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Audrey Claing - One of the best experts on this subject based on the ideXlab platform.
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ARF1 regulates adhesion of mda mb 231 invasive breast cancer cells through formation of focal adhesions
Cellular Signalling, 2015Co-Authors: S Schlienger, Rodrigo Alain Migueles Ramirez, Audrey ClaingAbstract:Adhesion complex formation and disassembly is crucial for maintaining efficient cell movement. During migration, several proteins act in concert to promote remodeling of the actin cytoskeleton and we have previously shown that in highly invasive breast cancer cells, this process is regulated by small GTP-binding proteins of the ADP-ribosylation factor (ARF) family. These are overexpressed and highly activated in these cells. Here, we report that one mechanism by which ARF1 regulates migration is by controlling assembly of focal adhesions. In cells depleted of ARF1, paxillin is no longer colocalized with actin at focal adhesion sites. In addition, we demonstrate that this occurs through the ability of ARF1 to regulate the recruitment of key proteins such as paxillin, talin and FAK to s1-integrin. Furthermore, we show that the interactions between paxillin and talin together and with FAK are significantly impaired in ARF1 knocked down cells. Our findings also indicate that ARF1 is essential for EGF-mediated phosphorylation of FAK and Src. Finally, we report that ARF1 can be found in complex with key focal adhesion proteins such as s1-integrin, paxillin, talin and FAK. Together our findings uncover a new mechanism by which ARF1 regulates cell migration and provide this GTPase as a target for the development of new therapeutics in triple negative breast cancer.
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the adaptor proteins p66shc and grb2 regulate the activation of the gtpases ARF1 and arf6 in invasive breast cancer cells
Journal of Biological Chemistry, 2014Co-Authors: Eric Haines, Caroline Saucier, Audrey ClaingAbstract:Signals downstream of growth factor receptors play an important role in mammary carcinogenesis. Recently, we demonstrated that the small GTPases ARF1 and ARF6 were shown to be activated downstream of the epidermal growth factor receptor (EGFR) and act as a key regulator of growth, migration, and invasion of breast cancer cells. However, the mechanism via which the EGFR recruits and activates ARF1 and ARF6 to transmit signals has yet to be fully elucidated. Here, we identify adaptor proteins Grb2 and p66Shc as important regulators mediating ARF activation. We demonstrate that ARF1 can be found in complex with Grb2 and p66Shc upon EGF stimulation of the basal-like breast cancer MDA-MB-231 cell line. However, we report that these two adaptors regulate ARF1 activation differently, with Grb2 promoting ARF1 activation and p66Shc blocking this response. Furthermore, we show that Grb2 is essential for the recruitment of ARF1 to the EGFR, whereas p66Shc hindered ARF1 receptor recruitment. We demonstrate that the negative regulatory role of p66Shc stemmed from its ability to block the recruitment of Grb2/ARF1 to the EGFR. Conversely, p66Shc potentiates ARF6 activation as well as the recruitment of this ARF isoform to the EGFR. Interestingly, we demonstrate that Grb2 is also required for the activation and receptor recruitment of ARF6. Additionally, we show an important role for p66Shc in modulating ARF activation, cell growth, and migration in HER2-positive breast cancer cells. Together, our results highlight a central role for adaptor proteins p66Shc and Grb2 in the regulation of ARF1 and ARF6 activation in invasive breast cancer cells.
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ARF1 regulates the rho mlc pathway to control egf dependent breast cancer cell invasion
Molecular Biology of the Cell, 2014Co-Authors: S Schlienger, Shirley Campbell, Audrey ClaingAbstract:Invasion of tumor cells is a key step in metastasis that depends largely on the ability of these cells to degrade the extracellular matrix. Although we have showed that the GTPase ADP-ribosylation factor 1 (ARF1) is overexpressed in highly invasive breast cancer cell lines and that epidermal growth factor stimulation can activate this ARF isoform to regulate migration as well as proliferation, the role of this small GTP-binding protein has not been addressed in the context of invasiveness. Here we report that modulation of ARF1 expression and activity markedly impaired the ability of M.D. Anderson-metastatic breast-231 cells, a prototypical highly invasive breast cancer cell line, to degrade the extracellular matrix by controlling metalloproteinase-9 activity. In addition, we demonstrate that this occurs through inhibition of invadopodia maturation and shedding of membrane-derived microvesicles, the two key structures involved in invasion. To further define the molecular mechanisms by which ARF1 controls invasiveness, we show that ARF1 acts to modulate RhoA and RhoC activity, which in turn affects myosin light-chain (MLC) phosphorylation. Together our findings underscore for the first time a key role for ARF1 in invasion of breast cancer cells and suggest that targeting the ARF/Rho/MLC signaling axis might be a promising strategy to inhibit invasiveness and metastasis.
