A lysine-rich cluster in the N-BAR domain of ARF GTPase-activating protein ASAP1 is necessary for binding and bundling actin filaments.

Actin filament maintenance is critical for both normal cell homeostasis and events associated with malignant transformation. The ADP-ribosylation factor GTPase-activating protein ASAP1 regulates the dynamics of filamentous actin-based structures, including stress fibers, focal adhesions, and circular dorsal ruffles. Here, we have examined the molecular basis for ASAP1 association with actin.

Rigosertib Induces Mitotic Arrest and Apoptosis in RAS-Mutated Rhabdomyosarcoma and Neuroblastoma.

Relapsed pediatric rhabdomyosarcomas (RMS) and neuroblastomas (NBs) have a poor prognosis despite multimodality therapy. In addition, the current standard of care for these cancers includes vinca alkaloids that have severe toxicity profiles, further underscoring the need for novel therapies for these malignancies. Here, we show that the small-molecule rigosertib inhibits the growth of RMS and NB cell lines by arresting cells in mitosis, which leads to cell death.

Interaction of the N terminus of ADP-ribosylation factor with the PH domain of the GTPase-activating protein ASAP1 requires phosphatidylinositol 4,5-bisphosphate.

Arf GAP with Src homology 3 domain, ankyrin repeat, and pleckstrin homology (PH) domain 1 (ASAP1) is a multidomain GTPase-activating protein (GAP) for ADP-ribosylation factor (ARF)-type GTPases. ASAP1 affects integrin adhesions, the actin cytoskeleton, and invasion and metastasis of cancer cells. ASAP1's cellular function depends on its highly-regulated and robust ARF GAP activity, requiring both the PH and the ARF GAP domains of ASAP1, and is modulated by phosphatidylinositol 4,5-bisphosphate (PIP).

Molecular Basis for Cooperative Binding of Anionic Phospholipids to the PH Domain of the Arf GAP ASAP1.

We have defined the molecular basis for association of the PH domain of the Arf GAP ASAP1 with phospholipid bilayers. Structures of the unliganded and dibutyryl PtdIns(4,5)P2-bound PH domain were solved. PtdIns(4,5)P2 made contact with both a canonical site (C site) and an atypical site (A site).

The Arf6 GTPase-activating proteins ARAP2 and ACAP1 define distinct endosomal compartments that regulate integrin α5β1 traffic.

Arf6 and the Arf6 GTPase-activating protein (GAP) ACAP1 are established regulators of integrin traffic important to cell adhesion and migration. However, the function of Arf6 with ACAP1 cannot explain the range of Arf6 effects on integrin-based structures. We propose that Arf6 has different functions determined, in part, by the associated Arf GAP.

Approaches to studying Arf GAPs in cells: in vitro assay with isolated focal adhesions.

The Arf GAPs are a family of proteins with a common catalytic function of hydrolyzing GTP bound to ADP-ribosylation factors (Arf) with proposed cellular functions that are diverse (Inoue and Randazzo, 2007; Kahn et al., 2008). Understanding the biochemistry of the Arf GAPs is valuable for designing and interpreting experiments using standard cell biology techniques described elsewhere.

GTP-binding protein-like domain of AGAP1 is protein binding site that allosterically regulates ArfGAP protein catalytic activity.

AGAPs are a subtype of Arf GTPase-activating proteins (GAPs) with 11 members in humans. In addition to the Arf GAP domain, the proteins contain a G-protein-like domain (GLD) with homology to Ras superfamily proteins and a PH domain. AGAPs bind to clathrin adaptors, function in post Golgi membrane traffic, and have been implicated in glioblastoma.

ArfGAP1 promotes COPI vesicle formation by facilitating coatomer polymerization.

The role of ArfGAP1 in COPI vesicle biogenesis has been controversial. In work using isolated Golgi membranes, ArfGAP1 was found to promote COPI vesicle formation. In contrast, in studies using large unilamellar vesicles (LUVs) as model membranes, ArfGAP1 functioned as an uncoating factor inhibiting COPI vesicle formation.

