scholarly journals Revisiting the tubulin cofactors and Arl2 in the regulation of soluble αβ-tubulin pools and their effect on microtubule dynamics

2017 ◽  
Vol 28 (3) ◽  
pp. 359-363 ◽  
Author(s):  
Jawdat Al-Bassam
Keyword(s):  
Microtubule Dynamics ◽  
Eukaryotic Cells ◽  
High Concentration ◽  
Adp Ribosylation ◽  
Adp Ribosylation Factor

Soluble αβ-tubulin heterodimers are maintained at high concentration inside eukaryotic cells, forming pools that fundamentally drive microtubule dynamics. Five conserved tubulin cofactors and ADP ribosylation factor–like 2 regulate the biogenesis and degradation of αβ-tubulins to maintain concentrated soluble pools. Here I describe a revised model for the function of three tubulin cofactors and Arl2 as a multisubunit GTP-hydrolyzing catalytic chaperone that cycles to promote αβ-tubulin biogenesis and degradation. This model helps explain old and new data indicating these activities enhance microtubule dynamics in vivo via repair or removal of αβ-tubulins from the soluble pools

scholarly journals ADP-ribosylation factor–like 4A interacts with Robo1 to promote cell migration by regulating Cdc42 activation

2019 ◽  
Vol 30 (1) ◽  
pp. 69-81 ◽  
Author(s):  
Tsai-Shin Chiang ◽  
Ming-Chieh Lin ◽  
Meng-Chen Tsai ◽  
Chieh-Hsin Chen ◽  
Li-Ting Jang ◽  
...  
Keyword(s):  
Cell Migration ◽  
Molecular Mechanisms ◽  
Small Gtpase ◽  
Dependent Manner ◽  
Amino Acid Residues ◽  
Adp Ribosylation ◽  
Promote Cell Migration ◽  
Cytoskeleton Remodeling ◽  
Adp Ribosylation Factor

Cell migration is a highly regulated event that is initiated by cell membrane protrusion and actin reorganization. Robo1, a single-pass transmembrane receptor, is crucial for neuronal guidance and cell migration. ADP-ribosylation factor (Arf)–like 4A (Arl4A), an Arf small GTPase, functions in cell morphology, cell migration, and actin cytoskeleton remodeling; however, the molecular mechanisms of Arl4A in cell migration are unclear. Here, we report that the binding of Arl4A to Robo1 modulates cell migration by promoting Cdc42 activation. We found that Arl4A interacts with Robo1 in a GTP-dependent manner and that the Robo1 amino acid residues 1394–1398 are required for this interaction. The Arl4A-Robo1 interaction is essential for Arl4A-induced cell migration and Cdc42 activation but not for the plasma membrane localization of Robo1. In addition, we show that the binding of Arl4A to Robo1 decreases the association of Robo1 with the Cdc42 GTPase-activating protein srGAP1. Furthermore, Slit2/Robo1 binding down-regulates the Arl4A-Robo1 interaction in vivo, thus attenuating Cdc42-mediated cell migration. Therefore, our study reveals a novel mechanism by which Arl4A participates in Slit2/Robo1 signaling to modulate cell motility by regulating Cdc42 activity.

scholarly journals Adp Ribosylation Factor-like Protein 2 (Arl2) Regulates the Interaction of Tubulin-Folding Cofactor D with Native Tubulin

2000 ◽  
Vol 149 (5) ◽  
pp. 1087-1096 ◽  
Author(s):  
Arunashree Bhamidipati ◽  
Sally A. Lewis ◽  
Nicholas J. Cowan
Keyword(s):  
Cultured Cells ◽  
Gtpase Activating Protein ◽  
Adp Ribosylation ◽  
Chaperone Protein ◽  
Ras Family ◽  
Mutant Forms ◽  
Adp Ribosylation Factor ◽  
Β Tubulin

