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Shamshad Cockcroft - One of the best experts on this subject based on the ideXlab platform.

  • A unique phosphatidylinositol 4-phosphate 5-kinase is activated by ADP-Ribosylation Factor in Plasmodium falciparum.
    International Journal for Parasitology, 2009
    Co-Authors: Werner Leber, Alison Skippen, Paul W. Bowyer, Quinton L Fivelman, Shamshad Cockcroft, David Baker
    Abstract:

    In eukaryotes, calcium signalling has been linked to hydrolysis of the phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). The final enzyme in the synthesis of this phosphoinositide, a Type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K), is activated by the small G protein ADP-Ribosylation Factor 1 (ARF1). In mammals, the ARF-PIP5K pathway is a key regulator of cell motility, secretion and cell signalling. We report the characterisation of a unique, putative bifunctional PIP5K in the human malaria parasite Plasmodium falciparum. The protein comprises a C-terminal, functional PIP5K domain with catalytic specificity for phosphatidylinositol 4-phosphate. The recombinant enzyme is activated by ARF1 but not phosphatidic acid. The protein also incorporates an unusual N-terminal domain with potential helix-loop-helix EF-hand-like motifs that is a member of the neuronal calcium sensor family (NCS). Intriguingly, NCS-1 has been shown to stimulate phosphatidylinositol 4-phosphate synthesis by activating mammalian and yeast phosphatidylinositol 4-kinase beta in vitro in a calcium-dependent manner. The unexpected physical attachment of an NCS-like domain to the plasmodial PIP5K might reflect a unique functional link between the calcium and PtdIns(4,5)P(2) pathways allowing modulation of PtdIns(4,5)P(2) production in response to changes in intracellular calcium concentrations within the parasite.

  • mechanism of ADP Ribosylation Factor stimulated phosphatidylinositol 4 5 bisphosphate synthesis in hl60 cells
    Journal of Biological Chemistry, 2002
    Co-Authors: Alison Skippen, David H Jones, Clive P Morgan, Shamshad Cockcroft
    Abstract:

    Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is required both as a substrate for the generation of lipid-derived second messengers as well as an intact lipid for many aspects of cell signaling, endo- and exocytosis, and reorganization of the cytoskeleton. ADP Ribosylation Factor (ARF) proteins regulate PI(4,5)P2 synthesis, and here we have examined whether this is due to direct activation of Type I phosphatidylinositol 4-phosphate (PIP) 5-kinase or indirectly by phosphatidate (PA) derived from phospholipase D (PLD) in HL60 cells. ARF1 and ARF6 are both expressed in HL60 cells and can be depleted from the cells by permeabilization. Both ARFs increased the levels of PIP2 (PI(4,5)P2, PI(3,5)P2, or PI(3,4)P2 isomers) at the expense of PIP when added back to permeabilized cells. The PIP2 could be hydrolyzed by phospholipase C, identifying it as PI(4,5)P2. However, the ARF1-stimulated pool of PI(4,5)P2 was accessible to the phospholipase C more efficiently in the presence of phosphatidylinositol transfer protein-α. To examine the role of PLD in the regulation of PI(4,5)P2 synthesis, we used butanol to diminish the PLD-derived PA. PI(4,5)P2 synthesis stimulated by ARF1 was not blocked by 0.5% butanol but could be blocked by 1.5% butanol. Although 0.5% butanol was optimal for maximal transphosphatidylation, PA production was still detectable. In contrast, 1.5% butanol was found to inhibit the activation of PLD by ARF1 and also decrease PIP levels by 50%. Thus the toxicity of 1.5% butanol prevented us from concluding whether PA was an important Factor in raising PI(4,5)P2 levels. To circumvent the use of alcohols, an ARF1 point mutant was identified (N52R-ARF1) that could selectively activate PIP 5-kinase α activity but not PLD activity. N52R-ARF1 was still able to increase PI(4,5)P2 levels but at reduced efficiency. We therefore conclude that both PA derived from the PLD pathway and ARF proteins, by directly activating PIP 5-kinase, contribute to the regulation of PI(4,5)P2 synthesis at the plasma membrane in HL60 cells.

  • 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, 2000
    Co-Authors: David H Jones, James B Morris, Clive P Morgan, Hisatake Kondo, Robin F Irvine, Shamshad Cockcroft
    Abstract:

    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.

  • 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 bisphospha
    Biochemical Journal, 2000
    Co-Authors: Gemma Way, Niamh Oluanaigh, Shamshad Cockcroft
    Abstract:

    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.

Richard A. Kahn - One of the best experts on this subject based on the ideXlab platform.

