GEP100/BRAG2: Activator of ADP-ribosylation factor 6 for regulation of cell adhesion and actin cytoskeleton via E-cadherin and α-catenin
Abstract
GEP100 (p100) was identified as an ADP-ribosylation factor (ARF) guanine nucleotide-exchange protein (GEP) that partially colocalized with ARF6 in the cell periphery. p100 preferentially accelerated guanosine 5[γ-thio]triphosphate (GTPγS) binding by ARF6, which participates in protein trafficking near the plasma membrane, including receptor recycling, cell adhesion, and cell migration. Here we report that yeast two-hybrid screening of a human fetal brain cDNA library using p100 as bait revealed specific interaction with α-catenin, which is known as a regulator of adherens junctions and actin cytoskeleton remodeling. Interaction of p100 with α-catenin was confirmed by coimmunoprecipitation of the endogenous proteins from human HepG2 or CaSki cells, although colocalization was difficult to demonstrate microscopically. α-Catenin enhanced GTPγS binding by ARF6 in vitro in the presence of p100. Depletion of p100 by small interfering RNA (siRNA) treatment in HepG2 cells resulted in E-cadherin content 3-fold that in control cells and blocked hepatocyte growth factor-induced redistribution of E-cadherin, consistent with a known role of ARF6 in this process. F-actin was markedly decreased in normal rat kidney (NRK) cells overexpressing wild-type p100, but not its GEP-inactive mutants, also consistent with the conclusion that p100 has an important role in the activation of ARF6 for its functions in both E-cadherin recycling and actin remodeling.
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GEP100 (p100) was described by Someya et al. (1) as an ≈100-kDa guanine nucleotide-exchange protein (GEP) that preferentially activated ADP-ribosylation factor 6 (ARF6) in vitro. The molecule itself was of interest because it contained, in addition to the ARF-activating Sec7 domain (with a nuclear localization sequence), a pleckstrin homology-like domain and an IQ-like motif, the functions of which remain to be demonstrated. In homogenates of human T98G neuroblastoma cells, p100 was entirely cytosolic. On confocal immunofluorescence microscopy, however, it appeared in punctate concentrations scattered throughout the cytoplasm colocalized, in part, with early endosomal antigen 1 (EEA1) in a perinuclear region and with ARF6 near the plasma membrane (1). ARF6 is known to function at the cell periphery in endocytosis (2–6), exocytosis/secretion (7–9), phagocytosis (10, 11), cell migration (12–14), adherens junction (AJ) turnover (12, 15), actin cytoskeleton remodeling (16–18), and in the activation of enzymes that modify membrane phospholipids (19, 20). Donaldson, in a 2003 review of the roles of ARF6 (21), commented on the “complex interplay between signal transduction, membrane traffic, and the cytoskeleton.” It seems that p100 may be a locus of some of that interplay as it is now implicated in regulation of several of those ARF6 actions.
ARF6 involvement in the internalization and recycling of diverse cell surface receptors has been described. Partial colocalization of ARF6 with transferrin receptor and participation in its clathrin-dependent internalization was observed (2, 22). Fcγ receptor-mediated phagocytosis by macrophages also was shown to be regulated by ARF6 (10, 11). ARF6 functions in the internalization of both agonist-stimulated G protein-coupled receptors, such as β2-adrenergic receptors via β-arrestin- and clathrin-dependent mechanisms (5, 6, 23) and M2-muscarinic acetylcholine receptors via β-arrestin- and clathrin-independent mechanisms (4). In addition, ARF6 regulation of recycling to the plasma membrane of integrins (24, 25), which mediate cell–cell adhesion in most solid tissues (12, 15, 26) and are crucial for cell migration, indicates that it can influence cell shape, migration, and scattering. ARF6 activation of phosphatidylinositol 4-phosphate 5-kinase (20), generates phosphatidylinositol 4,5-bisphosphate, a major plasma membrane phosphoinositide involved in membrane trafficking and actin rearrangement (18, 27–29). ARF6 also can activate phospholipase D, which catalyzes phosphatidylcholine hydrolysis, producing phosphatidic acid, another activator of phosphatidylinositol 4-phosphate 5-kinase (19). The activation of both phospholipase D and phosphatidylinositol 4-phosphate 5-kinase by ARF6 can amplify phosphatidylinositol bis-4,5-phosphate signals to affect membrane ruffling and trafficking, as well as actin rearrangement (24, 30–32).
