ABSTRACT
ßArrestin proteins shuttle between the cytosol and nucleus and have been shown to regulate G protein-coupled receptor signaling, actin remodeling, and gene expression. Here, we tested the hypothesis that ßarrestin1 regulates actin remodeling and cell migration through the small GTPase Rac. Depletion of ßarrestin1 promotes Rac activation, leading to the formation of multipolar protrusions and increased cell circularity, and overexpression of a dominant negative form of Rac reverses these morphological changes. Small interfering RNA library screen identifies RasGRF2 as a target of ßarrestin1. RasGRF2 gene and protein expression levels are elevated following depletion of ßarrestin1, and the consequent activation of Rac results in dephosphorylation of cofilin that can promote actin polymerization and formation of multipolar protrusions, thereby retarding cell migration and invasion. Together, these results suggest that ßarrestin1 regulates rasgrf2 gene expression and Rac activation to affect membrane protrusion and cell migration and invasion.
Subject(s)
Arrestins/metabolism , Cell Membrane Structures/metabolism , Gene Expression Regulation/physiology , Proto-Oncogene Proteins c-akt/metabolism , ras Guanine Nucleotide Exchange Factors/biosynthesis , Animals , Arrestins/genetics , Cell Membrane Structures/genetics , Cell Movement/physiology , Enzyme Activation/physiology , Gene Knockdown Techniques , HEK293 Cells , Humans , Mice , Phosphorylation/physiology , Proto-Oncogene Proteins c-akt/genetics , beta-Arrestins , ras Guanine Nucleotide Exchange Factors/geneticsABSTRACT
G protein-coupled receptor kinases (GRK) regulate diverse cellular functions ranging from metabolism to growth and locomotion. Here, we report an important contributory role for GRK5 in human prostate cancer. Inhibition of GRK5 kinase activity attenuated the migration and invasion of prostate cancer cells and, concordantly, increased cell attachment and focal adhesion formation. Mass spectrometric analysis of the phosphoproteome revealed the cytoskeletal-membrane attachment protein moesin as a putative GRK5 substrate. GRK5 regulated the subcellular distribution of moesin and colocalized with moesin at the cell periphery. We identified amino acid T66 of moesin as a principal GRK5 phosphorylation site and showed that enforcing the expression of a T66-mutated moesin reduced cell spreading. In a xenograft model of human prostate cancer, GRK5 silencing reduced tumor growth, invasion, and metastasis. Taken together, our results established GRK5 as a key contributor to the growth and metastasis of prostate cancer.
Subject(s)
G-Protein-Coupled Receptor Kinase 5/metabolism , Microfilament Proteins/metabolism , Prostatic Neoplasms/pathology , Animals , Antibodies/immunology , Cell Adhesion/genetics , Cell Movement/genetics , Focal Adhesions/pathology , G-Protein-Coupled Receptor Kinase 5/antagonists & inhibitors , G-Protein-Coupled Receptor Kinase 5/genetics , Humans , Kidney/pathology , Male , Mice , Mice, Nude , Microfilament Proteins/immunology , Neoplasm Invasiveness , Neoplasm Metastasis , Neoplasm Transplantation , Phosphorylation , RNA Interference , RNA, Small InterferingABSTRACT
AGAP2 [Arf (ADP-ribosylation factor) GAP (GTPase-activating protein) with GTP-binding-protein-like, ankyrin repeat and PH (pleckstrin homology) domains] is a multidomain Arf GAP that was shown to promote the fast recycling of transferrin receptors. In the present study we tested the hypothesis that AGAP2 regulates the trafficking of ß2-adrenergic receptors. We found that AGAP2 formed a complex with ß-arrestin1 and ß-arrestin2, proteins that are known to regulate ß2-adrenergic receptor signalling and trafficking. AGAP2 co-localized with ß-arrestin2 on the plasma membrane, and knockdown of AGAP2 expression reduced plasma membrane association of ß-arrestin2 upon ß2-adrenergic receptor activation. AGAP2 also co-localized with internalized ß2-adrenergic receptors on endosomes, and overexpression of AGAP2 slowed accumulation of ß2-adrenergic receptor in the perinuclear recycling endosomes. In contrast, knockdown of AGAP2 expression prevented the recycling of the ß2-adrenergic receptor back to the plasma membrane. In addition, AGAP2 formed a complex with endogenous ERK (extracellular-signal-regulated kinase) and overexpression of AGAP2 potentiated ERK phosphorylation induced by ß2-adrenergic receptors. Taken together, these results support the hypothesis that AGAP2 plays a role in the signalling and recycling of ß2-adrenergic receptors.
