Regulation of ASAP1 by phospholipids is dependent on the interface between the PH and Arf GAP domains

ASAP1 is an Arf GAP with a PH domain immediately N-terminal to the catalytic Arf GAP domain. PH domains are thought to regulate enzymes by binding to specific phosphoinositide lipids in membranes, thereby recruiting the enzyme to a site of action. Here, we have examined the functional relationship between the PH and Arf GAP domains. We found that GAP activity requires the cognate PH domain of ASAP1, leading us to hypothesize that the Arf GAP and PH domains directly interact to form the substrate binding site. This hypothesis was supported by the combined results of protection and hydrodynamic studies. We then examined the role of the PH domain in the regulation of Arf GAP activity. The results of saturation kinetics, limited proteolysis, FRET and fluorescence spectrometry support a model in which regulation of the GAP activity of ASAP1 involves a conformational change coincident with recruitment to a membrane surface, and a second conformational change following the specific binding of phosphatidylinositol 4,5-bisphosphate.     

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.     

Mutational analysis of the Arf1*GTP/Arf GAP interface reveals an Arf1 mutant that selectively affects the Arf GAP ASAP1

Arf1 is a GTP binding protein that functions at a number of cellular sites to control membrane traffic and actin remodeling. Arf1 is regulated by site-specific GTPase-activating proteins (GAPs). The combined results of crystallographic and biochemical studies have led to the proposal that Arf1 GAPs differ in the specific interface formed with Arf1. To test this hypothesis, we have used mutagenesis to examine the interaction of three Arf GAPs (ASAP1, AGAP1, and ArfGAP1) with switch 1, switch 2, and alpha helix3 of Arf1. The GAPs were similar in being affected by mutations in switch 1 and 2. However, effects of a mutation within alpha helix3 and specific mutations within switch 1 and 2 differed among the GAPs. The largest differences were observed with a change of isoleucine 46 to aspartate ([I46D]Arf1), which reduced ASAP1-induced catalysis by approximately 10,000-fold but had a 3-fold effect on AGAP1. The reduction was due to an isolated effect on the catalytic rate, k(cat). In vivo [I46D]Arf1 had no detectable effect on the Golgi apparatus but, instead, functioned as a constitutively active mutant in the cell periphery, affecting the localization of ASAP1 and paxillin. Based on our results, we conclude that the contribution of specific residues within switch 1 of Arf to binding and achieving a transition state toward GTP hydrolysis differs among Arf GAPs.     

ASAP3 is a focal adhesion-associated Arf GAP that functions in cell migration and invasion

ASAP3, an Arf GTPase-activating protein previously called DDEFL1 and ACAP4, has been implicated in the pathogenesis of hepatocellular carcinoma. We have examined in vitro and in vivo functions of ASAP3 and compared it to the related Arf GAP ASAP1 that has also been implicated in oncogenesis. ASAP3 was biochemically similar to ASAP1: the pleckstrin homology domain affected function of the catalytic domain by more than 100-fold; catalysis was stimulated by phosphatidylinositol 4,5-bisphosphate; and Arf1, Arf5, and Arf6 were used as substrates in vitro. Like ASAP1, ASAP3 associated with focal adhesions and circular dorsal ruffles. Different than ASAP1, ASAP3 did not localize to invadopodia or podosomes. Cells, derived from a mammary carcinoma and from a glioblastoma, with reduced ASAP3 expression had fewer actin stress fiber, reduced levels of phosphomyosin, and migrated more slowly than control cells. Reducing ASAP3 expression also slowed invasion of mammary carcinoma cells. In contrast, reduction of ASAP1 expression had no effect on migration or invasion. We propose that ASAP3 functions nonredundantly with ASAP1 to control cell movement and may have a role in cancer cell invasion. In comparing ASAP1 and ASAP3, we also found that invadopodia are dispensable for the invasive behavior of cells derived from a mammary carcinoma.     