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ARF1 controls rac1 signaling to regulate migration of mda mb 231 invasive breast cancer cells
Cellular Signalling, 2013Co-Authors: Sebastian Lewissaravalli, Shirley Campbell, Audrey ClaingAbstract:ADP-ribosylation factors (ARFs) are monomeric G proteins that regulate many cellular processes such as reorganization of the actin cytoskeleton. We have previously shown that ARF1 is overexpressed in highly invasive breast cancer cells and contribute to their enhanced migration. In this study, we propose to define the molecular mechanism by which ARF1 regulates this complex cellular response by investigating the role of this ARF GTPase on the activation process of Rac1, a Rho GTPase, associated with lamellipodia formation during cell migration. Here, we first show that inhibition of ARF1 or Rac1 expression markedly impacts the ability of MDA-MB-231 cells to migrate upon EGF stimulation. However, the effect of ARF1 depletion can be reversed by overexpression of the Rac1 active mutant, Rac1 Q(61)L. Depletion of ARF1 also impairs the ability of EGF stimulation to promote GTP-loading of Rac1. To further investigate the possible cross-talk between ARF1 and Rac1, we next examined whether they could form a complex. We observed that the two GTPases could directly interact independently of the nature of the nucleotide bound to them. EGF treatment however resulted in the association of Rac1 with its effector IRSp53, which was completely abrogated in ARF1 depleted cells. We present evidences that this ARF isoform is responsible for the plasma membrane targeting of both Rac1 and IRSp53, a step essential for lamellipodia formation. In conclusion, this study provides a new mechanism by which ARF1 regulates cell migration and identifies this GTPase as a promising pharmacological target to reduce metastasis formation in breast cancer patients.
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adp ribosylation factor 1 controls the activation of the phosphatidylinositol 3 kinase pathway to regulate epidermal growth factor dependent growth and migration of breast cancer cells
Journal of Biological Chemistry, 2008Co-Authors: Pierreluc Boulay, Mathieu Cotton, Paul Melancon, Audrey ClaingAbstract:Activation of intracellular signaling pathways by growth factors is one of the major causes of cancer development and progression. Recent studies have demonstrated that monomeric G proteins of the Ras family are key regulators of cell proliferation, migration, and invasion. Using an invasive breast cancer cell lines, we demonstrate that the ADP-ribosylation factor 1 (ARF1), a small GTPase classically associated with the Golgi, is an important regulator of the biological effects induced by epidermal growth factor. Here, we show that this ARF isoform is activated following epidermal growth factor stimulation and that, in MDA-MB-231 cells, ARF1 is found in dynamic plasma membrane ruffles. Inhibition of endogenous ARF1 expression results in the inhibition of breast cancer cell migration and proliferation. The underlying mechanism involves the activation of the phosphatidylinositol 3-kinase pathway. Our data demonstrate that depletion of ARF1 markedly impairs the recruitment of the phosphatidylinositol 3-kinase catalytic subunit (p110alpha) to the plasma membrane, and the association of the regulatory subunit (p85alpha) to the activated receptor. These results uncover a novel molecular mechanism by which ARF1 regulates breast cancer cell growth and invasion during cancer progression.
Bruno Antonny - One of the best experts on this subject based on the ideXlab platform.
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arfgap1 generates an ARF1 gradient on continuous lipid membranes displaying flat and curved regions
The EMBO Journal, 2010Co-Authors: Ernesto E Ambroggio, Benoit Sorre, Patricia Bassereau, Bruno Goud, Jeanbaptiste Manneville, Bruno AntonnyAbstract:ArfGAP1, which promotes GTP hydrolysis on the small G protein ARF1 on Golgi membranes, interacts preferentially with positively curved membranes through its amphipathic lipid packing sensor (ALPS) motifs. This should influence the distribution of ARF1-GTP when flat and curved regions coexist on a continuous membrane, notably during COPI vesicle budding. To test this, we pulled tubes from giant vesicles using molecular motors or optical tweezers. ARF1-GTP distributed on the giant vesicles and on the tubes, whereas ArfGAP1 bound exclusively to the tubes. Decreasing the tube radius revealed a threshold of R≈35 nm for the binding of ArfGAP1 ALPS motifs. Mixing catalytic amounts of ArfGAP1 with ARF1-GTP induced a smooth ARF1 gradient along the tube. This reflects that ARF1 molecules leaving the tube on GTP hydrolysis are replaced by new ARF1-GTP molecules diffusing from the giant vesicle. The characteristic length of the gradient is two orders of magnitude larger than a COPI bud, suggesting that ARF1-GTP diffusion can readily compensate for the localized loss of ARF1 during budding and contribute to the stability of the coat until fission.