  • the structure of ralf an ADP Ribosylation Factor guanine nucleotide exchange Factor from legionella pneumophila reveals the presence of a cap over the active site
    Journal of Biological Chemistry, 2005
    Co-Authors: Carlos J Amor, Xinjun Zhu, Jennifer Swails, Craig R Roy, Hiroki Nagai, Alyssa Ingmundson, Xiaodong Cheng, Richard A. Kahn
    Abstract:

    The Legionella pneumophila protein RalF is secreted into host cytosol via the Dot/Icm type IV transporter where it acts to recruit ADP-Ribosylation Factor (Arf) to pathogen-containing phagosomes in the establishment of a replicative organelle. The presence in RalF of the Sec7 domain, present in all Arf guanine nucleotide exchange Factors, has suggested that recruitment of Arf is an early step in pathogenesis. We have determined the crystal structure of RalF and of the isolated Sec7 domain and found that RalF is made up of two domains. The Sec7 domain is homologous to mammalian Sec7 domains. The C-terminal domain forms a cap over the active site in the Sec7 domain and contains a conserved folding motif, previously observed in adaptor subunits of vesicle coat complexes. The importance of the capping domain and of the glutamate in the "glutamic finger," conserved in all Sec7 domains, to RalF functions was examined using three different assays. These data highlight the functional importance of domains other than Sec7 in Arf guanine nucleotide exchange Factors to biological activities and suggest novel mechanisms of regulation of those activities.

  • functional genomic analysis of the ADP Ribosylation Factor family of gtpases phylogeny among diverse eukaryotes and function in c elegans
    The FASEB Journal, 2004
    Co-Authors: William G Kelly, John M Logsdon, Andrew M Schurko, Brian D Harfe, Katherine L Hillharfe, Richard A. Kahn
    Abstract:

    ADP-Ribosylation Factor (Arf) and Arf-like (Arl) proteins are a family of highly conserved 21 kDa GTPases that emerged early in the evolution of eukaryotes. These proteins serve regulatory roles in vesicular traffic, lipid metabolism, microtubule dynamics, development, and likely other cellular processes. We found evidence for the presence of 6 Arf family members in the protist Giardia lamblia and 22 members in mammals. A phylogenetic analysis was performed to delineate the evolutionary relationships among Arf family members and to attempt to organize them by both their evolutionary origins and functions in cells and/or organisms. The ∼100 protein sequences analyzed from animals, fungi, plants, and protists clustered into 11 groups, including Arfs, nine Arls, and Sar proteins. To begin functional analyses of the family in a metazoan model organism, we examined roles for all three C. elegans Arfs (Arf-1, Arf-3, and Arf-6) and three Arls (Arl-1, Arl-2, and Arl-3) by use of RNA-mediated interference (RNAi). ...

  • the escherichia coli heat labile toxin binds to golgi membranes and alters golgi and cell morphologies using ADP Ribosylation Factor dependent processes
    Journal of Biological Chemistry, 2001
    Co-Authors: Xinjun Zhu, Richard A. Kahn
    Abstract:

    The fate of the catalytic subunit of theEscherichia coli heat labile toxin (LTA1) was studied after expression in mammalian cells to assess the requirement for ADP-Ribosylation Factor (ARF) binding to localization and toxicity and ability to compete with endogenous ARF effectors. A progression in LTA1 localization from cytosol to binding Golgi stacks to condensation of Golgi membranes was found to correlate with the time and level of LTA1 expression. At the highest levels of LTA1 expression the staining of LTA and both extrinsic and lumenal Golgi markers all became diffuse, in a fashion reminiscent of the actions of brefeldin A. Thus, LTA1 binds to the Golgi and can alter its morphology in two distinct ways. However, point mutants of LTA1 that are defective in the ability to bind activated ARF were also unable to bind Golgi membranes or modify Golgi morphology. Co-expression of mutants of ARF3 that regained binding to these same mutant LTA1 proteins restored the localization and activities of the toxin. Thus, binding to ARF is required both for the localization of the toxin to the Golgi and for effects on Golgi membranes. A correlation was also seen between the ability of LTA mutants to bind ARF and the increase in cellular cAMP levels. These results demonstrate the importance of ARF binding to the toxicity and cellular effects of the ADP-ribosylating bacterial toxin and reveal that mutants defective in binding ARF retain basal ADP-Ribosylation activity but are the least toxic LTA1mutants yet described, making them the best candidates for development as mucosal adjuvants.

  • 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, 2000
    Co-Authors: Sutapa Mukherjee, Richard A. Kahn, Mariefrance Bader, James E Casanova, Vsevolod V Gurevich, Jonathan C R Jones, Scott R Frank, Evelyn T Maizels, Krzysztof Palczewski, Klaus Aktories
    Abstract:

    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.

