Arf GAPs: Gatekeepers of vesicle generation

Arf GAP proteins are a versatile and diverse group of proteins. They control the activity of the GTP-binding proteins of the ARF family by inducing the hydrolysis of GTP that is bound to Arf proteins. The best-studied role of Arf GAPs is in intracellular traffic. In this review, we will focus mainly on the Arf GAPs that play a role in vesicle formation, Arf GAP1, Arf GAP2 and Arf GAP3 and their yeast homologues, Gcs1p and Glo3p. We discuss the roles of Arf GAPs as regulators and effectors for Arf GTP-binding proteins.     

ACAPs are arf6 GTPase-activating proteins that function in the cell periphery

The GTP-binding protein ADP-ribosylation factor 6 (Arf6) regulates endosomal membrane trafficking and the actin cytoskeleton in the cell periphery. GTPase-activating proteins (GAPs) are critical regulators of Arf function, controlling the return of Arf to the inactive GDP-bound state. Here, we report the identification and characterization of two Arf6 GAPs, ACAP1 and ACAP2. Together with two previously described Arf GAPs, ASAP1 and PAP, they can be grouped into a protein family defined by several common structural motifs including coiled coil, pleckstrin homology, Arf GAP, and three complete ankyrin-repeat domains. All contain phosphoinositide-dependent GAP activity. ACAP1 and ACAP2 are widely expressed and occur together in the various cultured cell lines we examined. Similar to ASAP1, ACAP1 and ACAP2 were recruited to and, when overexpressed, inhibited the formation of platelet-derived growth factor (PDGF)-induced dorsal membrane ruffles in NIH 3T3 fibroblasts. However, in contrast with ASAP1, ACAP1 and ACAP2 functioned as Arf6 GAPs. In vitro, ACAP1 and ACAP2 preferred Arf6 as a substrate, rather than Arf1 and Arf5, more so than did ASAP1. In HeLa cells, overexpression of either ACAP blocked the formation of Arf6-dependent protrusions. In addition, ACAP1 and ACAP2 were recruited to peripheral, tubular membranes, where activation of Arf6 occurs to allow membrane recycling back to the plasma membrane. ASAP1 did not inhibit Arf6-dependent protrusions and was not recruited by Arf6 to tubular membranes. The additional effects of ASAP1 on PDGF-induced ruffling in fibroblasts suggest that multiple Arf GAPs function coordinately in the cell periphery.     

Arf GAPs as regulators of the actin cytoskeleton.

The Arf (ADP-ribosylation factor) GAPs (GTPase-activating proteins) are a family of proteins with a common catalytic domain that induces hydrolysis of GTP bound to Arf GTP-binding proteins. At least three groups of multidomain Arf GAPs affect the actin cytoskeleton and cellular activities, such as migration and movement, that depend on the cytoskeleton. One role of the Arf GAPs is to regulate membrane remodelling that accompanies actin polymerization. Regulation of membrane remodelling is mediated in part by the regulation of Arf proteins. However, Arf GAPs also regulate actin independently of effects on membranes or Arf. These functions include acting as upstream regulators of Rho family proteins and providing a scaffold for Rho effectors and exchange factors. With multiple functional elements, the Arf GAPs could integrate signals and biochemical activities that result in co-ordinated changes in actin and membranes necessary for a wide range of cellular functions.     

Arf GAPs as regulators of the actin cytoskeleton

The Arf (ADP-ribosylation factor) GAPs (GTPase-activating proteins) are a family of proteins with a common catalytic domain that induces hydrolysis of GTP bound to Arf GTP-binding proteins. At least three groups of multidomain Arf GAPs affect the actin cytoskeleton and cellular activities, such as migration and movement, that depend on the cytoskeleton. One role of the Arf GAPs is to regulate membrane remodelling that accompanies actin polymerization. Regulation of membrane remodelling is mediated in part by the regulation of Arf proteins. However, Arf GAPs also regulate actin independently of effects on membranes or Arf. These functions include acting as upstream regulators of Rho family proteins and providing a scaffold for Rho effectors and exchange factors. With multiple functional elements, the Arf GAPs could integrate signals and biochemical activities that result in co-ordinated changes in actin and membranes necessary for a wide range of cellular functions.     

Phosphoinositide-dependent activation of the ADP-ribosylation factor GTPase-activating protein ASAP1. Evidence for the pleckstrin homology domain functioning as an allosteric site