Subject(s)
Arrestins/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , GTP-Binding Proteins/metabolism , GTPase-Activating Proteins/metabolism , Receptors, Adrenergic, beta-2/metabolism , Cell Line, Tumor , Enzyme Activation/genetics , GTP-Binding Proteins/antagonists & inhibitors , GTP-Binding Proteins/physiology , GTPase-Activating Proteins/antagonists & inhibitors , GTPase-Activating Proteins/physiology , Gene Knockdown Techniques , HEK293 Cells , Humans , Protein Binding/genetics , Protein Transport/genetics , Receptors, Adrenergic, beta-2/biosynthesis , Receptors, Adrenergic, beta-2/genetics , Signal Transduction/genetics , beta-ArrestinsABSTRACT
ß(2)-Adrenergic receptors (ß(2)ARs) regulate cellular functions through G protein-transduced and ßArrestin-transduced signals. ß(2)ARs have been shown to regulate cancer cell migration, but the underlying mechanisms are not well understood. Here, we report that ß(2)AR regulates formation of focal adhesions, whose dynamic remodeling is critical for directed cell migration. ß(2)ARs induce activation of RhoA, which is dependent on ßArrestin2 but not G(s). ßArrestin2 forms a complex with p115RhoGEF, a guanine nucleotide exchange factor for RhoA that is well known to be activated by G(12/13)-coupled receptors. Our results show that ßArrestin2 forms a complex with p115RhoGEF in the cytosol in resting cells. Upon ß(2)AR activation, both ßArrestin2 and p115RhoGEF translocate to the plasma membrane, with concomitant activation of RhoA and formation of focal adhesions and stress fibers. Activation of RhoA and focal adhesion remodeling may explain, at least in part, the role of ß(2)ARs in cell migration. These results suggest that ßArrestin2 may serve as a convergence point for non-G(12/13) and non-G(q) protein-coupled receptors to activate RhoA.
Subject(s)
Arrestins/metabolism , Focal Adhesions/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Receptors, Adrenergic, beta-2/metabolism , Signal Transduction/physiology , rho GTP-Binding Proteins/metabolism , rhoA GTP-Binding Protein/metabolism , Activating Transcription Factor 6 , Animals , Arrestins/genetics , Basic-Leucine Zipper Transcription Factors/genetics , Basic-Leucine Zipper Transcription Factors/metabolism , Cell Movement/physiology , Enzyme Activation/physiology , Focal Adhesions/genetics , Guanine Nucleotide Exchange Factors/genetics , HEK293 Cells , Humans , Mice , Receptors, Adrenergic, beta-2/genetics , Rho Guanine Nucleotide Exchange Factors , beta-Arrestins , rho GTP-Binding Proteins/genetics , rhoA GTP-Binding Protein/geneticsABSTRACT
COPI (coat protein I) and the clathrin-AP-2 (adaptor protein 2) complex are well-characterized coat proteins, but a component that is common to these two coats has not been identified. The GTPase-activating protein (GAP) for ADP-ribosylation factor 1 (ARF1), ARFGAP1, is a known component of the COPI complex. Here, we show that distinct regions of ARFGAP1 interact with AP-2 and coatomer (components of the COPI complex). Selectively disrupting the interaction of ARFGAP1 with either of these two coat proteins leads to selective inhibition in the corresponding transport pathway. The role of ARFGAP1 in AP-2-regulated endocytosis has mechanistic parallels with its roles in COPI transport, as both its GAP activity and coat function contribute to promoting AP-2 transport.
