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First published online 31 October 2006
doi: 10.1242/jcs.03237

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ARAP2 effects on the actin cytoskeleton are dependent on Arf6-specific GTPase-activating-protein activity and binding to RhoA-GTP

Hye-Young Yoon¹, Koichi Miura^1,*, E. Jebb Cuthbert², Kathryn Kay Davis², Bijan Ahvazi³, James E. Casanova² and Paul A. Randazzo^1,

¹ Laboratory of Cellular Oncology, Center for Cancer Research, National Cancer Institute, Department of Health and Human Services, Building 37, Bethesda, MD 20892, USA
² Department of Microbiology, University of Virginia at Charlottesville, School of Medicine, Charlottesville, VA, USA
³ X-Ray Crystallography Facility, Office of Science and Technology, National Institute of Arthritis and Musculoskeletal and Skin Disease, National Institutes of Health, Bethesda, MD, USA

View larger version (97K): [in a new window]   Fig. 1. Characterization of the ArfGAP activity of ARAP2. (A) Schematic representation of ARAP2. ARAP2 accession no. NM_015230. SAM, sterile -motif; PH, pleckstrin-homology domain; ArfGAP, ArfGTPase-activating protein; RhoGAP, RhoGTPase-activating protein; A, ankyrin repeat; RA, Ras-associating domain; and Rab13, Rab13 effector. (B) Purification of recombinant ARAP2. Flag[467-930]ARAP2 was expressed in HEK 293 cells and purified by immunoprecipitation (lane 1) or immunoprecipitation followed by affinity purification on a PtdIns(3,4,5)P3 column (lane 2). The arrow points to [467-930]ARAP2 separated on a polyacrylamide gel. (C) In vitro Arf specificity of ARAP2. The ArfGAP assay was performed in the presence of 50 µM PA, 10 µM PtdIns(4,5)P2, 100 nM PtdIns(3,4,5)P3, with Arf1-GTP, Arf5-GTP or Arf6-GTP as substrate, and the indicated amount of purified ARAP2 (µM). (D) Activation of ArfGAP activity by PtdIns. The ArfGAP activity was measured as in the presence of 100 nM PtdIns(3,4,5)P3 [PI(3,4,5)P3], 10 µM PtdIns(4,5)P2 [PI(4,5)P2], 500 nM PtdIns(3,5)P2 [PI(3,5)P2], 500 nM PtdIns(3,4)P2 [PI(3,4)P2] or 500 nM PtdIns(3)P [PI(3)P] with or without 50 µM PA. Arf6 was used as a substrate. (E) Activation of ARAP2 ArfGAP activity by mixtures of PtdIns(3)P and PtdIns(4,5)P2. The ArfGAP assay was performed in the presence of varying concentration of PtdIns(3)P (PIP3) and in the absence (+) or presence (–) of 10 µM PtdIns(4,5)P2 [PI(4,5)P2]. (F) Localization of ARAP2 relative to and effect of overexpressed ARAP2 on epitope-tagged Arf proteins. U118 cells were transfected with plasmids encoding HA-tagged Arfs and, where indicated, Flag-tagged ARAP2, and stained with rabbit polyclonal Ab against ARAP2 or against the epitope tag and mouse monoclonal Ab to the HA-epitope followed by Texas-Red-conjugated anti-rabbit IgG and fluorescein-conjugated anti-mouse IgG. Arrows indicate endogenous ARAP2. Bars, 10 µm. (G,H) Effect of tetrafluoraluminate on relative ARAP2 and Arf6 (G) and Arf1 and Arf5 (H) localization. Where indicated, U118 cells expressing Arf6-HA, Arf1-HA or Arf5-HA and Flag-ARAP2 were treated with AlF –4 for 10 minutes, fixed and stained. Bars, 10 µm. (I) The effect of ARAP2 on in vivo Arf-GTP. Arf1 and Arf6 were coexpressed with ARAP2, AGAP1 or ACAP1. Cells were lysed and activated Arf was co-precipitated with GST-GGA. The precipitated Arf was detected by immunoblotting with monoclonal anti-HA Ab.