  • a family of ADP Ribosylation Factor effectors that can alter membrane transport through the trans golgi
    Molecular Biology of the Cell, 2000
    Co-Authors: Annette L Boman, Chunjiang Zhang, Xinjun Zhu, Richard A. Kahn
    Abstract:

    A family of three structurally related proteins were cloned from human cDNA libraries by their ability to interact preferentially with the activated form of human ADP-Ribosylation Factor 3 (ARF3) in two-hybrid assays. The specific and GTP-dependent binding was later confirmed through direct protein binding of recombinant proteins. The three proteins share large (’300 residues) domains at their N termini that are 60 ‐70% identical to each other and a shorter (73 residues) domain at their C termini with 70% homology to the C-terminal “ear” domain of g-adaptin. Although GGA1 is found predominantly as a soluble protein by cell fractionation, all three proteins were found to localize to the trans-Golgi network (TGN) by indirect immunofluorescence. The binding of GGAs to TGN was sensitive to brefeldin A, consistent with this being an ARF-dependent event. Thus, these proteins have been named Golgi-localizing, g-adaptin ear homology domain, ARF-binding proteins, or GGAs. The finding that overexpression of GGAs was sufficient to alter the distribution of markers of the TGN (TGN38 and mannose 6-phosphate receptors) led us to propose that GGAs are effectors for ARFs that function in the regulation of membrane traffic through the TGN.

Joel Moss - One of the best experts on this subject based on the ideXlab platform.

  • brefeldin a inhibited ADP Ribosylation Factor activator big2 regulates cell migration via integrin β1 cycling and actin remodeling
    Proceedings of the National Academy of Sciences of the United States of America, 2012
    Co-Authors: Xiaoyan Shen, Chun Chun Li, Angel M Aponte, Rong Fong Shen, Eric M Billings, Joel Moss, Martha Vaughan
    Abstract:

    Brefeldin A-inhibited guanine nucleotide-exchange protein (BIG)2 activates ADP-Ribosylation Factors, ∼20-kDa GTPase proteins critical for continuity of intracellular vesicular trafficking by accelerating the replacement of ADP-Ribosylation Factor-bound GDP with GTP. Mechanisms of additional BIG2 function(s) are less clear. Here, the participation of BIG2 in integrin β1 cycling through actin dynamics during cell migration was identified using small interfering RNA (siRNA) and difference gel electrophoresis analyses. After a 72-h incubation with BIG2 siRNA, levels of cytosolic Arp2, Arp3, cofilin-1, phosphocofilin, vinculin, and Grb2, known to be involved in the effects of integrin β1-extracellular matrix interactions on actin function and cell translocation, were increased. Treatment of HeLa cells with BIG2 siRNA resulted in perinuclear accumulation of integrin β1 and its delayed return to the cell surface. Motility of BIG2-depleted cells was simultaneously decreased, as were actin-based membrane protrusions and accumulations of Arp2, Arp3, cofilin, and phosphocofilin at the leading edges of migrating cells, in wound-healing assays. Taken together, these data reveal a mechanism(s) through which BIG2 may coordinate actin cytoskeleton mechanics and membrane traffic in cell migration via integrin β1 action and actin functions.

  • the guanine nucleotide exchange protein for ADP Ribosylation Factor 6 arf gep100 brag2 regulates phagocytosis of monocytic phagocytes in an arf6 dependent process
    Journal of Biological Chemistry, 2010
    Co-Authors: Akimasa Someya, Joel Moss, Isao Nagaoka
    Abstract:

    Phagocytosis is a complex multistep process requiring diverse signaling and regulatory molecules. ADP-Ribosylation Factor 6 (ARF6), a small GTPase, is known to regulate membrane trafficking and the actin cytoskeketon at the plasma membrane and functions as a regulatory molecule of phagocytosis. ARF activity is regulated by cycling between GDP-bound and GTP-bound forms. ARF activation is catalyzed by guanine nucleotide exchange Factors (GEFs) that facilitate GTP binding. We had earlier reported a 100-kDa ARF-GEF, termed ARF-guanine nucleotide exchange protein 100, GEP100, that preferentially activates ARF6 and was also described by Dunphy et al. (Dunphy, J. L., Moravec, R., Ly, K., Lasell, T. K., Melancon, P., and Casanova, J. E. (2006) Curr. Biol. 16, 315–320) as brefeldin A-resistant ARF-GEF2 (BRAG2). We have now examined a role for GEP100 in phagocytosis. Stable depletion of GEP100 decreased phagocytosis of serum-treated zymosan and IgG-coated latex beads by human monocyte-macrophage-like U937 cells differentiated with phorbol 12-myristate 13-acetate. Decrease of phagocytic activity by RNAi was not rescued by GEP100ΔSec7, a deletion mutant lacking the ARF-activating domain. GEP100-depleted cells also exhibited reduced F-actin fibers around internalized particles. Attachment of these particles to cells and amounts of C3bi and Fcγ receptors, however, were not affected by GEP100 depletion. On immunofluorescence microscopy, GEP100 and ARF6 were concentrated and partially colocalized around internalized particles. Phagocytosis by GEP100-depleted cells was not further affected by depletion of ARF6. Phagocytic activity of GEP100-depleted cells was, however, rescued by expression of the constitutively active ARF6Q67N mutant but not by the dominant-negative ARF6T27N mutant. These data are consistent with the conclusion that GEP100 functions in phagocytosis via its role in ARF6-dependent actin remodeling.