A PH Domain in the Arf GTPase-activating Protein (GAP) ARAP1 Binds Phosphatidylinositol 3,4,5-Trisphosphate and Regulates Arf GAP Activity Independently of Recruitment to the Plasma Membranes

ARAP1 is a phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P₃)-dependent Arf GTPase-activating protein (GAP) with five PH domains that regulates endocytic trafficking of the epidermal growth factor receptor (EGFR). Two tandem PH domains are immediately N-terminal of the Arf GAP domain, and one of these fits the consensus sequence for PtdIns(3,4,5)P₃ binding. Here, we tested the hypothesis that PtdIns(3,4,5)P₃-dependent recruitment mediated by the first PH domain of ARAP1 regulates the in vivo and in vitro function of ARAP1. We found that PH1 of ARAP1 specifically bound to PtdIns(3,4,5)P₃, but with relatively low affinity (≈1.6 μm), and the PH domains did not mediate PtdIns(3,4,5)P₃-dependent recruitment to membranes in cells. However, PtdIns(3,4,5)P₃ binding to the PH domain stimulated GAP activity and was required for in vivo function of ARAP1 as a regulator of endocytic trafficking of the EGFR. Based on these results, we propose a variation on the model for the function of phosphoinositide-binding PH domains. In our model, ARAP1 is recruited to membranes independently of PtdIns(3,4,5)P₃, the subsequent production of which triggers enzymatic activity.Pleckstrin homology (PH)² domains are a common structural motif encoded by the human genome (1, 2). Approximately 10% of PH domains bind to phosphoinositides. These PH domains are thought to mediate phosphoinositide-dependent recruitment to membranes (1–3). Most PH domains likely have functions other than or in addition to phosphoinositide binding. For example, PH domains have been found to bind to protein and DNA (4–12). In addition, some PH domains have been found to be structurally and functionally integrated with adjacent domains (13, 14). A small fraction of PH domain-containing proteins (about 9% of the human proteins) have multiple PH domains arranged in tandem, which have been proposed to function as adaptors but have only been examined in one protein (15, 16). Arf GTPase-activating proteins (GAPs) of the ARAP family are phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P₃)-dependent Arf GAPs with tandem PH domains (17, 18). The function of specific PH domains in regulating Arf GAP activity and for biologic activity has not been described.Arf GAPs are proteins that induce the hydrolysis of GTP bound to Arfs (19–23). The Arf proteins are members of the Ras superfamily of GTP-binding proteins (24–27). The six Arf proteins in mammals (five in humans) are divided into three classes based on primary sequence: Arf1, -2, and -3 are class 1, Arf4 and -5 are class 2, and Arf6 is class 3 (23, 24, 27–29). Class 1 and class 3 Arf proteins have been studied more extensively than class 2. They have been found to regulate membrane traffic and the actin cytoskeleton.The Arf GAPs are a family of proteins with diverse domain structures (20, 21, 23, 30). ARAPs, the most structurally complex of the Arf GAPs, contain, in addition to an Arf GAP domain, the sterile α motif (SAM), five PH, Rho GAP, and Ras association domains (17, 18, 31, 32). The first and second and the third and fourth PH domains are tandem (Fig. 1). The first and third PH domains of the ARAPs fit the consensus for PtdIns(3,4,5)P₃ binding (33–35). ARAPs have been found to affect actin and membrane traffic (21, 23). ARAP3 regulates growth factor-induced ruffling of porcine aortic endothelial cells (31, 36, 37). The function is dependent on the Arf GAP and Rho GAP domains. ARAP2 regulates focal adhesions, an actin cytoskeletal structure (17). ARAP2 function requires Arf GAP activity and a Rho GAP domain capable of binding RhoA·GTP. ARAP1 has been found to have a role in membrane traffic (18). The protein associates with pre-early endosomes involved in the attenuation of EGFR signals. The function of the tandem PH domains in the ARAPs has not been examined.Open in a separate windowFIGURE 1.ARAP1 binding to phospholipids. A, schematic of the recombinant proteins used in this study. Domain abbreviations: Ank, ankyrin repeat; PLCδ-PH, PH domain of phospholipase C δ; RA, Ras association motif; RhoGAP, Rho GTPase-activating domain. B, ARAP1 phosphoinositide binding specificity. 500 nm PH1-Ank recombinant protein was incubated with sucrose-loaded LUVs formed by extrusion through a 1-μm pore filter. LUVs contained PtdIns alone or PtdIns with 2.5 μm PtdIns(3,4,5)P₃, 2.5 μm PtdIns(3)P, 2.5 μm PtdIns(4)P, 2.5 μm PtdIns(5)P, 2.5 μm PtdIns(3,4)P₂, 2.5 μm PtdIns(3,5)P₂, or 2.5 μm PtdIns(4,5)P₂ with a total phosphoinositide concentration of 50 μm and a total phospholipid concentration of 500 μm. Vesicles were precipitated by ultracentrifugation, and associated proteins were separated by SDS-PAGE. The amount of precipitated protein was determined by densitometry of the Coomassie Blue-stained gels with standards on each gel. C, PtdIns(3,4,5)P₃-dependent binding of ARAP1 to LUVs. 1 μm PH1-Ank or ArfGAP-Ank recombinant protein was incubated with 1 mm sucrose-loaded LUVs formed by extrusion through a 1-μm pore size filter containing varying concentration of PtdIns(3,4,5)P₃. Precipitation of LUVs and analysis of associated proteins were performed as described in B. The average ± S.E. of three independent experiments is presented.Here we investigated the role of the first two PH domains of ARAP1 for catalysis and in vivo function. The first PH domain fits the consensus sequence for PtdIns(3,4,5)P₃ binding (33–35). The second does not fit a phosphoinositide binding consensus but is immediately N-terminal to the GAP domain. We have previously reported that the PH domain that occurs immediately N-terminal of the Arf GAP domain of ASAP1 is critical for the catalytic function of the protein (38, 39). We tested the hypothesis that the two PH domains of ARAP1 function independently; one recruits ARAP1 to PtdIns(3,4,5)P₃-rich membranes, and the other functions with the catalytic domain. As predicted, PH1 interacted specifically with PtdIns(3,4,5)P₃, and PH2 did not. However, both PH domains contributed to catalysis independently of recruitment to membranes. None of the PH domains in ARAP1 mediated PtdIns(3,4,5)P₃-dependent targeting to plasma membranes (PM). PtdIns(3,4,5)P₃ stimulated GAP activity, and the ability to bind PtdIns(3,4,5)P₃ was required for ARAP1 to regulate membrane traffic. We propose that ARAP1 is recruited independently of PtdIns(3,4,5)P₃ to the PM where PtdIns(3,4,5)P₃ subsequently regulates its GAP activity to control endocytic events.     