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lipid packing sensed by arfgap1 couples copi coat disassembly to membrane bilayer curvature
Nature, 2003Co-Authors: Joelle Bigay, Pierre Gounon, Sylviane Robineau, Bruno AntonnyAbstract:Protein coats deform flat lipid membranes into buds and capture membrane proteins to form transport vesicles1,2,3. The assembly/disassembly cycle of the COPI coat on Golgi membranes is coupled to the GTP/GDP cycle of the small G protein ARF1. At the heart of this coupling is the specific interaction of membrane-bound ARF1–GTP with coatomer, a complex of seven proteins that forms the building unit of the COPI coat4,5,6,7. Although COPI coat disassembly requires the catalysis of GTP hydrolysis in ARF1 by a specific GTPase-activating protein (ArfGAP1)8,9,10, the precise timing of this reaction during COPI vesicle formation is not known. Using time-resolved assays for COPI dynamics on liposomes of controlled size, we show that the rate of ArfGAP1-catalysed GTP hydrolysis in ARF1 and the rate of COPI disassembly increase over two orders of magnitude as the curvature of the lipid bilayer increases and approaches that of a typical transport vesicle. This leads to a model for COPI dynamics in which GTP hydrolysis in ARF1 is organized temporally and spatially according to the changes in lipid packing induced by the coat.
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binding site of brefeldin a at the interface between the small g protein adp ribosylation factor 1 ARF1 and the nucleotide exchange factor sec7 domain
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Sylviane Robineau, Marc Chabre, Bruno AntonnyAbstract:Sec7 domains (Sec7d) catalyze the exchange of guanine nucleotide on ARFs. Recent studies indicated that brefeldin A (BFA) inhibits Sec7d-catalyzed nucleotide exchange on ARF1 in an uncompetitive manner by trapping an early intermediate of the reaction: a complex between GDP-bound ARF1 and Sec7d. Using 3H-labeled BFA, we show that BFA binds to neither isolated Sec7d nor isolated ARF1–GDP, but binds to the transitory Sec7d–ARF1–GDP complex and stabilizes it. Two pairs of residues at positions 190–191 and 198–208 (Arno numbering) in Sec7d contribute equally to the stability of BFA binding, which is also sensitive to mutation of H80 in ARF1. The catalytic glutamic (E156) residue of Sec7d is not necessary for BFA binding. In contrast, BFA does not bind to the intermediate catalytic complex between nucleotide-free ARF1 and Sec7d. These results suggest that, on initial docking steps between ARF1–GDP and Sec7d, BFA inserts like a wedge between the switch II region of ARF1–GDP and a surface encompassing residues 190–208, at the border of the characteristic hydrophobic groove of Sec7d. Bound BFA would prevent the switch regions of ARF1–GDP from reorganizing and forming tighter contacts with Sec7d and thereby would maintain the bound GDP of ARF1 at a distance from the catalytic glutamic finger of Sec7d.
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a glutamic finger in the guanine nucleotide exchange factor arno displaces mg2 and the beta phosphate to destabilize gdp on ARF1
The EMBO Journal, 1998Co-Authors: Sophie Berauddufour, Marc Chabre, Sylviane Robineau, Jacqueline Cherfils, Pierre Chardin, Sonia Paris, Bruno AntonnyAbstract:The Sec7 domain of the guanine nucleotide exchange factor ARNO (ARNO-Sec7) is responsible for the exchange activity on the small GTP-binding protein ARF1. ARNO-Sec7 forms a stable complex with the nucleotide-free form of [Delta17]ARF1, a soluble truncated form of ARF1. The crystal structure of ARNO-Sec7 has been solved recently, and a site-directed mutagenesis approach identified a hydrophobic groove and an adjacent hydrophilic loop as the ARF1-binding site. We show that Glu156 in the hydrophilic loop of ARNO-Sec7 is involved in the destabilization of Mg2+ and GDP from ARF1. The conservative mutation E156D and the charge reversal mutation E156K reduce the exchange activity of ARNO-Sec7 by several orders of magnitude. Moreover, [E156K]ARNO-Sec7 forms a complex with the Mg2+-free form of [Delta17]ARF1-GDP without inducing the release of GDP. Other mutations in ARNO-Sec7 and in [Delta17]ARF1 suggest that prominent hydrophobic residues of the switch I region of ARF1 insert into the groove of the Sec7 domain, and that Lys73 of the switch II region of ARF1 forms an ion pair with Asp183 of ARNO-Sec7.