Subject(s)
Endocytosis/physiology , GTPase-Activating Proteins/physiology , Transcription Factor AP-2/physiology , Humans , Microscopy, Electron , Protein Transport , Receptors, Transferrin/metabolism , Transcription Factor AP-2/metabolismABSTRACT
Invasion of bladder epithelial cells by uropathogenic Escherichia coli (UPEC) contributes to antibiotic-resistant and recurrent urinary tract infections (UTIs), but this process is incompletely understood. In this paper, we provide evidence that the large guanosine triphosphatase dynamin2 and its partner, endothelial nitric oxide (NO) synthase (NOS [eNOS]), mediate bacterial entry. Overexpression of dynamin2 or treatment with the NO donor S-nitrosothiols increases, whereas targeted reduction of endogenous dynamin2 or eNOS expression with ribonucleic acid interference impairs, bacterial invasion. Exposure of mouse bladder to small molecule NOS inhibitors abrogates infection of the uroepithelium by E. coli, and, concordantly, bacteria more efficiently invade uroepithelia isolated from wild-type compared with eNOS(-/-) mice. E. coli internalization promotes rapid phosphorylation of host cell eNOS and NO generation, and dynamin2 S-nitrosylation, a posttranslational modification required for the bacterial entry, also increases during E. coli invasion. These findings suggest that UPEC escape urinary flushing and immune cell surveillance by means of eNOS-dependent dynamin2 S-nitrosylation and invasion of host cells to cause recurrent UTIs.
Subject(s)
Dynamin II/metabolism , Epithelial Cells/enzymology , Epithelial Cells/microbiology , Nitric Oxide Synthase Type III/metabolism , Urinary Bladder/microbiology , Urinary Bladder/pathology , Uropathogenic Escherichia coli/physiology , Animals , Cell Line, Tumor , Cysteine/metabolism , Female , Humans , Mice , Mice, Inbred C57BL , NitrosationABSTRACT
Androgen receptor (AR) signaling regulates the development and homeostasis of male reproductive organs, including the prostate. Deregulation of AR and AR coregulators, expression, or activity is involved in the initiation of prostate cancer and contributes to the transition of the disease to hormone-refractory stage. The ubiquitous betaArrestin proteins are now recognized as bona fide adapters and signal transducers with target effectors found in both the cytosol and nucleus. Here, we provide evidence that betaArrestin2 forms a complex with AR and acts as an AR corepressor in androgen-dependent prostate cancer cells. Accordingly, the forced overexpression of betaArrestin2 diminishes, and knockdown of betaArrestin2 expression with RNAi increases the androgen-induced prostate-specific antigen (PSA) gene expression. betaArrestin2 serves as an adapter, bringing into close proximity the Mdm2 E3 ligase and AR, thereby promoting AR ubiquitylation and degradation. Human prostate tissues evidence an inverse relationship between the expression of betaArrestin2 and AR activity: glands that express high levels of betaArrestin2 exhibit low expression of PSA, and those glands that express low levels of betaArrestin2 evidence elevated PSA levels. We conclude that betaArrestin2 acts as a corepressor of AR by serving as a scaffold for Mdm2 leading to the AR ubiquitylation and degradation.
Subject(s)
Arrestins/metabolism , Prostatic Neoplasms/metabolism , Receptors, Androgen/metabolism , Signal Transduction , Humans , Male , Prostate-Specific Antigen , Prostatic Neoplasms/pathology , Protein Stability , Proto-Oncogene Proteins c-mdm2/metabolism , Receptors, Androgen/analysis , Ubiquitination , beta-ArrestinsABSTRACT
Focal adhesions are specialized sites of cell attachment to the extracellular matrix where integrin receptors link extracellular matrix to the actin cytoskeleton, and they are constantly remodeled during cell migration. Focal adhesion kinase (FAK) is an important regulator of focal adhesion remodeling. AGAP2 is an Arf GTPase-activating protein that regulates endosomal trafficking and is overexpressed in different human cancers. Here we examined the regulation of the FAK activity and the focal adhesion remodeling by AGAP2. Our results show that FAK binds the pleckstrin homology domain of AGAP2, and the binding is independent of FAK activation following epidermal growth factor receptor stimulation. Overexpression of AGAP2 augments the activity of FAK, and concordantly, the knockdown of AGAP2 expression with RNA interference attenuates the FAK activity stimulated by epidermal growth factor or platelet-derived growth factor receptors. AGAP2 is localized to the focal adhesions, and its overexpression results in dissolution of the focal adhesions, whereas knockdown of its expression stabilizes them. The AGAP2-induced dissolution of the focal adhesions is independent of its GTPase-activating protein activity but may involve its N-terminal G protein-like domain. Our results indicate that AGAP2 regulates the FAK activity and the focal adhesion disassembly during cell migration.
