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.

Activation of a small GTP-binding protein by nucleoside diphosphate kinase.

Genes that encode nucleoside diphosphate kinases (NDKs) have been implicated as regulators of mammalian tumor metastasis and development in Drosophila melanogaster. However, the cellular pathways through which NDKs function are not known. One potential mechanism of regulation is phosphorylation of guanosine diphosphate (GDP) bound to regulatory guanosine triphosphate (GTP) binding proteins. NDK-catalyzed phosphorylation of bound GDP was investigated for the adenosine diphosphate ribosylation factor (ARF), a 21-kilodalton GTP-binding protein that functions in the protein secretion pathway. Bovine liver NDK, recombinant human NDK, and the protein product of the mouse gene nm23-1, which suppresses the metastatic potential of certain tumor cells, used ARF-GDP as a substrate, thereby allowing rapid and efficient production of activated ARF (ARF-GTP) in the absence of nucleotide exchange. These data are consistent with the proposed function of NDK as an activator of a small GTP-binding protein and provide a mechanism of activation for a regulatory GTP-binding protein that is independent of nucleotide exchange.

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.

Identification of a new Pyk2 target protein with Arf-GAP activity.

Protein tyrosine kinase Pyk2 is activated by a variety of G-protein-coupled receptors and by extracellular signals that elevate intracellular Ca2+ concentration. We have identified a new Pyk2 binding protein designated Pap. Pap is a multidomain protein composed of an N-terminal alpha-helical region with a coiled-coil motif, followed by a pleckstrin homology domain, an Arf-GAP domain, an ankyrin homology region, a proline-rich region, and a C-terminal SH3 domain. We demonstrate that Pap forms a stable complex with Pyk2 and that activation of Pyk2 leads to tyrosine phosphorylation of Pap in living cells. Immunofluorescence experiments demonstrate that Pap is localized in the Golgi apparatus and at the plasma membrane, where it is colocalized with Pyk2. In addition, in vitro recombinant Pap exhibits strong GTPase-activating protein (GAP) activity towards the small GTPases Arf1 and Arf5 and weak activity towards Arf6. Addition of recombinant Pap protein to Golgi preparations prevented Arf-dependent generation of post-Golgi vesicles in vitro. Moreover, overexpression of Pap in cultured cells reduced the constitutive secretion of a marker protein. We propose that Pap functions as a GAP for Arf and that Pyk2 may be involved in regulation of vesicular transport through its interaction with Pap.

Molecular aspects of the cellular activities of ADP-ribosylation factors.

Adenosine diphosphate-ribosylation factor (Arf) proteins are members of the Arf arm of the Ras superfamily of guanosine triphosphate (GTP)-binding proteins. Arfs are named for their activity as cofactors for cholera toxin-catalyzed adenosine diphosphate-ribosylation of the heterotrimeric G protein Gs. Physiologically, Arfs regulate membrane traffic and the actin cytoskeleton. Arfs function both constitutively within the secretory pathway and as targets of signal transduction in the cell periphery. In each case, the controlled binding and hydrolysis of GTP is critical to Arf function. The activities of some guanine nucleotide exchange factors (GEFs) and guanosine triphosphatase (GTPase)-activating proteins (GAPs) are stimulated by phosphoinositides, including phosphatidylinositol 3,4,5-trisphosphate (PIP3) and phosphatidylinositol 4,5-bisphosphate (PIP2), and phosphatidic acid (PA), likely providing both a means to respond to regulatory signals and a mechanism to coordinate GTP binding and hydrolysis. Arfs affect membrane traffic in part by recruiting coat proteins, including COPI and clathrin adaptor complexes, to membranes. However, Arf function likely involves many additional biochemical activities. Arf activates phospholipase D and phosphatidylinositol 4-phosphate 5-kinase with the consequent production of PA and PIP2, respectively. In addition to mediating Arf's effects on membrane traffic and the actin cytoskeleton, PA and PIP2 are involved in the regulation of Arf. Arf also works with Rho family proteins to affect the actin cytoskeleton. Several Arf-binding proteins suspected to be effectors have been identified in two-hybrid screens. Arf-dependent biochemical activities, actin cytoskeleton changes, and membrane trafficking may be integrally related. Understanding Arf's role in complex cellular functions such as protein secretion or cell movement will involve a description of the temporal and spatial coordination of these multiple Arf-dependent events.

Resolution of two ADP-ribosylation factor 1 GTPase-activating proteins from rat liver.