View larger version (42K): [in a new window]   Fig. 2. Interaction between ARAP2 with Rho family GTP-binding proteins. (A) In vitro tests for RhoGAP activity of ARAPs. The GAP activity of ARAP1 and Flag-tagged [875-1704]ARAP2 using His-tagged Cdc42 as a substrate was measured. (B-D) Effect of ARAP2 on in vivo levels of (B) RhoA-GTP, (C) Cdc42-GTP and (D) Rac1-GTP. ARAP2 or p190RhoGAP were expressed with RhoA-AU5, Cdc42-AU5 or Rac1-AU5, as indicated, in HEK 293 cells. RhoA-GTP, Cdc42-GTP and Rac1-GTP levels were measured by co-precipitation with GST-Rhotekin-RBD-coated beads (for RhoA) or GST-PAK-PBD-coated beads (for Cdc42 and Rac1) followed by immunoblotting. Expression levels of RhoA, Cdc42, Rac1, ARAP2 or p190RhoGAP in the total cell lysates are shown. (E) Quantitation of RhoA-GTP levels in cells expressing ARAP2. The experiment described in B was repeated and signals were quantified with a scanning densitometer. The results presented are normalized to the levels of RhoA-GTP in the absence of RhoGAP or ARAP2 and are the means and range of duplicate determinations. (F) Binding of Rho family proteins to ARAP2 in vivo. HEK 293 cells were co-transfected with ARAP2 and either AU5-tagged RhoA, Rac1, or Cdc42. Proteins were immunoprecipitated using anti-FLAG M2 gel. Co-precipitated Rho family proteins were detected by immunoblotting with an anti-AU5 Ab. Protein expression levels in the total cell lysates is shown. (G) Nucleotide dependence of Rho binding to ARAP2. Lysates of HEK 293 cells transiently expressing ARAP2 were incubated with GSTRhoA loaded with 100 µM GDPßS or GTPS or with GST. (H) Identification of the ARAP2 domain that interacts with RhoA. GST-RhoA–GTPS or GST was incubated with lysates of HEK 293 cells transfected with expression constructs encoding Flag-tagged fragment corresponding to amino acids 467-930 (PH1-ANK domain), 1114-1323 (RhoGAP domain), 1317-1433 (RA domain) of ARAP2. The amount of Flag-tagged protein co-precipitating with GST or GST-RhoA immobilized on beads was determined by immunoblotting. Expression levels of GST or GST-RhoA and the recombinant Flag tagged ARAP2 are shown. (I) Effect of changing arginine 728 to lysine on ARAP2 binding to RhoA. Lysates from cells expressing Flag-ARAP2 or Flag-[R728K]ARAP2 were incubated with GST-RhoA–GTPS or GST as a control, and interaction was determined as described in (F) and (G). A Coomassie-blue-stained acrylamide gel electrophoresis fractionation of GST and GST-RhoA. The levels of recombinant ARAP2 proteins in the cell lysates was determined by immunoblotting. (J) Effect of changing lysine 1190 to proline on ARAP2 binding to RhoA. Lysates from cells expressing Flag-ARAP2 or Flag-[K1190P]ARAP2 were incubated with GST or GST-RhoA–GTPS. The experiment was performed as described in (I). (K) Model of ARAP2 RhoGAP domain and predicted consequences of mutating lysine 1190 to proline. Ribbon image of the structure of the Rho-GDP-AlF–4 (purple)–RhoGAP (green) complex. The Mg2+ ion is shown in gray and the GDP molecule and AlF–4 are shown in ball-and-stick. The Asp side chains from switch II and a conserved lysine residue in RhoGAP protein are shown in the left panel. The lysine to proline mutation residue is highlighted in the right panel. The panel in the center is an enlarged view of salt-bridging interactions between Asp63 from the switch II region with the conserved lysine (proline mutation) residue in RhoGAP protein.

View larger version (69K): [in a new window]   Fig. 3. Cellular distribution of ARAP2. (A) Endogenous ARAP2. U118, MCF7 and MDA-MB-231 cells, as indicated, were stained for ARAP2 and paxillin. (B) Epitope-tagged ARAP2. U118 cells expressing Flag-ARAP2 were fixed and stained for the Flag epitope and paxillin. Arrows indicate FAs. Bars, 10 µm.

View larger version (20K): [in a new window]   Fig. 4. (A) Effect of ARAP2 on cell-spreading rate. U118 cells (control), U118 cells expressing epitope-tagged ARAP2 (ARAP2) or U118 cells treated with siRNA to reduce expression of ARAP2 (siRNA) were trypsinized from plastic flasks, triturated and reseeded on coverslips coated with a solution of 10 µg/ml fibronectin. Cells were fixed at the indicated times and stained for paxillin and either endogenous ARAP2 or Flag epitope. The cells were then examined by confocal microscopy. To quantify the cell spreading at the indicated time, fluorescent images of 50 randomly selected cells were captured using 20x 1.4 NA objectives and surface areas were determined using ImagePro® Plus 5.1 software package. Values are the mean ± standard error of the mean from two independent experiments. (B) Relative ARAP2 levels after siRNA treatment of U118 cells. Cells were either mock-transfected, or transfected with a control siRNA or siRNA targeting ARAP2 as indicated. Three days after transfection, the cells were lysed and analyzed by immunoblotting with specific antibodies to ARAP2.