Subject(s)
Cell Movement/physiology , Focal Adhesion Kinase 1/metabolism , Focal Adhesions/enzymology , GTP-Binding Proteins/metabolism , GTPase-Activating Proteins/metabolism , ADP-Ribosylation Factors/genetics , ADP-Ribosylation Factors/metabolism , Biological Transport/physiology , Cell Line , Endosomes/genetics , Endosomes/metabolism , Enzyme Activation/physiology , Focal Adhesion Kinase 1/genetics , Focal Adhesions/genetics , GTP-Binding Proteins/genetics , GTPase-Activating Proteins/genetics , Gene Knockdown Techniques , Humans , Neoplasms/genetics , Neoplasms/metabolism , Protein Structure, Tertiary/physiologyABSTRACT
Arf GAPs are a family of proteins with a common catalytic domain that induces hydrolysis of GTP bound to the small GTP-binding protein Arf. The proteins are otherwise structurally diverse. Several subtypes of Arf GAPs have been found to be targets of oncogenes and to control cell proliferation and cell migration. The latter effects are thought to be mediated by coordinating changes in actin remodeling and membrane traffic. In this chapter, we discuss Arf GAPs that have been linked to oncogenesis and the molecular mechanisms underlying the effects of these proteins in cancer cells. We also discuss the enzymology of the Arf GAPs related to possible targeted inhibition of specific subtypes of Arf GAPs.
Subject(s)
ADP-Ribosylation Factors/metabolism , GTPase-Activating Proteins/metabolism , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Neoplasms/pathology , ADP-Ribosylation Factors/chemistry , Animals , Cell Movement , Focal Adhesions , Humans , Models, Biological , Multigene Family , Neoplasm Invasiveness , Protein Structure, Tertiary , Signal TransductionABSTRACT
At the FASEB summer research conference on "Arf Family GTPases", held in Il Ciocco, Italy in June, 2007, it became evident to researchers that our understanding of the family of Arf GTPase activating proteins (ArfGAPs) has grown exponentially in recent years. A common nomenclature for these genes and proteins will facilitate discovery of biological functions and possible connections to pathogenesis. Nearly 100 researchers were contacted to generate a consensus nomenclature for human ArfGAPs. This article describes the resulting consensus nomenclature and provides a brief description of each of the 10 subfamilies of 31 human genes encoding proteins containing the ArfGAP domain.
Subject(s)
ADP-Ribosylation Factors/metabolism , GTPase-Activating Proteins/metabolism , Terminology as Topic , ADP-Ribosylation Factors/chemistry , ADP-Ribosylation Factors/genetics , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , GTPase-Activating Proteins/chemistry , GTPase-Activating Proteins/genetics , Humans , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Models, Molecular , Molecular Sequence Data , Multigene Family , Protein ConformationABSTRACT
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.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cell Movement , Focal Adhesions/metabolism , GTPase-Activating Proteins/metabolism , Neoplasm Proteins/metabolism , Neoplasms/metabolism , ADP-Ribosylation Factor 1/genetics , ADP-Ribosylation Factor 1/metabolism , ADP-Ribosylation Factor 6 , ADP-Ribosylation Factors/genetics , ADP-Ribosylation Factors/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Blood Proteins/genetics , Blood Proteins/metabolism , Cell Line, Tumor , Female , Focal Adhesions/genetics , GTPase-Activating Proteins/genetics , Humans , Mice , NIH 3T3 Cells , Neoplasm Invasiveness , Neoplasm Proteins/genetics , Neoplasms/genetics , Neoplasms/pathology , Phosphatidylinositol 4,5-Diphosphate/genetics , Phosphatidylinositol 4,5-Diphosphate/metabolism , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Structure, Tertiary , Sequence Homology, Amino AcidABSTRACT
