Identification and Characterization of RA-GEF-2, a Rap Guanine Nucleotide Exchange Factor That Serves as a Downstream Target of M-Ras
2001; Elsevier BV; Volume: 276; Issue: 45 Linguagem: Inglês
10.1074/jbc.m105760200
ISSN1083-351X
AutoresXianlong Gao, Takaya Satoh, Yanhong Liao, Chunhua Song, Chang-Deng Hu, Ken-ichi Kariya, Tohru Kataoka,
Tópico(s)Signaling Pathways in Disease
ResumoThe Ras family small GTPase Rap is regulated by an array of specific guanine nucleotide exchange factors (GEFs) in response to upstream stimuli. RA-GEF-1 was identified as a novel Rap GEF, which possesses a Ras/Rap1-associating (RA) domain. Here we report a protein closely related to RA-GEF-1, named RA-GEF-2. Like RA-GEF-1, a putative cyclic nucleotide monophosphate-binding domain, a Ras exchanger motif, a PSD-95/DlgA/ZO-1 domain, and an RA domain in addition to the GEF catalytic domain are found in RA-GEF-2. However, RA-GEF-2 displays a different tissue distribution profile from that of RA-GEF-1. RA-GEF-2 stimulates guanine nucleotide exchange of both Rap1 and Rap2, but not Ha-Ras. The RA domain of RA-GEF-2 binds to M-Ras in a GTP-dependent manner, but not to other Ras family GTPases tested, including Ha-Ras, N-Ras, Rap1A, Rap2A, R-Ras, RalA, Rin, Rit, and Rheb, in contrast to the RA domain of RA-GEF-1, which specifically binds to Rap1. In accordance with this, RA-GEF-2 colocalizes with activated M-Ras in the plasma membrane in COS-7 cells, suggesting a role of RA-GEF-2 in the regulation of Rap1 and Rap2, particularly in the plasma membrane. In fact, an increase in the level of the GTP-bound form of plasma membrane-located Rap1 was observed when coexpressed with RA-GEF-2 and activated M-Ras. Thus, RA-GEF-2 acts as a GEF for Rap1 and Rap2 downstream of M-Ras in the plasma membrane, whereas RA-GEF-1 exerts Rap GEF function in perinuclear compartments including the Golgi apparatus. The Ras family small GTPase Rap is regulated by an array of specific guanine nucleotide exchange factors (GEFs) in response to upstream stimuli. RA-GEF-1 was identified as a novel Rap GEF, which possesses a Ras/Rap1-associating (RA) domain. Here we report a protein closely related to RA-GEF-1, named RA-GEF-2. Like RA-GEF-1, a putative cyclic nucleotide monophosphate-binding domain, a Ras exchanger motif, a PSD-95/DlgA/ZO-1 domain, and an RA domain in addition to the GEF catalytic domain are found in RA-GEF-2. However, RA-GEF-2 displays a different tissue distribution profile from that of RA-GEF-1. RA-GEF-2 stimulates guanine nucleotide exchange of both Rap1 and Rap2, but not Ha-Ras. The RA domain of RA-GEF-2 binds to M-Ras in a GTP-dependent manner, but not to other Ras family GTPases tested, including Ha-Ras, N-Ras, Rap1A, Rap2A, R-Ras, RalA, Rin, Rit, and Rheb, in contrast to the RA domain of RA-GEF-1, which specifically binds to Rap1. In accordance with this, RA-GEF-2 colocalizes with activated M-Ras in the plasma membrane in COS-7 cells, suggesting a role of RA-GEF-2 in the regulation of Rap1 and Rap2, particularly in the plasma membrane. In fact, an increase in the level of the GTP-bound form of plasma membrane-located Rap1 was observed when coexpressed with RA-GEF-2 and activated M-Ras. Thus, RA-GEF-2 acts as a GEF for Rap1 and Rap2 downstream of M-Ras in the plasma membrane, whereas RA-GEF-1 exerts Rap GEF function in perinuclear compartments including the Golgi apparatus. Ras/Rap1-associating guanine nucleotide exchange factor PSD-95/DlgA/ZO-1 cyclic nucleotide monophosphate Ras exchanger motif rapid amplification of cDNA ends enhanced green fluorescence protein hemagglutinin Dulbecco's modified Eagle's medium Ral guanine nucleotide dissociation stimulator Rap1-interacting domain Ras-binding domain glutathione S-transferase