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.     

Specific regulation of the adaptor protein complex AP-3 by the Arf GAP AGAP1

Arf1 regulates membrane trafficking at several membrane sites by interacting with at least seven different vesicle coat proteins. Here, we test the hypothesis that Arf1-dependent coats are independently regulated by specific interaction with Arf GAPs. We find that the Arf GAP AGAP1 directly associates with and colocalizes with AP-3, a coat protein complex involved in trafficking in the endosomal-lysosomal system. Binding is mediated by the PH domain of AGAP1 and the delta and sigma3 subunits of AP-3. Overexpression of AGAP1 changes the cellular distribution of AP-3, and reduced expression of AGAP1 renders AP-3 resistant to brefeldin A. AGAP1 overexpression does not affect the distribution of other coat proteins, and AP-3 distribution is not affected by overexpression of other Arf GAPs. Cells overexpressing AGAP1 also exhibit increased LAMP1 trafficking via the plasma membrane. Taken together, these results support the hypothesis that AGAP1 directly and specifically regulates AP-3-dependent trafficking.     

The tyrosine kinase Pyk2 regulates Arf1 activity by phosphorylation and inhibition of the Arf-GTPase-activating protein ASAP1

Proline-rich tyrosine kinase 2 (Pyk2), a non-receptor tyrosine kinase structurally related to focal adhesion kinase, has been implicated in the regulation of mitogen-activated protein kinase cascades and ion channels, the induction of apoptosis, and in the modulation of the cytoskeleton. In order to understand how Pyk2 signaling mediates these diverse cellular functions, we performed a yeast two-hybrid screening using the C-terminal part of Pyk2 that contains potential protein-protein interaction sites as bait. A prominent binder of Pyk2 identified by this method was the Arf-GTPase-activating protein ASAP1. Pyk2-ASAP1 interaction was confirmed in pull-down as well as in co-immunoprecipitation experiments, and contact sites were mapped to the proline-rich regions of Pyk2 and the SH3 domain of ASAP1. Pyk2 directly phosphorylates ASAP1 on tyrosine residues in vitro and increases ASAP1 tyrosine phosphorylation when co-expressed in HEK293T cells. Phosphorylation of tyrosine 308 and 782 affects the phosphoinositide binding profile of ASAP1, and fluorimetric Arf-GTPase assays with purified proteins revealed an inhibition of ASAP1 GTPase-activating protein activity by Pyk2-mediated tyrosine phosphorylation. We therefore provide evidence for a functional interaction between Pyk2 and ASAP1 and a regulation of ASAP1 and hence Arf1 activity by Pyk2-mediated tyrosine phosphorylation.     

