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.     

ARAP1 regulates EGF receptor trafficking and signalling

The activation state of the EGF receptor (EGF-R) is tightly controlled in the cell so as to prevent excessive signalling, with the dangerous consequences that this would have on cell growth and proliferation. This control occurs at different levels, with a key level being the trafficking and degradation of the EGF-R itself. Multiple guanosine triphosphatases belonging to the Arf, Rab and Rho families and their accessory proteins have key roles in these processes. In this study, we have identified ARAP1, a multidomain protein with both Arf GTPase-activating protein (GAP) and Rho GAP activities, as a novel component of the machinery that controls the trafficking and signalling of the EGF-R. We show that ARAP1 localizes to multiple cell compartments, including the Golgi complex, as previously reported, and endosomal compartments, where it is enriched in the internal membranes of multivesicular bodies. ARAP1 distribution is controlled by its phosphorylation and by its interactions with the 3- and 4-phosphorylated phosphoinositides through its five PH domains. We provide evidence that ARAP1 controls the late steps of the endocytic trafficking of the EGF-R, with ARAP1 knockdown leading to EGF-R accumulation in a sorting/late endosomal compartment and to the inhibition of EGF-R degradation that is accompanied by prolonged signalling.     

ARAP1 regulates the ring size of circular dorsal ruffles through Arf1 and Arf5

Small guanosine triphosphatase (GTPase) ADP-ribosylation factors (Arfs) regulate membrane traffic and actin reorganization under the strict control of GTPase-activating proteins (GAPs). ARAP1 (Arf GAP with Rho GAP domain, ankyrin repeat, and PH domain 1) is an Arf GAP molecule with multiple PH domains that recognize phosphatidylinositol 3,4,5-trisphosphate. We found that growth factor stimulation induced localization of ARAP1 to an area of the plasma membrane inside the ring structure of circular dorsal ruffles (CDRs). Moreover, expression of ARAP1 increased the size of the CDR filamentous-actin ring in an Arf GAP activity-dependent manner, whereas smaller CDRs were formed by ARAP1 knockdown. In addition, expression of a dominant-negative mutant of Arf1 and Arf5, the substrates of ARAP1, expanded the size of CDRs, suggesting that the two Arf isoforms regulate ring structure downstream of ARAP1. Therefore our results reveal a novel molecular mechanism of CDR ring size control through the ARAP1-Arf1/5 pathway.     

ARAP2 Signals through Arf6 and Rac1 to Control Focal Adhesion Morphology

Focal adhesions (FAs) are dynamic structures that connect the actin cytoskeleton with the extracellular matrix. At least six ADP-ribosylation factor (Arf) GTPase-activating proteins (GAPs), including ARAP2 (an Arf6 GAP), are implicated in regulation of FAs but the mechanisms for most are not well defined. Although Rac1 has been reported to function downstream of Arf6 to control membrane ruffling and cell migration, this pathway has not been directly examined as a regulator of FAs. Here we test the hypothesis that ARAP2 promotes the growth of FAs by converting Arf6·GTP to Arf6·GDP thereby preventing the activation of the Rho family GTP-binding protein Rac1. Reduced expression of ARAP2 decreased the number and size of FAs in cells and increased cellular Arf6·GTP and Rac1·GTP levels. Overexpression of ARAP2 had the opposite effects. The effects of ARAP2 on FAs and Rac1 were dependent on a functional ArfGAP domain. Constitutively active Arf6 affected FAs in the same way as did reduced ARAP2 expression and dominant negative mutants of Arf6 and Rac1 reversed the effect of reduced ARAP2 expression. However, neither dominant negative Arf6 nor Rac1 had the same effect as ARAP2 overexpression. We conclude that changes in Arf6 and Rac1 activities are necessary but not sufficient for ARAP2 to promote the growth of FAs and we speculate that ARAP2 has additional functions that are effector in nature to promote or stabilize FAs.     

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.     

