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RA-GEF-1, a Guanine Nucleotide Exchange Factor for Rap1, Is Activated by Translocation Induced by Association with Rap1·GTP and Enhances Rap1-dependent B-Raf Activation

2001; Elsevier BV; Volume: 276; Issue: 30 Linguagem: Inglês

10.1074/jbc.m101737200

ISSN

1083-351X

Autores

Yanhong Liao, Takaya Satoh, Xianlong Gao, Taiguang Jin, Chang‐Deng Hu, Tohru Kataoka,

Tópico(s)

Cell death mechanisms and regulation

Resumo

We previously identified RA-GEF-1, a novel guanine nucleotide exchange factor (GEF) for Rap1 with the ability to associate with Rap1·GTP at its Ras/Rap1-associating (RA) domain. Because it possesses a PSD-95/DlgA/ZO-1 (PDZ) domain, it was also named PDZ-GEF. In this report, we have examined the role of the RA domain of this protein in Rap1-mediated cellular responses. A mutant of RA-GEF-1 (RA-GEF-1ΔRA) carrying a 21-residue deletion at its RA domain fully retains the in vitro GEF activity toward Rap1 but completely loses the Rap1 binding activity. In contrast, RA-GEF-1ΔRA, expressed in COS-7 cells, exhibits a 3-fold reduction in its in vivo GEF activity toward Rap1 compared with wild-type RA-GEF-1 as examined by the Rap1 pull-down assay. Correspondingly, when coexpressed with wild-type Rap1, RA-GEF-1ΔRA is unable to further activate B-Raf, whereas RA-GEF-1 stimulates B-Raf as efficiently as activated Rap1. Consistent with these observations, coexpression of activated Rap1 induces translocation of RA-GEF-1, which is otherwise located in the cytoplasm, to the perinuclear compartment, where Rap1 is also predominantly localized. This localization almost coincides with that of the Golgi apparatus, which was detected by anti-trans-Golgi-network 38 antibody. RA-GEF-1ΔRA fails to show the translocation. These results indicate that RA-GEF-1 defines a novel category of GEF that is translocated to a particular subcellular compartment by association with the GTP-bound form of a small GTPase and catalyzes activation of the GDP-bound form present in the compartment, thereby causing an amplification of cellular responses induced by the small GTPase. We previously identified RA-GEF-1, a novel guanine nucleotide exchange factor (GEF) for Rap1 with the ability to associate with Rap1·GTP at its Ras/Rap1-associating (RA) domain. Because it possesses a PSD-95/DlgA/ZO-1 (PDZ) domain, it was also named PDZ-GEF. In this report, we have examined the role of the RA domain of this protein in Rap1-mediated cellular responses. A mutant of RA-GEF-1 (RA-GEF-1ΔRA) carrying a 21-residue deletion at its RA domain fully retains the in vitro GEF activity toward Rap1 but completely loses the Rap1 binding activity. In contrast, RA-GEF-1ΔRA, expressed in COS-7 cells, exhibits a 3-fold reduction in its in vivo GEF activity toward Rap1 compared with wild-type RA-GEF-1 as examined by the Rap1 pull-down assay. Correspondingly, when coexpressed with wild-type Rap1, RA-GEF-1ΔRA is unable to further activate B-Raf, whereas RA-GEF-1 stimulates B-Raf as efficiently as activated Rap1. Consistent with these observations, coexpression of activated Rap1 induces translocation of RA-GEF-1, which is otherwise located in the cytoplasm, to the perinuclear compartment, where Rap1 is also predominantly localized. This localization almost coincides with that of the Golgi apparatus, which was detected by anti-trans-Golgi-network 38 antibody. RA-GEF-1ΔRA fails to show the translocation. These results indicate that RA-GEF-1 defines a novel category of GEF that is translocated to a