  • gep100 brag2 activator of ADP Ribosylation Factor 6 for regulation of cell adhesion and actin cytoskeleton via e cadherin and α catenin
    Proceedings of the National Academy of Sciences of the United States of America, 2006
    Co-Authors: Toyoko Hiroi, Joel Moss, Akimasa Someya, Walter C Thompson, Martha Vaughan
    Abstract:

    GEP100 (p100) was identified as an ADP-Ribosylation Factor (ARF) guanine nucleotide-exchange protein (GEP) that partially colocalized with ARF6 in the cell periphery. p100 preferentially accelerated guanosine 5[γ-thio]triphosphate (GTPγS) binding by ARF6, which participates in protein trafficking near the plasma membrane, including receptor recycling, cell adhesion, and cell migration. Here we report that yeast two-hybrid screening of a human fetal brain cDNA library using p100 as bait revealed specific interaction with α-catenin, which is known as a regulator of adherens junctions and actin cytoskeleton remodeling. Interaction of p100 with α-catenin was confirmed by coimmunoprecipitation of the endogenous proteins from human HepG2 or CaSki cells, although colocalization was difficult to demonstrate microscopically. α-Catenin enhanced GTPγS binding by ARF6 in vitro in the presence of p100. Depletion of p100 by small interfering RNA (siRNA) treatment in HepG2 cells resulted in E-cadherin content 3-fold that in control cells and blocked hepatocyte growth Factor-induced redistribution of E-cadherin, consistent with a known role of ARF6 in this process. F-actin was markedly decreased in normal rat kidney (NRK) cells overexpressing wild-type p100, but not its GEP-inactive mutants, also consistent with the conclusion that p100 has an important role in the activation of ARF6 for its functions in both E-cadherin recycling and actin remodeling.

  • ADP Ribosylation Factor domain protein 1 ard1 a multifunctional protein with ubiquitin e3 ligase gap and arf domains
    Methods in Enzymology, 2005
    Co-Authors: Alessandro Vichi, Joel Moss, Martha Vaughan
    Abstract:

    Abstract ADP‐Ribosylation Factor domain protein 1 (ARD1) is a multifunctional protein that belongs to the family of 20‐kDa ARF proteins. The ARD1 gene encodes a 64‐kDa protein with a structure comprising an 18‐kDa ADP‐Ribosylation Factor (ARF) domain at the C‐terminus (amino acids 403–574), and a 46‐kDa N‐terminal domain (amino acids 1–402) that contains, from the translation start site, a RING finger domain, two predicted B‐Boxes, and a coiled‐coil protein interaction motif, which places it among the TRIM (tripartite motif) or RBCC (RING, B‐Box, coiled‐coil) protein families. Recombinant ARD1 (amino acids 1–574) or its RING finger domain (amino acids 1–110) produced polyubiquitylated proteins when incubated in vitro with a mammalian E1, an E2 enzyme (UbcH6 or UbcH5a, ‐5b, or ‐5c), ATP, and ubiquitin. Via its C‐terminal ARF domain, recombinant ARD1 binds guanine nucleotides, through which it can enhance, in a GTP‐dependent manner, cholera toxin ADP‐ribosyltransferase activity. Unlike ARFs, ARD1, but not its ARF domain, exhibits significant GTPase activity. Hydrolysis of GTP bound to the C‐terminal ARF domain was stimulated by addition of the 46‐kDa N‐terminal domain (amino acids 1–402) via its GTPase activating protein (GAP) activity. The rate of GDP dissociation from the C‐terminal ARF domain in ARD1, is slowed by the adjacent 15 amino acids, which act as a GDP‐dissociation inhibitor (GDI) domain. Cytohesin‐1, known already as a guanine nucleotide‐exchange Factor (GEF) ARF activator, also specifically activated recombinant human ARD1, via activation of the ARF domain. Overexpressed ARD1 fusion proteins were associated with structures resembling lysosomes and Golgi membranes, as well as the nucleus, in different types of cells, and sequences potentially responsible for the intracellular localizations were identified.

