AF-6 Controls Integrin-mediated Cell Adhesion by Regulating Rap1 Activation through the Specific Recruitment of Rap1GTP and SPA-1

• DOI: 10.1074/jbc.m211888200
• Publication Date: 2003-04-18T19:34:12Z
• Authors: Li Su; Masakazu Hattori; Masaki Moriyama; Norihito Murata; Masashi Harazaki; Kozo Kaibuchi; Nagahiro Minato
• Keywords: Rap1; PDZ domain; Small GTPase; Immunoprecipitation

In the present study, we showed that SPA-1, a Rap1 GTPase-activating protein (GAP), was bound to cytoskeleton-anchoring AF-6. SPA-1 and AF-6 were co-immunoprecipitated in 293T cells transfected with both cDNAs as well normal thymocytes. vitro binding studies using truncated fragments their mutants suggested PDZ domain of via probable internal ligand motif within GAP-related domain. The conserved among GAPs, it shown rapGAP I comparably SPA-1. RapV12 also N-terminal domain, only presence AF-6, indicating they could be brought into close proximity cells. Immunostaining analysis revealed co-localized at cell attachment sites. HeLa expressing tetracycline-regulatory manner, expression inhibited endogenous Rap1GTP β1integrin-mediated adhesion fibronectin SPA-1-induced conditions, whereas affected neither them SPA-1-repressed conditions. These results control integrin-mediated by regulating activation through recruitment distinct domains. GTPase activating proteins transcription translation Ras/Rap1-binding N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine wild type doxycycline is member Ras family GTPases play roles regulation proliferation, differentiation, (reviewed Ref. 1Bos J.L. de Rooij J. Reedquist K.A. Nat. Rev. Mol. Cell Biol. 2001; 2: 369-377Crossref PubMed Scopus (508) Google Scholar). activated wide variety extracellular stimuli different kinds specific guanine nucleotide exchange factors, which are coupled various receptors or second messengers interaction motifs (1Bos amounts duration intracellular Rap1GTP, on other hand, controlled (GAPs).1 So far, two families GAPs identified including rapGAPs (I II) (SPA-1, E6TP1/SPAR/SPA-Ls), may have tissue distribution profiles (2Rubinfeld B. Munemitsu S. Clark R. Conroy L. Watt K. Crosier W.J. McCormick F. Polakis P. Cell. 1991; 65: 1033-1042Abstract Full Text PDF (201) Scholar, 3Kurachi H. Wada Y. Tsukamoto N. Maeda M. 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Block Kalbitzer H.R. Wittinghofer Herrmann 13556-13562Abstract (114) 16Boettner Govek E.E. Cross Van Aelst Proc. Natl. Acad. Sci. U. 97: 9064-9069Crossref (168) Scholar), tight-junction ZO-1 (17Yamamoto Kawano Taya Biochem. Biophys. Commun. 259: 103-107Crossref (53) actin-regulatory profilin (16Boettner subsets Eph-related receptor tyrosine kinases (18Hock Bohme Karn Holtrich Holland Pawson Rubsamen-Waigmann Strebhardt 1998; 95: 9779-9784Crossref (174) Although these features imply function molecular scaffold integrating signals related cytoskeletal reorganization, its exact remain seen. provide evidence binds substrate can activation. Thymocytes, splenic B obtained from BALB/c mice. 293T, HeLa, Caki-2 (human kidney cancer) maintained Dulbecco's modified Eagle's medium supplemented 10% fetal calf serum. stably cDNA (HeLa/Tet-SPA-1) pTRE vector (Clontech) before complete containing 10 ng/ml (Dox) (Sigma), unless indicated specifically. Antibodies used study included anti-SPA-1 anti-AF-6 (19Yamamoto Kano Mizoguchi Ide 139: 785-795Crossref (282) anti-Rap1, anti-Rap1 (Santa Cruz Biotechnology), anti-T7 antibodies (Novagen). Biotinylated antibody prepared EZ-link sulfo-NHS-LC biotin (Pierce). Anti-VLA-4 anti-VLA-5 provided Dr. Kinashi, Kyoto University, Kyoto, Japan. lacking (ΔRBD AF-6) deleting between NotI andAflII sites (1–128 residues) full-length cDNA. (ΔPDZ constructed fragment BamHI andSalI (910–1612 cDNA, PCR-amplified (910–990 1078–1612 ligated back consecutively. GRD (ΔGRD SPA-1) C3G CAAX box sequence C terminus (C3G-F) plasmids CaPO4 precipitation method Effectene Transfection Reagent (Qiagen). fragments, 1 (residues 1–211), 2 212–532), 3 538–680), 4 681–1038), 5 748–1038), amplified PCR subcloned intoBamHI/XhoI pSP73 vector. subfragments (fragment 2), G1 338–532), G2 (398–459), G3 435–489) fragment, M1 (V432A) M2 (F433A) generated site-directed mutagenesis kit (Stratagene Quick), intoBglII/EcoRI A 263–322) corresponding series 36–494), 495–909), 914–1129), 1130–1612), ΔPDZ KpnI SalI (IVTT) each performed TnTTMT7/SP6-coupled wheat germ extract system (Promega) [35S]methionine. assay follows. lysate pEF-BOS-AF-6 pSRα-SPA-1 immunoprecipitated anti-AF-6, anti-SPA-1, preimmune IgG followed A-Sepharose beads (Amersham Biosciences). labeled IVTT products above incubated conjugated h °C gentle rotation. extensively washed, eluted SDS sample buffer, electrophoresed regular SDS-PAGE Tricine-buffered smaller mass autoradiography. Cells lysed lysis buffer (0.5% Triton X-100, mm Tris-HCl, pH 7.6, 150 NaCl, protease inhibitor mixture), overnight rotation, then precipitated 30 min °C. After extensive washing, boiled, immunoblotting ECL detection To detect lysates (0.8–1 mg proteins) GST RalGDS-RBD glutathione-Sepharose 4B Biosciences) ice h, antibody. HeLa/Tet-SPA-1 pEF-BOS selected puromycin (Sigma) establish stable line (HeLa/Tet-SPA-1/AF-6). latter transiently T7-tagged pSRα cultured coverslips Dox either inductive (0.1 ng/ml) non-inductive (1 dose 24 h. rinsed Tris-buffered saline, fixed 3% paraformaldehyde, permeated 0.5% X-100/Tris-buffered blocked 2% bovine serum albumin/Tris-buffered AlexaFluor 546 anti-rabbit (Molecular Probes). further biotinylated 488 streptavidin anti-mouse IgG. Normal rabbit mouse primary stained analyzed confocal microscopy (Olympus). 96-Well flat-bottom plates coated μg/ml (FN) blocking albumin/phosphate-buffered saline 37 treated trypsin/EDTA, resuspended serum-free 0.02% albumin Hepes, rotated suspension plated triplicate onto FN-coated wells 0.5 × 105 per well. incubation °C, nonadherent removed gently aspiration, remaining adherent 3.7% paraformaldehyde staining crystal violet 20% methanol. washing distilled water, dye extracted solution (50% ethanol 50 sodium citrate, 4.5) measured enzyme-linked immunosorbent reader Devices). Absorbance uncoated subtracted show adhesion. Statistics done Student's t test. By yeast two-hybrid screening spleen library bait, potential SPA-1-binding (data not shown). therefore examined transient gene cells, expressed marginal if any. co-transfected cDNAs, (130 kDa) reciprocally (∼200 antibody, although tended degraded (Fig.1A). Neither investigated whether occurred physiologically As Fig. 1B, thymocytes abundantly lymphoid antibodies, respectively, It estimated around 40% 15% involved vitro. Among 2, residues 212–532) AF-6-coated beads, none (Fig.2A). shown, beads. Similar Fig.2A, 3, 914–1129) specifically SPA-1-coated confirm results, GRD-deletion mutant PDZ-deletion IVTT. 2B, ΔGRD failed proteins. strongly next intended investigate PDZ-mediated interaction. truncation 338–532) 398–459), another overlapping (Fig. 3B), suggesting site 398 434. peptide libraries, preferred class (φ-X-φ, where φ hydrophobic residue), residue −1 position (20Songyang Z. Fanning A.S. Fu Xu Marfatia S.M. Chishti A.H. Crompton Chan A.C. Anderson Cantley L.C. Science. 