Arf6 regulates RhoB subcellular localization to control cancer cell invasion

The ADP-ribosylation factor 6 (Arf6) is a small GTPase that regulates endocytic recycling processes in concert with various effectors. Arf6 controls cytoskeletal organization and membrane trafficking; however, the detailed mechanisms of regulation remain poorly understood. Here, we report that Arf6 forms a complex with RhoB. The interaction between RhoB and Arf6 is mediated by the GCI (glycine, cysteine, and isoleucine) residues (188–190) of RhoB. Specific targeting of Arf6 to plasma membrane or mitochondrial membranes promotes recruitment and colocalization of RhoB to these membrane microdomains. Arf6 depletion promotes the loss of RhoB from endosomal membranes and leads to RhoB degradation through an endolysosomal pathway. This results in defective actin and focal adhesion dynamics and increased 3D cell migration upon activation of the Met receptor tyrosine kinase. Our findings identify a novel regulatory mechanism for RhoB localization and stability by Arf6 and establish the strict requirement of Arf6 for RhoB-specific subcellular targeting to endosomes and biological functions.

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ARF6 regulates a plasma membrane pool of phosphatidylinositol(4,5)bisphosphate required for regulated exocytosis

The Journal of Cell Biology ◽

10.1083/jcb.200212142 ◽

2003 ◽

Vol 162 (4) ◽

pp. 647-659 ◽

Cited By ~ 166

Author(s):

Yoshikatsu Aikawa ◽

Thomas F.J. Martin

Keyword(s):

Plasma Membrane ◽

Pc12 Cells ◽

Membrane Trafficking ◽

Cell Types ◽

Neuroendocrine Cells ◽

Dense Core Vesicle ◽

Type I ◽

Endosomal Membranes ◽

Phosphatidylinositol 4 ◽

Adp Ribosylation Factor

ADP-ribosylation factor (ARF) 6 regulates endosomal plasma membrane trafficking in many cell types, but is also suggested to play a role in Ca2+-dependent dense-core vesicle (DCV) exocytosis in neuroendocrine cells. In the present work, expression of the constitutively active GTPase-defective ARF6Q67L mutant in PC12 cells was found to inhibit Ca2+-dependent DCV exocytosis. The inhibition of exocytosis was accompanied by accumulation of ARFQ67L, phosphatidylinositol 4,5-bisphosphate (PIP2), and the phosphatidylinositol 4-phosphate 5-kinase type I (PIP5KI) on endosomal membranes with their corresponding depletion from the plasma membrane. That the depletion of PIP2 and PIP5K from the plasma membrane caused the inhibition of DCV exocytosis was demonstrated directly in permeable cell reconstitution studies in which overexpression or addition of PIP5KIγ restored Ca2+-dependent exocytosis. The restoration of exocytosis in ARF6Q67L-expressing permeable cells unexpectedly exhibited a Ca2+ dependence, which was attributed to the dephosphorylation and activation of PIP5K. Increased Ca2+ and dephosphorylation stimulated the association of PIP5KIγ with ARF6. The results reveal a mechanism by which Ca2+ influx promotes increased ARF6-dependent synthesis of PIP2. We conclude that ARF6 plays a role in Ca2+-dependent DCV exocytosis by regulating the activity of PIP5K for the synthesis of an essential plasma membrane pool of PIP2.

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A Novel GTPase-activating Protein for ARF6 Directly Interacts with Clathrin and Regulates Clathrin-dependent Endocytosis

Molecular Biology of the Cell ◽

10.1091/mbc.e04-08-0683 ◽

2005 ◽

Vol 16 (4) ◽

pp. 1617-1628 ◽

Cited By ~ 57

Author(s):

Kenji Tanabe ◽

Tetsuo Torii ◽

Waka Natsume ◽

Sten Braesch-Andersen ◽

Toshio Watanabe ◽

...

Keyword(s):

Plasma Membrane ◽

Actin Cytoskeleton ◽

Heavy Chain ◽

Membrane Trafficking ◽

Novel Species ◽

Small Gtpase ◽

Transferrin Receptors ◽

Gtpase Activating Protein ◽

Multiple Functions ◽

Adp Ribosylation Factor

ADP-ribosylation factor 6 (Arf6) is a small-GTPase that regulates the membrane trafficking between the plasma membrane and endosome. It is also involved in the reorganization of the actin cytoskeleton. GTPase-activating protein (GAP) is a critical regulator of Arf function as it inactivates Arf. Here, we identified a novel species of GAP denoted as SMAP1 that preferentially acts on Arf6. Although overexpression of SMAP1 did not alter the subcellular distribution of the actin cytoskeleton, it did block the endocytosis of transferrin receptors. Knock down of endogenous SMAP1 also abolished transferrin internalization, which confirms that SMAP1 is needed for this endocytic process. SMAP1 overexpression had no effect on clathrin-independent endocytosis, however. Intriguingly, SMAP1 binds directly to the clathrin heavy chain via its clathrin-box and mutation studies revealed that its GAP domain and clathrin-box both contribute to the role SMAP1 plays in clathrin-dependent endocytosis. These observations suggest that SMAP1 may be an Arf6GAP that specifically regulates one of the multiple functions of Arf6, namely, clathrin-dependent endocytosis, and that it does so by binding directly to clathrin.