maltose-binding protein phospholipase C guanosine 5′-3-O-(thio)triphosphate The Ras family small GTPase Rap1 participates in the regulation of a wide variety of cellular responses, including proliferation, differentiation, lymphocyte aggregation, T-cell anergy, and platelet activation (1Bos J.L. EMBO J. 1998; 17: 6776-6782Crossref PubMed Scopus (288) Google Scholar, 2Bos J.L. de Rooij J. Reedquist K.A. Nat. Rev. Mol. Cell. Biol. 2001; 2: 369-377Crossref PubMed Scopus (512) Google Scholar). In contrast, the physiological function of Rap2, a close relative of Rap1, remains largely unknown. Diverse effectors for Rap1, almost all of which are common to Ras effectors, have been identified, although their roles in individual signaling pathways remain obscure. For activation of the effectors, the interaction of the effector region of Rap1 (amino acids 32–40) with the Ras/Rap1-binding or RA1 domain of the effectors is important (3Ponting C.P. Benjamin D.R. Trends Biochem. Sci. 1996; 21: 422-425Abstract Full Text PDF PubMed Scopus (179) Google Scholar). In addition, the second Ras/Rap1-binding site identified in several Ras/Rap1 effectors, including Raf-1, B-Raf, and yeast adenylyl cyclase, is required for proper effector activation (4Hu C.-D. Kariya K. Tamada M. Akasaka K. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1995; 270: 30274-30277Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 5Hu C.-D. Kariya K. Kotani G. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1997; 272: 11702-11705Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 6Okada T. Hu C.-D. Jin T.-G. Kariya K. Yamawaki-Kataoka Y. Kataoka T. Mol. Cell. Biol. 1999; 19: 6057-6064Crossref PubMed Scopus (72) Google Scholar, 7Shima F. Okada T. Kido M. Sen H. Tanaka Y. Tamada M. Hu C.-D. Yamawaki-Kataoka Y. Kariya K. Kataoka T. Mol. Cell. Biol. 2000; 20: 26-33Crossref PubMed Scopus (60) Google Scholar). Suppression of Ki-Ras-induced transformation by overexpressed Rap1 is thought to be ascribed to tight binding to the second Ras/Rap1-binding sites of Ras effectors such as Raf-1 without stimulating their activities (5Hu C.-D. Kariya K. Kotani G. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1997; 272: 11702-11705Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 6Okada T. Hu C.-D. Jin T.-G. Kariya K. Yamawaki-Kataoka Y. Kataoka T. Mol. Cell. Biol. 1999; 19: 6057-6064Crossref PubMed Scopus (72) Google Scholar). Ras family GTPases cycle between GTP-bound active and GDP-bound inactive states, serving as a molecular switch of intracellular signaling (8Lowy D.R. Willumsen B.M. Annu. Rev. Biochem. 1993; 62: 851-891Crossref PubMed Scopus (1126) Google Scholar, 9Katz M.E. McCormick F. Curr. Opin. Genet. Dev. 1997; 7: 75-79Crossref PubMed Scopus (276) Google Scholar). Conversion between GTP- and GDP-bound states is controlled by GEFs and GTPase-activating proteins (8Lowy D.R. Willumsen B.M. Annu. Rev. Biochem. 1993; 62: 851-891Crossref PubMed Scopus (1126) Google Scholar, 9Katz M.E. McCormick F. Curr. Opin. Genet. Dev. 1997; 7: 75-79Crossref PubMed Scopus (276) Google Scholar). Particularly, GEFs enhance the formation of the GTP-bound active conformation in response to upstream signals mediated by various cell surface receptors. To date, various GEFs for Rap1 have been identified in mammalian cells. C3G binds to the adaptor protein Crk, being involved in tyrosine kinase-dependent activation of Rap1(10). Epac/cAMP-GEF is activated through direct association with cAMP, thereby stimulating Rap-dependent signaling (11de Rooij J. Zwartkruis F.J. Verheijen M.H. Cool R.H. Nijman S.M. Wittinghofer A. Bos J.L. Nature. 1998; 396: 474-477Crossref PubMed Scopus (1633) Google Scholar, 12Kawasaki H. Springett G.M. Mochizuki N. Toki S. Nakaya M. Matsuda M. Housman D.E. Graybiel A.M. Science. 