Golgi-localized GAP for Cdc42 functions downstream of ARF1 to control Arp2/3 complex and F-actin dynamics

The small GTP-binding ADP-ribosylation factor 1 (ARF1) acts as a master regulator of Golgi structure and function through the recruitment and activation of various downstream effectors. It has been proposed that members of the Rho family of small GTPases also control Golgi function in coordination with ARF1, possibly through the regulation of Arp2/3 complex and actin polymerization on Golgi membranes. Here, we identify ARHGAP10--a novel Rho GTPase-activating protein (Rho-GAP) that is recruited to Golgi membranes through binding to GTP-ARF1. We show that ARHGAP10 functions preferentially as a GAP for Cdc42 and regulates the Arp2/3 complex and F-actin dynamics at the Golgi through the control of Cdc42 activity. Our results establish a role for ARHGAP10 in Golgi structure and function at the crossroads between ARF1 and Cdc42 signalling pathways.     

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.     

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.     

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.     

ARAP1 regulates endocytosis of EGFR

Signaling through the EGF receptor is regulated by endocytosis. ARAP1 is a protein with Arf guanosine triphosphatase-activating protein (GAP) and Rho GAP domains. We investigated the role of ARAP1 in EGF receptor endocytic trafficking. Following EGF treatment of cells, ARAP1 rapidly and transiently associated with the edge of the cell and punctate structures containing Rab5, rabaptin 5 and EGFR but not early embryonic antigen 1 (EEA1). EGF associated with the ARAP1-positive punctate structures prior to EEA1-positive early endosomes. Recruitment of ARAP1 to the punctate structures required active Rab5 and an additional signal from EGFR. Decreasing ARAP1 levels with small interfering RNA accelerated association of EGF with EEA1 endosomes and degradation of EGFR. Phosphorylation of extracellular-signal-regulated kinase (ERK) and c-Jun-amino-terminal kinase (JNK) was diminished and more transient in cells with reduced levels of ARAP1 than in controls. Based on these findings, we propose that ARAP1 regulates the endocytic traffic of EGFR and, consequently, the rate of EGFR signal attenuation.     

Identification of ARAP3, a novel PI3K effector regulating both Arf and Rho GTPases, by selective capture on phosphoinositide affinity matrices.

We show that matrices carrying the tethered homologs of natural phosphoinositides can be used to capture and display multiple phosphoinositide binding proteins in cell and tissue extracts. We present the mass spectrometric identification of over 20 proteins isolated by this method, mostly from leukocyte extracts: they include known and novel proteins with established phosphoinositide binding domains and also known proteins with surprising and unusual phosphoinositide binding properties. One of the novel PtdIns(3,4,5)P3 binding proteins, ARAP3, has an unusual domain structure, including five predicted PH domains. We show that it is a specific PtdIns(3,4,5)P3/PtdIns(3,4)P2-stimulated Arf6 GAP both in vitro and in vivo, and both its Arf GAP and Rho GAP domains cooperate in mediating PI3K-dependent rearrangements in the cell cytoskeleton and cell shape.     

Targeting of Arf-1 to the early Golgi by membrin, an ER-Golgi SNARE

Arf and Rab family GTPases regulate membrane traffic in cells, yet little is known about how they are targeted to distinct organelles. To identify sequences in Arf-1 necessary for Golgi targeting, we examined the localization of chimeras between Arf-1 and Arf-6. Here, we identify a 16-amino acid sequence in Arf-1 that specifies Golgi targeting and contains a motif (MXXE) that is important for Arf-1 binding to membrin, an ER-Golgi SNARE protein. The MXXE motif is conserved in all Arfs known to localize to the Golgi and enables Arf-1 to localize to the early Golgi. Arf-1 lacking these 16 aa can still localize to the late Golgi where it displays a more rapid Golgi-cytosol cycle than wild-type Arf-1. These studies suggest that membrin recruits Arf-1 to the early Golgi and reveal distinct kinetic cycles for Arf-1 at early and late Golgi determined by different sets of Arf regulators and effectors.     

Sorting of Golgi resident proteins into different subpopulations of COPI vesicles: a role for ArfGAP1.

