Centaurin-α1 Is an in Vivo Phosphatidylinositol 3,4,5-Trisphosphate-dependent GTPase-activating Protein for ARF6 That Is Involved in Actin Cytoskeleton Organization

Abstract

The ADP-ribosylation factor (ARF) 6 small GTPase regulates vesicle trafficking and cytoskeletal actin reorganization. The GTPase-activating proteins (GAPs) catalyze the formation of inactive ARF6 GDP . Centaurin-␣ 1 contains an ARF GAP and two pleckstrin homology (PH) domains, which bind the second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ). Here, we show that centaurin-␣ 1 specifically inhibits in vivo GTP loading of ARF6 and redistribution of ARF6 from the endosomal compartment to the plasma membrane, which are indicative of its activation. Centaurin-␣ 1 also inhibited cortical actin formation in a PIP 3 -dependent manner. Moreover, the constitutively active mutant of ARF6, but not that of ARF1, reverses the inhibition of cortical actin formation by centaurin-␣ 1 . An artificially plasma membrane-targeted centaurin-␣ 1 bypasses the requirement of PIP 3 for its involvement in ARF6 inactivation, suggesting that PIP 3 is required for recruitment of centaurin-␣ 1 to the plasma membrane but not for its activity. Together, these data suggest that centaurin-␣ 1 negatively regulates ARF6 activity by functioning as an in vivo PIP 3 -dependent ARF6 GAP. Phosphatidylinositol (PI)1 3-kinases phosphorylate the 3Ј position of the inositol ring of PI and its derivatives. The 3-phosphorylated PIs such as PIP 3 function as second messengers in the regulation of many cellular functions, including cell migration and vesicle transport (1). PIP 3 is localized in the cytosolic leaflet of the plasma membrane and acts as a site-specific signal for recruitment and/or activation of cytosolic proteins required for the formation of functional complexes at the plasma membrane. A large number of down stream targets have been identified for this lipid and used to characterize agonist activated PI 3-kinase associated cellular pathways. These include ARF regulators such as cytohesins, ARAP3 (ARF GAP, Rho GAP, ankyrin repeat, PH protein 3) and centaurin-␣ 1 (2).ARF family of small GTPases regulate vesicle trafficking by shuttling between an inactive GDP-and an active GTP-bound form (3). Among the known six-mammalian ARF isoforms (ARFs 1-6), ARF1 and ARF6 are the most distantly related and the best characterized. ARF1 localizes to the cytosol in GDP-bound form and to the Golgi membrane in GTP-bound form and regulates transport from the Golgi complex. In contrast, ARF6 localizes to endosomes in GDP-bound form and to the plasma membrane in GTP-bound form and regulates transport between these two organelles and cortical actin re-arrangements at the plasma membrane, which are vital for many cellular functions such as endocytosis, chemotaxis, and focal adhesion (4). ARF guanine-nucleotide exchange factors (GEFs) activate ARFs by catalyzing the release of ARF-bound GDP and permitting the subsequent binding of GTP. In contrast, ARF GAPs stimulate intrinsic ARF GTPase activity, resulting in the hydrolysis of ARF-bound GTP to GDP. Cytohesin 1-3 ARF GEFs recruit to the plasma membrane in agonist stimulated cells by binding PIP 3 and then...

ARF6 Activated by the LHCG Receptor through the Cytohesin Family of Guanine Nucleotide Exchange Factors Mediates the Receptor Internalization and Signaling

“…The active form of ARF6, ARF6-GTP, interacts with downstream effectors in the LHCGR signaling pathway to promote receptor internalization. In this study ARF6 activation by HCG-stimulated HLHCGR was determined using a GST-GGA3 PBD pulldown assay (23,25,52). HCG-stimulation of HLHCGR increased cellular levels of ARF6-GTP but not ARF1-GTP, while the Myr-ARF6 peptide but not Myr-ARF1 peptide inhibited HCG-stimulated ARF6 activation.…”

“…NM23-H1 H118C was generated by using QuickChange site-directed mutagenesis kit (Stratagene). GST-GGA3 protein binding domain (PBD) and GFP-cytohesin 2 plasmids described previously (23)(24)(25). EPS15 (EGFR pathway substrate clone 15) dominant negative (DN) mutant excised from pEGFP plasmid by EcoRI digestion and subcloned into the EcoRI site of pCMV-FLAG vector (26).…”

“…We used the GST-GGA3 PBD pulldown assay to assess if HCG-stimulation of HEK-HCGLHR cells causes ARF6 activation. The GST-GGA3 PBD specifically binds to the GTPbound form of ARF and therefore the fusion protein coupled to glutathione beads along with an ARF1-or ARF6-specific antibody has been used to detect activated endogenous ARF1 or ARF6 in cell lysates (23). Upon stimulation with HCG, the levels of endogenous ARF6-GTP, but not ARF1-GTP, are increased in HEK-HLHCGR cells (Fig.…”

Metastasis-associated Protein 1/Histone Deacetylase 4-Nucleosome Remodeling and Deacetylase Complex Regulates Phosphatase and Tensin Homolog Gene Expression and Function

“…In mammalian cells, localized PtdIns (3, 4, 5), P3 production, and PI3K activity control polarization and migration during chemotactic movement (47). Phosphatidylinositol-3,4,5-triphosphate also provides a docking site for regulators and effectors of the small GTPase Arf6, which regulates actin remodeling (48). AKT is the other well known effector of PI3K signaling.…”

RanBPM, a novel interaction partner of the brain‐specific protein p42IP4/centaurin α‐1

“…p42 IP4 can shuttle between cytosol, nucleus and plasma membrane, depending on the cell stimulation situation . Several interaction partners of p42 IP4 have been identified, such as casein kinase I (Dubois et al 2001(Dubois et al , 2002, nucleolin , protein kinase C , several ARFs (Thacker et al 2004;Venkateswarlu et al 2004) GAKIN (Venkateswarlu et al 2005;Horiguchi et al 2006), the peptidase nardilysin (Stricker et al 2006) or F-actin (Thacker et al 2004). It has been shown that p42 IP4 is a component of the neuronal phosphatidyl inositol 3-kinase cascade and is involved in dendritic differentiation/synaptic plasticity and/or transport of PtdIns(3,4,5)P 3 -containing vesicles along axons to regulate neuronal cell polarity (Horiguchi et al 2006;Moore et al 2007).…”