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. Using a combination of structural modeling, mutagenesis, and in vitro and cell-based assays, we identify a putative-binding interface between the N-Bin-Amphiphysin-Rvs (BAR) domain of ASAP1 and actin filaments. We found that neutralization of charges and charge reversal at positions 75, 76, and 79 of ASAP1 reduced the binding of ASAP1 BAR-pleckstrin homology tandem to actin filaments and abrogated actin bundle formation in vitro. In addition, overexpression of actin-binding defective ASAP1 BAR-pleckstrin homology [K75, K76, K79] mutants prevented cellular actin remodeling in U2OS cells. Exogenous expression of [K75E, K76E, K79E] mutant of full-length ASAP1 did not rescue the reduction of cellular actin fibers consequent to knockdown of endogenous ASAP1. Taken together, our results support the hypothesis that the lysine-rich cluster in the N-BAR domain of ASAP1 is important for regulating actin filament organization.

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. Our data indicate that rigosertib, like the vinca alkaloids, exerts its effects mainly by interfering with mitotic spindle assembly. Although rigosertib has the ability to inhibit oncogenic RAS signaling, we provide evidence that rigosertib does not induce cell death through inhibition of the RAS pathway in RAS-mutated RMS and NB cells. However, the combination of rigosertib and the MEK inhibitor trametinib, which has efficacy in RAS-mutated tumors, synergistically inhibits the growth of an RMS cell line, suggesting a new avenue for combination therapy. Importantly, rigosertib treatment delays tumor growth and prolongs survival in a xenograft model of RMS. In conclusion, rigosertib, through its impact on the mitotic spindle, represents a potential therapeutic for RMS.

The ArfGAP ASAP1 Controls Actin Stress Fiber Organization via Its N-BAR Domain.

ASAP1 is a multi-domain ArfGAP that controls cell migration, spreading, and focal adhesion dynamics. Although its GAP activity contributes to remodeling of the actin cytoskeleton, it does not fully explain all cellular functions of ASAP1. Here we find that ASAP1 regulates actin filament assembly directly through its N-BAR domain and controls stress fiber maintenance. ASAP1 depletion caused defects in stress fiber organization. Conversely, overexpression of ASAP1 enhanced actin remodeling. The BAR-PH fragment was sufficient to affect actin. ASAP1 with the BAR domain replaced with the BAR domain of the related ACAP1 did not affect actin. The BAR-PH tandem of ASAP1 bound and bundled actin filaments directly, whereas the presence of the ArfGAP and the C-terminal linker/SH3 domain reduced binding and bundling of filaments by BAR-PH. Together these data provide evidence that ASAP1 may regulate the actin cytoskeleton through direct interaction of the BAR-PH domain with actin filaments.

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 (PIP2). The mechanistic basis of PIP2-stimulated GAP activity is incompletely understood. Here, we investigated whether PIP2 controls binding of the N-terminal extension of ARF1 to ASAP1's PH domain and thereby regulates its GAP activity. Using [Δ17]ARF1, lacking the N terminus, we found that PIP2 has little effect on ASAP1's activity. A soluble PIP2 analog, dioctanoyl-PIP2 (diC8PIP2), stimulated GAP activity on an N terminus-containing variant, [L8K]ARF1, but only marginally affected activity on [Δ17]ARF1. A peptide comprising residues 2-17 of ARF1 ([2-17]ARF1) inhibited GAP activity, and PIP2-dependently bound to a protein containing the PH domain and a 17-amino acid-long interdomain linker immediately N-terminal to the first ß-strand of the PH domain. Point mutations in either the linker or the C-terminal α-helix of the PH domain decreased [2-17]ARF1 binding and GAP activity. Mutations that reduced ARF1 N-terminal binding to the PH domain also reduced the effect of ASAP1 on cellular actin remodeling. Mutations in the ARF N terminus that reduced binding also reduced GAP activity. We conclude that PIP2 regulates binding of ASAP1's PH domain to the ARF1 N terminus, which may partially regulate GAP activity.

Arf GAPs and molecular motors.