Different regulation of the Trio Dbl-Homology domains by their associated PH domains

Guanine nucleotide exchange factors for Rho-GTPases (Rho-GEFs) invariably share a catalytic Dbl-Homology (DH) domain associated with a Pleckstrin Homology (PH) domain, whose function in Rho-GEF activation is not well understood. Trio is the first member of an emerging family of Dbl proteins containing two Rho-GEF domains (GEFD1 and GEFD2). TrioGEFD1 activates the GTPases RhoG and Rac1, while TrioGEFD2 acts on RhoA. In this study, we have investigated the roles of the two PH domains of Trio in Rho-GEF activity. We show that TrioPH1 is required for GEFD1-mediated induction of actin cytoskeleton remodeling and JNK activation. TrioPH1 is involved both in the catalytic activity and in the subcellular localization of its associated DH domain, by acting as a cytoskeletal targeting signal. Moreover, TrioPH1 in association with DH2 activates the JNK pathway, by an unknown mechanism independent of DH2 catalytic activity. TrioPH2 does not behave as a targeting module in intact cells. TrioPH2 inhibits DH2-dependent stress fiber formation, which correlates with the TrioPH2-mediated inhibition of DH2 GEF activity. In addition, expression in the neuron-like PC12 cell line of the intact Trio protein deleted of each PH domain shows that only TrioPH1 is required for Trio-induced neurite outgrowth. Taken together, these data demonstrate that the two PH domains play a different role in the control of Trio Rho-GEF function.     

The Arf GAP ASAP1 provides a platform to regulate Arf4‐ and Rab11–Rab8‐mediated ciliary receptor targeting

Dysfunctional trafficking to primary cilia is a frequent cause of human diseases known as ciliopathies, yet molecular mechanisms for specific targeting of sensory receptors to cilia are largely unknown. Here, we show that the targeting of ciliary cargo, represented by rhodopsin, is mediated by a specialized system, the principal component of which is the Arf GAP ASAP1. Ablation of ASAP1 abolishes ciliary targeting and causes formation of actin‐rich periciliary membrane projections that accumulate mislocalized rhodopsin. We find that ASAP1 serves as a scaffold that brings together the proteins necessary for transport to the cilia including the GTP‐binding protein Arf4 and the two G proteins of the Rab family—Rab11 and Rab8—linked by the Rab8 guanine nucleotide exchange factor Rabin8. ASAP1 recognizes the FR ciliary targeting signal of rhodopsin. Rhodopsin FR‐AA mutant, defective in ASAP1 binding, fails to interact with Rab8 and translocate across the periciliary diffusion barrier. Our study implies that other rhodopsin‐like sensory receptors may interact with this conserved system and reach the cilia using the same platform.     

Mislocalization or reduced expression of Arf GTPase-activating protein ASAP1 inhibits cell spreading and migration by influencing Arf1 GTPase cycling

ADP-ribosylation factor (Arf) family of small GTP-binding proteins plays a central role in membrane trafficking and cytoskeletal remodeling. ASAP1 (Arf-GAP containing SH3, ankyrin repeats, and PH domain) is a phospholipid-dependent Arf GTPase-activating protein (Arf-GAP) that binds to protein-tyrosine kinases Src and focal adhesion kinase. Using affinity chromatography and mass spectrometry (MS), we identified the adaptor protein CD2-associated protein (CD2AP) as a candidate binding partner of ASAP1. Both co-immunoprecipitation and GST pull-down experiments confirmed that CD2AP stably interacts with ASAP1 through its N-terminal SH3 domains. Using a mislocalization strategy, we show that sequestration of endogenous ASAP1 to mitochondria with a CD2AP SH3-mito fusion protein (the three N-terminal SH3 domains of CD2AP fused to Listeria monocytogenes ActA mitochondria-targeting sequence) inhibited REF52 cell spreading and migration in response to fibronectin stimulation. Using an alternative strategy we show that suppressing ASAP1 expression with small interfering RNA duplexes also significantly retarded cell spreading and inhibited cell migration. Furthermore, abrogation of ASAP1 function using either small interfering RNAs or mislocalization approaches caused an increase of GTP loading on Arf1 and loss of paxillin from adhesions. These results taken together with our previous observations that overexpression of ASAP1 inhibits cell spreading and alters paxillin localization to adhesions (Liu, Y., Loijens, J. C., Martin, K. H., Karginov, A. V., and Parsons, J. T. (2002) Mol. Biol. Cell. 13, 2147-2156) suggest that the recruitment of certain adhesion components such as paxillin requires dynamic GTP/GDP turnover of Arf1 GTPase.     