1998; 282: 2275-2279Crossref PubMed Scopus (1178) Google Scholar). Another Rap GEF, CalDAGGEF1, which contains calcium- and diacylglycerol-binding motifs, has a role in Rap activation in response to these second messengers (13Kawasaki H. Springett G.M. Toki S. Canales J.J. Harlan P. Blumenstiel J.P. Chen E.J. Bany I.A. Mochizuki N. Ashbacher A. Matsuda M. Housman D.E. Graybiel A.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13278-13283Crossref PubMed Scopus (313) Google Scholar). Additionally, we and other groups recently identified a novel type of the Rap GEF, RA-GEF-1 (also termed PDZ-GEF1, nRapGEP, or CNrasGEF), which exhibits GEF activity toward Rap1 and Rap2, but not Ha-Ras (14Liao Y. Kariya K. Hu C.-D. Shibatohge M. Goshima M. Okada T. Watari Y. Gao X. Jin T.-G. Yamawaki-Kataoka Y. Kataoka T. J. Biol. Chem. 1999; 274: 37815-37820Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 15de Rooij J. Boenink N.M. van Triest M. Cool R.H. Wittinghofer A. Bos J.L. J. Biol. Chem. 1999; 274: 38125-38130Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 16Ohtsuka T. Hata Y. Ide N. Yasuda T. Inoue E. Inoue T. Mizoguchi A. Takai Y. Biochem. Biophys. Res. Commun. 1999; 265: 38-44Crossref PubMed Scopus (92) Google Scholar, 17Pham N. Cheglakov I. Koch C.A. de Hoog C.L. Moran M.F. Rotin D. Curr. Biol. 2000; 10: 555-558Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 18Liao Y. Satoh T. Gao X. Jin T.-G. Hu C.-D. Kataoka T. J. Biol. Chem. 2001; 276: 28478-28483Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). RA-GEF-1 contains putative cNMP-binding, REM, PDZ, and RA domains as well as the GEF catalytic domain. We and others detected no specific cAMP/cGMP binding to the cNMP-binding domain (14Liao Y. Kariya K. Hu C.-D. Shibatohge M. Goshima M. Okada T. Watari Y. Gao X. Jin T.-G. Yamawaki-Kataoka Y. Kataoka T. J. Biol. Chem. 1999; 274: 37815-37820Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 15de Rooij J. Boenink N.M. van Triest M. Cool R.H. Wittinghofer A. Bos J.L. J. Biol. Chem. 1999; 274: 38125-38130Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 16Ohtsuka T. Hata Y. Ide N. Yasuda T. Inoue E. Inoue T. Mizoguchi A. Takai Y. Biochem. Biophys. Res. Commun. 1999; 265: 38-44Crossref PubMed Scopus (92) Google Scholar, 18Liao Y. Satoh T. Gao X. Jin T.-G. Hu C.-D. Kataoka T. J. Biol. Chem. 2001; 276: 28478-28483Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), although Pham et al. (17Pham N. Cheglakov I. Koch C.A. de Hoog C.L. Moran M.F. Rotin D. Curr. Biol. 2000; 10: 555-558Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) reported cAMP binding to this domain and subsequent stimulation of Ras GEF activity. The RA domain of RA-GEF-1 binds to Rap1·GTP, suggesting that RA-GEF-1 plays an important role downstream of Rap1 as well (14Liao Y. Kariya K. Hu C.-D. Shibatohge M. Goshima M. Okada T. Watari Y. Gao X. Jin T.-G. Yamawaki-Kataoka Y. Kataoka T. J. Biol. Chem. 1999; 274: 37815-37820Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Indeed, the RA domain is required for translocation of RA-GEF-1 to the perinuclear compartments including the Golgi complex and for the full activation of Rap1, as evidenced by our recent observation that an RA domain mutation that abolishes Rap1 binding compromised RA-GEF-1-dependent activation of Rap1 in vivo(18Liao Y. Satoh T. Gao X. Jin T.-G. Hu C.