The ADP ribosylation factor-like proteins (Arls) are a family of small monomeric G proteins of unknown function. Here, we show that Arl2 interacts with the tubulin-specific chaperone protein known as cofactor D. Cofactors C, D, and E assemble the α/β- tubulin heterodimer and also interact with native tubulin, stimulating it to hydrolyze GTP and thus acting together as a β-tubulin GTPase activating protein (GAP). We find that Arl2 downregulates the tubulin GAP activity of C, D, and E, and inhibits the binding of D to native tubulin in vitro. We also find that overexpression of cofactors D or E in cultured cells results in the destruction of the tubulin heterodimer and of microtubules. Arl2 specifically prevents destruction of tubulin and microtubules by cofactor D, but not by cofactor E. We generated mutant forms of Arl2 based on the known properties of classical Ras-family mutations. Experiments using these altered forms of Arl2 in vitro and in vivo demonstrate that it is GDP-bound Arl2 that interacts with cofactor D, thereby averting tubulin and microtubule destruction. These data establish a role for Arl2 in modulating the interaction of tubulin-folding cofactors with native tubulin in vivo.

scholarly journals ADP Ribosylation Factor 1 Is Required for Synaptic Vesicle Budding in PC12 Cells

1997 ◽  
Vol 138 (3) ◽  
pp. 505-515 ◽  
Author(s):  
Victor Faúndez ◽  
Jim-Tong Horng ◽  
Regis B. Kelly
Keyword(s):  
Pc12 Cell ◽  
Cell Membranes ◽  
Brefeldin A ◽  
T Antigen ◽  
Gtpase Activity ◽  
Vesicle Budding ◽  
Adp Ribosylation ◽  
Adp Ribosylation Factor

Carrier vesicle generation from donor membranes typically progresses through a GTP-dependent recruitment of coats to membranes. Here we explore the role of ADP ribosylation factor (ARF) 1, one of the GTP-binding proteins that recruit coats, in the production of neuroendocrine synaptic vesicles (SVs) from PC12 cell membranes. Brefeldin A (BFA) strongly and reversibly inhibited SV formation in vivo in three different PC12 cell lines expressing vesicle-associated membrane protein–T Antigen derivatives. Other membrane traffic events remained unaffected by the drug, and the BFA effects were not mimicked by drugs known to interfere with formation of other classes of vesicles. The involvement of ARF proteins in the budding of SVs was addressed in a cell-free reconstitution system (Desnos, C., L. Clift-O'Grady, and R.B. Kelly. 1995. J. Cell Biol. 130:1041–1049). A peptide spanning the effector domain of human ARF1 (2–17) and recombinant ARF1 mutated in its GTPase activity, both inhibited the formation of SVs of the correct size. During in vitro incubation in the presence of the mutant ARFs, the labeled precursor membranes acquired different densities, suggesting that the two ARF mutations block at different biosynthetic steps. Cell-free SV formation in the presence of a high molecular weight, ARF-depleted fraction from brain cytosol was significantly enhanced by the addition of recombinant myristoylated native ARF1. Thus, the generation of SVs from PC12 cell membranes requires ARF and uses its GTPase activity, probably to regulate coating phenomena.

scholarly journals Exoenzyme S shows selective ADP-ribosylation and GTPase-activating protein (GAP) activities towards small GTPases in vivo

2002 ◽  
Vol 367 (3) ◽  
pp. 617-628 ◽  
Author(s):  
Maria L. HENRIKSSON ◽  
Charlotta SUNDIN ◽  
Anna L. JANSSON ◽  
Åke FORSBERG ◽  
Ruth H. PALMER ◽  
...  
Keyword(s):  
Small Gtpases ◽  
Small Gtpase ◽  
Eukaryotic Cells ◽  
Gtpase Activating Protein ◽  
Exoenzyme S ◽  
Adp Ribosylation ◽  
Intracellular Targeting ◽  
Gtp Binding ◽  
Adp Ribosyltransferase