Like all GTPases, ARF6 requires for its activation and inactivation, respectively, GEPs that accelerate GTP-binding and GTPase-activating proteins that enhance GTP hydrolysis. We assume that ARF6 activation for its several diverse functions depends on specific GEPs that will be present with it at the correct place and time. All of our observations are consistent with a potential role for p100 in multiple ARF6 actions. Chen et al. (33) recognized p100 as a shorter form of a protein termed Loner that was mutated in Drosophila embryos in which myoblasts failed to form myotubes. They determined that Loner was required to activate ARF6 for proper localization of Rac to initiate formation of a membrane fusion assembly. Our finding of p100 interaction with α-catenin in a yeast two-hybrid screen led us to investigate the possible involvement of p100 in ARF6-influenced processes related to cell adhesion and migration. As reported here, depletion of p100 or α-catenin with specific small interfering RNAs (siRNAs) did not alter cell content of the other, but p100 siRNA treatment increased E-cadherin content 3-fold and interfered with the “scatter” response to hepatocyte growth factor (HGF). In addition, overexpression of p100 resulted in disappearance of F-actin, implicating p100 in normal rat kidney (NRK) cells in ARF6 regulation of adhesion junction dynamics and actin remodeling.
Results
Interaction of p100 and α-catenin.
Yeast two-hybrid screening of a human fetal brain cDNA library using human p100 as bait yielded 62 clones representing 29 proteins; α-catenin is 1 of 4 that interacted specifically with p100 in mating assays.
On confocal laser-scanning immunofluorescence microscopy of HepG2 cells, endogenous p100 and α-catenin were distributed in punctate concentrations throughout the cytoplasm (Fig. 1A), but colocalization was not detected, presumably because only a fraction of total cell p100 is designated to activate the pool of ARF6 involved in regulating AJs, and most of the α-catenin is functioning in other molecular assemblies, e.g., related to actin dynamics. Endogenous p100 was immunoprecipitated from HepG2 and CaSki cells by antibodies (Abs) against α-catenin (Fig. 1B).
Fig. 1.
Effect of α-Catenin on Guanosine 5′-γ-(Thio)Triphosphate (GTPγS) Binding to ARF6.
Effects of α-catenin on GTPγS binding by ARF6 in vitro were evaluated in assays without or with p100 (Fig. 2). ARF6 alone bound a small amount of guanosine 5-[γ-(35S) thio]triphosphate ([35S]GTPγS) in 20-min assays; binding by α-catenin or p100 alone was not detected. p100 dramatically accelerated [35S]GTPγS binding by ARF6, and in its presence, α-catenin enhanced [35S]GTPγS binding significantly at the highest concentration tested, which alone was without effect (Fig. 2A). These effects were seen throughout the 20-min incubation (Fig. 2B).
Fig. 2.
Effects of p100 and α-Catenin siRNAs on These and Other Intracellular Proteins.
After incubation of HepG2 cells for 72 h with p100 siRNA, p100 protein was 5.2 ± 1.5% (n = 5) of that in lysates of untreated (N) cells (Fig. 3). It was not significantly altered in cells incubated for 72 h with vehicle alone or in those in which α-catenin was decreased ≈90% (9.7 ± 3.6% of N) after incubation with α-catenin siRNA. E-cadherin in cells incubated with p100 siRNA was 309 ± 79% (n = 5) of that in untreated cells, but amounts of α-catenin, ARF6, β-catenin, or GAPDH were not significantly altered. β-Catenin content was significantly lower (40.2 ± 12.1% of N) in cells incubated with α-catenin siRNA, whereas amounts of the other proteins were not altered (Fig. 3).
Fig. 3.
On immunofluorescence microscopy, elevated amounts of E-cadherin in CaSki cells that had been incubated with p100 siRNA were concentrated at the plasma membrane as in control cells, but the cells appeared to be larger, perhaps because of spreading (Fig. 4). Incubation of cells with HGF resulted in redistribution of E-cadherin from its proximity to plasma membranes to scattered punctate collections throughout the cytoplasm. In cells incubated with p100 siRNA, however, internalized E-cadherin was not seen after HGF treatment (Fig. 4).