Autoinhibition of Arf GTPase-activating Protein Activity by the BAR Domain in ASAP1

ASAP1 is an Arf GTPase-activating protein (GAP) that functions on membrane surfaces to catalyze the hydrolysis of GTP bound to Arf. ASAP1 contains a tandem of BAR, pleckstrin homology (PH), and Arf GAP domains and contributes to the formation of invadopodia and podosomes. The PH domain interacts with the catalytic domain influencing both the catalytic and Michaelis constants. Tandem BAR-PH domains have been found to fold into a functional unit. The results of sedimentation velocity studies were consistent with predictions from homology models in which the BAR and PH domains of ASAP1 fold together. We set out to test the hypothesis that the BAR domain of ASAP1 affects GAP activity by interacting with the PH and/or Arf GAP domains. Recombinant proteins composed of the BAR, PH, Arf GAP, and Ankyrin repeat domains (called BAR-PZA) and the PH, Arf GAP, and Ankyrin repeat domains (PZA) were compared. Catalytic power for the two proteins was determined using large unilamellar vesicles as a reaction surface. The catalytic power of PZA was greater than that of BAR-PZA. The effect of the BAR domain was dependent on the N-terminal loop of the BAR domain and was not the consequence of differential membrane association or changes in large unilamellar vesicle curvature. The K_m for BAR-PZA was greater and the k_cat was smaller than for PZA determined by saturation kinetics. Analysis of single turnover kinetics revealed a transition state intermediate that was affected by the BAR domain. We conclude that BAR domains can affect enzymatic activity through intraprotein interactions.The Bin, amphiphysin, RSV161/167 (BAR)² domain is a recently identified structural element in proteins that regulate membrane trafficking (1–7). The BAR superfamily comprises three subfamilies: F-BAR, I-BAR, and BAR. The BAR group can be further subdivided into BAR, N-BAR, PX-BAR, and BAR-pleckstrin homology (PH). The BAR group domains consist of three bundled α-helices that homodimerize to form a banana-shaped structure. The inner curved face can bind preferentially to surfaces with similar curvatures. As a consequence, BAR domains can function as membrane curvature sensors or as inducers of membrane curvature. BAR domains also bind to proteins (8, 9). Several proteins contain a BAR domain immediately N-terminal to a PH domain, which also mediates regulated membrane association (10–13). In the protein APPL1 (9), the BAR-PH domains fold together forming a binding site for the small GTP-binding protein Rab5. Arf GTPase-activating proteins (GAPs) are regulators of Arf family GTP-binding proteins (14–18). Two subtypes of Arf GAPs have N-terminal BAR and PH domains similar to that found in APPL1.Thirty-one genes encode Arf GAPs in humans (16–18). Each member of the family has an Arf GAP domain that catalyzes the hydrolysis of GTP bound to Arf family GTP-binding proteins. The Arf GAPs are otherwise structurally diverse. ASAP1 is an Arf GAP that affects membrane traffic and actin remodeling involved in cell movement and has been implicated in oncogenesis (19–22). ASAP1 contains, from the N terminus, BAR, PH, Arf GAP, Ankyrin repeat, proline-rich, and SH3 domains.ASAP1 contains a BAR domain immediately N-terminal to a PH domain. The PH domain of ASAP1 is functionally integrated with the Arf GAP domain and may form part of the substrate binding pocket (23, 24). The PH domain binds specifically to phosphatidylinositol 4,5-bisphosphate (PIP₂), a constituent of the membrane, leading to stimulation of GAP activity by a mechanism that is, in part, independent of recruitment to membranes (23, 25). The BAR domain of ASAP1 is critical for in vivo function of ASAP1, but the molecular functions of the BAR domain of ASAP1 have not been extensively characterized. Hypotheses related to membrane curvature have been examined. Recombinant ASAP1 can induce the formation of tubules from large unilamellar vesicles, which may be related to a function of ASAP1 in membrane traffic. The BAR domain might also regulate GAP activity of ASAP1. We have considered two mechanisms based on the known properties of BAR domains. First the BAR domain could regulate association of ASAP1 with membrane surfaces containing the substrate Arf1·GTP. The BAR domain could also affect GAP activity through an intramolecular association. In one BAR-PH protein that has been crystallized (APPL1), the two domains fold together to form a protein binding site (9). In ASAP1, the PH domain is functionally integrated with the GAP domain, raising the possibility that the BAR domain affects GAP activity by folding with the PH domain.Here we compared the kinetics of recombinant proteins composed of the PH, Arf GAP, and Ankyrin repeat (PZA)³ or BAR, PH, Arf GAP, and Ankyrin repeat (BAR-PZA) domains of ASAP1 to test the hypothesis that the BAR domain affects enzymatic activity. We found kinetic differences between the proteins that could not be explained by membrane association properties. The results were consistent with a model in which the BAR domain affects transition of ASAP1 through its catalytic cycle.     