We present evidence for two subpopulations of coatomer protein I vesicles, both containing high amounts of Golgi resident proteins but only minor amounts of anterograde cargo. Early Golgi proteins p24alpha2, beta1, delta1, and gamma3 are shown to be sorted together into vesicles that are distinct from those containing mannosidase II, a glycosidase of the medial Golgi stack, and GS28, a SNARE protein of the Golgi stack. Sorting into each vesicle population is Arf-1 and GTP hydrolysis dependent and is inhibited by aluminum and beryllium fluoride. Using synthetic peptides, we find that the cytoplasmic domain of p24beta1 can bind Arf GTPase-activating protein (GAP)1 and cause direct inhibition of ArfGAP1-mediated GTP hydrolysis on Arf-1 bound to liposomes and Golgi membranes. We propose a two-stage reaction to explain how GTP hydrolysis constitutes a prerequisite for sorting of resident proteins, yet becomes inhibited in their presence.     

Activation of type I phosphatidylinositol 4-phosphate 5-kinase isoforms by the Rho GTPases, RhoA, Rac1, and Cdc42

Type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) catalyzes the formation of the phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP(2)), which is implicated in many cellular processes. The Rho GTPases, RhoA and Rac1, have been shown previously to activate PIP5K and to bind PIP5K. Three type I PIP5K isoforms (Ialpha,Ibeta, and Igamma) have been identified; however, it is unclear whether these isoforms are differentially or even sequentially regulated by Rho GTPases. Here we show that RhoA and Rac1, as well as Cdc42, but not the Ras-like GTPases, RalA and Rap1A, markedly stimulate PIP(2) synthesis by all three PIP5K isoforms expressed in human embryonic kidney 293 cells, both in vitro and in vivo. RhoA-stimulated PIP(2) synthesis by the PIP5K isoforms was mediated by the RhoA effector, Rho-kinase. Stimulation of PIP5K isoforms by Rac1 and Cdc42 was apparently independent of and additive with RhoA- and Rho-kinase, as shown by studies with C3 transferase and Rho-kinase mutants. RhoA, and to a lesser extent Rac1, but not Cdc42, interacted in a nucleotide-independent form with all three PIP5K isoforms. Binding of PIP5K isoforms to GTP-bound, but not GDP-bound, RhoA could be displaced by Rho-kinase, suggesting a direct and constitutive PIP5K-Rho GTPase binding, which, however, does not trigger PIP5K activation. In summary, our findings indicate that synthesis of PIP(2) by the three PIP5K isoforms is controlled by RhoA, acting via Rho-kinase, as well as Rac1 and Cdc42, implicating that regulation of PIP(2) synthesis has a central position in signaling by these three Rho GTPases.     

Activation of rac and cdc42 video imaged by fluorescent resonance energy transfer-based single-molecule probes in the membrane of living cells

Rho family G proteins, including Rac and Cdc42, regulate a variety of cellular functions such as morphology, motility, and gene expression. We developed fluorescent resonance energy transfer-based probes which monitored the local balance between the activities of guanine nucleotide exchange factors and GTPase-activating proteins for Rac1 and Cdc42 at the membrane. These probes, named Raichu-Rac and Raichu-Cdc42, consisted of a Cdc42- and Rac-binding domain of Pak, Rac1 or Cdc42, a pair of green fluorescent protein mutants, and a CAAX box of Ki-Ras. With these probes, we video imaged the Rac and Cdc42 activities. In motile HT1080 cells, activities of both Rac and Cdc42 gradually increased toward the leading edge and decreased rapidly when cells changed direction. Under a higher magnification, we observed that Rac activity was highest immediately behind the leading edge, whereas Cdc42 activity was most prominent at the tip of the leading edge. Raichu-Rac and Raichu-Cdc42 were also applied to a rapid and simple assay for the analysis of putative guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) in living cells. Among six putative GEFs and GAPs, we identified KIAA0362/DBS as a GEF for Rac and Cdc42, KIAA1256 as a GEF for Cdc42, KIAA0053 as a GAP for Rac and Cdc42, and KIAA1204 as a GAP for Cdc42. In conclusion, use of these single-molecule probes to determine Rac and Cdc42 activity will accelerate the analysis of the spatiotemporal regulation of Rac and Cdc42 in a living cell.     