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role of protein phospholipid interactions in the activation of ARF1 by the guanine nucleotide exchange factor arno
Journal of Biological Chemistry, 1997Co-Authors: Sonia Paris, Marc Chabre, Joelle Bigay, Sylviane Robineau, Bruno Antonny, Sophie Berauddufour, Pierre ChardinAbstract:Arno is a 47-kDa human protein recently identified as a guanine nucleotide exchange factor for ADP ribosylation factor 1 (ARF1) with a central Sec7 domain responsible for the exchange activity and a carboxyl-terminal pleckstrin homology (PH) domain (Chardin, P., Paris, S., Antonny, B., Robineau, S., Beraud-Dufour, S., Jackson, C. L., and Chabre, M. (1996)Nature 384, 481–484). Binding of the PH domain to phosphatidylinositol 4,5-bisphosphate (PIP2) greatly enhances Arno-mediated activation of myristoylated ARF1. We show here that in the absence of phospholipids, Arno promotes nucleotide exchange on [Δ17]ARF1, a soluble mutant of ARF1 lacking the first 17 amino acids. This reaction is unaffected by PIP2, which suggests that the PIP2-PH domain interaction does not directly regulate the catalytic activity of Arno but rather serves to recruit Arno to membranes. Arno catalyzes the release of GDP more efficiently than that of GTP from [Δ17]ARF1, and a stable complex between Arno Sec7 domain and nucleotide-free [Δ17]ARF1 can be isolated. In contrast to [Δ17]ARF1, full-length unmyristoylated ARF1 is not readily activated by Arno in solution. Its activation requires the presence of phospholipids and a reduction of ionic strength and Mg2+ concentration. PIP2 is strongly stimulatory, indicating that binding of Arno to phospholipids is involved, but in addition, electrostatic interactions between phospholipids and the amino-terminal portion of unmyristoylated ARF1GDP seem to be important. We conclude that efficient activation of full-length ARF1 by Arno requires a membrane surface and two distinct protein-phospholipid interactions: one between the PH domain of Arno and PIP2, and the other between amino-terminal cationic residues of ARF1 and anionic phospholipids. The latter interaction is normally induced by insertion of the amino-terminal myristate into the bilayer but can also be artificially facilitated by decreasing Mg2+ and salt concentrations.
Marc Chabre - One of the best experts on this subject based on the ideXlab platform.
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Specificities for the Small G Proteins ARF1 and ARF6 of the Guanine Nucleotide Exchange Factors ARNO and EFA6
Journal of Biological Chemistry, 2001Co-Authors: Eric Macia, Marc Chabre, Michel FrancoAbstract:ARF1 and ARF6 are distant members of the ADP-ribosylation factor (ARF) small G-protein subfamily. Their distinct cellular functions must result from specificity of interaction with different effectors and regulators, including guanine nucleotide exchange factors (GEFs). ARF nucleotide-binding site opener (ARNO), and EFA6 are analogous ARF-GEFs, both comprising a catalytic "Sec7" domain and a pleckstrin homology domain. In vivo ARNO, like ARF1, is mostly cytosolic, with minor localizations at the Golgi and plasma membrane; EFA6, like ARF6, is restricted to the plasma membrane. However, depending on conditions, ARNO appears active on ARF6 as well as on ARF1. Here we analyze the origin of these ARF-GEF selectivities. In vitro, in the presence of phospholipid membranes, ARNO activates ARF1 preferentially and ARF6 slightly, whereas EFA6 activates ARF6 exclusively; the stimulation efficiency of EFA6 on ARF6 is comparable with that of ARNO on ARF1. These selectivities are determined by the GEFs Sec7 domains alone, without the pleckstrin homology and N-terminal domains, and by the ARF core domains, without the myristoylated N-terminal helix; they are not modified upon permutation between ARF1 and ARF6 of the few amino acids that differ within the switch regions. Thus selectivity for ARF1 or ARF6 must depend on subtle folding differences between the ARFs switch regions that interact with the Sec7 domains.