Active Arf6 recruits ARNO/cytohesin GEFs to the PM by binding their PH domains

ARNO is a soluble guanine nucleotide exchange factor (GEF) for the Arf family of GTPases. Although in biochemical assays ARNO prefers Arf1 over Arf6 as a substrate, its localization in cells at the plasma membrane (PM) suggests an interaction with Arf6. In this study, we found that ARNO activated Arf1 in HeLa and COS-7 cells resulting in the recruitment of Arf1 on to dynamic PM ruffles. By contrast, Arf6 was activated less by ARNO than EFA6, a canonical Arf6 GEF. Remarkably, Arf6 in its GTP-bound form recruited ARNO to the PM and the two proteins could be immunoprecipitated. ARNO binding to Arf6 was not mediated through the catalytic Sec7 domain, but via the pleckstrin homology (PH) domain. Active Arf6 also bound the PH domain of Grp1, another ARNO family member. This interaction was direct and required both inositol phospholipids and GTP. We propose a model of sequential Arf activation at the PM whereby Arf6-GTP recruits ARNO family GEFs for further activation of other Arf isoforms.     

AlFx affects the formation of focal complexes by stabilizing the Arf-GAP ASAP1 in a complex with Arf1

Aluminum fluoride (AlFx) is known to activate directly the alpha subunit of G-proteins but not the homologous small GTP-binding proteins. However, AlFx can stabilize complexes formed between Ras, RhoA or Cdc42 and their corresponding GTPase-activating proteins (GAPs). Here, we demonstrate that Arf1GDP can be converted into an active conformation by AlFx to form a complex with the Arf-GAP ASAP1 in vitro and in vivo. Within this complex ASAP1, which GAP activity is inoperative, can still alter the recruitment of paxillin to the focal complexes, thus indicating that ASAP1 interferes with focal complexes independently of its GAP activity.     

Distinct subcellular localisations of the putative inositol 1,3,4,5-tetrakisphosphate receptors GAP1 and GAP1 result from the GAP1 PH domain directing plasma membrane targeting

Inositol 1,3,4,5-tetrakisphosphate (IP₄), is a ubiquitous inositol phosphate that has been suggested to function as a second messenger. Recently, we purified and cloned a putative IP₄ receptor, termed GAP1^IP4BP[1], which is also a member of the GAP1 family of GTPase-activating proteins for the Ras family of GTPases. A homologue of GAP1^IP4BP, called GAP1^m, has been identified [2] and here we describe the cloning of a GAP1^m cDNA from a human circulating-blood cDNA library. We found that a deletion mutant of GAP1^m, in which the putative phospholipid-binding domains (C2A and C2B) have been removed, binds to IP₄ with a similar affinity and specificity to that of the corresponding GAP1^IP4BP mutant. Expression studies of the proteins in either COS-7 or HeLa cells showed that, whereas GAP1^IP4BP is located solely at the plasma membrane, GAP1^m seems to have a distinct perinuclear localisation. By mutational analysis, we have shown that the contrast in subcellular distribution of these two closely related proteins may be a function of their respective pleckstrin homology (PH) domains. This difference in localisation has fundamental significance for our understanding of the second messenger functions of IP₄.     