-D. Kataoka T. J. Biol. Chem. 2001; 276: 28478-28483Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Herein, we describe a novel member of the RA-GEF family, designated RA-GEF-2, whose structural features are intimately related to RA-GEF-1. Like RA-GEF-1, RA-GEF-2 exhibits GEF activity toward Rap1 and Rap2, but not Ha-Ras. However, unlike RA-GEF-1, the RA domain of RA-GEF-2 specifically binds to M-Ras·GTP, which causes the translocation of RA-GEF-2 to the plasma membrane, where M-Ras exists. Correspondingly, activation of Rap1 that localized in the plasma membrane was observed following coexpression of RA-GEF-2 and activated M-Ras. A genomic clone (AC004227) that encodes an open reading frame whose predicted amino acid sequence is highly homologous to RA-GEF-1 was identified through a search of the GenBank™ data base with the BLAST program. The predicted partial coding sequence inAC004227 was amplified from a cDNA library synthesized from human fetal brain mRNA (Invitrogen) using two gene-specific primers. Upstream and downstream sequences were obtained by 5′- and 3′-RACE using the same library by a Marathon cDNA amplification procedure (CLONTECH). The complete nucleotide sequence was confirmed by isolating and sequencing multiple clones, and the encoded protein was designated RA-GEF-2. The full-length RA-GEF-2 cDNA was subcloned into the mammalian expression vectors pFLAG-CMV2 (Sigma) and pcDNA3.1HisB (Invitrogen), generating pFLAG-CMV2-RA-GEF-2 and pcDNA3.1HisB-RA-GEF-2, respectively. The cDNA for RA-GEF-2ΔRA, RA-GEF-2 lacking the N-terminal portion of the RA domain (amino acids 749–779), was constructed by the polymerase chain reaction and subcloned into pFLAG-CMV2, generating pFLAG-CMV2-GEF-2ΔRA. cDNAs for EGFP-tagged RA-GEF-2 and RA-GEF-2ΔRA were constructed by the polymerase chain reaction and subcloned into the mammalian expression vector pCMV2, generating pCMV2-EGFP-GEF-2 and pCMV2-EGFP-RA-GEF-2ΔRA, respectively. cDNAs for wild-type and activated M-Ras were subcloned into pFLAG-CMV2, generating pFLAG-CMV2-M-Ras and pFLAG-CMV2-M-Ras71L, respectively. The cDNA for HA-tagged M-Ras71L was subcloned into the mammalian expression vector pEF-BOS (19Mizushima S. Nagata S. Nucleic Acids Res. 1990; 18: 5322Crossref PubMed Scopus (1499) Google Scholar), generating pEF-BOS-HA-M-Ras71L. The cDNA for R-Ras was kindly provided by Shintaro Iwashita (Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan). COS-7 cells were cultured in DMEM supplemented with 10% fetal calf serum. Expression plasmids were introduced into COS-7 cells by using GenePORTER (Gene Therapy System) or Superfect (Qiagen) according to the manufacturer's protocol. A human adult multiple tissue blot membrane containing 2 μg each of poly(A)+ RNA from various tissues (CLONTECH) was probed with a32P-radiolabeled RNA probe (specific activity = 5 × 107 cpm/μg) corresponding to RA-GEF-1 (nucleotides 3450–4500) or RA-GEF-2 (nucleotides 3425–4384) under stringent conditions. A FLAG-tagged RA-GEF-2 fragment (amino acids 403–1276), designated FLAG-RA-GEF-2-(403–1276), was expressed in Spodoptera frugiperda Sf9 cells and affinity-purified with anti-FLAG M2 resin (Sigma). Ras family small GTPases except Ha-Ras and Rap1A were expressed in Escherichia coli as 6×His-tagged proteins and purified with TALON metal affinity resin (CLONTECH). Ha-Ras and Rap1A were purified from Sf9 cells as previously described (4Hu C.-D. Kariya K. Tamada M. Akasaka K. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1995; 270: 30274-30277Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 5Hu C.-D. Kariya K. Kotani G. Shirouzu M. Yokoyama S. Kataoka T. J. Biol. Chem. 1997; 272: 11702-11705Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 20Kuroda Y. Suzuki N. Kataoka T. Science. 1993; 259: 683-686Crossref PubMed Scopus (120) Google Scholar).In vitro GEF assays were performed essentially as described (14Liao Y. Kariya K. Hu C.-D. Shibatohge M. Goshima M. Okada T. Watari Y. Gao X. Jin T.