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binding site of brefeldin a at the interface between the small g protein adp ribosylation factor 1 ARF1 and the nucleotide exchange factor sec7 domain
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Sylviane Robineau, Marc Chabre, Bruno AntonnyAbstract:Sec7 domains (Sec7d) catalyze the exchange of guanine nucleotide on ARFs. Recent studies indicated that brefeldin A (BFA) inhibits Sec7d-catalyzed nucleotide exchange on ARF1 in an uncompetitive manner by trapping an early intermediate of the reaction: a complex between GDP-bound ARF1 and Sec7d. Using 3H-labeled BFA, we show that BFA binds to neither isolated Sec7d nor isolated ARF1–GDP, but binds to the transitory Sec7d–ARF1–GDP complex and stabilizes it. Two pairs of residues at positions 190–191 and 198–208 (Arno numbering) in Sec7d contribute equally to the stability of BFA binding, which is also sensitive to mutation of H80 in ARF1. The catalytic glutamic (E156) residue of Sec7d is not necessary for BFA binding. In contrast, BFA does not bind to the intermediate catalytic complex between nucleotide-free ARF1 and Sec7d. These results suggest that, on initial docking steps between ARF1–GDP and Sec7d, BFA inserts like a wedge between the switch II region of ARF1–GDP and a surface encompassing residues 190–208, at the border of the characteristic hydrophobic groove of Sec7d. Bound BFA would prevent the switch regions of ARF1–GDP from reorganizing and forming tighter contacts with Sec7d and thereby would maintain the bound GDP of ARF1 at a distance from the catalytic glutamic finger of Sec7d.
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a glutamic finger in the guanine nucleotide exchange factor arno displaces mg2 and the beta phosphate to destabilize gdp on ARF1
The EMBO Journal, 1998Co-Authors: Sophie Berauddufour, Marc Chabre, Sylviane Robineau, Jacqueline Cherfils, Pierre Chardin, Sonia Paris, Bruno AntonnyAbstract:The Sec7 domain of the guanine nucleotide exchange factor ARNO (ARNO-Sec7) is responsible for the exchange activity on the small GTP-binding protein ARF1. ARNO-Sec7 forms a stable complex with the nucleotide-free form of [Delta17]ARF1, a soluble truncated form of ARF1. The crystal structure of ARNO-Sec7 has been solved recently, and a site-directed mutagenesis approach identified a hydrophobic groove and an adjacent hydrophilic loop as the ARF1-binding site. We show that Glu156 in the hydrophilic loop of ARNO-Sec7 is involved in the destabilization of Mg2+ and GDP from ARF1. The conservative mutation E156D and the charge reversal mutation E156K reduce the exchange activity of ARNO-Sec7 by several orders of magnitude. Moreover, [E156K]ARNO-Sec7 forms a complex with the Mg2+-free form of [Delta17]ARF1-GDP without inducing the release of GDP. Other mutations in ARNO-Sec7 and in [Delta17]ARF1 suggest that prominent hydrophobic residues of the switch I region of ARF1 insert into the groove of the Sec7 domain, and that Lys73 of the switch II region of ARF1 forms an ion pair with Asp183 of ARNO-Sec7.
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role of protein phospholipid interactions in the activation of ARF1 by the guanine nucleotide exchange factor arno
Journal of Biological Chemistry, 1997Co-Authors: Sonia Paris, Marc Chabre, Joelle Bigay, Sylviane Robineau, Bruno Antonny, Sophie Berauddufour, Pierre ChardinAbstract:Arno is a 47-kDa human protein recently identified as a guanine nucleotide exchange factor for ADP ribosylation factor 1 (ARF1) with a central Sec7 domain responsible for the exchange activity and a carboxyl-terminal pleckstrin homology (PH) domain (Chardin, P., Paris, S., Antonny, B., Robineau, S., Beraud-Dufour, S., Jackson, C. L., and Chabre, M. (1996)Nature 384, 481–484). Binding of the PH domain to phosphatidylinositol 4,5-bisphosphate (PIP2) greatly enhances Arno-mediated activation of myristoylated ARF1. We show here that in the absence of phospholipids, Arno promotes nucleotide exchange on [Δ17]ARF1, a soluble mutant of ARF1 lacking the first 17 amino acids. This reaction is unaffected by PIP2, which suggests that the PIP2-PH domain interaction does not directly regulate the catalytic activity of Arno but rather serves to recruit Arno to membranes. Arno catalyzes the release of GDP more efficiently than that of GTP from [Δ17]ARF1, and a stable complex between Arno Sec7 domain and nucleotide-free [Δ17]ARF1 can be isolated. In contrast to [Δ17]ARF1, full-length unmyristoylated ARF1 is not readily activated by Arno in solution. Its activation requires the presence of phospholipids and a reduction of ionic strength and Mg2+ concentration. PIP2 is strongly stimulatory, indicating that binding of Arno to phospholipids is involved, but in addition, electrostatic interactions between phospholipids and the amino-terminal portion of unmyristoylated ARF1GDP seem to be important. We conclude that efficient activation of full-length ARF1 by Arno requires a membrane surface and two distinct protein-phospholipid interactions: one between the PH domain of Arno and PIP2, and the other between amino-terminal cationic residues of ARF1 and anionic phospholipids. The latter interaction is normally induced by insertion of the amino-terminal myristate into the bilayer but can also be artificially facilitated by decreasing Mg2+ and salt concentrations.