  • beta arrestin mediated ADP Ribosylation Factor 6 activation and beta 2 adrenergic receptor endocytosis
    Journal of Biological Chemistry, 2001
    Co-Authors: Audrey Claing, Nicolas Vitale, William E. Miller, Joel Moss, Wei Chen, Richard T Premont
    Abstract:

    Abstract β-Arrestins are multifunctional adaptor proteins known to regulate internalization of agonist-stimulated G protein-coupled receptors by linking them to endocytic proteins such as clathrin and AP-2. Here we describe a previously unappreciated mechanism by which β-arrestin orchestrates the process of receptor endocytosis through the activation of ADP-Ribosylation Factor 6 (ARF6), a small GTP-binding protein. Involvement of ARF6 in the endocytic process is demonstrated by the ability of GTP-binding defective and GTP hydrolysis-deficient mutants to inhibit internalization of the β2-adrenergic receptor. The importance of regulation of ARF6 function is shown by the ability of the ARF GTPase-activating protein GIT1 to inhibit and of the ARF nucleotide exchange Factor, ARNO, to enhance receptor endocytosis. Endogenous β-arrestin is found in complex with ARNO. Upon agonist stimulation of the receptor, β-arrestin also interacts with the GDP-liganded form of ARF6, thereby facilitating ARNO-promoted GTP loading and activation of the G protein. Thus, the agonist-driven formation of a complex including β-arrestin, ARNO, and ARF6 provides a molecular mechanism that explains how the agonist-stimulated receptor recruits a small G protein necessary for the endocytic process and controls its activation.

Paul A. Randazzo - One of the best experts on this subject based on the ideXlab platform.

  • agap1 an endosome associated phosphoinositide dependent ADP Ribosylation Factor gtpase activating protein that affects actin cytoskeleton
    Journal of Biological Chemistry, 2002
    Co-Authors: Zhongzhen Nie, Katherine T Stanley, Stacey Stauffer, Kerry M Jacques, Dianne S Hirsch, Jiro Takei, Paul A. Randazzo
    Abstract:

    We have identified three members of the AGAP subfamily of ASAP family ADP-Ribosylation Factor GTPase-activating proteins (Arf GAPs). In addition to the Arf GAP domain, these proteins contain GTP-binding protein-like, ankyrin repeat and pleckstrin homology domains. Here, we have characterized the ubiquitously expressed AGAP1/KIAA1099. AGAP1 had Arf GAP activity toward Arf1>Arf5>Arf6. Phosphatidylinositol 4,5-bisphosphate and phosphatidic acid synergistically stimulated GAP activity. As found for other ASAP family Arf GAPs, the pleckstrin homology domain was necessary for activity. Deletion of the GTP-binding protein-like domain affected lipid dependence of Arf GAP activity. In vivo effects of AGAP1 were distinct from other ASAP family Arf GAPs. Overexpressed AGAP1 induced the formation of and was associated with punctate structures containing the endocytic markers transferrin and Rab4. AP1 was redistributed from the trans-Golgi to the punctate structures. Like other ASAP family members, AGAP1 overexpression inhibited the formation of PDGF-induced ruffles. However, distinct from other ASAP family members, AGAP1 also induced the loss of actin stress fibers. Thus, AGAP1 is a phosphoinositide-dependent Arf GAP that impacts both the endocytic compartment and actin.

  • phosphoinositide dependent activation of the ADP Ribosylation Factor gtpase activating protein asap1 evidence for the pleckstrin homology domain functioning as an allosteric site
    Journal of Biological Chemistry, 2000
    Co-Authors: Jeanelle L Kam, Jenny Clark, Peter P Roller, Stacey Stauffer, Koichi Miura, Trevor R Jackson, James M Gruschus, Rajindra Aneja, Paul A. Randazzo
    Abstract:

    Abstract The ADP-Ribosylation Factor (Arf) family of GTP-binding proteins are regulators of membrane traffic and the actin cytoskeleton. Both negative and positive regulators of Arf, the centaurin β family of Arf GTPase-activating proteins (GAPs) and Arf guanine nucleotide exchange Factors, contain pleckstrin homology (PH) domains and are activated by phosphoinositides. To understand how the activities are coordinated, we have examined the role of phosphoinositide binding for Arf GAP function using ASAP1/centaurin β4 as a model. In contrast to Arf exchange Factors, phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) specifically activated Arf GAP. D3 phosphorylated phosphoinositides were less effective. Activation involved PtdIns-4,5-P2binding to the PH domain; however, in contrast to the Arf exchange Factors and contrary to predictions based on the current paradigm for PH domains as independently functioning recruitment signals, we found the following: (i) the PH domain was dispensable for targeting to PDGF-induced ruffles; (ii) activation and recruitment could be uncoupled; (iii) the PH domain was necessary for activity even in the absence of phospholipids; and (iv) the Arf GAP domain influenced localization and lipid binding of the PH domain. Furthermore, PtdIns-4,5-P2 binding to the PH domain caused a conformational change in the Arf GAP domain detected by limited proteolysis. Thus, these data demonstrate that PH domains can function as allosteric sites. In addition, differences from the published properties of the Arf exchange Factors suggest a model in which feedforward and feedback loops involving lipid metabolites coordinate GTP binding and hydrolysis by Arf.