Aeromonas exoenzyme T of Aeromonas salmonicida is a bifunctional protein that targets the host cytoskeleton

Type III protein secretion has been shown recently to be important in the virulence of the fish pathogen Aeromonas salmonicida. The ADP-ribosylating toxin Aeromonas exoenzyme T (AexT) is one effector protein targeted for secretion via this system. In this study, we identified muscular and nonmuscular actin as substrates of the ADP-ribosylating activity of AexT. Furthermore, we show that AexT also functions as a GTPase-activating protein (GAP), displaying GAP activity against monomeric GTPases of the Rho family, specifically Rho, Rac, and Cdc42. Transfection of fish cells with wild type AexT resulted in depolymerization of the actin cytoskeleton and cell rounding. Point mutations within either the GAP or the ADP-ribosylating active sites of AexT (Arg-143 as well as Glu-398 and Glu-401, respectively) abolished enzymatic activity, yet did not prevent actin filament depolymerization. However, inactivation of the two catalytic sites simultaneously did. These results suggest that both the GAP and ADP-ribosylating domains of AexT contribute to its biological activity. This is the first bacterial virulence factor to be described that has a specific actin ADP-ribosylation activity and GAP activity toward Rho, Rac, and Cdc42, both enzymatic activities contributing to actin filament depolymerization.     

Understanding the catalytic mechanism of GTPase-activating proteins: demonstration of the importance of switch domain stabilization in the stimulation of GTP hydrolysis

Cdc42, a member of the Rho family of GTP-binding proteins, has been implicated in a variety of biological activities, including the organization of the actin cytoskeleton, changes in cell morphology and motility, intracellular trafficking, cell cycle progression, and cellular transformation. The cycling of Cdc42 between its on (GTP-bound) and off (GDP-bound) states is essential for its stimulation of cell growth and transformation, with an important aspect of this cycle being the regulation of the GTP hydrolytic activity of Cdc42 by its GTPase-activating protein (Cdc42GAP). On the basis of the structural determinations of the Cdc42-Cdc42GAP complex, as well as the Ras-RasGAP complex, it has been proposed that an arginine residue provided by the GAP (called the "arginine finger") stabilizes charges developing on the guanine nucleotide during the transition state for GTP hydrolysis and is an important contributor to GAP-stimulated catalysis. However, the 85 kDa regulatory subunit (p85) of the phosphoinositide 3-kinase (PI-3K) is homologous with the Cdc42GAP and contains the essential arginine residue, but is ineffective as a GAP. This argues that the introduction of the arginine finger is insufficient for GAP activity and that the GAP must fulfill an additional function, one possibility being the engagement and stabilization of the conformationally sensitive switch regions of Cdc42. In the study presented here, we have tested this idea by examining three residues within the Cdc42GAP, which are missing in the GAP homology domain of the 85 kDa regulatory subunit (p85) of the PI 3-kinase and are involved in specific interactions with switch domain residues of Cdc42. We show that the mutation of all three residues, as well as individual mutations of each of these residues, yields GAPs that are defective in stimulating GTP hydrolysis. We further demonstrate that the switch I residue tyrosine 32 plays an important role in GAP interactions and in the regulation of both intrinsic and GAP-stimulated GTP hydrolysis. Taken together, these findings indicate that stabilizing the switch domains of GTP-binding proteins is an important part of GAP-stimulated catalysis, and that the inability of p85 to participate in these interactions may at least in part explain its ineffectiveness as a GAP.     

Detection of Rho GEF and GAP activity through a sensitive split luciferase assay system

Rho GTPases regulate the assembly of cellular actin structures and are activated by GEFs (guanine-nucleotide-exchange factors) and rendered inactive by GAPs (GTPase-activating proteins). Using the Rho GTPases Cdc42, Rac1 and RhoA, and the GTPase-binding portions of the effector proteins p21-activated kinase and Rhophilin1, we have developed split luciferase assays for detecting both GEF and GAP regulation of these GTPases. The system relies on purifying split luciferase fusion proteins of the GTPases and effectors from bacteria, and our results show that the assays replicate GEF and GAP specificities at nanomolar concentrations for several previously characterized Rho family GEFs (Dbl, Vav2, Trio and Asef) and GAPs [p190, Cdc42 GAP and PTPL1-associated RhoGAP]. The assay detected activities associated with purified recombinant GEFs and GAPs, cell lysates expressing exogenous proteins, and immunoprecipitates of endogenous Vav1 and p190. The results demonstrate that the split luciferase system provides an effective sensitive alternative to radioactivity-based assays for detecting GTPase regulatory protein activities and is adaptable to a variety of assay conditions.