The ADP-ribosylation factor (Arf) family of GTP-binding proteins are regulators of membrane traffic and the actin cytoskeleton. Both negative and positive regulators of Arf, the centaurin beta family of Arf GTPase-activating proteins (GAPs) and Arf guanine nucleotide exchange factors, contain pleckstrin homology (PH) domains and are activated by phosphoinositides. To understand how the activities are coordinated, we have examined the role of phosphoinositide binding for Arf GAP function using ASAP1/centaurin beta4 as a model. In contrast to Arf exchange factors, phosphatidylinositol 4, 5-bisphosphate (PtdIns-4,5-P(2)) specifically activated Arf GAP. D3 phosphorylated phosphoinositides were less effective. Activation involved PtdIns-4,5-P(2) binding to the PH domain; however, in contrast to the Arf exchange factors and contrary to predictions based on the current paradigm for PH domains as independently functioning recruitment signals, we found the following: (i) the PH domain was dispensable for targeting to PDGF-induced ruffles; (ii) activation and recruitment could be uncoupled; (iii) the PH domain was necessary for activity even in the absence of phospholipids; and (iv) the Arf GAP domain influenced localization and lipid binding of the PH domain. Furthermore, PtdIns-4,5-P(2) binding to the PH domain caused a conformational change in the Arf GAP domain detected by limited proteolysis. Thus, these data demonstrate that PH domains can function as allosteric sites. In addition, differences from the published properties of the Arf exchange factors suggest a model in which feedforward and feedback loops involving lipid metabolites coordinate GTP binding and hydrolysis by Arf.     

Arf GAPs: multifunctional proteins that regulate membrane traffic and actin remodelling

The ADP-ribosylation factor (Arf) Arf GTPase-activating proteins (GAPs) are a family of proteins that induce hydrolysis of GTP bound to Arf. A conserved domain containing a zinc finger motif mediates catalysis. The substrate, Arf.GTP, affects membrane trafficking and actin remodelling. Consistent with activity as an Arf regulator, the Arf GAPs affect both of these pathways. However, the Arf GAPs are likely to have Arf-independent activities that contribute to their cellular functions. Structures of the Arf GAPs are diverse containing catalytic, protein-protein interaction and lipid interaction domains in addition to the Arf GAP domain. Some Arf GAPs have been identified and characterized on the basis of activities other than Arf GAP. Here, we describe the Arf GAP family, enzymology of some members of the Arf GAP family and known functions of the proteins. The results discussed illustrate roles for both Arf-dependent and -independent activities in the regulation of cellular architecture.     

A BAR domain in the N terminus of the Arf GAP ASAP1 affects membrane structure and trafficking of epidermal growth factor receptor

BACKGROUND: Arf GAPs are multidomain proteins that function in membrane traffic by inactivating the GTP binding protein Arf1. Numerous Arf GAPs contain a BAR domain, a protein structural element that contributes to membrane traffic by either inducing or sensing membrane curvature. We have examined the role of a putative BAR domain in the function of the Arf GAP ASAP1. RESULTS: ASAP1's N terminus, containing the putative BAR domain together with a PH domain, dimerized to form an extended structure that bound to large unilamellar vesicles containing acidic phospholipids, properties that define a BAR domain. A recombinant protein containing the BAR domain of ASAP1, together with the PH and Arf GAP domains, efficiently bent the surface of large unilamellar vesicles, resulting in the formation of tubular structures. This activity was regulated by Arf1*GTP binding to the Arf GAP domain. In vivo, the tubular structures induced by ASAP1 mutants contained epidermal growth factor receptor (EGFR) and Rab11, and ASAP1 colocalized in tubular structures with EGFR during recycling of receptor. Expression of ASAP1 accelerated EGFR trafficking and slowed cell spreading. An ASAP1 mutant lacking the BAR domain had no effect. CONCLUSIONS: The N-terminal BAR domain of ASAP1 mediates membrane bending and is necessary for ASAP1 function. The Arf dependence of the bending activity is consistent with ASAP1 functioning as an Arf effector.     

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. In one model, Arf GAP1 functions in a ternary complex with coat proteins and is inhibited by cargo proteins. In another model, Arf GAP1 is recruited to a membrane surface that has defects created by the increased membrane curvature that accompanies transport vesicle formation. Here we have used kinetic and mutational analysis to test predictions of models of regulation of Arf GAP1. We found that Arf GAP1 has a similar affinity for Arf1.GTP as another Arf GAP, ASAP1, but the catalytic rate is approximately 0.5% that of ASAP1. Coatomer stimulated Arf GAP1 activity; however, different from that predicted from the current model, coatomer affected the K(m) and not the k(cat) values. Effects of most mutations in Arf GAP1 paralleled those in ASAP1. Mutation of an arginine that aligned with an arginine presumed to be catalytic in ASAP1 abrogated activity. Peptide from the cytoplasmic tail of cargo proteins inhibited Arf GAP1; however, the unrelated Arf GAP ASAP1 was also inhibited. The curvature of the lipid bilayer had a small effect on activity of Arf GAP1 under the conditions of our experiments. We conclude that coatomer is an allosteric regulator of Arf GAP1. The relevance of the results to the two models of Arf GAP1-mediated regulation of Arf1 is discussed.     