Intracellular targeting of the Pseudomonas aeruginosa toxins exoenzyme S (ExoS) and exoenzyme T (ExoT) initially results in disruption of the actin microfilament structure of eukaryotic cells. ExoS and ExoT are bifunctional cytotoxins, with N-terminal GTPase-activating protein (GAP) and C-terminal ADP-ribosyltransferase activities. We show that ExoS can modify multiple GTPases of the Ras superfamily in vivo. In contrast, ExoT shows no ADP-ribosylation activity towards any of the GTPases tested in vivo. We further examined ExoS targets in vivo and observed that ExoS modulates the activity of several of these small GTP-binding proteins, such as Ras, Rap1, Rap2, Ral, Rac1, RhoA and Cdc42. We suggest that ExoS is the major ADP-ribosyltransferase protein modulating small GTPase function encoded by P. aeruginosa. Furthermore, we show that the GAP activity of ExoS abrogates the activation of RhoA, Cdc42 and Rap1.

scholarly journals ADP-Ribosylation Factor 1 (ARF1) Regulates Recruitment of the AP-3 Adaptor Complex to Membranes

1998 ◽  
Vol 142 (2) ◽  
pp. 391-402 ◽  
Author(s):  
Chean Eng Ooi ◽  
Esteban C. Dell'Angelica ◽  
Juan S. Bonifacino
Keyword(s):  
Membrane Trafficking ◽  
Brefeldin A ◽  
Membrane Association ◽  
Coat Proteins ◽  
Nucleotide Exchange ◽  
Membrane Recruitment ◽  
Adp Ribosylation ◽  
Adp Ribosylation Factor

Small GTP-binding proteins such as ADP- ribosylation factor 1 (ARF1) and Sar1p regulate the membrane association of coat proteins involved in intracellular membrane trafficking. ARF1 controls the clathrin coat adaptor AP-1 and the nonclathrin coat COPI, whereas Sar1p controls the nonclathrin coat COPII. In this study, we demonstrate that membrane association of the recently described AP-3 adaptor is regulated by ARF1. Association of AP-3 with membranes in vitro was enhanced by GTPγS and inhibited by brefeldin A (BFA), an inhibitor of ARF1 guanine nucleotide exchange. In addition, recombinant myristoylated ARF1 promoted association of AP-3 with membranes. The role of ARF1 in vivo was examined by assessing AP-3 subcellular localization when the intracellular level of ARF1-GTP was altered through overexpression of dominant ARF1 mutants or ARF1- GTPase-activating protein (GAP). Lowering ARF1-GTP levels resulted in redistribution of AP-3 from punctate membrane-bound structures to the cytosol as seen by immunofluorescence microscopy. In contrast, increasing ARF1-GTP levels prevented redistribution of AP-3 to the cytosol induced by BFA or energy depletion. Similar experiments with mutants of ARF5 and ARF6 showed that these other ARF family members had little or no effect on AP-3. Taken together, our results indicate that membrane recruitment of AP-3 is promoted by ARF1-GTP. This finding suggests that ARF1 is not a regulator of specific coat proteins, but rather is a ubiquitous molecular switch that acts as a transducer of diverse signals influencing coat assembly.

scholarly journals The Cholera Toxin A13 Subdomain Is Essential for Interaction with ADP-Ribosylation Factor 6 and Full Toxic Activity but Is Not Required for Translocation from the Endoplasmic Reticulum to the Cytosol

2006 ◽  
Vol 74 (4) ◽  
pp. 2259-2267 ◽  
Author(s):  
Ken Teter ◽  
Michael G. Jobling ◽  
Danielle Sentz ◽  
Randall K. Holmes
Keyword(s):  
Endoplasmic Reticulum ◽  
Cholera Toxin ◽  
Toxic Activity ◽  
Hydrophobic Domain ◽  
Vesicular Traffic ◽  
Adp Ribosylation ◽  
Quality Control Mechanism ◽  
Adp Ribosylation Factor