particular subcellular compartment by association with the GTP-bound form of a small GTPase and catalyzes activation of the GDP-bound form present in the compartment, thereby causing an amplification of cellular responses induced by the small GTPase. Ras/Rap1-associating guanine nucleotide exchange factor cyclic nucleotide monophosphate PSD-95/DlgA/ZO-1 hemagglutinin enhanced green fluorescence protein maltose-binding protein Dulbecco's modified Eagle's medium glutathione S-transferase Rap1-interacting domain Ras family small GTPases have been implicated as a molecular switch that directs cell proliferation and differentiation by cycling between GTP-bound and GDP-bound forms (1Lowy D.R. Willumsen B.M. Annu. Rev. Biochem. 1993; 62: 851-891Crossref PubMed Scopus (1127) Google Scholar, 2Bos J.L. EMBO J. 1998; 17: 6776-6782Crossref PubMed Scopus (288) Google Scholar). The GTP-bound form is active in that it directly binds to and activates specific effector molecules (3Katz M.E. McCormick F. Curr. Opin. Genet. Dev. 1997; 7: 75-79Crossref PubMed Scopus (276) Google Scholar). The effector region of Ras family GTPases (amino acids 32–40 in human Ha-Ras) is involved in the interaction with effectors (1Lowy D.R. Willumsen B.M. Annu. Rev. Biochem. 1993; 62: 851-891Crossref PubMed Scopus (1127) Google Scholar). The Ras-binding domain of the serine/threonine kinase Raf-1, one of the best characterized Ras effectors, interacts directly with the effector region of Ras in a GTP-dependent manner. On the other hand, the RA1 domain, which is also responsible for binding to Ras, was identified in several Ras effectors such as RalGDS and AF-6/Afadin (4Ponting C.P. Benjamin D.R. Trends Biochem. Sci. 1996; 21: 422-425Abstract Full Text PDF PubMed Scopus (179) Google Scholar). The tertiary structure of the Ras-binding domain of Raf-1 is similar to that of the RA domain of RalGDS, although no obvious homology was found in their amino acid sequences (5Nassar N. Horn G. Herrmann C. Scherer A. McCormick F. Wittinghofer A. Nature. 1995; 375: 554-560Crossref PubMed Scopus (561) Google Scholar, 6Huang L. Weng X. Hofer F. Martin G.S. Kim S.H. Nat. Struct. Biol. 1997; 4: 609-615Crossref PubMed Scopus (66) Google Scholar, 7Huang L. Hofer F. Martin G.S. Kim S.H. Nat. Struct. Biol. 1998; 5: 422-426Crossref PubMed Scopus (205) Google Scholar). The Ras family consists of ∼20 members, including Ha-Ras, Ki-Ras, N-Ras, Rap1A, Rap1B, Rap2A, Rap2B, R-Ras, R-Ras2/TC21, R-Ras3/M-Ras, RalA, and RalB. Among them, Rap1 was originally characterized as an antagonist of Ki-Ras-induced transformation and is thus termed Krev-1 as well (8Noda M. Biochim. Biophys. Acta. 1993; 1155: 97-109PubMed Google Scholar). The ability of Rap1 to block transformation is likely ascribed to its competitive binding to Ras effectors because Rap1 shares the effector region with Ras and in fact associates with a subset of Ras effectors such as Raf-1 without stimulating their activities. This property of Rap1 is attributable to its greatly enhanced interaction with the cysteine-rich domain, a second Ras-binding site, of Raf-1 (9Hu 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, 10Okada 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). In mammalian cells, including fibroblasts, platelets, T and B lymphocytes, and neutrophils, Rap1 is activated in response to a diverse array of extracellular stimuli. Interleukin-2 gene transcription in T cells and insulin-induced mitogen-activated protein kinase activation in Chinese hamster ovary cells, for instance, are