ADP-ribosylation factor 1 (ARF1) is a 21 kDa GTP-binding protein that regulates multiple steps in membrane traffic. Here, two ARF1 GTPase-activating proteins (GAPs) from rat liver were resolved. The GAPs were antigenically distinct. One reacted with a polyclonal antibody raised against the GAP catalytic peptide previously purified by Makler et al. [Makler, Cukierman, Rotman, Admon and Cassel (1995) J. Biol. Chem. 270, 5232-5237], and here is referred to as GAP1. The other GAP (GAP2) did not react with the antibody. These GAPs differed in phospholipid dependencies. GAP1 was activated 3-7-fold by the acid phospholipids phosphatidylinositol 4, 5-bisphosphate (PIP2), phosphatidic acid (PA) and phosphatidylserine (PS). In contrast, GAP2 was stimulated 20-40-fold by PIP2. PA and PS had no effect by themselves but PA increased GAP2 activity in the presence of PIP2. The GAPs were otherwise similar in activity. In the presence of phosphoinositides, the Km of GAP1 for ARF1-GTP was estimated to be 8.1+/-1.6 microM and the dissociation constant for ARF1-guanosine 5',3-O-(thio)triphosphate (GTP[S]) was 7.4+/-2.2 microM. GAP2 was similar with a Km for ARF1-GTP of 5.4+/-1.2 microM and a dissociation constant for ARF1-GTP[S] of 4.8+/-0.3 microM. Similarly, no differences were found in substrate preferences. Both GAP1 and GAP2 used ARF1 and ARF5 as substrates but not ARF6 or ARF-like protein-2. The potential role of multiple ARF GAPs in the independent regulation of ARF at specific steps in membrane traffic is discussed.

Functional interaction of ADP-ribosylation factor 1 with phosphatidylinositol 4,5-bisphosphate.

The relationship between ADP-ribosylation factor (Arf) 1 and phosphoinositides, which have been independently implicated as regulators of membrane traffic, was examined. Because both Arf-dependent phospholipase D and Arf1 GTPase-activating protein (GAP) require phosphatidylinositol 4,5-bisphosphate (PIP2), Arf1 complexed with PIP2 has been proposed to interact with target proteins. This hypothesis was tested using Arf1 GAP as a model system. Arf1 was shown to bind to PIP2 in Triton X-100 micelles with a Kd of 45 +/- 13 microM. Arf1 also bound phosphatidic acid but with 10-fold lower affinity. PIP2 binding was specifically disrupted by mutating lysines 15, 16, and 181 and arginine 178 to leucines. Decreased PIP2 binding resulted in an increased EC50 of PIP2 for activation of Arf GAP. None of the mutations that decreased PIP2 binding affected binding to or activation of GAP by phosphatidic acid, which acts at a functionally distinct site. These data support the hypothesis that PIP2 binding to Arf1 promotes interaction with Arf GAP. The implications of lipid-directed protein-protein interactions for membrane traffic are discussed.

In vivo and in vitro phosphorylation studies of numatrin, a cell cycle regulated nuclear protein, in insulin-stimulated NIH 3T3 HIR cells.

Numatrin, a nuclear matrix protein has been implicated to be involved in mitogenesis of normal and malignant cells (Feuerstein and Mond, J. Biol. Chem. 262, 11389, 1987) and was later found to be identical to the nuclear phosphoprotein, B23. To study whether phosphorylation of numatrin is regulated by mitogenic stimulation, we examined the effect of phosphorylation of numatrin in the insulin-responsive cells, NIH 3T3 HIR. We found that an increase in phosphorylation of numatrin was associated with stimulation of the cells with insulin for 4 h and that the level of phosphorylation remained elevated after 8 h. By this time there was no increase in numatrin abundance as shown by Coom-massie blue stain and Western blot analysis. The induction in phosphorylation of numatrin could not be detected after 30 min stimulation with insulin, thus, indicating that the increase in phosphorylation of numatrin is not a rapid event. Analysis of the phosphopeptides by thin layer chromatography indicated four peptides that were phosphorylated in numatrin (one major and three minor). Stimulation with insulin was associated primarily with an increase in phosphorylation of the minor phosphopeptides. The phosphopeptide map of numatrin was identical after 4, 8, 17, 24, and 32 h stimulation with insulin, indicating that identical sites are phosphorylated at different phases of the cell cycle. In a search for the protein kinase which is involved in phosphorylation of numatrin we found that numatrin is a most prominent substrate for the cell cycle regulated cdc2 (p 34) kinase. However, the major phosphopeptides which were phosphorylated by this kinase did not comigrate with either of the phosphopeptides phosphorylated in insulin-stimulated intact cells. This may indicate that it is unlikely that cdc2 kinase may account for the mechanism(s) associated with phosphorylation of numatrin by insulin under physiological conditions.