Fig. 4.
Effects of p100 siRNA on HepG2 cell adhesion to collagen-coated wells were assessed by counting cells remaining in medium at intervals after replating (Fig. 5). Cells that had been incubated with p100 siRNA became attached to the type 1 collagen-coated surface significantly more slowly than those treated with nontarget siRNA, or with vehicle alone, which is difficult to relate to E-cadherin function.
Fig. 5.
F-Actin in Cells Overexpressing p100.
The disappearance of F-actin from NRK cells overexpressing wild-type (WT) p100–GFP was dramatic and was not seen in cells overexpressing GFP or any GFP-tagged p100 mutants (p100E498A, p100ΔSec7, or p100ΔPH) (Fig. 6D). The staining pattern of F-actin in cells overexpressing p100ΔPH, i.e., without the PH domain, was, however, somewhat altered. Amounts of total actin were similar in all cells whether or not the appearance of F-actin was altered (Fig. 6B). From NRK cells overexpressing WT p100–GFP, but not GFP alone, anti-GFP Abs precipitated endogenous α-catenin (Fig. 6C).
Fig. 6.
Discussion
Finding that p100 interacted with α-catenin in a yeast two-hybrid screen suggested, of course, its involvement as an activator in at least some of the known actions of ARF6 near the cell surface, such as actin remodeling (16–18), cell migration (12–14), and AJ function (12, 15). The dramatic increase in E-cadherin content that we found in HepG2 cells depleted of p100 by siRNA treatment was not associated with changes in amount of α-catenin, nor did similar depletion of α-catenin alter amounts of p100 or E-cadherin. E-cadherin distribution in the p100-depleted cells resembled microscopically that in controls, although the cells did appear larger, consistent with spreading and flattening. In AJs, α-catenin interacts with E-cadherin indirectly through its association with β-catenin, which does interact with E-cadherin. It also binds to actin (34), and α-catenin was long considered a linker between AJs and actin fibers. Recently, it was reported (35, 36) that α-catenin binding to actin and β-catenin is, in effect, mutually exclusive, i.e., α-catenin monomers preferentially bind β-catenin and thereby the E-cadherin complex, whereas dimeric α-catenin binds actin filaments. In this way, α-catenin would serve as a molecular switch coordinating functions of AJs in cell–cell interaction and the actin cytoskeleton in cell motility (35, 36)
Cadherins, Ca2+-dependent, homophilic, adhesion proteins present in the cells of most solid tissues, are critical in the structure of AJs, which regulate cell–cell adhesion (37, 38). Cell-surface E-cadherin in epithelial cells is partially internalized and recycled back to the plasma membrane (39). ARF6 has important functions in both cell adhesion and E-cadherin recycling (12, 15, 26, 40). Overexpression of constitutively active ARF6(Q67L) resulted in loss of AJs and ruffling of basolateral membranes of Madin–Darby canine kidney (MDCK) cells (18). In contrast, overexpression of dominant-negative ARF6(T27N) blocked HGF-induced internalization of E-cadherin, which requires ARF6–GTP (18). ARF6 and Rac1 functions were both required in cell scattering, and an ARF6-dependent decrease in Rac1–GTP was necessary for HGF-induced cell–cell dissociation, which depends on prior disassembly of AJs (40). ARF6 activation by p100 appeared to be required for internalization of E-cadherin by HepG2 cells, resulting in its accumulation at the plasma membrane of p100-depleted cells, and interfered with its internalization in response to HGF. We found that E-cadherin levels were elevated in cells after siRNA depletion of p100 content, where it appeared increased in both cell interior and plasma membrane, with clearly impaired redistribution in response to HGF. Along with changes in their E-cadherin localization, the p100-depleted cells became attached to collagen-coated wells more slowly than did cells treated with other siRNAs. This result seems likely due to the requirement of p100-activated ARF6 for maintenance of a cell surface adhesion function other than that of E-cadherin in AJs. It is consistent with known roles of ARF6 in cell–matrix and cell–cell interactions (21), as well as with the recent report of Dunphy et al. (41) implicating BRAG2, a form of which is identical to p100, in recycling of β1-integrin.
