Proteomic Identification and Functional Characterization of a Novel ARF6 GTPase-activating Protein, ACAP4

• DOI: 10.1074/mcp.m600050-mcp200
• Publication Date: 2006-06-01T00:40:58Z
• Authors: Zhiyou Fang; Yong Miao; Xia Ding; Hui Deng; Siqi Liu; Fengsong Wang; Rihong Zhou; Charles Watson; Chuanhai Fu; Qicong Hu; James W. Lillard; Michael Powell; Yong Chen; John G. Forte; Xuebiao Yao
• Keywords: ADP ribosylation factor; Pleckstrin homology domain; Membrane ruffling

ARF6 GTPase is a conserved regulator of membrane trafficking and actin-based cytoskeleton dynamics at the leading edge migrating cells. A key determinant function lifetime GTP-bound active state, which orchestrated by GTPase-activating protein (GAP) GTP-GDP exchanging factor. However, very little known about molecular mechanisms underlying ARF6-mediated cell migration. To systematically analyze proteins that regulate activity during migration, we performed proteomic analysis selectively bound to using mass spectrometry identified novel ARF6-specific GAP, ACAP4. ACAP4 encodes 903 amino acids contains two coiled coils, one pleckstrin homology domain, GAP motif, ankyrin repeats. Our biochemical characterization demonstrated has phosphatidylinositol 4,5-bisphosphate-dependent specific for ARF6. The co-localization with occurred in ruffling membranes formed upon AIF4 epidermal growth factor stimulation. overexpression limited recruitment ruffles absence Expression GTP hydrolysis-resistant ARF6Q67L resulted accumulations cytoplasmic membrane, suggesting hydrolysis required ARF6-dependent remodeling. Significantly depletion small interfering RNA or inhibition overexpressing GAP-deficient suppressed migration wound healing, demonstrating importance Thus, our study sheds new light on biological During coordination between traffic, substrate adhesion, actin reorganization protrusive occur cells (e.g. Ref. 1Jaffe A.B. Hall A. Rho GTPases: biochemistry biology.Annu. Rev. Cell Dev. Biol. 2003; 21: 247-269Crossref Scopus (2353) Google Scholar). 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Long Wu Bi Q. Fan Forte Teng Yao PALS1 specifies localization ezrin apical gastric parietal cells.J. 2005; 280: 13584-13592Abstract (40) GST-ARF6Q67L was affinity matrix isolate HeLa cells, whereas GST-ARF6T27N control. Briefly interphase extracted lysis buffer containing 50 mm Tris-HCl (pH 6.9), 100 NaCl, 0.1% Triton X-100 plus proteinase inhibitor mixture (pepstatin A, leupeptin, aprotinin, chymostatin final concentrations 5 μg/ml each). lysates clarified Eppendorf centrifuge 13,000 rpm 10 min. resulting supernatant incubated 30 μl amylase-ARF6 beads room temperature 2 h. then washed three times followed boiling 1× sample buffer. samples resolved 6–16% gradient SDS-polyacrylamide gel, band interest removed spectrometric analyses (14Zhou Cao Watson Miao Characterization kinase A-mediated phosphorylation activation.J. 35651-35659Abstract (77) In-gel digestion done essentially P1 excised, chopped fragments razor blade, destained, subjected porcine trypsin (50–100 ng/digestion; Promega, Madison, WI). Peptides recovered extractions 50% acetonitrile 5% trifluoroacetic acid desalted concentrated C18 ZipTips (Millipore Corp., Bedford, MA), eluting peptides (v/v) acetonitrile/water. All supernatants pooled SpeedVac brought back up 25 acetonitrile, acid. peptide mix stored −20 °C until analysis. Aliquots unseparated tryptic digests co-crystallized cyano-4-hydroxycinnamic analyzed MALDI delayed extraction reflectron TOF instrument (Voyager DE-STR spectrometer; Perseptive Biosystems, Framingham, MA) equipped nitrogen laser. Measurements positive ionization mode. spectra externally calibrated standard (Sigma). Some PSD acquired TofSpec