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Rala and the exocyst control Pvr trafficking and signaling to ensure lymph gland homeostasis in Drosophila melanogaster

10.1101/2020.05.26.086140 ◽

2020 ◽

Author(s):

Helene Knævelsrud ◽

Caroline Baril ◽

Gwenaëlle Gavory ◽

Jorrit M. Enserink ◽

Marc Therrien

Keyword(s):

Receptor Tyrosine Kinase ◽

Membrane Trafficking ◽

Lymph Gland ◽

Small Gtpase ◽

Loss Of Function ◽

Hematopoietic Progenitors ◽

Hematopoietic Organ ◽

Differentiated Cells ◽

Epistasis Analysis ◽

Guanine Exchange Factor

AbstractThe balance between hematopoietic progenitors and differentiated hemocytes is finely tuned during development. In the larval hematopoietic organ of Drosophila, called the lymph gland, the receptor tyrosine kinase Pvr signals from differentiated cells to maintain a pool of undifferentiated progenitors. However, little is known about the processes that support Pvr function. The small GTPase Ral is involved in the regulation of several membrane trafficking events. Drosophila has a single Ral protein, Rala, which has been implicated in the development of various tissues. Here, we investigated the involvement of Rala in the larval fly hematopoietic system. We discovered that the loss of Rala activity phenocopies Pvr loss of function by promoting hemocyte progenitor differentiation. Moreover, using epistasis analysis, we found that the guanine exchange factor RalGPS lies upstream of Rala in this event, whereas the exocyst and Rab11 are acting downstream. Strikingly, the loss of Rala activity leads to a considerable accumulation of Pvr at the plasma membrane, hence suggesting a trafficking defect and reduced Pvr function. Consistent with this hypothesis, Rala loss of function phenotype in the lymph gland is fully suppressed by constitutive STAT activity, which normally mediates Pvr function in the lymph gland. Together, our findings unravel a novel RalGPS-Rala-exocyst-Rab11 axis for the maintenance of lymph gland homeostasis through Pvr.

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Faculty Opinions recommendation of ADP-ribosylation factor-like GTPase ARFRP1 is required for trans-Golgi to plasma membrane trafficking of E-cadherin.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1122979.580087 ◽

2008 ◽

Author(s):

Kermit Carraway

Keyword(s):

Plasma Membrane ◽

Membrane Trafficking ◽

Adp Ribosylation ◽

Adp Ribosylation Factor ◽

E Cadherin

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Novel and Converging Ways of NOX2 and SOD3 in Trafficking and Redox Signaling in Macrophages

Antioxidants ◽

10.3390/antiox10020172 ◽

2021 ◽

Vol 10 (2) ◽

pp. 172

Author(s):

Steen Vang Petersen ◽

Nanna Bach Poulsen ◽

Cecilie Linneberg Matthiesen ◽

Frederik Vilhardt

Keyword(s):

Hydrogen Peroxide ◽

Membrane Trafficking ◽

Spatial Organization ◽

Redox Signaling ◽

Small Gtpase ◽

Paracrine Signaling ◽

Tissue Macrophage ◽

Storage Vesicles ◽

Superoxide Dismutase 3 ◽

Macrophage Populations

Macrophages and related tissue macrophage populations use the classical NADPH oxidase (NOX2) for the regulated production of superoxide and derived oxidants for pathogen combat and redox signaling. With an emphasis on macrophages, we discuss how sorting into secretory storage vesicles, agonist-responsive membrane trafficking, and segregation into sphingolipid and cholesterol-enriched microdomains (lipid rafts) determine the subcellular distribution and spatial organization of NOX2 and superoxide dismutase-3 (SOD3). We discuss how inflammatory activation of macrophages, in part through small GTPase Rab27A/B regulation of the secretory compartments, mediates the coalescence of these two proteins on the cell surface to deliver a focalized hydrogen peroxide output. In interplay with membrane-embedded oxidant transporters and redox sensitive target proteins, this arrangement allows for the autocrine and paracrine signaling, which govern macrophage activation states and transcriptional programs. By discussing examples of autocrine and paracrine redox signaling, we highlight why formation of spatiotemporal microenvironments where produced superoxide is rapidly converted to hydrogen peroxide and conveyed immediately to reach redox targets in proximal vicinity is required for efficient redox signaling. Finally, we discuss the recent discovery of macrophage-derived exosomes as vehicles of NOX2 holoenzyme export to other cells.