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myristoylation facilitated binding of the g protein ARF1gdp to membrane phospholipids is required for its activation by a soluble nucleotide exchange factor
Journal of Biological Chemistry, 1996Co-Authors: Michel Franco, Marc Chabre, Pierre Chardin, Sonia ParisAbstract:Abstract We have investigated the role of N-myristoylation in the activation of bovine ADP-ribosylation factor 1 (ARF1). We previously showed that myristoylation allows some spontaneous GDP-to-GTP exchange to occur on ARF1 at physiological Mg levels in the presence of phospholipid vesicles (Franco, M., Chardin, P., Chabre, M., and Paris, S.(1995) J. Biol. Chem. 270, 1337-1341). Here, we report that this basal nucleotide exchange can be accelerated (by up to 5-fold) by addition of a soluble fraction obtained from bovine retinas. This acceleration is totally abolished by brefeldin A (IC = 2 μM) and by trypsin treatment of the retinal extract, as expected for an ARF-specific guanine nucleotide exchange factor. To accelerate GDP release from ARF1, this soluble exchange factor absolutely requires myristoylation of ARF1 and the presence of phospholipid vesicles. The retinal extract also stimulates guanosine 5′-3-O-(thio)triphosphate (GTPS) release from ARF1 in the presence of phospholipids, but in this case myristoylation of ARF is not required. These observations, together with our previous findings that both myristoylated and nonmyristoylated forms of ARF but only the myristoylated form of ARF bind to membrane phospholipids, suggest that (i) the retinal exchange factor acts only on membrane-bound ARF, (ii) the myristate is not involved in the protein-protein interaction between ARF1 and the exchange factor, and (iii) N-myristoylation facilitates both spontaneous and catalyzed GDP-to-GTP exchange on ARF1 simply by facilitating the binding of ARF to membrane phospholipids.
Shamshad Cockcroft - One of the best experts on this subject based on the ideXlab platform.
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type i phosphatidylinositol 4 phosphate 5 kinase directly interacts with adp ribosylation factor 1 and is responsible for phosphatidylinositol 4 5 bisphosphate synthesis in the golgi compartment
Journal of Biological Chemistry, 2000Co-Authors: David H Jones, James B Morris, Clive P Morgan, Hisatake Kondo, Robin F Irvine, Shamshad CockcroftAbstract:Abstract Phosphatidylinositol (PtdIns) 4,5-bisphosphate is involved in many aspects of membrane traffic, but the regulation of its synthesis is only partially understood. Golgi membranes contain PI 4-kinase activity and a pool of phosphatidylinositol phosphate (PIP), which is further increased by ADP-ribosylation factor 1 (ARF1). COS7 cells were transfected with α and β forms of PI 4-kinase, and only membranes from COS7 cells transfected with PI 4-kinase β increased their content of PIP when incubated with ARF1. PtdIns(4,5)P2 content in Golgi membranes was nonexistent but could be increased to a small extent upon adding either cytosol or Type I or Type II PIP kinases. However, when ARF1 was present, PtdIns(4,5)P2 levels increased dramatically when membranes were incubated in the presence of cytosol or Type I, but not Type II, PIP kinase. To examine whether ARF1 could directly activate Type I PIP 5-kinase, we used an in vitro assay consisting of phosphatidycholine-containing liposomes, ARF1, and PIP 5-kinase. ARF1 increased Type I PIP 5-kinase activity in a guanine nucleotide-dependent manner, identifying this enzyme as a direct effector for ARF1.