View larger version (83K): [in a new window]   Fig. 5. ARAP2 and the cytoskeleton. (A) Effect of expression levels of ARAP2 on cytoskeletal markers in U118 cells. Untreated cells (control, left panel), cells expressing Flag tagged ARAP2 (middle panel), or cells with reduced expression of ARAP2 (ARAP2-siRNA, right panel) were replated on fibronectin-coated slide for 4 hr in serum free media, fixed and stained for ARAP2 and either paxillin, vinculin, phosphotyrosine (pY), or actin. Bars 10 µm. (B) ARAP2 protein levels in cells transfected with siRNA and siRNA-resistant plasmids. U118 cells were transfected with siRNA directed to the 3'UTR of the ARAP2 message and plasmids directing expression of the indicated proteins. Cell lysates were analyzed by immunoblotting using antiserum (1185) raised against ARAP2 and an antiFLAG antibody. Bars, 10 µm.

View larger version (185K): [in a new window]   Fig. 6. ARAP2 and the cytoskeleton. (A,B) Role of ArfGAP and RhoGAP domains in effects of ARAP2 on cytoskeleton. Effect of replacing endogenous ARAP2 with recombinant ARAP2 on stress fiber (A) and focal adhesion (B) formation. U118 cells were transfected with empty vector, Flag-ARAP2, Flag-[R728K]ARAP2, or Flag-[K1190]ARAP2 with (right panel) or without (left panel) ARAP2 siRNA. Three days after transfection, cells were seeded onto fibronectin-coated slides, fixed, and stained with polyclonal anti-ARAP2 Ab and either Rhodamine-phalloidin to visualize actin (A) or monoclonal anti-paxillin Ab to visualize focal adhesions (B). (C) Effect of [Q67L]Arf6 on stress fibers in U118 cells. Cells were transfected with either plasmids for the expression of wild type or [Q67L]Arf6-HA. After 24 hours the cells were replated on fibronectin-coated coverslips, incubated for 6 hours in serum-free medium and then stained for epitope-tagged Arf6 and actin. (D) Effect of ARAP2 expression level on stress fibers induced by [Q63L]RhoA. Cells were co-transfected with siRNA targeting ARAP2 and an expression plasmid for [Q63L]RhoA, fixed and stained for RhoA and polymerized actin. Bars, 10 µm.

View larger version (67K): [in a new window]   Fig. 7. Relationship of ROK and ARAP2. (A,B) Effect of ROK on cells with reduced ARAP2 expression. U118 cells were transfected with a plasmid for expression of HA tagged-ROK and ARAP2-siRNA (right panels) and stained using polyclonal anti-HA and either Rhodamine-phalloidin for actin (A) or a monoclonal Ab for paxillin (B). (C) Effect of activated ROK on FA formation in cells with reduced ARAP2 levels. The experiment was similar to that described in B but using GFP-[1-478]ROK instead of full-length ROK. (D,E) Effect of ARAP2 on cells with inhibited ROK activity. U118 cells were transfected with a plasmid for the expression of ARAP2. Twenty-four hours later, the cells were treated with Y27632, a specific ROK inhibitor. The cells were fixed and stained for ARAP2 and either actin (C) or paxillin (D) as indicated. Arrows point to focal contacts. Bars, 10 µm.

View larger version (70K): [in a new window]   Fig. 8. Characterization of antibody for ARAP2. (A) ARAP2 protein levels in cultured cells. ARAP2 was detected by western blotting using antibody 1185, which was raised against amino acid residues 1689-1704 of ARAP2 as described in Materials and Methods. HEK 293, human embryonic kidney 293 cells; U118, glioblastoma cells; U87, glioblastoma cells; Jurkat, T lymphocyte cells; MCF-7, breast adenocarcinoma cells; MDA-MB-231, advanced breast adenocarcinoma cells; LNCAP, prostate cancer cells. A lysate from cells transiently transfected with a plasmid directing expression of Flag-ARAP2 was used a positive control. Actin was used as a control for normalization. In the middle panel, the signal was blocked by incubating the antibody with the peptide (10 µg/ml) against which the antibody was raised. (B) Effect of siRNA treatment of cells on proteins detected by antiserum 1185. The indicated cell lines were treated for 72 hours with a irrelevant siRNA (control siRNA) or siRNA targeting the 3'UTR of ARAP2 and then lysed for immunoblotting. (C) Characterization of antiserum 1185 against ARAP2 in immunofluorescence. The effect of peptide treatment of antibody and siRNA treatment of cells was examined in the indicated panels. The cells were counterstained for paxillin. Note that the signal for paxillin is easily detected on siRNA treatment but is also altered by the siRNA as described in the article. (D) Antibodies affinity-purified from 1185 and 1187 antisera. Rabbit anti-ARAP2 antisera raised against the synthetic peptides RSRTLPKELQDEQILK, residues 1689-1704 of ARAP2 (antiserum 1185), and ANVHKTKKNDDPSKDY, residues 78-93 of ARAP2 (antiserum 1187) were affinity-purified and used for immunoblotting of lysates from MCF7. (E) Immunofluorescence with affinity purified antibodies. Immunofluorescence of MCF7 cells was performed as in (C) but with affinity-purifed antibodes from antisera 1185 and 1187.

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