Invadopodia are Src-induced cellular structures that are thought to mediate tumor invasion. ASAP1, an Arf GTPase-activating protein (GAP) containing Src homology 3 (SH3) and Bin, amphiphysin, and RVS161/167 (BAR) domains, is a substrate of Src that controls invadopodia. We have examined the structural requirements for ASAP1-dependent formation of invadopodia and related structures in NIH 3T3 fibroblasts called podosomes. We found that both predominant splice variants of ASAP1 (ASAP1a and ASAP1b) associated with invadopodia and podosomes. Podosomes were highly dynamic, with rapid turnover of both ASAP1 and actin. Reduction of ASAP1 levels by small interfering RNA blocked formation of invadopodia and podosomes. Podosomes were formed in NIH 3T3 fibroblasts in which endogenous ASAP1 was replaced with either recombinant ASAP1a or ASAP1b. ASAP1 mutants that lacked the Src binding site or GAP activity functioned as well as wild-type ASAP1 in the formation of podosomes. Recombinant ASAP1 lacking the BAR domain, the SH3 domain, or the Src phosphorylation site did not support podosome formation. Based on these results, we conclude that ASAP1 is a critical target of tyrosine kinase signaling involved in the regulation of podosomes and invadopodia and speculate that ASAP1 may function as a coincidence detector of simultaneous protein association through the ASAP1 SH3 domain and phosphorylation by Src.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cell Membrane Structures/enzymology , Proto-Oncogene Proteins pp60(c-src)/metabolism , Adaptor Proteins, Signal Transducing/chemistry , Amino Acid Motifs , Animals , Cell Line, Tumor , Cortactin/metabolism , GTPase-Activating Proteins/metabolism , Humans , Mice , Mutant Proteins/metabolism , NIH 3T3 Cells , Phosphopeptides/metabolism , Phosphorylation , Protein Binding , Protein Isoforms/metabolism , Protein Structure, Tertiary , Protein Transport , Tyrosine/metabolismABSTRACT
The GTPase Arf6 regulates multiple cellular processes, including endocytosis, secretion, phagocytosis, cell adhesion, and cell migration [1, 2]. The Arf6-specific GAP ACAP1 is a negative regulator of Arf6-mediated signaling [3-7]. However, regulation of ACAP1- and Arf6-mediated signaling by other cellular proteins is not well understood. GULP/CED-6 is a phosphotyrosine binding (PTB)-domain-containing adaptor protein linked to engulfment of apoptotic cells [8-13] and to cholesterol homeostasis [14]. Here, we identify a novel role for GULP as a positive regulator of Arf6. Knockdown of GULP decreased cellular Arf6-GTP, whereas GULP overexpression increased cellular Arf6-GTP. At the mechanistic level, GULP influenced Arf6 at four levels. First, GULP bound directly to GDP-bound Arf6 via its PTB domain. Second, GULP associated with the Arf6-GAP ACAP1 at endogenous levels. Third, GULP reversed the Arf6-GTP decrease induced by ACAP1, and countered the ACAP1-mediated inhibition of cell migration. Fourth, GULP, ACAP1, and GDP-bound Arf6 were part of a tripartite complex, suggesting sequestration of ACAP1 as one mechanism of GULP action. Taken together, these data identify GULP as a modifier of cellular Arf6-GTP through regulation of ACAP1. Because PTB-domain-containing adaptor proteins influence endocytosis and trafficking of membrane proteins and cell migration [15, 16], our data support a model wherein PTB-domain-containing adaptor proteins regulate Arf family proteins.