Those researchers found that overexpression of epitope-tagged BRAG2a/p100 (or BRAG2b) increased amounts of ARF6–GTP in cell lysates, whereas mutants incapable of activating ARF6 were without effect. Overexpressed BRAG2/p100 in both HeLa and MDCK cells was detected in nuclei and increased after incubation of cells with leptomycin B (41). This observation is consistent with the presence of a nuclear localization sequence, which had been identified in p100, although we had not seen the endogenous protein in T98G cell nuclei (1), and its nuclear function is not readily apparent. Overexpression of HA–BRAG2a or -2b (but not an E498A mutant incapable of activating ARF6) markedly decreased amounts of F-actin seen microscopically in HeLa cells, without changing total actin content (41). The findings in NRK cells reported here are quite similar, but possible differences in the appearance of F-actin when p100 lacked the PH domain require additional investigation, as do other potential effects of p100 mutants. Dunphy et al. (41) described the accumulation of overexpressed BRAG2 (not mutant E498A) in clusters or patches at the plasma membrane where F-actin was also concentrated, and those effects were enhanced by phorbol myristoyl acetate. In their studies, “knock-down” of BRAG2 in HeLa cells resulted in increased amounts of β1-integrin on the cell surface, which correlated with increased cell spreading, whereas cell-surface β1-integrin and cell spreading were decreased in cells treated with ARF6 siRNA. The authors suggested that ARF6 activated by BRAG2 served specifically for β1-integrin internalization, whereas a different activator of ARF6 was required for its recycling (41).
The endocytosis of E-cadherin that accompanies AJ disassembly in MDCK cells (26) required Nm23-H1, a nucleoside diphosphate kinase (NDPK), earlier described as a suppressor of tumor metastasis (26). ARF6–GTP recruited Nm23–H1 to the cytoplasmic face of AJs where it was necessary for dynamin-catalyzed fission of clathrin-coated endocytic vesicles that remove E-cadherin and also sequestered Tiam 1, a GEP for Rac1, thereby interfering with Rac1 activation (26). The altered appearance of E-cadherin that we saw in p100-depleted cells responding to HGF may reflect a role of p100 in the activation of ARF6 for at least some of the steps of AJ dissociation and E-cadherin recycling in addition to its endocytosis.
In addition to p100/BRAG2, EFA6 (42) and cytohesin/ARNO (43) are reported to be brefeldin A (BFA)-insensitive activators of ARF6. EFA6 was initially implicated in regulation of transferrin receptor endocytosis, endosomal membrane recycling, and actin cytoskeleton remodeling related to membrane ruffling (42). Its role in E-cadherin recruitment and actin rearrangement for generation of tight junctions (TJs), which follows AJ assembly, and establishment of cell polarity was later described (44). EFA6 and p100 may have analogous functions in parallel pathways that regulate, respectively, TJ and AJ dynamics, including their interactions with the actin cytoskeleton. ARNO had been described as an activator of ARF1 (43) before Frank et al. (45) described its activation of ARF6 and location at the plasma membrane, commenting on the relationship of their findings to the report of cytohesin-1 as a regulator of αLβ2 integrin function at the cell surface (46). Santy and Casanova (13) later showed that ARNO was responsible for ARF6 activation in MDCK cells, where its overexpression enhanced migration by inducing formation of lamellipodia and separation of cell–cell contacts. Activation of Rac and phospholipase D also was observed. Inhibition of phospholipase D activity diminished cell motility, but not Rac activation, i.e., it appeared that phosphatidic acid production was not necessary for Rac activation but was important for the migratory behavior (13). Whether or not p100 is involved in those processes remains to be determined.