-G. Yamawaki-Kataoka Y. Kataoka T. J. Biol. Chem. 1999; 274: 37815-37820Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Small GTPases preloaded with [3H]GDP (5,000 cpm/pmol) were incubated with 10 pmol of FLAG-RA-GEF-2-(403–1276) in 50 μl of reaction buffer containing 20 mm Tris-HCl, pH 7.4, 3 mm MgCl2, 50 mm NaCl, 10 mm 2-mercaptoethanol, 5% glycerol, 5 mg/ml bovine serum albumin, and 1.5 mm GTP for 20 min. The reaction was terminated by adding ice-cold stop buffer (20 mm Tris-HCl, pH 8.0, 100 mm NaCl, 5 mm MgCl2). The reaction mixture was subjected to the filter binding assay (14Liao Y. Kariya K. Hu C.-D. Shibatohge M. Goshima M. Okada T. Watari Y. Gao X. Jin T.-G. Yamawaki-Kataoka Y. Kataoka T. J. Biol. Chem. 1999; 274: 37815-37820Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), and [3H]GDP trapped on the membrane was quantitated by liquid scintillation counting. For GTP-binding assays, small GTPases preloaded with GDP were incubated with FLAG-RA-GEF-2-(403–1276) in the presence of [35S]GTPγS (12,000 cpm/pmol), and [35S]GTPγS bound to the GTPases was quantitated as described above. Post-translationally modified FLAG-tagged M-Ras was purified from COS-7 cells as follows. COS-7 cells transfected with pFLAG-CMV2-M-Ras were sonicated in sonication buffer (20 mmTris-HCl, pH 7.4, 100 mm NaCl, 2.5 mmMgCl2, 1 mm EDTA, 10 μg/ml aprotinin, 1 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride) and centrifuged at 100,000 × g for 1 h. The pellet was resuspended and incubated for 1 h on ice in sonication buffer containing 0.2% Triton X-100. The 15,000 × gsupernatant was subjected to affinity purification of FLAG-M-Ras using anti-FLAG M2 resin. For examining the effect of M-Ras on GEF activity of RA-GEF-2 toward Rap1A in vitro, 6×His-M-Ras (purified from E. coli) or FLAG-M-Ras (purified from COS-7 cells) was preloaded with GTPγS and incubated at 4 °C for 2 h with full-length FLAG-RA-GEF-2 purified from Sf9 cells. Subsequently, the mixture was added to 4 pmol of [3H]GDP-preloaded Rap1A at a total volume of 200 μl, and GEF assays were performed as described above. COS-7 cells were transfected with pFLAG-CMV2-RA-GEF-2 and either pEF-BOS-HA-Rap1A (14Liao Y. Kariya K. Hu C.-D. Shibatohge M. Goshima M. Okada T. Watari Y. Gao X. Jin T.-G. Yamawaki-Kataoka Y. Kataoka T. J. Biol. Chem. 1999; 274: 37815-37820Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) or pEF-BOS-HA-Ha-Ras (14Liao Y. Kariya K. Hu C.-D. Shibatohge M. Goshima M. Okada T. Watari Y. Gao X. Jin T.-G. Yamawaki-Kataoka Y. Kataoka T. J. Biol. Chem. 1999; 274: 37815-37820Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) by using the GenePORTER transfection reagent (Gene Therapy System). After transfection, cells were incubated in DMEM supplemented with 10% fetal bovine serum for 24 h and then serum-starved for another 16 h. After serum starvation, cells were harvested in phosphate-buffered saline and lysed in lysis buffer (50 mm Tris-HCl, pH 7.4, 200 mm NaCl, 2.5 mm MgCl2, 10% glycerol, 1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 1 mmleupeptin). Cell lysates were cleared by centrifugation (15,000 ×g for 15 min) and subjected to pull-down assays by the use of GST-RalGDS-RID (for Rap1A) or GST-Raf-1-RBD (for Ha-Ras). GTP-bound forms of HA-Rap1A and HA-Ha-Ras were detected by SDS-polyacrylamide gel electrophoresis and immunoblotting by using anti-HA antibody (12CA5,Roche Molecular Biochemicals). Rap1A- or Ha-Ras-bound GDP/GTP ratios were measured essentially as described (14Liao Y. Kariya K. Hu C.-D. Shibatohge M. Goshima M. Okada T. Watari Y. Gao X. Jin T.-G. Yamawaki-Kataoka Y. Kataoka T. J. Biol. Chem. 1999; 274: 37815-37820Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Briefly, COS-7 cells were transfected with pcDNA3.1HisB-RA-GEF-2 and either pFLAG-CMV2-Rap1A (14Liao Y. Kariya K. Hu C.