Subject(s)
ADP-Ribosylation Factors/metabolism , Adaptor Proteins, Signal Transducing/metabolism , GTPase-Activating Proteins/metabolism , ADP-Ribosylation Factor 6 , Animals , Cell Line , Cricetinae , Guanosine Diphosphate/metabolism , HeLa Cells , Humans , Mice , Protein Structure, TertiaryABSTRACT
The selective transfer of material between membrane-delimited organelles is mediated by protein-coated vesicles. In many instances, formation of membrane trafficking intermediates is regulated by the GTP-binding protein Arf. Binding and hydrolysis of GTP by Arf was originally linked to the assembly and disassembly of vesicle coats. Arf GTPase-activating proteins (GAPs), a family of proteins that induce hydrolysis of GTP bound to Arf, were therefore proposed to regulate the disassembly and dissociation of vesicle coats. Following the molecular identification of Arf GAPs, the roles for GAPs and GTP hydrolysis have been directly examined. GAPs have been found to bind cargo and known coat proteins as well as directly contribute to vesicle formation, which is consistent with the idea that GAPs function as subunits of coat proteins rather than simply Arf inactivators. In addition, GTP hydrolysis induced by GAPs occurs largely before vesicle formation and is required for sorting. These results are the primary basis for modifications to the classical model for the function of Arf in transport vesicle formation, including a recent proposal that Arf has a proofreading, rather than a structural, role.
Subject(s)
ADP-Ribosylation Factors/metabolism , GTPase-Activating Proteins/metabolism , Intracellular Membranes/metabolism , Biological Transport , COP-Coated Vesicles/metabolism , Models, BiologicalABSTRACT
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.
Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/physiology , Cell Membrane/ultrastructure , ErbB Receptors/metabolism , GTPase-Activating Proteins/chemistry , GTPase-Activating Proteins/physiology , ADP-Ribosylation Factors/physiology , Adaptor Proteins, Signal Transducing/analysis , Amino Acid Sequence , Animals , GTPase-Activating Proteins/analysis , Mice , Models, Biological , Models, Molecular , Molecular Sequence Data , Protein Structure, Tertiary , Protein Transport , Sequence AlignmentABSTRACT
The core complex of Coat Protein I (COPI), known as coatomer, is sufficient to induce coated vesicular-like structures from liposomal membrane. In the context of biological Golgi membrane, both palmitoyl-coenzyme A (p-coA) and ARFGAP1, a GTPase-activating protein (GAP) for ADP-Ribosylation Factor 1, also participate in vesicle formation, but how their roles may be linked remains unknown. Moreover, whether COPI vesicle formation from Golgi membrane requires additional factors also remains unclear. We now show that Brefeldin-A ADP-Ribosylated Substrate (BARS) plays a critical role in the fission step of COPI vesicle formation from Golgi membrane. This role of BARS requires its interaction with ARFGAP1, which is in turn regulated oppositely by p-coA and nicotinamide adenine dinucleotide, which act as cofactors of BARS. Our findings not only identify a new factor needed for COPI vesicle formation from Golgi membrane but also reveal a surprising mechanism by which the roles of p-coA and GAP are linked in this process.
Subject(s)
Coat Protein Complex I/metabolism , Cytoplasmic Vesicles/metabolism , DNA-Binding Proteins/physiology , Golgi Apparatus/physiology , Phosphoproteins/physiology , Acyltransferases/physiology , Alcohol Oxidoreductases , Animals , COS Cells , Chlorocebus aethiops , Cytoplasmic Vesicles/ultrastructure , GTPase-Activating Proteins/metabolism , Golgi Apparatus/ultrastructure , Humans , Mutation , NAD/physiology , Palmitoyl Coenzyme A/physiologyABSTRACT
ADP ribosylation factors (Arf) regulate membrane trafficking at multiple intracellular sites by recruiting coat proteins to membranes. The site-specific regulation of Arf is thought to be mediated by regulatory proteins including the guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Here, we test this hypothesis by comparing the site of action of the Arf GAP AGAP2 to the closely related AGAP1. AGAP1 has previously been found to associate with the adaptor protein complex AP-3 and regulate the function of AP-3 endosomes. We found that AGAP2 directly interacted with AP-1. AGAP2 colocalized with AP-1, transferrin receptor and Rab4 on endosomes. Overexpression of AGAP2 changed the intracellular distribution of AP-1 and promoted Rab4-dependent fast recycling of transferrin. Based on these results, we concluded that the closely related Arf GAPs, AGAP1 and AGAP2, distinguish between these related heterotetrameric adaptor protein complexes to specifically regulate AP-3 endosomes and AP-1 recycling endosomes.
