This study revealed that Salmonella bacteria exploit a remarkable interplay between Arf GEFs and GAPs to direct cycles of Arf GTPase activation and inactivation. These cycles drive Salmonella cytoskeleton remodeling and enable intracellular infections. IMPORTANCE To initiate infections, the Salmonella bacterial pathogen remodels the mammalian actin cytoskeleton and invades host cells by subverting host Arf GEFs that activate Arf1 and Arf6 GTPases. Cellular Arf GAPs deactivate Arf GTPases and negatively regulate cell processes, but whether they target Arfs during infection is unknown. Here, we uncovered an important role for the Arf GAP family in Salmonella invasion. Surprisingly, we found that Arf1 and Arf6 GAPs cooperate with their Arf GEF counterparts to facilitate cycles of Arf GTPase activation and inactivation, which direct pathogen invasion. This report illustrates that GAP proteins promote actin-dependent processes and are not necessarily restricted to negatively regulating cellular signaling. It uncovers a remarkable interplay between Arf GEFs and GAPs that is exploited by Salmonella to establish infection and expands our understanding of Arf GTPase-regulated cytoskeleton remodeling.
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arf6 coordinates actin assembly through the wave complex a mechanism usurped by salmonella to invade host cells
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Daniel Humphreys, Anthony C Davidson, Peter J Hume, Laura E Makin, Vassilis KoronakisAbstract:ADP ribosylation factor (Arf) 6 anchors to the plasma membrane, where it coordinates membrane trafficking and cytoskeleton remodelling, but how it assembles actin filaments is unknown. By reconstituting membrane-associated actin assembly mediated by the WASP family veroprolin homolog (WAVE) regulatory complex (WRC), we recapitulated an Arf6-driven actin polymerization pathway. We show that Arf6 is divergent from other Arf members, as it was incapable of directly recruiting WRC. We demonstrate that Arf6 triggers actin assembly at the membrane indirectly by recruiting the Arf guanine nucleotide exchange factor (GEF) ARNO that activates Arf1 to enable WRC-dependent actin assembly. The pathogen Salmonella usurped Arf6 for host cell invasion by recruiting its canonical GEFs EFA6 and BRAG2. Arf6 and its GEFs facilitated membrane ruffling and pathogen invasion via ARNO, and triggered actin assembly by generating an Arf1–WRC signaling hub at the membrane in vitro and in cells. This study reconstitutes Arf6-dependent actin assembly to reveal a mechanism by which related Arf GTPases orchestrate distinct steps in the WRC cytoskeleton remodelling pathway.
Juliette Ferlin - One of the best experts on this subject based on the ideXlab platform.
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etude de la voie de signalisation gbf1 arf au cours de la replication virale
2018Co-Authors: Juliette FerlinAbstract:Depuis une decennie, GBF1 a emerge comme etant un facteur cellulaire essentiel a la replication de plusieurs virus a ARN de polarite positive (ARN(+)), issus de differentes familles. GBF1 est un facteur d’echange nucleotidique de petites proteines G de la famille Arf, connu pour son action regulatrice des etapes precoces de la voie de secretion. En etudiant le virus de l’hepatite C (VHC) comme virus modele, nous avons montre que le role de GBF1 dans la replication virale est distinct de sa fonction regulatrice de la voie de secretion. En effet,GBF1 participe a la replication du VHC par l’intermediaire de la paire Arf4 et ARF5, alors que c’est la paire Arf1et Arf4 qui est impliquee dans la voie secretion. Pour determiner si ce mecanisme d’action est conserve parmi les virus a ARN(+), nous avons d’abord montre que GBF1 est impliquee dans l’infection par le virus de la fievre jaune (YFV), le virus sindbis (SINV), le coronavirus 229E humain (HCoV-229E) et le coxsackievirus B4(CVB4). Puis, nos resultats indiquent que les infections YFV, SINV et HCoV-229E dependent, tout comme le VHC, des proteines Arf4 et ARF5. Toutefois, le YFV et le SINV utiliseraient egalement une autre paire d’Arf,Arf1 et Arf4, lors de leur cycle viral. Par ailleurs, l’infection par le coxsackievirus B4 (CVB4) est dependante de GBF1 mais ne semble pas dependre des proteines Arf. Bien que GBF1 soit un facteur crucial pour la replication des virus ARN(+), son mecanisme d’action ne semble donc pas etre conserve.La paire Arf4-ARF5 semble impliquee dans la replication de plusieurs virus a ARN(+). Cependant, ces deux Arf ont ete tres peu etudiees, contrairement a la proteine Arf1. Nous faisons l’hypothese que la paire Arf4-ARF5 agit en regulant une serie d’effecteurs specifiques qui sont importants pour la replication virale. Nos resultats montrent que la depletion simultanee de Arf4 et de ARF5 perturbe la morphologie de l’appareil de Golgi, qui devient condense, et des gouttelettes lipidiques (GL), qui s’accumulent et grossissent en peripherie cellulaire.Toutefois, une analyse lipidomique de cellules depletees de Arf4 et ARF5 montre une composition lipidique inchangee, ce qui suggere un effet sur la morphologie des GL et non pas sur le metabolisme des lipides. Une analyse transcriptomique nous a permis de mettre en evidence une serie de proteines, dont l’expression est modulee, suite a la depletion de Arf4 et ARF5. Nous avons evalue l’implication potentielle de certaines d’entre elles dans l’infection du VHC, mais aucune ne s’est revelee importante pour ce virus. En conclusion, nos resultats ont permis de mettre en evidence de nouvelles fonctions de GBF1, mediees par la paire de proteines Arf4 et ARF5. Arf4 et ARF5 sont impliquees dans la regulation de la morphologie de l’appareil de Golgi et des GL ainsi que dans la replication de plusieurs virus a ARN(+). Il reste a evaluer si ces fonctions sont independantes les unes des autres ou liees entre elles et quels effecteurs specifiques elles font intervenir.
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Investigation of the role of GBF1 in the replication of positive-sense single-stranded RNA viruses
Journal of General Virology, 2018Co-Authors: Juliette Ferlin, Rayan Farhat, Sandrine Belouzard, Laurence Cocquerel, Antoine Bertin, Didier Hober, Jean Dubuisson, Yves RouilléAbstract:GBF1 has emerged as a host factor required for the replication of positive-sense single-stranded RNA viruses of different families, but its mechanism of action is still unknown. GBF1 is a guanine nucleotide exchange factor for Arf family members. Recently, we identified Arf4 and ARF5 (class II Arfs) as host factors required for the replication of hepatitis C virus (HCV), a GBF1-dependent virus. To assess whether a GBF1/class II Arf pathway is conserved among positive-sense single-stranded RNA viruses, we investigated yellow fever virus (YFV), Sindbis virus (SINV), coxsackievirus B4 (CVB4) and human coronavirus 229E (HCoV-229E). We found that GBF1 is involved in the replication of these viruses. However, using siRNA or CRISPR-Cas9 technologies, it was seen that the depletion of Arf1, Arf3, Arf4 or ARF5 had no impact on viral replication. In contrast, the depletion of Arf pairs suggested that class II Arfs could be involved in HCoV-229E, YFV and SINV infection, as for HCV, but not in CVB4 infection. In addition, another Arf pair, Arf1 and Arf4, appears to be essential for YFV and SINV infection, but not for infection by other viruses. Finally, CVB4 infection was not inhibited by any combination of Arf depletion. We conclude that the mechanism of action of GBF1 in viral replication appears not to be conserved, and that a subset of positive-sense single-stranded RNA viruses from different families might require class II Arfs for their replication.