ARFGAP1 promotes AP-2-dependent endocytosis.

COPI (coat protein I) and the clathrin-AP-2 (adaptor protein 2) complex are well-characterized coat proteins, but a component that is common to these two coats has not been identified. The GTPase-activating protein (GAP) for ADP-ribosylation factor 1 (ARF1), ARFGAP1, is a known component of the COPI complex. Here, we show that distinct regions of ARFGAP1 interact with AP-2 and coatomer (components of the COPI complex).

ARAP1 association with CIN85 affects epidermal growth factor receptor endocytic trafficking.

Background Information: ARAP1 is an Arf (ADP-ribosylation factor)-directed GAP (GTPase-activating protein) that inhibits the trafficking of EGFR (epidermal growth factor receptor) to the early endosome. To further understand the function of ARAP1, we sought to identify proteins that interact with ARAP1.

Results: Here we report that ARAP1 associates with the CIN85 (Cbl-interacting protein of 85 kDa).

Regulation of membrane trafficking and organ separation by the NEVERSHED ARF-GAP protein.

Cell separation, or abscission, is a highly specialized process in plants that facilitates remodeling of their architecture and reproductive success. Because few genes are known to be essential for organ abscission, we conducted a screen for mutations that alter floral organ shedding in Arabidopsis. Nine recessive mutations that block shedding were found to disrupt the function of an ADP-ribosylation factor-GTPase-activating protein (ARF-GAP) we have named NEVERSHED (NEV).

Contribution of AZAP-Type Arf GAPs to cancer cell migration and invasion.

Arf GAPs are a family of proteins with a common catalytic domain that induces hydrolysis of GTP bound to the small GTP-binding protein Arf. The proteins are otherwise structurally diverse. Several subtypes of Arf GAPs have been found to be targets of oncogenes and to control cell proliferation and cell migration.

Dynamic interaction between Arf GAP and PH domains of ASAP1 in the regulation of GAP activity.

ASAP family Arf GAPs induce the hydrolysis of GTP bound to the Ras superfamily protein Arf1, regulate cell adhesion and migration and have been implicated in carcinogenesis. The ASAP proteins have a core catalytic domain of PH, Arf GAP and Ank repeat domains. The PH domain is necessary for both biological and catalytic functions of ASAP1 and has been proposed to be integrally folded with the Arf GAP domain.

Kinetic analysis of Arf GAP1 indicates a regulatory role for coatomer.

Arf GAPs are a family of enzymes that catalyze the hydrolysis of GTP bound to Arf. Arf GAP1 is one member of the family that has a critical role in membrane traffic at the Golgi apparatus. Two distinct models for the regulation of Arf GAP1 in membrane traffic have been proposed.

Regulation of Arf6 and ACAP1 signaling by the PTB-domain-containing adaptor protein GULP.

The GTPase Arf6 regulates multiple cellular processes, including endocytosis, secretion, phagocytosis, cell adhesion, and cell migration [1, 2]. The Arf6-specific GAP ACAP1 is a negative regulator of Arf6-mediated signaling [3-7]. However, regulation of ACAP1- and Arf6-mediated signaling by other cellular proteins is not well understood.

Kinetic analysis of GTP hydrolysis catalysed by the Arf1-GTP-ASAP1 complex.

Arf (ADP-ribosylation factor) GAPs (GTPase-activating proteins) are enzymes that catalyse the hydrolysis of GTP bound to the small GTP-binding protein Arf. They have also been proposed to function as Arf effectors and oncogenes. We have set out to characterize the kinetics of the GAP-induced GTP hydrolysis using a truncated form of ASAP1 [Arf GAP with SH3 (Src homology 3) domain, ankyrin repeats and PH (pleckstrin homology) domains 1] as a model.