Arf GTPase-activating proteins (Arf GAPs) were first identified as regulators of the small GTP-binding proteins ADP-ribosylation factors (Arfs). The Arf GAPs are a large family of proteins in metazoans, outnumbering the Arfs that they regulate. The members of the Arf GAP family have complex domain structures and some have been implicated in particular cellular functions, such as cell migration, or with particular pathologies, such as tumor invasion and metastasis. The specific effects of Arfs sometimes depend on the Arf GAP involved in their regulation. These observations have led to speculation that the Arf GAPs themselves may affect cellular activities in capacities beyond the regulation of Arfs. Recently, 2 Arf GAPs, ASAP1 and AGAP1, have been found to bind directly to and influence the activity of myosins and kinesins, motor proteins associated with filamentous actin and microtubules, respectively. The Arf GAP-motor protein interaction is critical for cellular behaviors involving the actin cytoskeleton and microtubules, such as cell migration and other cell movements. Arfs, then, may function with molecular motors through Arf GAPs to regulate microtubule and actin remodeling.

ARAP2 inhibits Akt independently of its effects on focal adhesions.

BACKGROUND INFORMATION: ARAP2, an Arf GTPase-activating protein (Arf GAP) that binds to adaptor protein with PH domain, PTB domain and leucine zipper motifs 1 (APPL1), regulates focal adhesions (FAs). APPL1 affects FA dynamics by regulating Akt. Here, we tested the hypothesis that ARAP2 affects FAs in part by regulating Akt through APPL1. RESULTS: We found that ARAP2 controlled FA dynamics dependent on its enzymatic Arf GAP activity. In some cells, ARAP2 also regulated phosphoAkt (pAkt) levels. However, ARAP2 control of FAs did not require Akt and conversely, the effects on pAkt were independent of FAs. Reducing ARAP2 expression reduced the size and number of FAs in U118, HeLa and MDA-MB-231 cells. Decreasing ARAP2 expression increased pAkt in U118 cells and HeLa cells and overexpressing ARAP2 decreased pAkt in U118 cells; in contrast, ARAP2 had no effect on pAkt in MDA-MB-231 cells. An Akt inhibitor did not block the effect of reduced ARAP2 on FAs in U118. Furthermore, the effect of ARAP2 on Akt did not require Arf GAP activity, which is necessary for effects on FAs and integrin traffic. Altering FAs by other means did not induce the same changes in pAkt as those seen by reducing ARAP2 in U118 cells. In addition, we discovered that ARAP2 and APPL1 had co-ordinated effects on pAkt in U118 cells. Reduced APPL1 expression, as for ARAP2, increased pAkt in U118 and the effect of reduced APPL1 expression was reversed by overexpressing ARAP2. Conversely, the effect of reduced ARAP2 expression was reversed by overexpressing APPL1. ARAP2 is an Arf GAP that has previously been reported to affect FAs by regulating Arf6 and integrin trafficking and to bind to the adaptor proteins APPL1. Here, we report that ARAP2 suppresses pAkt levels in cells co-ordinately with APPL1 and independently of GAP activity and its effect on the dynamic behaviour of FAs. CONCLUSIONS: We conclude that ARAP2 affects Akt signalling in some cells by a mechanism independent of FAs or membrane traffic. SIGNIFICANCE: Our results highlight an Arf GAP-independent function of ARAP2 in regulating Akt activity and distinguish the effect of ARAP2 on Akt from that on FAs and integrin trafficking, which requires regulation of Arf6.

Direct Functional Interaction of the Kinesin-13 Family Member Kinesin-like Protein 2A (Kif2A) and Arf GAP with GTP-binding Protein-like, Ankyrin Repeats and PH Domains1 (AGAP1).

The molecular basis for control of the cytoskeleton by the Arf GTPase-activating protein AGAP1 has not been characterized. AGAP1 is composed of G-protein-like (GLD), pleckstrin homology (PH), Arf GAP, and ankyrin repeat domains. Kif2A was identified in screens for proteins that bind to AGAP1. The GLD and PH domains of AGAP1 bound the motor domain of Kif2A. Kif2A increased GAP activity of AGAP1, and a protein composed of the GLD and PH domains of AGAP1 increased ATPase activity of Kif2A. Knockdown (KD) of Kif2A or AGAP1 slowed cell migration and accelerated cell spreading. The effect of Kif2A KD on spreading could be rescued by expression of Kif2A-GFP or FLAG-AGAP1, but not by Kif2C-GFP. The effect of AGAP1 KD could be rescued by FLAG-AGAP1, but not by an AGAP1 mutant that did not bind Kif2A efficiently, ArfGAP1-HA or Kif2A-GFP. Taken together, the results support the hypothesis that the Kif2A·AGAP1 complex contributes to control of cytoskeleton remodeling involved in cell movement.