Proteins that contain Sec7 domains, which activate ARF GTPases by accelerating replacement of bound GDP by GTP, are present in all eukaryotic cells. In a phylogenetic analysis of Sec7 domain proteins, Cox et al. (47) described the ARF6-activating BRAG2/p100, EFA, and cytohesin/ARNO groups as the only ones that are found exclusively in animals. Perhaps each coevolved with other proteins to serve in similarly structured, parallel pathways, through which dynamic mechanical, metabolic, and signaling processes associated with cell–cell and cell–matrix interactions are controlled. Those vital functions all require continual temporal and spatial regulation to assure precise coordination and integration throughout the life of an organism. It will be necessary to understand better each of the BRAG2/p100, EFA6, and cytohesin/ARNO pathways for inside-out and outside-in signaling before we can begin to relate them. In addition to those GEPs, BIG2 was identified as critical in E-cadherin transport from Golgi to plasma membranes in an investigation of autosomal recessive mutations that caused defective human embryonic brain development (48). BIG2 is a brefeldin A-inhibited activator of ARF1–3, previously implicated in delivery of transferrin receptor from recycling endosome to the cell surface (49). The findings of Sheen et al. (48) emphasize the major importance of vesicular trafficking in differentiation and development.
The elevated E-cadherin content of cells depleted of p100 in our studies was striking, but the mechanism through which it occurs is unknown, as is the presumably different process through which β-catenin was decreased in cells treated with α-catenin siRNA. Our data are consistent with roles for p100 in an ARF6-dependent E-cadherin recycling pathway and E-cadherin/α-catenin/β-catenin-mediated effects on cell adhesion and structure. Learning how p100 levels are controlled and characterization of the endogenous p100 molecules are now of major interest. We need to understand how the production of alternatively spliced forms of BRAG2 described by Dunphy et al. (41) is regulated, as well as how these forms differ in function. Recognition of BRAG2/p100 involvement in functions resembling several better-studied actions of EFA6 and cytohesin/ARNO with ARF6, adhesion molecules, and the actin cytoskeleton provides many new questions.
Materials and Methods
Materials.
FBS, medium, nonessential amino acids, Nupage Bis-Tris gels, and Escherichia coli-competent cells [one shot TOP10 and BL21 (DE3)] were purchased from Invitrogen; HGF and protease inhibitors were purchased from Sigma; and [35S]GTPγS (1,250 Ci/mmol; 1 Ci = 37 GBq) was purchased from NEN. cDNA encoding KIAA0763 (GenBank accession no. AB018306; GI 3882246) was kindly provided by T. Nagase (Kazusa DNA Research Institute, Chiba, Japan).
Cell Culture.
All cells (purchased from American Type Culture Collection) were incubated in an atmosphere of 5% CO2/95% air at 37°C. HepG2 human liver carcinoma cells, used in most experiments, were grown in minimum essential medium with 10% FBS and 0.1 mM nonessential amino acids on collagen-coated plastic plates (BD Biosciences, San Diego). Human epidermoid carcinoma CaSki cells were grown in RPMI medium 1640 with 10% FBS. NRK cells, used for overexpression of GFP-tagged WT and mutant p100, were grown in Dulbecco’s modified Eagle’s medium with 10% FBS. All media contained penicillin G (100 units/ml) and streptomycin (100 μg/ml).
p100 and α-Catenin siRNA Experiments.
HepG2 cells (50–60% confluent) were incubated for 72 h with p100 or α-catenin siRNA (siGENOME Smartpool reagent), according to the manufacturer’s instructions (Dharmacon Research, Lafayette, CO). Control cells were incubated with vehicle (DharmaFECT siRNA Transfection reagents no. 4) alone or with nontarget siRNA (siGENOME Smartpool reagent), but not all control data are shown. For immunoblotting analyses, cells were washed with PBS, harvested, and homogenized in lysis buffer (150 mM NaCl in 50 mM Tris·HCl, pH 7.5) containing benzamidine (16 μg/ml), phenanthroline, aprotinin, leupeptin, and pepstatin A (each 10 μg/ml), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1% Triton X-100 (0.2 ml per dish).
Confocal Microscopy.
HepG2 and CaSki cells grown on type 1 collagen-coated four-well CultureSlide (BD Biosciences) were usually fixed (20 min, room temperature) with 3% paraformaldehyde in PBS, washed with PBS, permeabilized (4 min) with 0.1% Triton X-100 in PBS, washed with PBS, and incubated (1 h, room temperature) with PBS containing 10% goat serum and 3% BSA. Cells were washed with PBS and incubated (overnight at 4°C) with anti-p100 polyclonal Abs (1/20 dilution), anti-α-catenin monoclonal Abs (1/20 dilution, α-E-catenin; Santa Cruz Biotechnology), or anti-E-cadherin monoclonal Abs (1/200 dilution; BD Biosciences), washed with PBS, and incubated (1 h at room temperature) with FITC-conjugated anti-mouse IgG (1/100 dilution; Vector Laboratories) or Texas red-conjugated or FITC-conjugated anti-rabbit IgG (1/100 dilution; Vector Laboratories). After washing with PBS, cells were mounted in Vectashield (Vector Laboratories) and inspected with a confocal microscope (LSM 510; Zeiss).