Arf GTPase-activating protein ASAP1 interacts with Rab11 effector FIP3 and regulates pericentrosomal localization of transferrin receptor-positive recycling endosome

ADP-ribosylation factors (Arfs) and Arf GTPase-activating proteins (GAPs) are key regulators of membrane trafficking and the actin cytoskeleton. The Arf GAP ASAP1 contains an N-terminal BAR domain, which can induce membrane tubulation. Here, we report that the BAR domain of ASAP1 can also function as a protein binding site. Two-hybrid screening identified FIP3, which is a putative Arf6- and Rab11-effector, as a candidate ASAP1 BAR domain-binding protein. Both coimmunoprecipitation and in vitro pulldown assays confirmed that ASAP1 directly binds to FIP3 through its BAR domain. ASAP1 formed a ternary complex with Rab11 through FIP3. FIP3 binding to the BAR domain stimulated ASAP1 GAP activity against Arf1, but not Arf6. ASAP1 colocalized with FIP3 in the pericentrosomal endocytic recycling compartment. Depletion of ASAP1 or FIP3 by small interfering RNA changed the localization of transferrin receptor, which is a marker of the recycling endosome, in HeLa cells. The depletion also altered the trafficking of endocytosed transferrin. These results support the conclusion that ASAP1, like FIP3, functions as a component of the endocytic recycling compartment.     

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. We tested this idea by comparing the Arf6 GAPs ARAP2 and ACAP1. We found that ARAP2 and ACAP1 had opposing effects on apparent integrin β1 internalization. ARAP2 knockdown slowed, whereas ACAP1 knockdown accelerated, integrin β1 internalization. Integrin β1 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.     

Ancient Complexity,Opisthokont Plasticity,and Discovery of the 11th Subfamily of Arf GAP Proteins

The organelle paralogy hypothesis is one model for the acquisition of nonendosymbiotic organelles, generated from molecular evolutionary analyses of proteins encoding specificity in the membrane traffic system. GTPase activating proteins (GAPs) for the ADP‐ribosylation factor (Arfs) GTPases are additional regulators of the kinetics and fidelity of membrane traffic. Here we describe molecular evolutionary analyses of the Arf GAP protein family. Of the 10 subfamilies previously defined in humans, we find that 5 were likely present in the last eukaryotic common ancestor. Of the 3 most recently derived subfamilies, 1 was likely present in the ancestor of opisthokonts (animals and fungi) and apusomonads (flagellates classified as the sister lineage to opisthokonts), while 2 arose in the holozoan lineage. We also propose to have identified a novel ancient subfamily (ArfGAPC2), present in diverse eukaryotes but which is lost frequently, including in the opisthokonts. Surprisingly few ancient domains accompanying the ArfGAP domain were identified, in marked contrast to the extensively decorated human Arf GAPs. Phylogenetic analyses of the subfamilies reveal patterns of single and multiple gene duplications specific to the Holozoa, to some degree mirroring evolution of Arf GAP targets, the Arfs. Conservation, and lack thereof, of various residues in the ArfGAP structure provide contextualization of previously identified functional amino acids and their application to Arf GAP biology in general. Overall, our results yield insights into current Arf GAP biology, reveal complexity in the ancient eukaryotic ancestor and integrate the Arf GAP family into a proposed mechanism for the evolution of nonendosymbiotic organelles.     

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 ≈ 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.     

Arf1 dissociates from the clathrin adaptor GGA prior to being inactivated by Arf GTPase-activating proteins

The effectors of monomeric GTP-binding proteins can influence interactions with GTPase-activating proteins (GAPs) in two ways. In one case, effector and GAP binding to the GTP-binding protein is mutually exclusive. In another case, the GTP-binding protein bound to an effector is the substrate for the GTPase-activating protein. Here predictions for these two mechanisms were tested for the Arf1 effector GGA and ASAP family Arf GAPs. GGA inhibited Arf GAP activity of ASAP1, AGAP1, ARAP1, and Arf GAP1 and inhibited binding of Arf1.GTPgammaS to AGAP1 with K(i) values correlating with the K(d) for the GGA.Arf1 complex. ASAP1 blocked Arf1.GTPgammaS binding to GGA with a K(i) similar to the K(d) for the ASAP.Arf1.GTPgammaS complex. No interaction of GGA with ASAP1 was detected. Consistent with GGA sequestering Arf from GAPs, overexpression of GGA slowed the rate of Arf dissociation from the Golgi apparatus following treatment with brefeldin A. Mutational analysis revealed the amino-terminal alpha-helix and switch I of Arf1 contributed to interaction with both GGA and GAPs. These data exclude the mechanism previously documented for Arf GAP1/coatomer in which Arf1 is inactivated in a tripartite complex. Instead, termination of Arf1 signals mediated through GGA require that Arf1.GTP dissociates from GGA prior to interaction with GAP and consequent hydrolysis of GTP.     