The Arf GTPase-activating Protein, ASAP1, Binds Nonmuscle Myosin 2A to Control Remodeling of the Actomyosin Network.

ASAP1 regulates F-actin-based structures and functions, including focal adhesions (FAs) and circular dorsal ruffles (CDRs), cell spreading and migration. ASAP1 function requires its N-terminal BAR domain. We discovered that nonmuscle myosin 2A (NM2A) directly bound the BAR-PH tandem of ASAP1in vitro ASAP1 and NM2A co-immunoprecipitated and colocalized in cells. Knockdown of ASAP1 reduced colocalization of NM2A and F-actin in cells. Knockdown of ASAP1 or NM2A recapitulated each other's effects on FAs, cell migration, cell spreading, and CDRs. The NM2A-interacting BAR domain contributed to ASAP1 control of cell spreading and CDRs. Exogenous expression of NM2A rescued the effect of ASAP1 knockdown on CDRs but ASAP1 did not rescue NM2A knockdown defect in CDRs. Our results support the hypothesis that ASAP1 is a positive regulator of NM2A. Given other binding partners of ASAP1, ASAP1 may directly link signaling and the mechanical machinery of cell migration.

Simple in vitro assay of Arf GAPs and preparation of Arf proteins as substrates.

Defining the interaction of Arf GAPs with specific Arfs is important for understanding their functions in the endocytic system. Cell-based approaches have been valuable for identifying Arfs and Arf GAPs active in the endocytic compartment; however, the cell-based assays have some limitations in establishing relationships among the Arfs and ArfGAPs. Here we describe a simple in vitro assay that will provide a means for comparing Arfs as substrates and serve to complement cell-based studies.

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). We hypothesized cooperative binding of PtdIns(4,5)P2 to the C site and a nonspecific anionic phospholipid to the A site. PtdIns(4,5)P2 dependence of binding to large unilamellar vesicles and GAP activity was sigmoidal, consistent with cooperative sites. In contrast, PtdIns(4,5)P2 binding to the PH domain of PLC δ1 was hyperbolic. Mutation of amino acids in either the C or A site resulted in decreased PtdIns(4,5)P2-dependent binding to vesicles and decreased GAP activity. The results support the idea of cooperative phospholipid binding to the C and A sites of the PH domain of ASAP1. We propose that the mechanism underlies rapid switching between active and inactive ASAP1.

The Arf6 GTPase-activating proteins ARAP2 and ACAP1 define distinct endosomal compartments that regulate integrin α5ß1 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. We tested this idea by comparing the Arf6 GAPs ARAP2 and ACAP1. We found that ARAP2 and ACAP1 had opposing effects on apparent integrin ß1 internalization. ARAP2 knockdown slowed, whereas ACAP1 knockdown accelerated, integrin ß1 internalization. Integrin ß1 association with adaptor protein containing a pleckstrin homology (PH) domain, phosphotyrosine-binding (PTB) domain, and leucine zipper motif (APPL)-positive endosomes and EEA1-positive endosomes was affected by ARAP2 knockdown and depended on ARAP2 GAP activity. ARAP2 formed a complex with APPL1 and colocalized with Arf6 and APPL in a compartment distinct from the Arf6/ACAP1 tubular recycling endosome. In addition, although ACAP1 and ARAP2 each colocalized with Arf6, they did not colocalize with each other and had opposing effects on focal adhesions (FAs). ARAP2 overexpression promoted large FAs, but ACAP1 overexpression reduced FAs. Taken together, the data support a model in which Arf6 has at least two sites of opposing action defined by distinct Arf6 GAPs.

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. The following briefly reviews some common approaches for in vivo studies of Arf GAPs and discusses the use of isolated cellular organelles to complement in vivo experiments. Detailed protocols for examining the activity of Arf GAPs in whole cell lysates and in association with isolated focal adhesions are provided.

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. The regulation of AGAPs is largely unexplored. Other enzymes containing GTP binding domains are regulated by nucleotide binding. However, nucleotide binding to AGAPs has not been detected. Here, we found that neither nucleotides nor deleting the GLD of AGAP1 affected catalysis, which led us to hypothesize that the GLD is a protein binding site that regulates GAP activity. Two-hybrid screens identified RhoA, Rac1, and Cdc42 as potential binding partners. Coimmunoprecipitation confirmed that AGAP1 and AGAP2 can bind to RhoA. Binding was mediated by the C terminus of RhoA and was independent of nucleotide. RhoA and the C-terminal peptide from RhoA increased GAP activity specifically for the substrate Arf1. In contrast, a C-terminal peptide from Cdc42 neither bound nor activated AGAP1. Based on these results, we propose that AGAPs are allosterically regulated through protein binding to the GLD domain.