  • the amino terminus of ADP Ribosylation Factor arf 1 is essential for interaction with gs and arf gtpase activating protein
    Journal of Biological Chemistry, 1994
    Co-Authors: Paul A. Randazzo, Takeshi Terui, Stacey Sturch, Richard A. Kahn
    Abstract:

    Abstract The role of the amino terminus in the actions of ADP-Ribosylation Factor 1 (ARF1) was examined by comparing wild type ARF1, a 13-residue NH2-terminal deletion mutant ([delta 13]ARF1), and a 17-residue NH2-terminal deletion mutant ([delta 17]ARF1). The amino-terminal 13 residues of ARF1 are required for coFactor activity in the ADP-Ribosylation by cholera toxin when Gs is the substrate. This is in marked contrast to the finding that coFactor activity is the same for wild type and [delta 13]ARF1 when agmatine is substrate (Hong, J.-X., Haun, R. S., Tsai, S.-C., Moss, J., and Vaughan, M. (1994) J. Biol. Chem. 269, 9743-9745). These data support the conclusion that ARF1 interacts with both cholera toxin and Gs and that the amino terminus of ARF1 is required specifically for binding Gs. Surprisingly, this result also clearly revealed that the two principal assays for ARF activity, coFactor activity for cholera toxin using either Gs or agmatine as substrates, used for over 10 years in different laboratories, can yield quite different results. While both NH2-terminal deletion mutants failed to support the ADP-Ribosylation of Gs by cholera toxin, [delta 13]ARF1, but not [delta 17]ARF1, inhibited the activity of the wild type protein. The GTPase activity of [delta 13]ARF1 was activated to a small extent by ARF GTPase-activating protein (GAP), whereas that of [delta 17]ARF1 was unaffected. We conclude that residues 14-17 are involved in the interaction of ARF with both cholera toxin and ARF GAP. The co-purifying nucleotides, nucleotide exchange kinetics, and dependence of exchange on phospholipids for the mutant proteins were all different from the wild type ARF1 proteins. The importance of monitoring the nucleotide binding to ARF proteins under the conditions used in the ARF assay and expressing ARF activities as specific activities, normalized to GTP binding sites, particularly when comparisons between different proteins or preparations are made, is discussed.

  • activation of ADP Ribosylation Factor by golgi membranes evidence for a brefeldin a and protease sensitive activating Factor on golgi membranes
    Journal of Biological Chemistry, 1993
    Co-Authors: Paul A. Randazzo, Yun Chung Yang, C Rulka, Richard A. Kahn
    Abstract:

    Abstract Recent evidence has implicated ADP-Ribosylation Factor (ARF) proteins as critical regulators of the protein secretory pathway, particularly in the endoplasmic reticulum-Golgi pathway. We have examined whether Golgi membranes contain activators of ARF and the consequences of ARF activation and acylation on its membrane association. Two means were used to assess ARF activation. First, guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) binding to protein was found to be greater when ARF and Golgi were incubated together than when either was incubated alone. These data suggested that ARF GTP gamma S was formed. This was confirmed by showing that the GTP gamma S-bound protein functioned as a coFactor for cholera toxin-stimulated ADP-Ribosylation of Gs alpha, a reaction for which activated ARF is a necessary coFactor. Trypsin treatment of Golgi, an inhibitory ARF peptide, and brefeldin A each inhibited Golgi-mediated activation by approximately 70%, demonstrating that a specific protein interaction is required for the majority of the ARF activation. This ARF-activating protein is a strong candidate for the molecular target for brefeldin A. The ubiquitous nature of ARF proteins and their importance in both the exocytic and endocytic pathways may explain the effects of brefeldin A on both exocytic and endocytic membrane traffic in animal cells. A protease-insensitive activation of ARF by Golgi could also be demonstrated and was the dominant activity observed in submicromolar concentrations of magnesium. We believe this to be the lipid-mediated process described previously for purified ARF proteins. ARF activation resulted in tight association of ARF with phospholipid vesicles. Vesicle association required amino-terminal myristoylation of ARF whereas activation did not. These studies indicate that the brefeldin A-sensitive ARF-activating protein and other Factors that determine the level of activation of ARF in animal cells are fundamental regulators of membrane traffic in animal cells.