Ryanodine receptor regulation by intramolecular interaction between cytoplasmic and transmembrane domains

Ryanodine receptors (RyR) function as Ca(2+) channels that regulate Ca(2+) release from intracellular stores to control a diverse array of cellular processes. The massive cytoplasmic domain of RyR is believed to be responsible for regulating channel function. We investigated interaction between the transmembrane Ca(2+)-releasing pore and a panel of cytoplasmic domains of the human cardiac RyR in living cells. Expression of eGFP-tagged RyR constructs encoding distinct transmembrane topological models profoundly altered intracellular Ca(2+) handling and was refractory to modulation by ryanodine, FKBP12.6 and caffeine. The impact of coexpressing dsRed-tagged cytoplasmic domains of RyR2 on intracellular Ca(2+) phenotype was assessed using confocal microscopy coupled with parallel determination of in situ protein: protein interaction using fluorescence resonance energy transfer (FRET). Dynamic interactions between RyR cytoplasmic and transmembrane domains were mediated by amino acids 3722-4610 (Interacting or "I"-domain) which critically modulated intracellular Ca(2+) handling and restored RyR sensitivity to caffeine activation. These results provide compelling evidence that specific interaction between cytoplasmic and transmembrane domains is an important mechanism in the intrinsic modulation of RyR Ca(2+) release channels.     

ASAP1, a Phospholipid-Dependent Arf GTPase-Activating Protein That Associates with and Is Phosphorylated by Src