Subject(s)
ADP-Ribosylation Factors/metabolism , Adaptor Protein Complex 1/metabolism , Adaptor Protein Complex 3/metabolism , GTP-Binding Proteins/metabolism , GTPase-Activating Proteins/metabolism , ras Proteins/metabolism , ADP-Ribosylation Factors/genetics , Amino Acid Sequence , Animals , Cell Line , GTP-Binding Proteins/genetics , GTPase-Activating Proteins/genetics , Gene Expression Regulation , HeLa Cells , Humans , Mice , Molecular Sequence Data , NIH 3T3 Cells , Receptors, Transferrin/metabolism , ras Proteins/geneticsABSTRACT
ADP-ribosylation factors (Arfs) are Ras-like GTP-binding proteins that regulate membrane traffic and actin remodeling. Arf function requires GTP hydrolysis but Arf lacks GTPase activity; consequently, Arf function is dependent on Arf GTPase-activating proteins (GAPs). The Arf GAPs are a structurally diverse group of at least 16 proteins. Several Arf GAPs use a single Arf isoform. However, due to structural differences, the conditions supporting productive interactions between Arf and different Arf GAPs vary. Here, we describe preparation and basic properties of three Arf GAPs. We use these proteins to illustrate assays for Arf GAP activity. Conditions that optimize activity for each GAP are discussed. These methods can be used for the further characterization of Arf-Arf GAP interaction that is necessary for understanding the function of Arf in cellular physiology.
Subject(s)
ADP-Ribosylation Factors/analysis , Adaptor Proteins, Signal Transducing/analysis , GTP Phosphohydrolase Activators/analysis , GTPase-Activating Proteins/analysis , ADP-Ribosylation Factor 1/metabolism , Animals , Fluorescence , Guanosine Triphosphate/metabolism , Humans , Phosphorus Radioisotopes , Tryptophan/chemistryABSTRACT
ARAP3 is a GTPase activating protein (GAP) for Rho and Arf GTPases that is implicated in phosphoinositide 3-kinase (PI 3-kinase) signalling pathways controlling lamellipodia formation and actin stress fibre assembly. We have identified ARAP3 as a phosphorylated target of protein tyrosine kinases. In cells, ARAP3 was tyrosine phosphorylated when co-expressed with Src-family kinases (SFKs), upon stimulation with growth factors and during adhesion to the extracellular matrix (ECM) substrate fibronectin. Adhesion-induced phosphorylation of ARAP3 was suppressed by selective inhibitors of Src-family kinases and PI 3-kinase and by a Src dominant interfering mutant. Inducible expression of ARAP3 in HEK293 epithelial cells resulted in increased cell rounding, membrane process formation and cell clustering on ECM substrates. In contrast, ARAP3 dramatically slowed the kinetics of cell spreading on fibronectin but had no effect on cell adhesion. These effects of ARAP3 required a functional Rho GAP domain and were associated with reduced cellular levels of active RhoA and Rac1 but did not require the sterile alpha motif (SAM) or Arf GAP domains. Mutation of two phosphorylation sites, Y1399 and Y1404, enhanced some ARAP3 activities, suggesting that ARAP3 may be negatively regulated by phosphorylation on these tyrosine residues. These results implicate ARAP3 in integrin-mediated tyrosine kinase signalling pathways controlling Rho GTPases and cell spreading.
Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , GTPase-Activating Proteins/chemistry , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cell Adhesion , Cell Line , Cell Membrane/metabolism , Cell Movement , DNA/metabolism , DNA, Complementary/metabolism , Extracellular Matrix/metabolism , Fibronectins/metabolism , GTP Phosphohydrolases/metabolism , GTPase-Activating Proteins/metabolism , Gene Expression Regulation , Genes, Dominant , Humans , Immunoblotting , Immunoprecipitation , Kinetics , Mice , Mutation , NIH 3T3 Cells , Phosphorylation , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Signal Transduction , Time Factors , Tyrosine/chemistry , Tyrosine/metabolism , cdc42 GTP-Binding Protein/metabolism , rac1 GTP-Binding Protein/metabolism , rhoA GTP-Binding Protein/chemistry , src-Family Kinases/metabolismABSTRACT
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.