SE MALDI-TOF spectrometer (Micromass, Manchester, UK). Data base interrogations based experimentally determined masses carried out MS-Fit, data interrogation MS-Tag; software programs developed University California San Francisco Mass Spectrometry Facility available World Wide Web prospector.ucsf.edu. Both National Center Biotechnology Information Swiss-Prot searched. Search parameters included putative tolerance 100–200 ppm. Pleckstrin (PH), (ANK) repeat domains program Pfam run Sanger ProfileScan Institut Suisse Recherche Experimentale sue le Cancer server (15Kondo Yano Nagayama Mazaki paxillin-binding PAG3/Papα/KIAA0400, bearing activity, involved paxillin focal adhesions migration.Mol. Cell. 11: 1315-1327Crossref (99) As previously, predicted ANK repeats agree, large extent, published structure Pyk2-associating (16Mandiyan Andreev Schlessinger Hubbard S.R. Crystal ARF-GAP PYK2-associated β.EMBO 1999; 18: 6890-6898Crossref Coiled coils COILS (17Lupas Van Dyke Stock Predicting sequences.Science. 1991; 252: 1162-1164Crossref (3471) accessed ExPASy Biology Server Swiss Institute Bioinformatics MTIDK (matrix derived myosins, tropomyosins, intermediate filaments, desmosomal proteins, kinesines). 28-residue window detect potential coil domains, 21-residue ends. probability 0.6 less than 25% change weighting cutoff. ACAP1 ACAP2 sequences aligned module within Genetics Computer Group (NCBI) server. alignments ClustalW NCBI Human clones ARF5 obtained cDNA library, gift Drs. Julie Harish Radhakrishna (National Institutes Health). purchased Open Biosystem (Huntsville, AL). Point mutations introduced QuikChange method (Stratagene). following mammalian constructs generated HA (Sigma), 3×FLAG pEGFP-N1 (BD Biosciences): (Q71L) (N126I) mutants, (Q67L) (T27N) p3×FLAG-tagged full-length ACAP4, (R469G), deletion (ΔPH ΔGAP). Site-directed mutation biology protocol. DNA sequencing. Recombinant adenoviruses expressing GFP-tagged (wild type R469G mutant) AdMax system (Microbix Biosystems) (18Zhou Zhu Kodani Hauser P. Phosphorylation threonine 567 produces secretory phenotype repolarizes cell.J. 118: 4381-4391Crossref (53) inserted fuse GFP sequence. ACAP4-GFP sequence amplified PCR shuttle vector, pDC311, pDC311/ACAP4-GFP. By co-transfection kidney (HEK) 293 pDC311/ACAP4-GFP pBHGloxDE1,3Cre Biosystems; adenovirus type-5 genome) CellPhect transfection kit (Amersham Biosciences), recombination plasmids led infectious virus production. single viral colony isolated, amplified, titrated. −80 °C. expressed bacteria proteins. ARF1, ARF5, cDNAs cloned pGEX-2T vector Biosciences). One liter LB (10 g tryptone, yeast extract, pH 7.4) inoculated transformed GST-ARF1, GST-ARF5, GST-ARF6 mutants). When optical density (A600) culture reached 0.7 1.0, induced addition 0.5 isopropyl β-d-thiogalactopyranoside. Bacteria harvested centrifugation 3 h after induction, resuspended PBS inhibitors (leupeptin, pepstatin, chymostatin; μg/ml), sonicated four bursts s each probe-tip sonicator. solution cleared insoluble material 10,000 × 20 soluble fraction applied column packed glutathione-agarose (Sigma) extensive washes (20× bead volume). either pulldown assay eluted glutathione gel filtration (Econ-Pac DG; Bio-Rad) remove assays. estimated 90% pure SDS-PAGE; major contaminants degraded ARFs. Protein Bradford assay. 