presumed to be regulated by both positive and negative actions on Raf-1 exerted by Ras and Rap1, respectively (2Bos J.L. EMBO J. 1998; 17: 6776-6782Crossref PubMed Scopus (288) Google Scholar). However, Rap1 is rapidly activated after various stimulations without affecting the Ras signaling pathway (11Posern G. Weber C.K. Rapp U.R. Feller S.M. J. Biol. Chem. 1998; 273: 24297-24300Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 12Zwartkruis F.J. Wolthuis R.M. Nabben N.M. Franke B. Bos J.L. EMBO J. 1998; 17: 5905-5912Crossref PubMed Scopus (191) Google Scholar). Therefore, it is feasible that Rap1 also exerts its own physiological function other than the modulation of Ras-dependent pathways. In nerve growth factor-triggered signaling in PC12 pheochromocytoma cells, Rap1 is reported to be involved in B-Raf activation, leading to the sustained activation of extracellular signal-regulated kinases that is required for neuronal differentiation (13Vossler M.R. Yao H. York R.D. Pan M.G. Rim C.S. Stork P.J. Cell. 1997; 89: 73-82Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar, 14York R.D. Yao H. Dillon T. Ellig C.L. Eckert S.P. McCleskey E.W. Stork P.J. Nature. 1998; 392: 622-626Crossref PubMed Scopus (762) Google Scholar). Rap1-dependent activation of B-Raf, but not Raf-1, was observed in COS-7 cells as well (10Okada 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). Furthermore, the role of Rap1 in integrin-mediated leukocyte adhesion has recently been delineated. An active form of Rap1 potently induced the activation of integrins and subsequent cell aggregation (15Katagiri K. Hattori M. Minato N. Irie S. Takatsu K. Kinashi T. Mol. Cell. Biol. 2000; 20: 1956-1969Crossref PubMed Scopus (287) Google Scholar, 16Reedquist K.A. Ross E. Koop E.A. Wolthuis R.M. Zwartkruis F.J. van Kooyk Y. Salmon M. Buckley C.D. Bos J.L. J. Cell Biol. 2000; 148: 1151-1158Crossref PubMed Scopus (367) Google Scholar), whereas a dominant-negative form of Rap1 and GTPase-activating proteins for Rap1, RapGAP, and SPA-1 inhibited cell adhesion triggered by extracellular stimulations including T-cell receptor or CD31 ligation (15Katagiri K. Hattori M. Minato N. Irie S. Takatsu K. Kinashi T. Mol. Cell. Biol. 2000; 20: 1956-1969Crossref PubMed Scopus (287) Google Scholar, 16Reedquist K.A. Ross E. Koop E.A. Wolthuis R.M. Zwartkruis F.J. van Kooyk Y. Salmon M. Buckley C.D. Bos J.L. J. Cell Biol. 2000; 148: 1151-1158Crossref PubMed Scopus (367) Google Scholar, 17Tsukamoto N. Hattori M. Yang H. Bos J.L. Minato N. J. Biol. Chem. 1999; 274: 18463-18469Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Lipopolysaccharide-induced activation of integrins in macrophages also requires Rap1 (18Schmidt A. Caron E. Hall A. Mol. Cell. Biol. 2001; 21: 438-448Crossref PubMed Scopus (84) Google Scholar). Mechanisms underlying the regulation of Rap1 remain largely unknown. Like Ras, Rap1 is believed to be activated by specific GEFs such as smgGDS (19Mizuno T. Kaibuchi K. Yamamoto T. Kawamura M. Sakoda T. Fujioka H. Matsuura Y. Takai Y. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6442-6446Crossref PubMed Scopus (169) Google Scholar), C3G (20Gotoh T. Hattori S. Nakamura S. Kitayama H. Noda M. Takai Y. Kaibuchi K. Matsui H. Hatase O. Takahashi H. Kurata T. Matsuda M. Mol. Cell. Biol. 1995; 15: 6746-6753Crossref PubMed Scopus (336) Google Scholar), Epac/cAMP-GEF (21de 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 (1634) Google Scholar, 22Kawasaki 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 (1179) Google Scholar) and CalDAGGEFI (23Kawasaki 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). Recently, we identified a novel type of Rap1 GEF in humans (Hs-RA-GEF; referred to from here on as RA-GEF-1) and Caenorhabditis elegans (Ce-RA-GEF) (24Liao 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). Other groups also reported a molecule identical to RA-GEF-1 and designated it PDZ-GEF1 (25de 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), nRapGEP (26Ohtsuka 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), and CNrasGEF (27Pham 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). RA-GEF-1 contains the cNMP-binding, Ras exchanger motif and PDZ and RA domains as well as the GEF catalytic domain. The RA domain of RA-GEF-1 associates directly with the GTP-bound form of Rap1, whereas it exhibits no detectable binding to Ha-Ras. On the other hand, RA-GEF-1 shows GEF activity toward Rap1 and Rap2, but not Ha-Ras. However, regulatory mechanisms of GEF activity remained obscure. We and others detected no specific cAMP/cGMP binding to the cNMP-binding domain of RA-GEF-1 (24Liao 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, 25de 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, 26Ohtsuka 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), although Pham et al. (27Pham 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. Here we investigated the role of the RA domain in the regulation of RA-GEF-1. We show that a deletion mutation within the RA domain that virtually abolishes Rap1 binding significantly diminished GEF activity in the cell, whereas it did not affect GEF activity in vitro. Additionally, Rap1-dependent translocation of RA-GEF-1 to the perinuclear compartment was observed, which was totally abolished by the RA domain mutation. Hence, GEF activity of RA-GEF-1in vivo is likely to be enhanced through the RA domain-mediated translocation to the perinuclear region, where Rap1 exerts its function. Anti-Rap1A (sc-65) and anti-B-Raf (sc-166) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-HA (12CA5; Roche Molecular Biochemicals), anti-FLAG (M2; Sigma), anti-trans-Golgi-network 38 (Transduction Laboratories), and tetramethylrhodamine-conjugated goat anti-mouse IgG (T2762; Molecular Probes) antibodies were purchased from the indicated commercial suppliers. Horseradish peroxidase-conjugated anti-mouse immunoglobulin (NA9310) and anti-rabbit Ig (NA9340) antibodies were purchased from Amersham Pharmacia Biotech. [2,8-3H]cAMP and [8-3H]cGMP were purchased from Moravek Biochemicals. [γ-32P]ATP was purchased from Amersham Pharmacia Biotech. The full-length human RA-GEF-1 cDNA (KIAA0313) was kindly provided by Takahiro Nagase (Kazusa DNA Research Institute, Chiba, Japan). pcDNA3.1/HisC-RA-GEF-1 was described previously (24Liao 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). pcDNA3.1/HisC-RA-GEF-1ΔRA, which encodes a mutant (designated RA-GEF-1ΔRA) containing an internal deletion (amino acids 606–626) of the RA domain, was generated by oligonucleotide-directed mutagenesis. For expressing EGFP- fused RA-GEF-1 and RA-GEF-1ΔRA in mammalian cells, an EGFP cDNA was subcloned into pcDNA3.1/HisC-RA-GEF-1 and pcDNA3.1/HisC-RA-GEF-1ΔRA, yielding pcDNA3.1/HisC-EGFP-RA-GEF-1 and pcDNA3.1/HisC-EGFP-RA-GEF-1ΔRA, respectively. pFLAG-CMV2-RA-GEF-1, pFLAG-CMV2-RA-GEF-1ΔRA, pBlueBacIII-FLAG-RA-GEF-1, and pBlueBacIII-FLAG-RA-GEF-1ΔRA were constructed by subcloning N-terminally FLAG-tagged RA-GEF-1 and RA-GEF-1ΔRA coding sequences into pCMV2 (Sigma) and pBlueBacIII (PharMingen), respectively. Polypeptides corresponding to RA domains of RA-GEF-1 and RA-GEF-1ΔRA (amino acids 539–709 and 539–688, respectively) were expressed as MBP-fusion proteins (MBP-RAWT and MBP-RAMUT, respectively) in Escherichia coli AG1 cells by using pMal-c2 (New England Biolabs). pEF-BOS-HA-Rap1AWT, pEF-BOS-HA-Rap1AV12, pH8-FLAG-B-Raf, pGEX2-MEK-His6, and pGEX5-kinase-negative extracellular signal-regulated kinase were described previously (10Okada 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, 24Liao 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). COS-7 cells were maintained in DMEM supplemented with 10% (v/v) fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 µg/ml). Transfections were performed using Superfect (Qiagen) or GENEPORTER (Gene Therapy System) transfection reagents according to the manufacturer's protocols. The post-translationally modified form of Rap1A was purified from Spodoptera frugiperda Sf9 cells infected with baculovirus overexpressing Rap1A as described previously (9Hu 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). The in vitro binding assay was carried out by incubating 20 µl of amylose resin carrying MBP-RAWT or MBP-RAMUTwith guanosine 5′-O-(3-thiotriphosphate)- or guanosine 5′-O-(2-thiodiphosphate)-loaded Rap1A in a total volume of 100 µl of buffer A (20 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1 mmdithiothreitol, 5 mm MgCl2, and 0.1% Lubrol PX). After incubation at 4 °C for 2 h, resin was washed, and bound proteins were eluted with buffer A containing 10 mmmaltose and subjected to SDS-polyacrylamide gel electrophoresis. Immunoblot detection was performed using anti-Rap1A antibody (sc-65) and enhanced chemiluminescence reagents (Roche Molecular Biochemicals). FLAG-RA-GEF-1 and FLAG-RA-GEF-1ΔRA were expressed in Sf9 cells and affinity-purified with agarose resin conjugated with the anti-FLAG antibody M2 (Sigma). GEF assays were performed as described previously (24Liao 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, 2 pmol of Rap1A loaded with [3H]GDP (5,000 cpm/pmol) was incubated with 1 µg of FLAG-RA-GEF-1 or FLAG-RA-GEF-1ΔRA in 50 µl of reaction buffer containing 20 mm Tris-HCl, pH 7.4, 3 mmMgCl2, 50 mm NaCl, 10 mm2-mercaptoethanol, 5% glycerol, 5 mg/ml bovine serum albumin, and 3 mm unlabeled GTP for the indicated periods. The reaction was terminated by the addition of 2 ml of ice-cold stop buffer containing 20 mm Tris-HCl, pH 8.0, 100 mm NaCl, and 5 mm MgCl2, and the reaction mixture was subjected to filtration through a nitrocellulose membrane (0.22-µm pore size). After washing with stop buffer, the membrane-trapped radioactivity was measured by liquid scintillation counting. GST-RalGDS-RID, a GST-fusion protein of RID of human RalGDS, was described previously (24Liao 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). In vivoGEF activities of RA-GEF-1 and RA-GEF-1ΔRA toward Rap1A were examined by the RalGDS-RID pull-down assay as described previously (24Liao 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 (50% confluent) in 100-mm plates were cotransfected with pEF-BOS-HA-Rap1AWT (1 µg) and either pFLAG-CMV2-RA-GEF-1 or pFLAG-CMV2-RA-GEF-1ΔRA (2 µg) by using the GENEPORTER reagent. COS-7 cells transfected with pEF-BOS-HA-Rap1AV12 served as a positive control. After a 4-h incubation with the transfection mixture, cells were incubated in DMEM containing 10% fetal bovine serum for 26 h. Subsequently, cells were washed once with phosphate-buffered saline and starved for another 16 h in DMEM containing 0.1% fetal