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ADP-Ribosylation Factor (ARF) Interaction Is Not Sufficient for Yeast GGA Protein Function or Localization

Molecular Biology of the Cell ◽

10.1091/mbc.e02-02-0078 ◽

2002 ◽

Vol 13 (9) ◽

pp. 3078-3095 ◽

Cited By ~ 30

Author(s):

Annette L. Boman ◽

Paul D. Salo ◽

Melissa J. Hauglund ◽

Nicole L. Strand ◽

Shelly J. Rensink ◽

...

Keyword(s):

Membrane Trafficking ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Protein Localization ◽

Carboxypeptidase Y ◽

Minor Role ◽

Temperature Sensitive ◽

Adp Ribosylation ◽

And Function ◽

Adp Ribosylation Factor

Golgi-localized γ-ear homology domain, ADP-ribosylation factor (ARF)-binding proteins (GGAs) facilitate distinct steps of post-Golgi traffic. Human and yeast GGA proteins are only ∼25% identical, but all GGA proteins have four similar domains based on function and sequence homology. GGA proteins are most conserved in the region that interacts with ARF proteins. To analyze the role of ARF in GGA protein localization and function, we performed mutational analyses of both human and yeast GGAs. To our surprise, yeast and human GGAs differ in their requirement for ARF interaction. We describe a point mutation in both yeast and mammalian GGA proteins that eliminates binding to ARFs. In mammalian cells, this mutation disrupts the localization of human GGA proteins. Yeast Gga function was studied using an assay for carboxypeptidase Y missorting and synthetic temperature-sensitive lethality between GGAs andVPS27. Based on these assays, we conclude that non-Arf-binding yeast Gga mutants can function normally in membrane trafficking. Using green fluorescent protein-tagged Gga1p, we show that Arf interaction is not required for Gga localization to the Golgi. Truncation analysis of Gga1p and Gga2p suggests that the N-terminal VHS domain and C-terminal hinge and ear domains play significant roles in yeast Gga protein localization and function. Together, our data suggest that yeast Gga proteins function to assemble a protein complex at the late Golgi to initiate proper sorting and transport of specific cargo. Whereas mammalian GGAs must interact with ARF to localize to and function at the Golgi, interaction between yeast Ggas and Arf plays a minor role in Gga localization and function.

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Angiotensin II Impairs Endothelial Nitric-oxide Synthase Bioavailability under Free Cholesterol-enriched Conditions via Intracellular Free Cholesterol-rich Membrane Microdomains

Journal of Biological Chemistry ◽

10.1074/jbc.m112.448522 ◽

2013 ◽

Vol 288 (20) ◽

pp. 14497-14509 ◽

Cited By ~ 13

Author(s):

Eisuke Amiya ◽

Masafumi Watanabe ◽

Norihiko Takeda ◽

Tetsuya Saito ◽

Taro Shiga ◽

...

Keyword(s):

Angiotensin Ii ◽

Endothelial Dysfunction ◽

Free Cholesterol ◽

Small Gtpase ◽

Membrane Microdomains ◽

No Production ◽

Aortic Endothelial Cells ◽

Modified Ldl ◽

Rich Domain ◽

Endothelial Nitric Oxide

Vascular endothelial function is impaired in hypercholesterolemia partly because of injury by modified LDL. In addition to modified LDL, free cholesterol (FC) is thought to play an important role in the development of endothelial dysfunction, although the precise mechanisms remain to be elucidated. The aim of this study was to clarify the mechanisms of endothelial dysfunction induced by an FC-rich environment. Loading cultured human aortic endothelial cells with FC induced the formation of vesicular structures composed of FC-rich membranes. Raft proteins such as phospho-caveolin-1 (Tyr-14) and small GTPase Rac were accumulated toward FC-rich membranes around vesicular structures. In the presence of these vesicles, angiotensin II-induced production of reactive oxygen species (ROS) was considerably enhanced. This ROS shifted endothelial NOS (eNOS) toward vesicle membranes and vesicles with a FC-rich domain trafficked toward perinuclear late endosomes/lysosomes, which resulted in the deterioration of eNOS Ser-1177 phosphorylation and NO production. Angiotensin II-induced ROS decreased the bioavailability of eNOS under the FC-enriched condition.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e18-01-0001 ◽

2019 ◽

Vol 30 (1) ◽

pp. 69-81 ◽

Cited By ~ 2

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.

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