Differences between AGAP1, ASAP1 and Arf GAP1 in substrate recognition: interaction with the N-terminus of Arf1

The Arf GAPs are a structurally diverse group of proteins that catalyze the hydrolysis of GTP bound to Arf1. Here, we directly compare the role of amino acids 2-17 of Arf1, a GTP- and phospholipid-sensitive switch, for interaction with three Arf GAPs: Arf GAP1, AGAP1 and ASAP1. Sequestration of amino acids 2-17 with an antibody inhibited interaction with the three tested Arf GAPs. Examination of Arf1 mutants also indicated that [2-17]Arf1 is a critical structural determinant of interaction with all three Arf GAPs; however, the effect of specific mutations differed among the GAPs. Compared to wild-type Arf1, Arf1 with the amino terminal 13 ([Delta13]Arf1) and 17 amino acids ([Delta17]Arf1) deleted had 200- and 4000-fold reduced interaction with ASAP1 and 150-fold reduced interaction with AGAP1. In contrast, deletion of the amino terminus of Arf reduced interaction with Arf GAP1 by 5-fold. By analysis of point mutants, we found that lysines 15 and 16 had a greater contribution to productive interaction between Arf1, ASAP1 and AGAP1 than between Arf1 and Arf GAP1. Leucine 8 contributed to the interaction with Arf GAP1 but not with ASAP1 and AGAP1. Amino acids 2-17 of Arf1, isolated from the protein, inhibited GAP activity of Arf GAP1, ASAP1 and AGAP1 and bound directly to ASAP1. Taken together, our results indicate that (i) Arf GAPs interact with amino acids 2-17 of Arf1 and (ii) each subgroup of Arf GAPs has a unique interface with Arf1.     

AGAP1, an endosome-associated,phosphoinositide-dependent ADP-ribosylation factor GTPase-activating protein that affects actin cytoskeleton

We have identified three members of the AGAP subfamily of ASAP family ADP-ribosylation factor GTPase-activating proteins (Arf GAPs). In addition to the Arf GAP domain, these proteins contain GTP-binding protein-like, ankyrin repeat and pleckstrin homology domains. Here, we have characterized the ubiquitously expressed AGAP1/KIAA1099. AGAP1 had Arf GAP activity toward Arf1>Arf5>Arf6. Phosphatidylinositol 4,5-bisphosphate and phosphatidic acid synergistically stimulated GAP activity. As found for other ASAP family Arf GAPs, the pleckstrin homology domain was necessary for activity. Deletion of the GTP-binding protein-like domain affected lipid dependence of Arf GAP activity. In vivo effects of AGAP1 were distinct from other ASAP family Arf GAPs. Overexpressed AGAP1 induced the formation of and was associated with punctate structures containing the endocytic markers transferrin and Rab4. AP1 was redistributed from the trans-Golgi to the punctate structures. Like other ASAP family members, AGAP1 overexpression inhibited the formation of PDGF-induced ruffles. However, distinct from other ASAP family members, AGAP1 also induced the loss of actin stress fibers. Thus, AGAP1 is a phosphoinositide-dependent Arf GAP that impacts both the endocytic compartment and actin.     

ELMOD2 is an Arl2 GTPase-activating protein that also acts on Arfs

Regulatory GTPases in the Ras superfamily employ a cycle of alternating GTP binding and hydrolysis, controlled by guanine nucleotide exchange factors and GTPase-activating proteins (GAPs), as essential features of their actions in cells. Studies of these GAPs and guanine nucleotide exchange factors have provided important insights into our understanding of GTPase signaling and biology. Within the Ras superfamily, the Arf family is composed of 30 members in mammals, including 22 Arf-like (Arl) proteins. Much less is known about the mechanisms of cell regulation by Arls than by Arfs. We report the purification from bovine testis of an Arl2 GAP and its identity as ELMOD2, a protein with no previously described function. ELMOD2 is one of six human proteins that contain an ELMO domain, and a second member, ELMOD1, was also found to have Arl2 GAP activity. Surprisingly, ELMOD2 also exhibited GAP activity against Arf proteins even though it does not contain the canonical Arf GAP sequence signature. The broader specificity of ELMOD2, as well as the previously described role for ELMO1 and ELMO2 in linking Arf6 and Rac1 signaling, suggests that ELMO family members may play a more general role in integrating signaling pathways controlled by Arls and other GTPases.     

ARAP3 is a PI3K- and rap-regulated GAP for RhoA

Rho and Arf family small GTPases are well-known regulators of cellular actin dynamics. We recently identified ARAP3, a member of the ARAP family of dual GTPase activating proteins (GAPs) for Arf and Rho family GTPases, in a screen for PtdIns(3,4,5)P(3) binding proteins. PtdIns(3,4,5)P(3) is the lipid product of class I phosphoinositide 3OH-kinases (PI3Ks) and is a signaling molecule used by growth factor receptors and integrins in the regulation of cell dynamics. We report here that as a Rho GAP, ARAP3 prefers RhoA as a substrate and that it can be activated in vitro by the direct binding of Rap proteins to a neighbouring Ras binding domain (RBD). This activation by Rap is GTP dependent and specific for Rap versus other Ras family members. We found no evidence for direct regulation of ARAP3's Rho GAP activity by PtdIns(3,4,5)P(3) in vitro, but PI3K activity was required for activation by Rap in a cellular context, suggesting that PtdIns(3,4,5)P(3)-dependent translocation of ARAP3 to the plasma membrane may be required for further activation by Rap. Our results indicate that ARAP3 is a Rap-effector that plays an important role in mediating PI3K-dependent crosstalk between Ras, Rho, and Arf family small GTPases.     