275: 73-77Crossref (1209) noticed stretch 432–434, IVF, fitted predicted motif, β-sheet immediately turn Chou-Fasman secondary structure prediction 3A). possible involvement this G2, (V433A), (F434A), IVTT, 3B, barely markedly reduced efficiency compared G1, Val-433 Phe-434 required known (IVF VVF rapGAP), 3B). Furthermore, 3C, renal cancer endogenously shared feature GAPs. relation those (HeLa/Tet-SPA-1/AF-6), repressed undetectable level 1.0 while 0.1 Dox, diffusely cortical area cytosol little (Fig.4A). On localized predominantly HeLa/Tet-SPA-1/AF-6 4B). significant proportion 4C). Strong nuclear nonspecific, because detected untransfected When express regions 4D). noted become slender, detachment dish days, much earlier than (see below). 4E, under condition. possibility more directly. without absence 5A), conforming general consensus (21Ahmadian M.R. Mittal Hall 408: 315-318Crossref (65) however, along 4A). ΔRBD RapV12, hardly co-immunoprecipitation simultaneously domains, leading efficient Rap1GTP. effect Expression amount transfection C3G-F dose-dependent manner Additional significantly enhanced 0.3 μg resulted inhibition generation still remained 4B, enhancing deletion finally effects adhered (FN)-coated dependent VLA4 andVLA5 (Fig.6A). alone 6A). However, co-transfection greater nor adhesion, Rap1. experiments vector, day ng/ml. At 6B, left). Under condition, slight marginally statistical insignificance right). induced, concomitantly reduced, decrease (Fig.6B). condition level, 6B). contribute SPA-1-mediated inactivation. demonstrated vice versa but thymocytes, physiological.In Most interactions occur recognition short (22Kim Niethammer Rothschild Jan Y.N. 1995; 378: 85-88Crossref (892) 23Brenman J.E. Chao D.S. Gee S.H. McGee A.W. Craven S.E. Santillano D.R. Wu Huang Xia Peters M.F. Froehner S.C. Bredt 84: 757-767Abstract (1424) exceptional (24Xu X.Z. Choudhury Li X. Montell 142: 545-555Crossref (195) 25Cuppen Gerrits Pepers Wieringa Hendriks W. 9: 671-683Crossref (126) 26van Huizen Miller Chen D.M. Lai Z.C. Raab R.W. Stark W.S. Shortridge R.D. EMBO 17: 2285-2297Crossref (97) 27Hillier B.J. Christopherson K.S. Prehoda K.E. Lim W.A. 284: 812-815Crossref (461) neuronal nitric-oxide synthase syntrophin region, sharp β-finger mimicked pseudo-peptide (27Hillier (φ-X-φ) libraries contained IVF 432–434), PDZ, mutations Ala minimal (G2) AF-6in Marginally retained activity F434A due residual alanine anchoring residue. β-finger-like structure. suggested, did prove, E6TP1, RKR positions 421–423 LIG, attenuated vivo, affect all. 2L. Su, Hattori, Minato, unpublished observations. polarized epithelial cadherin-based cell-cell tight adherens junctions, junction might role non-polarized focally matrix. cytosol. irrespective previous Rap1V12 Collectively (RapV12) independent sites, central RBD, respectively. p120RasGAP RBD competitive RasGTP (15Linnemann Independent prevent interference Rather, vivo facilitated them. β2 integrins cell-matrix inactivating levels FN plays Integrin-mediated adhesions regulated dynamically during cellular functions. immune system, leukocyte movement migration involve spatio-temporally organized (28Flaishon Lantner Hershkoviz Levo Shachar I. 276: 46701-46706Abstract (26) 29Abbitt K.B. Rainger G.E. Nash G.B. Immunol. Methods. 239: 109-119Crossref (68) Also, responses depends intimate synapse, extent profoundly modes intensity (30Sims T.N. Dustin M.L. 186: 100-117Crossref (120) played crucial LFA-1/ICAM-1 partly our 3K. Kometani, observations.using transgenic mice essential differentiation expansion vivo. thus seems fine systems. thank Drs. assistance assay.