19Randazzo Kahn R.A. dependent phospholipids.J. 1994; 269: 10758-10763Abstract ARFs, concentration μm, loaded [α-32P]GTP incubation 40 min HEPES, 7.4, X-100, 1 EDTA, 2.5 MgCl2, KCl, DTT supplemented phosphoenolpyruvate, 1.25 pyruvate kinase, 20–40 μCi [α-32P]GTP, phosphatidylcholine. preloaded ARFs diluted 1:9 reaction GTP, mixed micelles phospholipids (PIP2 (PA)) X-100. Reactions initiated mixing ARF-[α-32P]GTP FLAG-ACAP4 terminated diluting ice-cold Tris, 8.0, DTT. added 0.3 μm avoid saturation enzyme. Protein-bound nucleotide trapped nitrocellulose filters 19), ml m formic This treatment shown release 95–100% filter-bound radiolabel without changing nature radiolabeled Nucleotides fractionated thin layer chromatography polyethyleneimine cellulose plate LiCl, Quantification GDP scintillation counting PhosphorImager Phospholipids solubilized micelles. PA) 95:5 chloroform/methanol dried under stream nitrogen. prepare liposomes, lipids sonication G112SP1 ultrasonic cleaner (Laboratory Supplies Co., Hicksville, NY) micelles, 1% (v/v). experiments revealed half-maximal achieved 10–20 PIP2 presence PA. reported normalized amount quantified Western blot cultured Advanced DMEM (Invitrogen) 10% fetal bovine serum. derivatives transfected Lipofectamine 2000 FuGENE (Roche Applied Science) synchronized (20Yao Abrieau Zheng Sullivan K.F. Cleveland D.W. CENP-E forms attachment spindle microtubules kinetochores mitotic checkpoint.Nat. 484-491Crossref (307) Double-stranded 19-nucleotide duplex targeted (5′-TCTCACACAGCACGATAAA-3′) Dharmacon Research Inc. (Boulder, CO) silencing control, targeting cyclophilin scrambled used. (siRNA) duplexes grown ∼40% confluency serum 37 CO2 siRNA duplex. Pilot nm 36 maximal suppression expression. this condition healing Adenoviral infections postplating. Cells infected control alone recombinant adenoviral incorporating wild GFP-ACAP4 GFP-ACAP4R469G mutant. Infection executed 106 particles/ml viruses. Cultures 12 changed fresh medium We chose experimental conditions level intensity measured Spex fluorometer general appearance Direct observation subsequent immunostaining 92 ± 4% constructs. ∼45% CO2. HA-tagged their constructs, respectively, Science). collected 22 transfection, prepared (50 7.2, 150 EGTA, X-100) 16,000 4 antibody-coupled agarose beads. five boiled SDS-PAGE After SDS-PAGE, transferred blotting tag, HEK transiently express Twenty-fours coupled FLAG (M2) antibodies (40 DTT) fixed formaldehyde 0.025% glutaraldehyde Coverslips permeabilized 0.2% blocked 0.05% Tween (TPBS) BSA primary humidified chamber TPBS. Texas Red-conjugated goat anti-mouse IgG FITC-conjugated anti-rabbit (Jackson Immunoresearch) secondary visualization appropriate antigens. stained 4′,6-diamidino-2-phenylindole Slides examined Zeiss Axiovert200 microscope, images Image-5 (Carl Zeiss; 20Yao changes For assay, confluent coverslips starved overnight 0.01% BSA. marring linear scratch sterile pipette tip, stimulated 20% indicated. Images taken 10× objective inverted microscope (Axiovert200) Axiocam-HS digital camera Zeiss). Samples membrane. Proteins probed 125I-protein Immunoreactive signals visualized autoradiography Kodak BioMAX MS film 6–8 intensifying screen Anti-ACAP4 antibody immunizing rabbits His-tagged protein. rabbit GST-tagged (21Yao Anderson K.L. microtubule-dependent motor centromere-associated E (CENP-E) integral component kinetochore corona fibers centromeres microtubules.J. 1997; 139: 435-447Crossref (189) In some cases, mouse ACAP4-immunized mice immunofluorescence blotting. specificity tested lysates. other follows: monoclonal ant-ARF6 (Novus Biologicals, Inc; 1:1,000), anti-FLAG M2 (Sigma; 1:2,000), anti-ezrin 6H11(1:5,000; 14Zhou anti-ACAP1 (1:1,000; 22Dai Bos Porcionatto Premont R.T. Bourgoin 7: 771-776Abstract (86) Alexa Fluor 488-conjugated phalloidin (Molecular Probes; 1:500), conjugated horseradish peroxidase, IgG, rhodamine-conjugated Immunoresearch). Cycling GTP- GDP-bound various effectors modulates dynamics. binding results conformational change, leads further understand "active" matrix-immobilized search extracts bind distinguish putat