  • the amino terminus of ADP Ribosylation Factor arf is a critical determinant of arf activities and is a potent and specific inhibitor of protein transport
    Journal of Biological Chemistry, 1992
    Co-Authors: Richard A. Kahn, C Rulka, Paul A. Randazzo, Tito Serafini, O Weiss, Jenny Clark, Mylene Amherdt, Peter P Roller, Lelio Orci
    Abstract:

    Deletion of the amino-terminal 17 residues from human ADP-Ribosylation Factor (ARF) resulted in a protein ([delta 1-17]mARF1p) devoid of ARF activity but which retained the ability to bind guanine nucleotides with high affinity. Unlike the wild type, the binding of guanine nucleotides to this deletion mutant was found to be independent of added phospholipids. A chimeric protein was produced, consisting of 10% (the amino-terminal 17 amino acids) human ARF1p and 90% ARL1p, an ARF-like protein (55% identical protein sequence) from Drosophila. This chimera was found to have ARF activity, lacking in the parental ARL1 protein. Thus, the amino terminus of ARF1p was shown to be a critical component of ARF activity. A synthetic peptide, derived from the amino terminus of ARF1p, has no ARF activity. Rather, the peptide was found to be a specific inhibitor of ARF activities. This peptide was also found to be a potent and specific inhibitor of both an in vitro intra-Golgi transport assay and the guanosine 5'-3-O-(thio)triphosphate-stimulated accumulation of coated vesicles and buds from Golgi preparations. We conclude that ARF is required for the budding of coated vesicles from the Golgi stacks and serves a regulatory role in protein secretion through the Golgi in eukaryotic cells.

Nicolas Vitale - One of the best experts on this subject based on the ideXlab platform.

  • ADP Ribosylation Factor 6 regulates mammalian myoblast fusion through phospholipase d1 and phosphatidylinositol 4 5 bisphosphate signaling pathways
    Molecular Biology of the Cell, 2010
    Co-Authors: Annesophie Bach, Nicolas Vitale, Sandrine Enjalbert, Franck Comunale, Stephane Bodin, Sophie Charrasse, Cecile Gauthierrouviere
    Abstract:

    Myoblast fusion is an essential step during myoblast differentiation that remains poorly understood. M-cadherin-dependent pathways that signal through Rac1 GTPase activation via the Rho-guanine nucleotide exchange Factor (GEF) Trio are important for myoblast fusion. The ADP-Ribosylation Factor (ARF)6 GTPase has been shown to bind to Trio and to regulate Rac1 activity. Moreover, Loner/GEP(100)/BRAG2, a GEF of ARF6, has been involved in mammalian and Drosophila myoblast fusion, but the specific role of ARF6 has been not fully analyzed. Here, we show that ARF6 activity is increased at the time of myoblast fusion and is required for its implementation in mouse C2C12 myoblasts. Specifically, at the onset of myoblast fusion, ARF6 is associated with the multiproteic complex that contains M-cadherin, Trio, and Rac1 and accumulates at sites of myoblast fusion. ARF6 silencing inhibits the association of Trio and Rac1 with M-cadherin. Moreover, we demonstrate that ARF6 regulates myoblast fusion through phospholipase D (PLD) activation and phosphatidylinositol 4,5-bis-phosphate production. Together, these data indicate that ARF6 is a critical regulator of C2C12 myoblast fusion and participates in the regulation of PLD activities that trigger both phospholipids production and actin cytoskeleton reorganization at fusion sites.

  • calcium regulated exocytosis of dense core vesicles requires the activation of ADP Ribosylation Factor arf 6 by arf nucleotide binding site opener at the plasma membrane
    Journal of Cell Biology, 2002
    Co-Authors: Nicolas Vitale, Naoko Morinaga, Sylvette Chasserotgolaz, Michael A Frohman, Yannick Bailly, Mariefrance Bader
    Abstract:

    The ADP Ribosylation Factor (ARF) GTP binding proteins are believed to mediate cytoskeletal remodeling and vesicular trafficking along the secretory pathway. Here we show that ARF6 is specifically associated with dense-core secretory granules in neuroendocrine PC12 cells. Stimulation with a secretagogue triggers the recruitment of secretory granules to the cell periphery and the concomitant activation of ARF6 by the plasma membrane-associated guanine nucleotide exchange Factor, ARF nucleotide binding site opener (ARNO). Expression of the constitutively inactive ARF6(T27N) mutant inhibits secretagogue-dependent exocytosis from PC12 cells. Using a mutant of ARF6 specifically impaired for PLD1 stimulation, we find that ARF6 is functionally linked to phospholipase D (PLD)1 in the exocytotic machinery. Finally, we show that ARNO, ARF6, and PLD1 colocalize at sites of exocytosis, and we demonstrate direct interaction between ARF6 and PLD1 in stimulated cells. Together, these results provide the first direct evidence that ARF6 plays a role in calcium-regulated exocytosis in neuroendocrine cells, and suggest that ARF6-stimulated PLD1 activation at the plasma membrane and consequent changes in membrane phospholipid composition are critical for formation of the exocytotic fusion pore.