-D. Shibatohge M. Goshima M. Okada T. Watari Y. Gao X. Jin T.-G. Yamawaki-Kataoka Y. Kataoka T. J. Biol. Chem. 1999; 274: 37815-37820Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) or pFLAG-CMV2-Ha-Ras (14Liao Y. Kariya K. Hu C.-D. Shibatohge M. Goshima M. Okada T. Watari Y. Gao X. Jin T.-G. Yamawaki-Kataoka Y. Kataoka T. J. Biol. Chem. 1999; 274: 37815-37820Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). After serum starvation, cells were washed with phosphate-free DMEM and labeled with [32P]orthophosphate (7.4 MBq/ml of culture medium) for 4 h. FLAG-Rap1A and FLAG-Ha-Ras were immunoprecipitated from cleared cell lysates with anti-FLAG M2 resin and eluted with the FLAG peptide (Sigma). Guanine nucleotides bound to FLAG-Rap1A and FLAG-Ha-Ras were released by heating the eluate at 68 °C for 20 min in denaturing buffer (14Liao Y. Kariya K. Hu C.-D. Shibatohge M. Goshima M. Okada T. Watari Y. Gao X. Jin T.-G. Yamawaki-Kataoka Y. Kataoka T. J. Biol. Chem. 1999; 274: 37815-37820Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) and separated on a polyethyleneimine-cellulose plate. Radioactivities of GDP and GTP spots were quantified by using the BAS2000 bioimaging analyzer (Fujix, Tokyo, Japan). For analyzing the effect of M-Ras on GEF activity of RA-GEF-2 toward Rap1A in vivo, COS-7 cells were transfected with a combination of pEF-BOS-HA-Rap1A, pFLAG-CMV2-M-Ras71L, and either pFLAG-CMV2-RA-GEF-2 or pFLAG-CMV2-RA-GEF-2ΔRA by using the GenePORTER transfection reagent. In vivo GEF activity was detected by pull-down assays as described above. Polypeptides corresponding to the RA domains and their flanking regions of RA-GEF-2 (amino acids 683–853) and RA-GEF-2ΔRA (amino acids 683–822) were expressed as MBP fusion proteins (MBP-RAWT and MBP-RAMUT, respectively) in E. coli by using pMal-c2 (New England Biolabs). The interaction of the RA domain of RA-GEF-2 with Ras family small GTPases was assessed essentially as described previously (14Liao Y. Kariya K. Hu C.-D. Shibatohge M. Goshima M. Okada T. Watari Y. Gao X. Jin T.-G. Yamawaki-Kataoka Y. Kataoka T. J. Biol. Chem. 1999; 274: 37815-37820Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Small GTPases (6 pmol) preloaded with GTPγS or GDP were incubated at 4 °C for 2 h with MBP-RAWT or MBP-RAMUT(50 pmol) immobilized on amylose resin in 100 μl of binding buffer (14Liao Y. Kariya K. Hu C.-D. Shibatohge M. Goshima M. Okada T. Watari Y. Gao X. Jin T.-G. Yamawaki-Kataoka Y. Kataoka T. J. Biol. Chem. 1999; 274: 37815-37820Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). After extensive washing with binding buffer, bound proteins were eluted from the resin with 10 mm maltose and subjected to SDS-polyacrylamide gel electrophoresis followed by immunoblotting. Ha-Ras and Rap1A were detected with anti-Ha-Ras antibody (F235, Oncogene Science Inc.) and anti-Rap1A antibody (sc-65, Santa Cruz Biotechnology), respectively. Other GTPases were detected with anti-6×His antibody (CLONTECH). For quantitative in vitro association assays, 6×His-M-Ras was loaded with [35S]GTPγS (3, 500 cpm/pmol) or [3H]GDP (1, 100 cpm/pmol) and incubated with MBP-RAWT (25 pmol) as described above except that unlabeled GTPγS or GDP (0.1 mm), respectively, was included in the binding reaction. Radioactivities eluted from amylose resin with 10 mmmaltose were counted. COS-7 cells cultivated in 20 culture plates (100-mm diameter) were harvested by centrifugation (600 × g for 5 min) and washed with phosphate-buffered saline. Preparation of the plasma membrane fraction was performed essentially as described (21Hubbard A.L. Wall D.A. Ma A. J. Cell Biol. 1983; 96: 217-229Crossref PubMed Scopus (204) Google Scholar). Cell pellets were resuspended in 0.25m STM (0.25 m sucrose, 5 