ABSTRACT Cholera toxin (CT) moves from the plasma membrane to the endoplasmic reticulum (ER) by retrograde vesicular traffic. In the ER, the catalytic CTA1 polypeptide dissociates from the rest of the toxin and enters the cytosol by a process that involves the quality control mechanism of ER-associated degradation (ERAD). The cytosolic CTA1 then ADP ribosylates Gsα, resulting in adenylate cyclase activation and intoxication of the target cell. It is hypothesized that the C-terminal A13 subdomain of CTA1 plays two crucial roles in the intoxication process: (i) it contains a hydrophobic domain that triggers the ERAD mechanism and (ii) it facilitates interaction with the cytosolic ADP-ribosylation factors (ARFs) that serve as allosteric activators of CTA1. In this study, we examined the role(s) of the CTA13 subdomain in CT intoxication. Full-length CTA1 constructs and truncated CTA1 constructs lacking the A13 subdomain were generated and used to conduct two-hybrid studies of interactions with ARF6, in vitro enzyme assays, in vivo toxicity assays, and in vivo processing/degradation assays. Direct, plasmid-mediated expression of CTA1 constructs in the ER or cytosol of transfected CHO cells was used to perform the in vivo assays. With these methods, we found that the A13 subdomain of CTA1 is important both for interaction with ARF6 and for full expression of enzyme activity in vivo. Surprisingly, however, the A13 subdomain was not required for ERAD-mediated passage of CTA1 from the ER to the cytosol. A possible alternative trigger for CTA1 to activate the ERAD mechanism is discussed.

scholarly journals Remodeling of the Actin Cytoskeleton Is Coordinately Regulated by Protein Kinase C and the ADP-Ribosylation Factor Nucleotide Exchange Factor ARNO

1998 ◽  
Vol 9 (11) ◽  
pp. 3133-3146 ◽  
Author(s):  
Scott R. Frank ◽  
Jessica C. Hatfield ◽  
James E. Casanova
Keyword(s):  
Actin Cytoskeleton ◽  
Catalytic Domain ◽  
Nucleotide Exchange ◽  
Ph Domain ◽  
Surface Receptors ◽  
Adp Ribosylation ◽  
Adp Ribosylation Factor ◽  
Actin Rearrangement

ARNO is a member of a family of guanine-nucleotide exchange factors with specificity for the ADP-ribosylation factor (ARF) GTPases. ARNO possesses a central catalytic domain with homology to yeast Sec7p and an adjacent C-terminal pleckstrin homology (PH) domain. We have previously shown that ARNO localizes to the plasma membrane in vivo and efficiently catalyzes ARF6 nucleotide exchange in vitro. In addition to a role in endocytosis, ARF6 has also been shown to regulate assembly of the actin cytoskeleton. To determine whether ARNO is an upstream regulator of ARF6 in vivo, we examined the distribution of actin in HeLa cells overexpressing ARNO. We found that, while expression of ARNO leads to disassembly of actin stress fibers, it does not result in obvious changes in cell morphology. However, treatment of ARNO transfectants with the PKC agonist phorbol 12-myristate 13-acetate results in the dramatic redistribution of ARNO, ARF6, and actin into membrane protrusions resembling lamellipodia. This process requires ARF activation, as actin rearrangement does not occur in cells expressing a catalytically inactive ARNO mutant. PKC phosphorylates ARNO at a site immediately C-terminal to its PH domain. However, mutation of this site had no effect on the ability of ARNO to regulate actin rearrangement, suggesting that phosphorylation of ARNO by PKC does not positively regulate its activity. Finally, we demonstrate that an ARNO mutant lacking the C-terminal PH domain no longer mediates cytoskeletal reorganization, indicating a role for this domain in appropriate membrane localization. Taken together, these data suggest that ARNO represents an important link between cell surface receptors, ARF6, and the actin cytoskeleton.

scholarly journals ADP-Ribosylation Factor 6 Regulates Tumorigenic and Invasive Properties In vivo

2009 ◽  
Vol 69 (6) ◽  
pp. 2201-2209 ◽  
Author(s):  
Vandhana Muralidharan-Chari ◽  
Holly Hoover ◽  
James Clancy ◽  
Jill Schweitzer ◽  
Mark A. Suckow ◽  
...  
Keyword(s):  
Adp Ribosylation ◽  
Adp Ribosylation Factor

scholarly journals Identification of centaurin-α1 as a potential in vivo phosphatidylinositol 3,4,5-trisphosphate-binding protein that is functionally homologous to the yeast ADP-ribosylation factor (ARF) GTPase-activating protein, Gcs1