Arf GTPase-activating proteins and their potential role in cell migration and invasion

Cell migration is central to normal physiology in embryogenesis, the inflammatory response and wound healing. In addition, the acquisition of a motile and invasive phenotype is an important step in the development of tumors and metastasis. Arf GTPase-activating proteins (GAPs) are nonredundant regulators of specialized membrane surfaces implicated in cell migration. Part of Arf GAP function is mediated by regulating the ADP ribosylation factor (Arf) family GTP-binding proteins. However, Arf GAPs can also function independently of their GAP enzymatic activity, in some cases working as Arf effectors. In this commentary, we discuss examples of Arf GAPs that function either as regulators of Arfs or independently of the GTPase activity to regulate membrane structures that mediate cell adhesion and movement.Key words: Arf GAP, Arf, effector, ADP-ribosylation factor, GTPase-activating protein, focal adhesions, podosomes, invadopodia, cell migrationCell migration involves adhesive structures in which the cell membrane is integrated with the actin cytoskeleton.¹ Cells acquire a spatial asymmetry to enable them to turn intracellular generated forces into a net cell body translocation. With the asymmetry, there is a clear distinction between the cell front and rear. Active membrane processes, including lamellipodia and filopodia, take place primarily around the cell front. Extension of both filopodia and lamellipodia is coupled with local actin polymerization, which generates protrusive force. In some cells, focal complexes form at the leading edge of lamellipodia and filopodia. Focal complexes are specialized surfaces of the plasma membrane that mediate attachment to the substratum, providing traction and allowing the cell edge to protrude. Focal complexes mature with cell migration to form another specialized surface in the plasma membrane, focal adhesions (FAs). FAs localize to the termini of stress fiber bundles and serve in longer-term anchorage at the rear of the cell.² A contractile force is generated at the rear of the cell by the myosin motors to move the cell forward and cell-substratum (extracellular matrix) attachments are released to retract the cell rear. In some cells, podosomes are adhesive structures that mediate cell migration and sometimes invasion.The structures involved in cell migration that are affected by Arf GAPs are FAs, podosomes and invadopodia. FAs contain multiple proteins, including integrins, which are transmembrane proteins.³ The extracellular part of integrins binds to the extracellular matrix. The cytoplasmic domains of integrins associate with multiple signaling proteins as well as proteins that are part of the actin cytoskeleton, thereby coordinating signaling events involved in cell migration and linking the extracellular matrix to the cytoskeleton. Cytoplasmic proteins critical to the function of FAs and that are often used as markers of FAs include vinculin, paxillin and focal adhesion kinase. At least five distinct Arf GAPs have been found to associate with FAs, including GIT1, GIT2, ASAP1, ASAP3 and ARAP2.⁴Podosomes and invadopodia are related structures induced by action of Src (reviewed in ref. ⁵). They contain some proteins in common with FAs, but do have some differences that likely reflect different function and/or regulation. For example, podosomes contain ASAP1 but not ASAP3.⁶ Podosomes and invadopodia have not been examined for the presence of other Arf GAPs. Like FAs, podosomes and invadopodia mediate adhesion to extracellular surfaces. In addition, they are points of degradation of the extracellular matrix and may transfer tension along the extracellular matrix to enable the cell to move. Consistent with the function in motility, podosomes and invadopodia are dynamic structures, turning over in minutes. Podosomes are found in normal physiology of cells including smooth muscle cells, osteoclasts and macrophages and in Src-transformed fibroblasts. Invadopodia are observed in transformed cells, such as cells derived from breast cancers.Two families of GTP-binding proteins within the Ras superfamily, Rho and Arf, are involved in both actin and membrane remodeling. RhoA regulates stress fibers (bundles of actin filaments that traverse the cell and are linked to the extracellular matrix through FAs) and the assembly of FAs.⁷ Rac1 regulates membrane ruffling and lamellipodia formation.⁸ Cdc42 regulates filopodia formation.⁹ Activation of Cdc42 has been shown to lead to the sequential activation of Rac1 and then RhoA in growth factor stimulated fibroblasts.Arf proteins regulate membrane traffic and the actin cytoskeleton.¹⁰ There are six mammalian Arf proteins, divided into three classes based on their amino-acid sequence. Arf12 and 3 are class I, Arf4 and Arf5 are class II and Arf6 is the single member of the class III group. Arf1 and Arf6 have been the most extensively studied. Most work has focused on Arf1 function in the Golgi apparatus and endocytic compartments although Arf1 has been found to affect paxillin recruitment to FAs and trafficking of epidermal growth factor receptor from the plasma membrane. Arf6 affects the endocytic pathway and the peripheral actin cytoskeleton.The function of Rho and Arf family proteins depends on a cycle of binding and hydrolyzing GTP. However, Rho and Arf family proteins have slow intrinsic nucleotide exchange. Rho family proteins have slow intrinsic GTPase activity and Arf family proteins have no detectable intrinsic GTPase activity. The cycle of GTP binding and hydrolysis is driven by accessory proteins called guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Rho family proteins are also regulated by guanine nucleotide dissociation inhibitors, which prevent spontaneous activation in the cytoplasm.Arf GAPs are enzymes that catalyze the hydrolysis of GTP bound to Arf proteins, thereby converting Arf•GTP to Arf•GDP. Thirty-one genes in human encode proteins with Arf GAP domains (Fig. 1). The Arf GAP family is divided into ten subgroups based on domain structure and phylogenetic analysis.