  • beta arrestin mediated ADP Ribosylation Factor 6 activation and beta 2 adrenergic receptor endocytosis
    Journal of Biological Chemistry, 2001
    Co-Authors: Audrey Claing, Nicolas Vitale, William E. Miller, Joel Moss, Wei Chen, Richard T Premont
    Abstract:

    Abstract β-Arrestins are multifunctional adaptor proteins known to regulate internalization of agonist-stimulated G protein-coupled receptors by linking them to endocytic proteins such as clathrin and AP-2. Here we describe a previously unappreciated mechanism by which β-arrestin orchestrates the process of receptor endocytosis through the activation of ADP-Ribosylation Factor 6 (ARF6), a small GTP-binding protein. Involvement of ARF6 in the endocytic process is demonstrated by the ability of GTP-binding defective and GTP hydrolysis-deficient mutants to inhibit internalization of the β2-adrenergic receptor. The importance of regulation of ARF6 function is shown by the ability of the ARF GTPase-activating protein GIT1 to inhibit and of the ARF nucleotide exchange Factor, ARNO, to enhance receptor endocytosis. Endogenous β-arrestin is found in complex with ARNO. Upon agonist stimulation of the receptor, β-arrestin also interacts with the GDP-liganded form of ARF6, thereby facilitating ARNO-promoted GTP loading and activation of the G protein. Thus, the agonist-driven formation of a complex including β-arrestin, ARNO, and ARF6 provides a molecular mechanism that explains how the agonist-stimulated receptor recruits a small G protein necessary for the endocytic process and controls its activation.

  • β arrestin mediated ADP Ribosylation Factor 6 activation and β2 adrenergic receptor endocytosis
    Journal of Biological Chemistry, 2001
    Co-Authors: Audrey Claing, Nicolas Vitale, William E. Miller, Joel Moss, Richard T Premont, Wei Chen, Robert J Lefkowitz
    Abstract:

    β-Arrestins are multifunctional adaptor proteins known to regulate internalization of agonist-stimulated G protein-coupled receptors by linking them to endocytic proteins such as clathrin and AP-2. Here we describe a previously unappreciated mechanism by which β-arrestin orchestrates the process of receptor endocytosis through the activation of ADP-Ribosylation Factor 6 (ARF6), a small GTP-binding protein. Involvement of ARF6 in the endocytic process is demonstrated by the ability of GTP-binding defective and GTP hydrolysis-deficient mutants to inhibit internalization of the β2-adrenergic receptor. The importance of regulation of ARF6 function is shown by the ability of the ARF GTPase-activating protein GIT1 to inhibit and of the ARF nucleotide exchange Factor, ARNO, to enhance receptor endocytosis. Endogenous β-arrestin is found in complex with ARNO. Upon agonist stimulation of the receptor, β-arrestin also interacts with the GDP-liganded form of ARF6, thereby facilitating ARNO-promoted GTP loading and activation of the G protein. Thus, the agonist-driven formation of a complex including β-arrestin, ARNO, and ARF6 provides a molecular mechanism that explains how the agonist-stimulated receptor recruits a small G protein necessary for the endocytic process and controls its activation.

  • β2 adrenergic receptor regulation by git1 a g protein coupled receptor kinase associated ADP Ribosylation Factor gtpase activating protein
    Proceedings of the National Academy of Sciences of the United States of America, 1998
    Co-Authors: Richard T Premont, Nicolas Vitale, Walter A Patton, Audrey Claing, Jennifer L Freeman, Julie A Pitcher, Robert J Lefkowitz
    Abstract:

    G protein-coupled receptor activation leads to the membrane recruitment and activation of G protein-coupled receptor kinases, which phosphorylate receptors and lead to their inactivation. We have identified a novel G protein-coupled receptor kinase-interacting protein, GIT1, that is a GTPase-activating protein (GAP) for the ADP Ribosylation Factor (ARF) family of small GTP-binding proteins. Overexpression of GIT1 leads to reduced β2-adrenergic receptor signaling and increased receptor phosphorylation, which result from reduced receptor internalization and resensitization. These cellular effects of GIT1 require its intact ARF GAP activity and do not reflect regulation of GRK kinase activity. These results suggest an essential role for ARF proteins in regulating β2-adrenergic receptor endocytosis. Moreover, they provide a mechanism for integration of receptor activation and endocytosis through regulation of ARF protein activation by GRK-mediated recruitment of the GIT1 ARF GAP to the plasma membrane.