mmTris-HCl, pH 7.4, 1 mm MgCl2, 10 μg/ml aprotinin, 1 μg/ml leupeptin, 1 mmphenylmethylsulfonyl fluoride), homogenized using a PotterElvehjem homogenizer, and centrifuged at 280 × g for 5 min. The supernatant was further centrifuged at 1,500 × g for 10 min, and the pellet was resuspended in 0.25 m STM. 2m STM (2 m sucrose, 5 mm Tris-HCl, pH 7.4, 1 mm MgCl2) was added to adjust the sucrose concentration to 1.42 m. The suspension was transferred to a centrifugation tube and overlaid with 0.25m STM. After centrifugation (82,000 × gfor 1 h), the pellicle at the 0.25 m/1.42m interface was collected, resuspended in 0.25m STM, and centrifuged at 1,500 × g for 10 min. The final pellet (the plasma membrane fraction) was resuspended in 150 μl of lysis buffer and subjected to the pull-down assay. Alkaline phosphodiesterase (a marker enzyme for the plasma membrane) and mannosidase II (a marker enzyme for the Golgi apparatus) activities were determined as described by Storrie and Madden (22Storrie B. Madden E.A. Methods Enzymol. 1990; 182: 203-225Crossref PubMed Scopus (497) Google Scholar) to estimate the purity of the fractions. COS-7 cells cultured in two-well slide chambers (Nalge Nunc) were transfected with expression plasmids by using the Superfect transfection reagent (Qiagen). After transfection, cells were incubated in DMEM supplemented with 10% fetal bovine serum for 8 h and then serum-starved for another 18 h. Following fixation with 3.7% formaldehyde and permeabilization with 0.2% Triton X-100, cells were stained with mouse anti-HA antibody (12CA5) and rhodamine-conjugated anti-mouse IgG (T2762, Molecular Probes) antibody. Localization of HA-M-Ras71L, HA-Ha-Ras12V, EGFP-Rap1A, EGFP-RA-GEF-2, and EGFP-RA-GEF-2ΔRA was examined under a confocal laser microscope (MRC-1024; Bio-Rad). Through a BLAST search of the GenBank data base, we identified a genomic clone (AC004227) encoding an open reading frame closely related to RA-GEF-1, designated RA-GEF-2. Two gene-specific primers designed on the basis of the AC004227 sequence were employed to isolate a partial cDNA clone encoding RA-GEF-2. Subsequently, the full-length cDNA was reconstructed from 5′- and 3′-RACE products obtained from a human fetal brain cDNA library. The putative start codon matched the Kozak consensus sequence and was preceded by in-frame stop codons. The full-length open reading frame was composed of 4,530 nucleotides, encoding a protein of 1,509 amino acids, and was divided into four contiguous genomic clones (AC005218,AC005576, AC004227, and AC004622 from the N terminus to the C terminus) derived from chromosome 5. RA-GEF-1 and RA-GEF-2 were highly homologous in five domains identified by ISREC ProfileScan (76.2, 80.0, 71.4, 81.6, and 85.9% identity in cNMP-binding, REM, PDZ, RA, and GEF domains, respectively), whereas both N-terminal and C-terminal regions were rather divergent (Fig. 1). Three regions (named structurally conserved regions 1–3), which are highly conserved among diverse Ras/Rap GEFs, were also identified in the GEF domain of RA-GEF-2 (Fig. 1 C). Compared with RA-GEF-1, RA-GEF-2 has 145-amino acid extension at its N terminus, but lacks the C-terminal 102 amino acid residues. In particular, the C-terminal region was least homologous (47.5% identity). We next examined mRNA levels for RA-GEF-1 and RA-GEF-2 in various human tissues using a multiple tissue blot membrane (Fig. 2). Probes derived from divergent regions were used under stringent hybridization conditions to compare the distribution of the transcripts. A single band of ∼8 kilobases was detected for both probes. The RA-GEF-2 transcript was abundant in heart, brain, placenta, lung, and liver, but barely detectable in skeletal muscle, kidney, and pancreas, whereas