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). Selectively disrupting the interaction of ARFGAP1 with either of these two coat proteins leads to selective inhibition in the corresponding transport pathway. The role of ARFGAP1 in AP-2-regulated endocytosis has mechanistic parallels with its roles in COPI transport, as both its GAP activity and coat function contribute to promoting AP-2 transport.

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). Arg86 and Arg90 of ARAP1 and the SH3 (Src homology 3) domains of CIN85 are necessary for the interaction. We found that a mutant of ARAP1 with reduced affinity for CIN85 does not efficiently rescue the effect of reduced ARAP1 expression on EGFR trafficking to the early endosome. Reduced expression of CIN85 has a similar effect as reduced expression of ARAP1 on traffic of the EGFR. Cbl proteins regulate the endocytic trafficking of the EGFR by mediating ubiquitination of the EGFR. Overexpression of ARAP1 reduced ubiquitination of the EGFR by Cbl and slowed Cbl-dependent degradation of the EGFR. Reduced expression of ARAP1 accelerated degradation of EGFR but did not affect the level of ubiquitination of the receptor that was detected. CONCLUSION: ARAP1 interaction with CIN85 regulates endocytic trafficking of the EGFR and affects ubiquitination of EGFR. We propose a model in which the ARAP1-CIN85 complex drives exit of EGF-EGFR-Cbl complex from a pre-early endosome into a pathway distinct from the early endosome/lysosome pathway.

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. We set out to discriminate between these models. First, we reexamined the effect of ArfGAP1 on LUVs. We found that ArfGAP1 increased the efficiency of coatomer-induced deformation of LUVs. Second, ArfGAP1 and peptides from cargo facilitated self-assembly of coatomer into spherical structures in the absence of membranes, reminiscent of clathrin self-assembly. Third, in vivo, ArfGAP1 overexpression induced the accumulation of vesicles and allowed normal trafficking of a COPI cargo. Taken together, these data support the model in which ArfGAP1 promotes COPI vesicle formation and membrane traffic and identify a function for ArfGAP1 in the assembly of coatomer into COPI.

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). As predicted by its homology to the yeast Age2 ARF-GAP and transcriptional profile, NEV influences other aspects of plant development, including fruit growth. Co-localization experiments carried out with NEV-specific antiserum and a set of plant endomembrane markers revealed that NEV localizes to the trans-Golgi network and endosomes in Arabidopsis root epidermal cells. Interestingly, transmission electron micrographs of abscission zone regions from wild-type and nev flowers reveal defects in the structure of the Golgi apparatus and extensive accumulation of vesicles adjacent to the cell walls. Our results suggest that NEV ARF-GAP activity at the trans-Golgi network and distinct endosomal compartments is required for the proper trafficking of cargo molecules required for cell separation.

Arf GAP2 is positively regulated by coatomer and cargo.

Arf GAP2 is one of four Arf GAPs that function in the Golgi apparatus. We characterized the kinetics of Arf GAP2 and its regulation. Purified Arf GAP2 had little activity compared to purified Arf GAP1. Of the potential regulators we examined, coatomer had the greatest effect, stimulating activity one to two orders of magnitude. The effect was biphasic, with half-maximal activation observed at 50 nM coatomer and activation peaking at approximately 150 nM coatomer. Activation by coatomer was greater for Arf GAP2 than has been reported for Arf GAP1. The effects of phosphoinositides and changes in vesicle curvature on GAP activity were small compared to coatomer; however, both increased coatomer-dependent activity. Peptides from p24 cargo proteins increased Arf GAP2 activity by an additional 2- to 4-fold. The effect of cargo peptide was dependent on coatomer. Overexpressing the cargo protein p25 decreased cellular Arf1*GTP levels. The differential sensitivity of Arf GAP1 and Arf GAP2 to coatomer could coordinate their activities. Based on the common regulatory features of Arf GAP1 and 2, we propose a mechanism for cargo selection in which GTP hydrolysis triggered by cargo binding to the coat protein is coupled to coat polymerization.