The Non-Catalytic Carboxyl-Terminal Domain of ARFGAP1 Regulates Actin Cytoskeleton Reorganization by Antagonizing the Activation of Rac1

University of Birmingham, United Kingdom
PLoS ONE (Impact Factor: 3.23). 04/2011; 6(4):e18458. DOI: 10.1371/journal.pone.0018458
Source: PubMed

ABSTRACT The regulation of the actin cytoskeleton and membrane trafficking is coordinated in mammalian cells. One of the regulators of membrane traffic, the small GTP-binding protein ARF1, also activates phosphatidylinositol kinases that in turn affect actin polymerization. ARFGAP1 is a GTPase activating protein (GAP) for ARF1 that is found on Golgi membranes. We present evidence that ARFGAP1 not only serves as a GAP for ARF1, but also can affect the actin cytoskeleton.
As cells attach to a culture dish foci of actin appear prior to the cells flattening and spreading. We have observed that overexpression of a truncated ARFGAP1 that lacks catalytic activity for ARF, called GAP273, caused these foci to persist for much longer periods than non-transfected cells. This phenomenon was dependent on the level of GAP273 expression. Furthermore, cell spreading after re-plating or cell migration into a previously scraped area was inhibited in cells transfected with GAP273. Live cell imaging of such cells revealed that actin-rich membrane blebs formed that seldom made protrusions of actin spikes or membrane ruffles, suggesting that GAP273 interfered with the regulation of actin dynamics during cell spreading. The over-expression of constitutively active alleles of ARF6 and Rac1 suppressed the effect of GAP273 on actin. In addition, the activation of Rac1 by serum, but not that of RhoA or ARF6, was inhibited in cells over-expressing GAP273, suggesting that Rac1 is a likely downstream effector of ARFGAP1. The carboxyl terminal 65 residues of ARFGAP1 were sufficient to produce the effects on actin and cell spreading in transfected cells and co-localized with cortical actin foci.
ARFGAP1 functions as an inhibitor upstream of Rac1 in regulating actin cytoskeleton. In addition to its GAP catalytic domain and Golgi binding domain, it also has an actin regulation domain in the carboxyl-terminal portion of the protein.

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Available from: Michael G Roth, Sep 26, 2015
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    • "Besides promoting GTP hydrolysis in Arf1, it has been implicated in the regulation of COPI vesicle biogenesis and in protein transport from the ER to the cell surface.63,64 When a truncation mutant of ArfGAP1 (lacking the N-terminal 141 amino acids) was expressed, cells displayed impaired flattening and spreading, suggesting a defect in the actin cytoskeleton.65 Mechanistically, this truncated mutant of ArfGAP1, suppressed the activity of Rac1 and thereby exerted its negative effect on actin nucleation, which translated into an inhibition of cell migration.65 "
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    • "For R475X, its affinity to TRAPPC2 is reduced to a level below the detection limit of this experiment. Myc-TRAPPC2 failed to pull down negative control protein GFP-GAP273, a GFP fusion protein containing the carboxyl terminus 273 residues of ARFGAP1 [36], [37]. ARFGAP1 does not interact with the TRAPP complex (unpublished data). "
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