RA-GEF-1 was expressed in all tissues tested. A FLAG-tagged RA-GEF-2 fragment (amino acids 403–1276) containing the GEF domain was expressed in Sf9 cells and purified to near homogeneity. By using this protein, in vitro GEF activities toward various Ras family proteins were examined. RA-GEF-2 showed GEF activity toward Rap1A and Rap2A, but not toward other Ras family members including Ha-Ras, N-Ras, M-Ras, R-Ras, RalA, Rin, Rit, and Rheb, as determined by [3H]GDP-releasing assays (Fig. 3 A). [35S]GTPγS-binding assays were also performed for Rap1A and Rap2A, revealing that GEF activity toward Rap2A was higher than that toward Rap1A (Fig. 3 B). Next, in vivo GEF activities of RA-GEF-2 toward Rap1A and Ha-Ras were examined by pull-down assays (Fig. 3 C). Rap1A·GTP and Ha-Ras·GTP were affinity-precipitated by GST-RalGDS-RID and GST-Raf-1-RBD, respectively, from COS-7 cells that expressed increasing amounts of RA-GEF-2 and either Rap1A or Ha-Ras, and then were detected by immunoblotting. In parallel with the in vitro activity, RA-GEF-2 caused the accumulation of Rap1A·GTP, but not Ha-Ras·GTP. RA-GEF-2-dependent increase in the level of Rap1A·GTP, but not Ha-Ras·GTP, was also shown by measuring the ratio of Rap1A- or Ha-Ras-bound GDP and GTP (Fig. 3 D). Collectively, RA-GEF-2, like RA-GEF-1, acts as a specific GEF for Rap1 and Rap2. The RA domain of RA-GEF-1 binds to Rap1A in a GTP-dependent manner (14Liao Y. Kariya K. Hu C.-D. Shibatohge M. Goshima M. Okada T. Watari Y. Gao X. Jin T.-G. Yamawaki-Kataoka Y. Kataoka T. J. Biol. Chem. 1999; 274: 37815-37820Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), suggesting that the RA domain of RA-GEF-2 may also bind to Rap1A·GTP. To test this possibility, we employed in vitroassociation assays using an MBP fusion construct, named MBP-RAWT, consisting of the RA domain and its flanking region of RA-GEF-2. Unexpectedly, MBP-RAWT bound to Rap1A only weakly compared with the binding of the RA-GEF-1 RA domain, and this binding was GTP-independent (Fig. 4 A). Thus, we next examined the binding activity of MBP-RAWT to a variety of Ras family proteins. Among Ras family members tested, only M-Ras associated with MBP-RAWT in a GTP-dependent manner, and virtually no binding was detected for other Ras family proteins, such as Ha-Ras, N-Ras, Rap2A, R-Ras, RalA, Rin, Rit, and Rheb (Fig. 4 A). Quantitative analyses further confirmed that the association between MBP-RAWT and M-Ras was dose- and GTP-dependent (Fig. 4 B). Additionally, the binding of M-Ras·GTP was abolished when a 31-amino acid deletion was introduced within the RA domain, suggesting that these highly conserved residues are crucial (Fig. 4 C). RA-GEF-1 is translocated to the perinuclear compartments, including the Golgi apparatus, upon interaction with Rap1A·GTP (18Liao Y. Satoh T. Gao X. Jin T.-G. Hu C.-D. Kataoka T. J. Biol. Chem. 2001; 276: 28478-28483Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Thereafter, RA-GEF-1 serves as an amplifier of Rap1A signaling by yielding the GTP-bound form of Rap1A through the action of the GEF domain (18Liao Y. Satoh T. Gao X. Jin T.-G. Hu C.-D. Kataoka T. J. Biol. Chem. 2001; 276: 28478-28483Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). In view of the observations that the RA domain of RA-GEF-2, unlike that of RA-GEF-1, specifically binds to M-Ras·GTP, it is feasible that RA-GEF-2 colocalizes with M-Ras in the cell and plays a role downstream of M-Ras. As an initial step to clarify this point, localization of Rap1A, M-Ras, and RA-GEF-2 was examined by immunofluorescence microscopy. Although Rap1A was localized mainly in the perinuclear region as reported previously (23Beranger F. Goud B. Tav
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