1999 ◽  
Vol 340 (2) ◽  
pp. 359-363 ◽  
Author(s):  
Kanamarlapudi VENKATESWARLU ◽  
Paru B. OATEY ◽  
Jeremy M. TAVARÉ ◽  
Trevor R. JACKSON ◽  
Peter J. CULLEN
Keyword(s):  
Plasma Membrane ◽  
Growth Factor ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Dominant Negative ◽  
Gtpase Activating Protein ◽  
Adp Ribosylation ◽  
Adp Ribosylation Factor

Centaurin-α is a 46 kDa in vitro binding protein for the lipid second messenger PtdIns(3,4,5)P3. In this report we have addressed whether centaurin-α1, a human homologue of centaurin-α, binds PtdIns(3,4,5)P3in vivo and furthermore, identified a potential physiological function for centaurin-α1. Using confocal microscopy of live PC12 cells, transiently transfected with a chimera of green fluorescent protein (GFP) fused to the N-terminus of centaurin-α1 (GFP-centaurin-α1), we demonstrated the rapid plasma membrane recruitment of cytosolic GFP-centaurin-α1 following stimulation with either nerve growth factor or epidermal growth factor. This recruitment was dependent on the centaurin-α1 pleckstrin homology domains and was blocked by the PtdIns(4,5)P2 3-kinase (PI 3-kinase) inhibitors wortmannin (100 nM) and LY294002 (50 μM), and also by co-expression with a dominant negative p85. Functionally, we demonstrated that centaurin-α1 could complement a yeast strain deficient in the ADP-ribosylation factor (ARF) GTPase-activating protein Gcs1; a complementation that was blocked by mutagenesis of conserved cysteine residues within the ARF GTPase-activating protein analogous domain of centaurin-α1. Taken together, our data demonstrated that centaurin-α1 could potentially function as an ARF GTPase-activating protein that, on agonist stimulation, was recruited to the plasma membrane possibly through an ability to interact with PtdIns(3,4,5)P3.

Arf family GTPases: roles in membrane traffic and microtubule dynamics

2005 ◽  
Vol 33 (6) ◽  
pp. 1269-1272 ◽  
Author(s):  
R.A. Kahn ◽  
L. Volpicelli-Daley ◽  
B. Bowzard ◽  
P. Shrivastava-Ranjan ◽  
Y. Li ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Family Members ◽  
Membrane Traffic ◽  
Microtubule Dynamics ◽  
Database Mining ◽  
Vesicle Traffic ◽  
Adp Ribosylation ◽  
Adp Ribosylation Factor ◽  
In Cells

Database mining and phylogenetic analysis of the Arf (ADP-ribosylation factor) superfamily revealed the presence in mammals of at least 22 members, including the six Arfs, two Sars and 14 Arl (Arf-like) proteins. At least six Arf family members were found in very early eukaryotes, including orthologues of Arf, Sar, Arl2, Arl3, Arl6 and Arl8. While roles for Arfs in membrane traffic are well known, those for most of the Arls remain unknown. Depletion in cells of the most closely related human Arf proteins, Arf1–Arf5, reveals specificities among their cellular roles and suggests that they may function in pairs at different steps in endocytic and secretory membrane traffic. In addition, recent results from a number of laboratories suggest that several of the Arl proteins may be involved in different aspects of microtubule-dependent functions. Thus, a second major role for Arf family GTPases, that of regulating microtubules, is emerging. Because membrane traffic is often dependent upon movement of vesicles along microtubules this raises the possibility that these two fundamental functions of Arf family members, regulation of vesicle traffic and microtubule dynamics, diverged from one function of Arfs in the earliest cells that has continued to branch and allow additional levels of regulation.

Sign in / Sign up

Export Citation Format

Share Document