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activation of exocytosis by cross linking of the ige receptor is dependent on adp ribosylation factor 1 regulated phospholipase d in rbl 2h3 mast cells evidence that the mechanism of activation is via regulation of phosphatidylinositol 4 5 bisphosphate synthesis
Biochemical Journal, 2000Co-Authors: Gemma Way, Niamh Oluanaigh, Shamshad CockcroftAbstract:The physiological stimulus to exocytosis in mast cells is the cross-linking of the high-affinity IgE receptor, FcepsilonR1, with antigen. We demonstrate a novel function for ADP-ribosylation factor 1 (ARF1) in the regulation of antigen-stimulated secretion using cytosol-depleted RBL-2H3 mast cells for reconstitution of secretory responses. When antigen is used as the stimulus, ARF1 also reconstitutes phospholipase D activation. Using ethanol to divert the phosphatidic acid (the product of phospholipase D activity) to phosphatidylethanol causes inhibition of ARF1-reconstituted secretion. In addition. ARF1 causes an increase in phosphatidylinositol 4,5-bisphosphate (PIP(2)) levels at the expense of phosphatidylinositol 4-monophosphate. The requirement for PIP(2) in exocytosis was confirmed by using phosphatidylinositol transfer protein (PITPalpha) to increase PIP(2) levels. Exocytosis, restored by either ARF1 or PITPalpha, was inhibited when PIP(2) levels were depleted by phospholipase Cdelta1. We conclude that the function of ARF1 and PITPalpha is to increase the local synthesis of PIP(2), the function of which in exocytosis is likely to be linked to lipid-protein interactions, whereby recruitment of key components of the exocytotic machinery are targeted to the appropriate membrane compartment.
Pierre Chardin - One of the best experts on this subject based on the ideXlab platform.
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Structure-based discovery of an inhibitor of Arf activation by Sec7 domains through targeting of protein-protein complexes.
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Julien Viaud, Jeanchristophe Zeeh, Mahel Zeghouf, Jacqueline Cherfils, Bernard Guibert, Hélène Barelli, André Padilla, Pierre Chardin, Catherine A Royer, Alain ChavanieuAbstract:Small molecules that produce nonfunctional protein-protein complexes are an alternative to competitive inhibitors for the inhibition of protein functions. Here we target the activation of the small GTP-binding protein ARF1, a major regulator of membrane traffic, by the Sec7 catalytic domain of its guanine nucleotide exchange factor ARNO. The crystal structure of the ARF1-GDP/ARNO complex, which initiates the exchange reaction, was used to discover an inhibitor, LM11, using in silico screening of a flexible pocket near the ARF1/ARNO interface. Using fluorescence kinetics and anisotropy, NMR spectroscopy and mutagenesis, we show that LM11 acts following a noncompetitive mechanism in which the inhibitor targets both ARF1-GDP and the ARF1-GDP/ARNO complex and produces a nonfunctional Arf-GDP/ARNO complex whose affinity is similar to that of the native complex. In addition, LM11 recognizes features of both Arf and ARNO near the Arf/Sec7 interface, a characteristic reminiscent of the paradigm interfacial inhibitor Brefeldin A. We then show that LM11 is a cell-active inhibitor that impairs Arf-dependent trafficking structures at the Golgi. Furthermore, LM11 inhibits ARNO-dependent migration of Madin-Darby canine kidney (MDCK) cells, demonstrating that ARNO is a target of LM11 in cells. Remarkably, LM11 inhibits the activation of ARF1 but not Arf6 in vitro, pointing to a possible synergy between ARF1 and Arf6 activation by ARNO in cell migration. Our design method shows that flexible regions in protein-protein complexes provide drugable sites with the potential to develop novel tools for investigating and inhibiting signaling pathways.
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a glutamic finger in the guanine nucleotide exchange factor arno displaces mg2 and the beta phosphate to destabilize gdp on ARF1
The EMBO Journal, 1998Co-Authors: Sophie Berauddufour, Marc Chabre, Sylviane Robineau, Jacqueline Cherfils, Pierre Chardin, Sonia Paris, Bruno AntonnyAbstract:The Sec7 domain of the guanine nucleotide exchange factor ARNO (ARNO-Sec7) is responsible for the exchange activity on the small GTP-binding protein ARF1. ARNO-Sec7 forms a stable complex with the nucleotide-free form of [Delta17]ARF1, a soluble truncated form of ARF1. The crystal structure of ARNO-Sec7 has been solved recently, and a site-directed mutagenesis approach identified a hydrophobic groove and an adjacent hydrophilic loop as the ARF1-binding site. We show that Glu156 in the hydrophilic loop of ARNO-Sec7 is involved in the destabilization of Mg2+ and GDP from ARF1. The conservative mutation E156D and the charge reversal mutation E156K reduce the exchange activity of ARNO-Sec7 by several orders of magnitude. Moreover, [E156K]ARNO-Sec7 forms a complex with the Mg2+-free form of [Delta17]ARF1-GDP without inducing the release of GDP. Other mutations in ARNO-Sec7 and in [Delta17]ARF1 suggest that prominent hydrophobic residues of the switch I region of ARF1 insert into the groove of the Sec7 domain, and that Lys73 of the switch II region of ARF1 forms an ion pair with Asp183 of ARNO-Sec7.