Membrane trafficking is regulated in part by small GTP-binding proteins of the ADP-ribosylation factor (Arf) family. Arf function depends on the controlled exchange and hydrolysis of GTP. We have purified and cloned two variants of a 130-kDa phosphatidylinositol 4,5-biphosphate (PIP₂)-dependent Arf1 GTPase-activating protein (GAP), which we call ASAP1a and ASAP1b. Both contain a pleckstrin homology (PH) domain, a zinc finger similar to that found in another Arf GAP, three ankyrin (ANK) repeats, a proline-rich region with alternative splicing and SH3 binding motifs, eight repeats of the sequence E/DLPPKP, and an SH3 domain. Together, the PH, zinc finger, and ANK repeat regions possess PIP₂-dependent GAP activity on Arf1 and Arf5, less activity on Arf6, and no detectable activity on Arl2 in vitro. The cDNA for ASAP1 was independently identified in a screen for proteins that interact with the SH3 domain of the tyrosine kinase Src. ASAP1 associates in vitro with the SH3 domains of Src family members and with the Crk adapter protein. ASAP1 coprecipitates with Src from cell lysates and is phosphorylated on tyrosine residues in cells expressing activated Src. Both coimmunoprecipitation and tyrosine phosphorylation depend on the same proline-rich class II Src SH3 binding site required for in vitro association. By directly interacting with both Arfs and tyrosine kinases involved in regulating cell growth and cytoskeletal organization, ASAP1 could coordinate membrane remodeling events with these processes.Membrane traffic, the transfer of material between membrane-bound compartments, is needed for such diverse cellular processes as secretion, endocytosis, and changes in cell shape that accompany cell growth, division, and migration (reviewed in references 84, 85, and 87). It is mediated by transport vesicles that are formed by budding from a donor membrane. The process of budding is driven by the assembly of a proteinaceous coat. Once the vesicle is formed, the coat must dissociate to permit fusion with an acceptor membrane and the consequent delivery of the vesicle’s contents. These steps are regulated in part by the Arf family of small GTP-binding proteins (reviewed in references 8, 23, 61, and 63). Arfs are highly conserved and are found in eukaryotes ranging from yeast to humans. The mammalian Arf family is divided into several classes based largely on sequence similarity: class I (Arfs 1 through 3), class II (Arfs 4 and 5), class III (Arf6), and the more distantly related Arf-like (Arl) class. By linking GTP binding and hydrolysis to coat assembly and disassembly, Arfs regulate membrane trafficking at a number of sites. Arf1 has been implicated in endoplasmic reticulum-to-Golgi and intra-Golgi transport, endosome-to-endosome fusion, and synaptic vesicle formation (8, 23, 28, 61, 63, 66). Arf6 has been implicated in regulation of membrane traffic between the plasma membrane and a specialized endocytic compartment, and its function has been linked to cytoskeletal reorganization (25, 26, 71, 73, 74). The specific sites of action of the other Arf family members are not known.The hydrolysis of GTP on Arf requires a GTPase-activating protein (GAP) (19, 61). With multiple Arfs and multiple sites of action, the existence of several unique Arf GAPs had been anticipated. A number of activities have been purified or partially purified from mammalian sources, including rat liver (19, 57, 77), rat spleen (21), and bovine brain (79), and two Arf GAP activities from rat liver have been resolved (77). They have similar Arf specificities but differ in their lipid dependencies. One of the Arf GAPs (ArfGAP/ArfGAP1, hereafter referred to as ArfGAP1) which functions in the Golgi is activated by dioleoglycerols (3, 4, 19, 40). ArfGAP1, in common with a yeast Arf GAP, GCS1 (72), contains a zinc finger domain which is required for activity (19). The second Arf GAP (ArfGAP2) is specifically activated by phosphatidylinositol 4,5-bisphosphate (PIP₂) and phosphatidic acid (PA). Based on lipid requirements, ArfGAP2 was speculated to function at the plasma membrane and be regulated independently of ArfGAP1 (77). ArfGAP1 and ArfGAP2 were antigenically distinct and, therefore, likely to be distinct gene products; however, prior to this study, only ArfGAP1 had been cloned (19).Src, a cytoplasmic tyrosine kinase with N-terminal Src homology 3 (SH3) and SH2 domains, transduces signals important for cell growth and cytoskeletal organization (12, 68, 91). A number of studies suggest that Src is also involved in regulating membrane traffic. Src associates primarily with endosomal membranes and in several cell types has been localized to specialized secretory vesicles, including synaptic vesicles (5, 20, 34, 46, 54, 69, 81). Overexpression of Src accelerates endocytosis (95). In addition, Src associates with or phosphorylates several proteins involved in membrane trafficking (5, 31, 43, 65).Here, we report the purification and cloning of a PIP₂-dependent Arf GAP, ASAP1. ASAP1 contains a zinc finger domain similar to that required for GAP activity in ArfGAP1 and GCS1. ASAP1 also contains a number of domains that are likely to be involved in regulation and/or localization: a pleckstrin homology (PH) domain, three ankyrin (ANK) repeats, a proline-rich region with SH3 binding motifs, and an SH3 domain. In addition, ASAP1 was identified independently as a binding protein for Src and was found to be phosphorylated on tyrosine in cells that express activated Src. ASAP1 also associated with the adapter protein c-Crk in vitro. ASAP1 was localized to the cytoplasm and the cell edge likely associated with the plasma membrane. We propose that ASAP1, by binding both Src and PIP₂, could coordinate membrane trafficking with cell growth or actin cytoskeleton remodeling.     

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.     

Dynamic interaction between soluble tubulin and C-terminal domains of N-methyl-D-aspartate receptor subunits

The cytoplasmic C-terminal domains (CTs) of the NR1 and NR2 subunits of the NMDA receptor have been implicated in its anchoring to the subsynaptic cytoskeleton. Here, we used affinity chromatography with glutathione S-transferase-NR1-CT and -NR2B-CT fusion proteins to identify novel binding partner(s) of these NMDA receptor subunits. Upon incubation with rat brain cytosolic protein fraction, both NR1-CT and NR2B-CT, but not glutathione S-transferase, specifically bound tubulin. The respective fusion proteins also bound tubulin purified from brain, suggesting a direct interaction between the two binding partners. In tubulin polymerization assays, NR1-CT and NR2B-CT significantly decreased the rate of microtubule formation without destabilizing preformed microtubules. Moreover, only minor fractions of either fusion protein coprecipitated with the newly formed microtubules. Consistent with these findings, ultrastructural analysis of the newly formed microtubules revealed a limited association only with the CTs of the NR1 and NR2B. These data suggest a direct interaction of the NMDA receptor channel subunit CTs and tubulin dimers or soluble forms of tubulin. The efficient modulation of microtubule dynamics by the NR1 and NR2 cytoplasmic domains suggests a functional interaction of the receptor and the subsynaptic cytoskeletal network that may play a role during morphological adaptations, as observed during synaptogenesis and in adult CNS plasticity.