Immunoblotting Analyses.
Proteins (10 μg) were separated by SDS/PAGE (4–12% gel) and transferred to poly(vinylidene difluoride) membranes. Blots were incubated with anti-p100 polyclonal (1/500 dilution), anti-α-catenin monoclonal (1/500 dilution), anti-E-cadherin monoclonal (1/2,500 dilution), anti-ARF6 monoclonal (1/200 dilution; Chemicon International, Temecula, CA), anti-β-catenin monoclonal (1/500 dilution; BD Biosciences), or anti-GAPDH polyclonal (1/2,000 dilution; Abcam, Inc., Cambridge, MA) Abs, followed by horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (Promega) and development using SuperSignal West Pico Chemiluminescent Substrate (Pierce). Band density was quantified by the nih image program (http://rsb.info.nih.gov/nih-image).
Immunoprecipitation.
HepG2 or CaSki cell proteins (100 μg) were incubated with anti-α-catenin monoclonal Ab or mouse IgG (2 μg) for 1 h, then overnight with protein A/G-agarose (50 μl). Agarose was washed twice with lysis buffer, once with 300 mM NaCl in 50 mM Tris·HCl (pH 7.5), and twice with 0.1% Triton X-100 in 10 mM Tris·HCl (pH 7.5). Bound proteins, extracted by boiling beads for 3 min in loading buffer, were separated by SDS/PAGE (4–12% gel) and transferred to poly(vinylidene difluoride) membranes for reaction with anti-p100 polyclonal Abs (1/500 dilution).
NRK cells (4 × 106 cells), 24 h after transfection, were washed twice with PBS, suspended in lysis buffer, and, after 30 min on ice, homogenized (30 strokes) in a Dounce homogenizer. The homogenate was centrifuged (2,000 × g, 15 min), and the supernatant (100 μg) was incubated (4°C, 1 h) with 50 μl of protein A/G-agarose, which was discarded, then incubated with anti-GFP Living Colors Full-Length Aequorea victoria polyclonal Ab (2 μg; Clontech) for 1 h, followed by addition of 50 μl of protein A/G-agarose and incubation overnight. Bound proteins were eluted for immunoblotting with anti-α-catenin monoclonal Ab (1/500 dilution).
Abbreviations:
- AJ
- adherens junction
- ARF
- ADP-ribosylation factor
- GTPγS
- guanosine 5′-γ-(thio)triphosphate
- [35S]GTPγS
- 5-[γ-(35S) thio]triphosphate
- HGF
- hepatocyte growth factor
- NRK
- normal rat kidney
- p100
- GEP100
- siRNA
- small interfering RNA.
Acknowledgments
We thank Dr. Christian Combs (National Heart, Lung, and Blood Institute Confocal Microscopy Core Facility) and Dr. Zu-Xi Yu (National Heart, Lung, and Blood Institute Pathology Core Facility) for invaluable help and Dr. Julie G. Donaldson (Laboratory of Cell Biology, National Heart, Lung, and Blood Institute) for manuscript review. This work was supported by the Intramural Research Program of the National Heart, Lung, and Blood Institute, National Institutes of Health.
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Published online: July 11, 2006
Published in issue: July 11, 2006
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Acknowledgments
We thank Dr. Christian Combs (National Heart, Lung, and Blood Institute Confocal Microscopy Core Facility) and Dr. Zu-Xi Yu (National Heart, Lung, and Blood Institute Pathology Core Facility) for invaluable help and Dr. Julie G. Donaldson (Laboratory of Cell Biology, National Heart, Lung, and Blood Institute) for manuscript review. This work was supported by the Intramural Research Program of the National Heart, Lung, and Blood Institute, National Institutes of Health.
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Conflict of interest statement: No conflicts declared.
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