¹¹ Six subgroups contain the Arf GAP domain at the N-terminus of the protein. Four groups contain a tandem of a PH, Arf GAP and Ankyrin repeat domains. The Arf GAP nomenclature is mostly based on the protein domain structure. For instance, the ASAP first identified, ASAP1, contains Arf GAP, SH3, Ank repeat and PH domains; ARAPs contain Arf GAP, Rho GAP, Ank repeat and PH domains; ACAPs contain Arf GAP, coiled-coil (later identified as BAR domain), Ank repeat and PH domains; and AGAPs contain Arf GAP, GTP-binding protein-like, Ank repeat and PH domains.Open in a separate windowFigure 1Domain structure of the Arf GAP family. The schematic representation of the ten groups of proteins containing the Arf GAP domain is not drawn to scale. Abbreviations used are: ALPS, ArfGAP1 lipid-packing sensor domain; Ank, Ankyrin repeats; Arf GAP, Arf GTPase activating domain; BAR, Bin/Amphiphysin/Rvs domain; CALM, CALM binding domain; CB, clathrin box; CC, coiled-coiled domain; FG repeats, multiple copies of the XXFG motif; GLD, GTP-binding protein-like domain; PBS, paxillin binding site; PH, pleckstrin homology domain; Pro (PxxP)₃, cluster of three proline-rich (PxxP) motifs; Pro (D/ELPPKP)₈, eigth tandem Prolin-rich (D/ELPPKP) motifs; RA, Ras association motif; Rho GAP, Rho GTPase activating domain; SAM, sterile α-motif; SH3, Src homology 3 domain; SHD, Spa homology domain. *ASAP2 and ASAP3 lack the Pro (D/ELPPKP)₈ motifs. ASAP3 has no SH3 domain. ^&AGAP2 has a splice variant with three N-terminal PxxP motifs, called PIKE-L. @ARAP2 has an inactive Rho GAP domain.The subcellular localization and function of a number of Arf GAPs have been identified. Arf GAP1, Arf GAP2 and Arf GAP3 are found in the Golgi apparatus where they control membrane traffic by regulating Arf1•GTP levels.^12,13 Arf GAP1 has also been proposed to directly contribute to the formation of transport intermediates.¹⁴ SMAPs and AGAP1 and AGAP2 are associated with endosomes and regulate endocytic trafficking.^14,15 ASAPs, ARAPs and Gits are associated with FAs. ASAPs, ARAPs and ACAPs are found in actin-rich membrane ruffles. ASAP1 is also found in invadopodia and podosomes.⁴ We propose that common to all Arf GAPs is that they laterally organize membranes, which maintain surfaces of specialized functions such as FAs and podosomes/invadopodia. Some Arf GAPs function primarily as Arf effectors with the turnover rate of the specialized membrane surface being determined by the catalytic rate of the GAP. Other Arf GAPs function as Arf regulators that integrate several signals.ASAP1 is an example of an Arf GAP that may function as an Arf effector to regulate podosomes and invadopodia. ASAP1 is encoded by a gene on the short arm of chromosome 8. The gene is amplified in aggressive forms of uveal melanoma and cell migration rates correlate with ASAP1 expression levels in uveal melanoma¹⁶ and other cell types. ASAP1 function depends on cycling among four cellular locations, cytosol, FAs, lamellipodia and podosomes/invadopodia. ASAP1 is necessary for the formation of podosomes/invadopodia.^17,18The structural features of ASAP1 that are required to support podosome formation have been examined.^17,18 ASAP1 contains, from the N-terminus, BAR, PH, Arf GAP, Ank repeat, proline rich and SH3 domains (Fig. 2A).¹⁹ There are two major isoforms, ASAP1a and ASAP1b that differ in the proline rich domain. ASAP1a contains three SH3 binding motifs within the proline rich region including an atypical SH3 binding motif with 6 consecutive prolines. The atypical SH3 binding motif is absent in ASAP1b (Fig. 2A). ASAP1 also has a highly conserved tyrosine between the Ank repeat and proline rich domains that is a site of phosphorylation by the oncogene Src.¹⁸Open in a separate windowFigure 2ASAP1 function in podosome and invadopodia formation. (A) Domain structure of ASAP1 splice variants. ASAP1a contains three proline-rich motifs, P1, P2 and P3. P1 and P3 contain a typical (PxxP) motif. P2 contains six prolines. ASAP1b contains only P1 and P3. (B) Model of ASAP1 functioning as an Arf effector to regulate podosome and invadopodia formation. ASAP1 integrates signals from Src, PIP₂ and Arf•GTP. For abbreviations of the domain structure of ASAP1 see Figure 1. Other abbreviations: PIP₂, phosphoinositides 4,5-biphosphate; Arf1, ADP-ribosylation factor 1.The BAR domain is a bundle of 3 α-helices that homodimerizes to form a boomerang-shaped structure.^20,21 BAR domains sense or induce membrane curvature.²⁰ ASAP1 has been found to induce curvature dependent on its BAR domain.²² BAR domains are also protein binding sites.²¹ The BAR domain of ASAP1 binds to FIP3, a Rab11 and Arf6 binding proteins.²³ Arf6-dependent targeting of ASAP1 is likely mediated by FIP3.²³ Deletion of or introduction of point mutations into the BAR domain render ASAP1 inactive in supporting podosome formation. The relative role of membrane tubulation and protein binding in mediating the effect of the BAR domain on podosome formation has not been explored.The SH3 domain of ASAP1 binds to focal adhesion kinase²⁴ and pyk2.²⁵ Either deletion of or introduction of point mutations into the SH3 domain abrogates the ability of ASAP1 to support podosome formation.¹⁸ The molecular basis for the function of the SH3 domain in podosome formation is not known. The proline rich domain binds to Src¹⁹ and CrkL.²⁶ Whether it also binds to cortactin has not been resolved. Reports also conflict regarding the importance of the proline rich domain for podosomes/invadopodia formation.^17,18Three signals impinge on ASAP1 to drive podosome formation (Fig. 2B). A conserved tyrosine between the Ank and proline rich motifs is phosphorylated by Src.^18,25 Mutation of the tyrosine to phenylalanine results in a protein that functions as a dominant negative blocking podosome formation. ASAP1 with the tyrosine changed to glutamate can support podosome formation, but the mutant ASAP1 is not sufficient to drive podosome formation.¹⁸ Based on these results, phosphorylation of the conserved tyrosine is necessary but not sufficient to support podosome formation. Phosphatidylinositol 4,5-bisphosphate (PIP₂) binds to the PH domain, which stimulates GAP activity in vitro.