Characterization of the growth of murine fibroblasts that express human insulin receptors. II. Interaction of insulin with other growth factors.

The effects of insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and insulin on DNA synthesis were studied in murine fibroblasts transfected with an expression vector containing human insulin receptor cDNA (NIH 3T3/HIR) and the parental NIH 3T3 cells. In NIH 3T3/HIR cells, individual growth factors in serum-free medium stimulated DNA synthesis with the following relative efficacies: insulin greater than or equal to 10% fetal calf serum greater than PDGF greater than IGF-1 much greater than EGF. In comparison, the relative efficacies of these factors in stimulating DNA synthesis by NIH 3T3 cells were 10% fetal calf serum greater than PDGF greater than EGF much greater than IGF-1 = insulin. In NIH 3T3/HIR cells, EGF was synergistic with 1-10 ng/ml insulin but not with 100 ng/ml insulin or more. Synergy of PDGF or IGF-1 with insulin was not detected. In the parental NIH 3T3 cells, insulin and IGF-1 were found to be synergistic with EGF (1 ng/ml), PDGF (100 ng/ml), and PDGF plus EGF. In NIH 3T3/HIR cells, the lack of interaction of insulin with other growth factors was also observed when the percentage of cells synthesizing DNA was examined. Despite insulin's inducing only 60% of NIH 3T3/HIR cells to incorporate thymidine, addition of PDGF, EGF, or PDGF plus EGF had no further effect. In contrast, combinations of growth factors resulted in 95% of the parental NIH 3T3 cells synthesizing DNA. The independence of insulin-stimulated DNA synthesis from other mitogens in the NIH 3T3/HIR cells is atypical for progression factor-stimulated DNA synthesis and is thought to be partly the result of insulin receptor expression in an inappropriate context or quantity.

GTP hydrolysis by ADP-ribosylation factor is dependent on both an ADP-ribosylation factor GTPase-activating protein and acid phospholipids.

ADP-ribosylation factor (ARF) is a 21-kDa GTP binding protein that regulates eukaryotic membrane traffic. Both the binding and hydrolysis of GTP by ARF have been shown to be necessary for this function. However, purified mammalian ARF lacks intrinsic GTPase activity (< 0.0015 min-1). We document the presence, in bovine brain extracts, of a protein with the predicted properties for an ARF GTPase-activating protein (ARF GAP). This activity was highly dependent on phospholipids. An acid phospholipid fraction from bovine brain (containing primarily phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate, phosphatidylinositol, and phosphatidylserine) had no effect on intrinsic GTPase activity of purified ARF but increased the ARF GAP activity of bovine brain homogenates about 8-fold. This dependence on acid phospholipids was retained after > 100-fold purification of ARF GAP, making it, likely, an inherent property of this reaction. PIP2 alone stimulated ARF GAP activity up to 30-fold with a half-maximal effect at 100-300 microM but had no effect on the GTPase rate of ARF alone. Phosphatidylinositol 4-phosphate was also active but had only 50% of the maximal effect and twice the EC50 of PIP2. Phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, and diacylglycerol either alone or in the presence of ARF GAP do not stimulate ARF GTPase activity. ARF proteins have been identified recently as regulators of phospholipase D. The product of the phospholipase D reaction, phosphatidic acid, stimulated ARF GAP approximately 5-fold and reduced the PIP2 concentration needed for GAP stimulation about 6-fold. The substrate of phospholipase D, phosphatidylcholine, inhibited ARF GAP activity, but this inhibition seen with phosphatidylcholine was partially reversed by phosphatidic acid. A feedback loop for the coordinate regulation of phospholipase D and ARF activities is proposed.

Regulatory GTP-binding proteins (ADP-ribosylation factor, Gt, and RAS) are not activated directly by nucleoside diphosphate kinase.

The expression of nucleoside diphosphate kinase (NDK) genes has been implicated as a negative regulator of murine and human tumor metastases and is critical to proper development in Drosophila melanogaster. Molecular mechanisms for the role(s) of NDK in these complex processes have not yet been elucidated, but several reports have suggested that these and many other signal transduction pathways may be activated by NDK acting directly on a regulatory GTP-binding protein(s). To test this hypothesis, we examined the ability of NDK to catalyze the phosphorylation of the GDP bound to the following three members of the superfamily of regulatory GTP-binding proteins: Gt, Ha-ras p21, and ARF. We have found no evidence to support the hypothesis that NDK can directly activate any GTP-binding protein. Rather, evidence is presented which clearly shows that all of the GTP formed upon incubation of GTP-binding proteins with NDK is the result of NDK utilizing free GDP as substrate. The GDP bound to the regulatory proteins is not a substrate for NDK under conditions in which free nucleotides are rapidly and efficiently phosphorylated. The importance of appropriate controls for dissociation of GDP from the regulatory proteins both during the NDK reaction and during the analysis of product is demonstrated. We believe there is currently no experimental evidence to support the hypothesis that NDK can directly activate a regulatory GTP-binding protein.