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role of protein phospholipid interactions in the activation of ARF1 by the guanine nucleotide exchange factor arno
Journal of Biological Chemistry, 1997Co-Authors: Sonia Paris, Marc Chabre, Joelle Bigay, Sylviane Robineau, Bruno Antonny, Sophie Berauddufour, Pierre ChardinAbstract:Arno is a 47-kDa human protein recently identified as a guanine nucleotide exchange factor for ADP ribosylation factor 1 (ARF1) with a central Sec7 domain responsible for the exchange activity and a carboxyl-terminal pleckstrin homology (PH) domain (Chardin, P., Paris, S., Antonny, B., Robineau, S., Beraud-Dufour, S., Jackson, C. L., and Chabre, M. (1996)Nature 384, 481–484). Binding of the PH domain to phosphatidylinositol 4,5-bisphosphate (PIP2) greatly enhances Arno-mediated activation of myristoylated ARF1. We show here that in the absence of phospholipids, Arno promotes nucleotide exchange on [Δ17]ARF1, a soluble mutant of ARF1 lacking the first 17 amino acids. This reaction is unaffected by PIP2, which suggests that the PIP2-PH domain interaction does not directly regulate the catalytic activity of Arno but rather serves to recruit Arno to membranes. Arno catalyzes the release of GDP more efficiently than that of GTP from [Δ17]ARF1, and a stable complex between Arno Sec7 domain and nucleotide-free [Δ17]ARF1 can be isolated. In contrast to [Δ17]ARF1, full-length unmyristoylated ARF1 is not readily activated by Arno in solution. Its activation requires the presence of phospholipids and a reduction of ionic strength and Mg2+ concentration. PIP2 is strongly stimulatory, indicating that binding of Arno to phospholipids is involved, but in addition, electrostatic interactions between phospholipids and the amino-terminal portion of unmyristoylated ARF1GDP seem to be important. We conclude that efficient activation of full-length ARF1 by Arno requires a membrane surface and two distinct protein-phospholipid interactions: one between the PH domain of Arno and PIP2, and the other between amino-terminal cationic residues of ARF1 and anionic phospholipids. The latter interaction is normally induced by insertion of the amino-terminal myristate into the bilayer but can also be artificially facilitated by decreasing Mg2+ and salt concentrations.
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myristoylation facilitated binding of the g protein ARF1gdp to membrane phospholipids is required for its activation by a soluble nucleotide exchange factor
Journal of Biological Chemistry, 1996Co-Authors: Michel Franco, Marc Chabre, Pierre Chardin, Sonia ParisAbstract:Abstract We have investigated the role of N-myristoylation in the activation of bovine ADP-ribosylation factor 1 (ARF1). We previously showed that myristoylation allows some spontaneous GDP-to-GTP exchange to occur on ARF1 at physiological Mg levels in the presence of phospholipid vesicles (Franco, M., Chardin, P., Chabre, M., and Paris, S.(1995) J. Biol. Chem. 270, 1337-1341). Here, we report that this basal nucleotide exchange can be accelerated (by up to 5-fold) by addition of a soluble fraction obtained from bovine retinas. This acceleration is totally abolished by brefeldin A (IC = 2 μM) and by trypsin treatment of the retinal extract, as expected for an ARF-specific guanine nucleotide exchange factor. To accelerate GDP release from ARF1, this soluble exchange factor absolutely requires myristoylation of ARF1 and the presence of phospholipid vesicles. The retinal extract also stimulates guanosine 5′-3-O-(thio)triphosphate (GTPS) release from ARF1 in the presence of phospholipids, but in this case myristoylation of ARF is not required. These observations, together with our previous findings that both myristoylated and nonmyristoylated forms of ARF but only the myristoylated form of ARF bind to membrane phospholipids, suggest that (i) the retinal exchange factor acts only on membrane-bound ARF, (ii) the myristate is not involved in the protein-protein interaction between ARF1 and the exchange factor, and (iii) N-myristoylation facilitates both spontaneous and catalyzed GDP-to-GTP exchange on ARF1 simply by facilitating the binding of ARF to membrane phospholipids.