²⁷ ASAP1 with mutations in the PH domain that abrogate binding, does not support podosome formation (Jian, Bharti and Randazzo PA, unpublished observations). Point mutations in the PH domain affect both the K_m and the k_cat for GAP activity. The effect of mutating the PH domain on the ability of ASAP1 to support podosome formation may be consequent to changes in binding Arf1•GTP; it is not likely the result of loss of GAP activity. ASAP1 with a point mutation in the GAP domain that prevents GAP activity but not Arf1•GTP binding is able to support podosome formation whereas a point mutant of ASAP1 that cannot bind Arf1•GTP does not (Jian, Bharti and Randazzo PA, unpublished observations).¹⁸ These data support the idea that ASAP1 integrates three signals, (1) PIP₂, (2) Src and (3) Arf1•GTP. In response to the signals, ASAP1 functions as a scaffold and directly alters the lipid bilayer to create a domain within the plasma membrane that becomes a podosome. In this model, ASAP1 is functioning as an Arf effector and the GAP activity may regulate the turnover of podosomes.ASAP3, another ASAP-type protein, is found in FAs.⁶ Reducing ASAP3 expression also reduces cell migration and invasion of Arf GAPs in cell migration mammary carcinoma cells through matrigel. Although ASAP3 does not affect the ability to form FAs, it does affect stress fiber formation and may affect focal adhesion maturation (Ha, Chen and Randazzo PA, unpublished observations).⁶ The molecular mechanisms underlying the effects of ASAP3 on the cytoskeleton are being examined including the possibility that, like ASAP1, ASAP3 integrates several signals and functions as an Arf effector.ARAPs are examples of Arf GAP family proteins that function as Arf regulators. In common with ASAPs, they integrate a number of signaling pathways and affect the actin cytoskeleton. Three genes encode ARAPs in humans.¹¹ Each of the ARAPs is comprised of a SAM, five PH, Arf GAP, Rho GAP, Ank repeat and Ras association domains. Two of the five PH domains have the consensus sequence for binding to the signaling lipid phosphoinositide 3,4,5-triphosphate (PIP₃); however, when examined for ARAP1, PIP₃ was not involved in membrane targeting (Campa F, Balla and Randazzo PA, unpublished observations).Examination of the role of ARAP2 in FA formation has provided information about the function of the GAP activity in the cellular function of an Arf GAP. ARAP2 selectively uses Arf6 as a substrate and, different from ARAP1 and ARAP3, has an inactive Rho GAP domain. The Rho GAP domain, however, retains the ability to selectively bind to RhoA•GTP. Also different from ARAP1 and ARAP3, ARAP2 associates with FAs. Cells with reduced expression of ARAP2, consequent to siRNA treatment, have fewer FAs and stress fibers and more focal complexes than control cells. The formation of FAs and stress fibers can be restored by expressing recombinant wild type ARAP2. A mutant of ARAP2 that lacks Arf GAP activity, while retaining the ability to bind to Arf6•GTP, cannot restore FA and stress fiber formation. Similarly, expression of a mutant of ARAP2 that is not able to bind RhoA•GTP cannot reverse the effect of reducing expression of endogenous ARAP2.²⁸ These results support the idea that ARAP2 functions as an Arf GAP that is an effector of RhoA.The model of ARAP2 functioning as a RhoA effector can explain the effects of ARAP2 on FAs (Fig. 3). Arf6•GTP is involved in the formation of Rac1•GTP.²⁹ Rac1•GTP drives lamellipodia and focal complex formation. The conversion of focal complexes to FAs is accompanied by an increase in RhoA•GTP and a decrease in Rac1•GTP. ARAP2 could function to mediate the reciprocal changes in RhoA and Rac1. RhoA•GTP formation leads to the activation of ARAP2. As a consequence of Arf6 GAP activity, Arf6•GTP is converted to Arf6•GDP. With reduced Arf6•GTP, Rac1•GTP concentration also decreases.Open in a separate windowFigure 3Model of ARAP2 as an Arf regulator that controls focal adhesion formation. In this model, ARAP2 functions as a RhoA effector. The inactive Rho GAP domain of ARAP2 binds to RhoA•GTP, which contributes to activation of Arf6 GAP activity. ARAP2 hydrolyzes its substrate Arf6•GTP into Arf6•GDP. Subsequent to Arf6•GTP hydrolysis, Rac1•GTP concentration decreases. For abbreviations of the domain structure of ARAP2 see Figure 1.The Arf GAP activity of other ARAPs may also be critical for cellular functions of the protein. Furthermore, the Rho GAP activity is slow for ARAP1 and ARAP3. It is possible that ARAP1 and ARAP3 can function as Rho effectors with an active Rho GAP domain analogously to ASAP1 functioning as an Arf effector. Further definition of the cellular function of ARAP1 and ARAP3 will provide opportunities to test this idea.We have provided two examples of Arf GAPs that affect cell adhesion and migration. In one case, the Arf GAP appears to function as an Arf effector. In the other case, the Arf GAP functions as a regulator of Arf. The difference in function was discerned using Arf GAP mutants. If functioning as an Arf effector, an Arf GAP mutant that can bind Arf•GTP but not induce hydrolysis can reverse the effect of reduced endogenous Arf GAP, whereas a mutant that cannot bind Arf•GTP cannot replace endogenous Arf GAP. When working as an Arf regulator, a mutant that can bind Arf•GTP but not induce GTP hydrolysis cannot replace endogenous Arf GAP. Whether functioning as an effector or regulator, the rate of GAP activity determines the turnover rate of a specialized membrane surface maintained by Arf.The Arf GAPs have specific sites of action within cells. Some contribute to malignancy, such as ASAP1, ASAP3, AGAP2 and SMAP1.³⁰ The molecular basis of cellular function of each Arf GAPs is distinct. Here, we describe one Arf GAP that functions as an Arf effector and another that functions as an Arf regulator. Each class of Arf GAP has distinct sets of protein binding partners. Furthermore, catalytic mechanism differs among the GAPs. Because of these differences, Arf GAPs may be useful therapeutic targets for cancer therapy.     