Effects of acid phospholipids on ARF activities: potential roles in membrane traffic.

ADP-ribosylation factors are a family of approximately 21 kDa GTP binding proteins which have been implicated as ubiquitous regulators of multiple steps in both exocytic and endocytic membrane traffic in mammals and yeast. Reversible membrane associations are thought to be an essential component in the physiological actions of ARF and are regulated by GTP binding. ARFs are unique among the superfamily of GTP binding proteins in having a strict dependence on phospholipids for nucleotide exchange. In addition, ARF proteins were found to bind phospatidylinositol 4,5-bisphosphate (PIP2) specifically. PIP2 was found to increase the rate of GDP dissociation and stabilize the nucleotide-free form of the protein. The previously described requirements for PIP2 in the ARF stimulated phospholipase D (PLD) activity and ARF GTPase activating protein (ARF GAP) assays provide the basis for a model in which PIP2 acts as a cofactor in one or more ARF pathways. There are potentially two distinct phospholipid binding sites each of which are coupled to the nucleotide binding site of ARFs.

Effects of acid phospholipids on nucleotide exchange properties of ADP-ribosylation factor 1. Evidence for specific interaction with phosphatidylinositol 4,5-bisphosphate.

ADP-ribosylation factors (ARFs) have been implicated as ubiquitous regulators of multiple steps in both exocytic and endocytic membrane traffic in yeast and mammalian cells. More specific interactions have also been described for ARF proteins with an ARF-specific GTP-ase-activating protein and as activators of phospholipase D activity. These protein interactions have defined requirements for phosphatidylinositol 4,5-bisphosphate (PIP2). Direct interactions between ARF1 and PIP2 or other phospholipids were tested by examining effects on guanine nucleotide binding kinetics. PIP2 (400 microM) increased the rate of GDP dissociation > 100-fold. Several other acid phospholipids had more modest effects (4-7-fold) on GDP dissociation rates, while other phospholipids had no effect. PIP2 also had the greatest effect on the rate of binding of guanosine 5'-(gamma-thio)triphosphate (GTP gamma S), increasing it almost 100-fold at early time points. However, at later times (> 5 min), PIP2 caused a paradoxical loss of nucleotide binding to ARF1. PIP2 was found to stabilize the nucleotide-free form of ARF1 as subsequent dilution of PIP2 allowed ARF1 to bind GTP gamma S to high stoichiometry. The demonstration of direct interaction between ARF1 and PIP2 provides the basis for a model in which PIP2 acts as a cofactor in some of the interactions between ARF1 and other proteins.

Activation of ADP-ribosylation factor by Golgi membranes. Evidence for a brefeldin A- and protease-sensitive activating factor on Golgi membranes.

Recent evidence has implicated ADP-ribosylation factor (ARF) proteins as critical regulators of the protein secretory pathway, particularly in the endoplasmic reticulum-Golgi pathway. We have examined whether Golgi membranes contain activators of ARF and the consequences of ARF activation and acylation on its membrane association. Two means were used to assess ARF activation. First, guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) binding to protein was found to be greater when ARF and Golgi were incubated together than when either was incubated alone. These data suggested that ARF GTP gamma S was formed. This was confirmed by showing that the GTP gamma S-bound protein functioned as a cofactor for cholera toxin-stimulated ADP-ribosylation of Gs alpha, a reaction for which activated ARF is a necessary cofactor. Trypsin treatment of Golgi, an inhibitory ARF peptide, and brefeldin A each inhibited Golgi-mediated activation by approximately 70%, demonstrating that a specific protein interaction is required for the majority of the ARF activation. This ARF-activating protein is a strong candidate for the molecular target for brefeldin A. The ubiquitous nature of ARF proteins and their importance in both the exocytic and endocytic pathways may explain the effects of brefeldin A on both exocytic and endocytic membrane traffic in animal cells. A protease-insensitive activation of ARF by Golgi could also be demonstrated and was the dominant activity observed in submicromolar concentrations of magnesium. We believe this to be the lipid-mediated process described previously for purified ARF proteins. ARF activation resulted in tight association of ARF with phospholipid vesicles. Vesicle association required amino-terminal myristoylation of ARF whereas activation did not. These studies indicate that the brefeldin A-sensitive ARF-activating protein and other factors that determine the level of activation of ARF in animal cells are fundamental regulators of membrane traffic in animal cells.