Substrate specificities and activities of AZAP family Arf GAPs in vivo

The ADP-ribosylation factor (Arf) GTPases are important regulators of vesicular transport in eukaryotic cells. Like other GTPases, the Arfs require guanine nucleotide exchange factors to facilitate GTP loading and GTPase-activating proteins (GAPs) to promote GTP hydrolysis. Whereas there are only six mammalian Arfs, the human genome encodes over 20 proteins containing Arf GAP domains. A subset of these, referred to as AZAPs (Randazzo PA, Hirsch DS. Cell Signal 16: 401-413, 2004), are characterized by the presence of at least one NH(2)-terminal pleckstrin homology domain and two or more ankyrin repeats following the GAP domain. The substrate specificities of these proteins have been previously characterized by using in vitro assay systems. However, a limitation of such assays is that they may not accurately represent intracellular conditions, including posttranslational modifications, or subcellular compartmentalization. Here we present a systematic analysis of the GAP activity of seven AZAPs in vivo, using an assay for measurement of cellular Arf-GTP (Santy LC, Casanova JE. J Cell Biol 154: 599-610, 2001). In agreement with previous in vitro results, we found that ACAP1 and ACAP2 have robust, constitutive Arf6 GAP activity in vivo, with little activity toward Arf1. In contrast, although ARAP1 was initially reported to be an Arf1 GAP, we found that it acts primarily on Arf6 in vivo. Moreover, this activity appears to be regulated through a mechanism involving the NH(2)-terminal sterile-alpha motif. AGAP1 is unique among the AZAPs in its specificity for Arf1, and this activity is dependent on its NH(2)-terminal GTPase-like domain. Finally, we found that expression of AGAP1 induces a surprising reciprocal activation of Arf6, which suggests that regulatory cross talk exists among Arf isoforms.