bovine serum. Cells were then harvested and lysed in 1 ml of lysis buffer A (50 mm Tris-HCl, pH 7.4, 200 mm NaCl, 2.5 mm MgCl2, 10% glycerol, 1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, and 1 mmleupeptin). Soluble cellular extracts (250 µl) were incubated with 5 µg of GST-RalGDS-RID immobilized on glutathione-agarose resin. After a 60-min incubation at 4 °C, resin was washed four times with lysis buffer A. Bound proteins were eluted with 10 mm glutathione and subjected to SDS-polyacrylamide gel electrophoresis. Immunoblot detection was performed using anti-HA antibody (12CA5) and enhanced chemiluminescence reagents (Roche Molecular Biochemicals). COS-7 cells (60–70% confluent) in 100-mm plates were cotransfected with a combination of pH8-FLAG-B-Raf (0.5 µg), pEF-BOS-HA-Rap1AWT (1 µg), and either pFLAG-CMV2-RA-GEF-1 or pFLAG-CMV2-RA-GEF-1ΔRA (2 µg) by using the Superfect reagent. COS-7 cells cotransfected with pH8-FLAG-B-Raf and pEF-BOS-HA-Rap1AV12 served as a positive control. After a 4-h incubation with the transfection mixture, cells were incubated in DMEM containing 10% fetal bovine serum for 8 h. Subsequently, cells were washed once with phosphate-buffered saline and starved for another 24 h in DMEM containing 0.1% fetal bovine serum. Cells were then harvested and lysed by sonication in 300 µl of lysis buffer B (20 mm Tris-HCl, pH 7.4, 137 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, 10 µg/ml leupeptin, 20 mm β-glycerophosphate, 1 mm sodium vanadate, and 1% Triton X-100). Soluble cellular extracts were prepared by centrifugation at 100,000 ×g for 1 h at 4 °C, and FLAG-B-Raf in extracts was immunoprecipitated with anti-FLAG M2 resin (Sigma). B-Raf kinase activity was examined by incubating immunoprecipitates in the presence of GST-tagged mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (0.1 µg) and GST-kinase-negative extracellular signal-regulated kinase (1 µg) in 50 µl of the kinase reaction mixture (20 mm Tris-HCl, pH 7.4, 10 mmMgCl2, 20 mm β-glycerophosphate, and 50 µm [γ-32P]ATP (3,000 cpm/pmol)) for 20 min at 30 °C as described previously (10Okada 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). After incubation, reaction mixtures were fractionated by SDS-polyacrylamide gel electrophoresis. Phosphorylated proteins were visualized and quantified using the BAS2000 bioimaging analyzer (Fujix, Tokyo, Japan). HA-Rap1AWT, FLAG-RA-GEF-1, FLAG-RA-GEF-1ΔRA, and FLAG-B-Raf in extracts were fractionated by SDS-polyacrylamide gel electrophoresis and detected by immunoblotting using anti-HA (12CA5), anti-FLAG (M2), and anti-B-Raf (sc-166) antibodies, respectively, and enhanced chemiluminescence reagents (Roche Molecular Biochemicals). COS-7 or Rat-1 cells were seeded on a 2-well chamber slide (Nalge Nunc) and transfected with pEFBOSHA-Rap1AV12 (0.5 µg) in combination with either pcDNA3.1/HisCEGFP-RA-GEF-1 or pcDNA3.1/HisC-EGFP-RA-GEF-1ΔRA (0.5 µg) by using the Superfect reagent. After a 4-h incubation with the transfection mixture, cells were incubated in DMEM containing 10% fetal bovine serum for 8 h. Subsequently, cells were washed once with phosphate-buffered saline and starved for another 16 h in DMEM containing 0.1% fetal bovine serum. After fixation with 4% paraformaldehyde and permeabilization with 0.2% Triton X-100, cells were stained with anti-HA (12CA5) and tetramethylrhodamine-conjugated goat anti-mouse IgG (T2762) antibodies. Subcellular localization of EGFP-RA-GEF-1, EGFP-RA-GEF-1ΔRA, and HA-Rap1AV12 was analyzed under a confocal laser microscope (MRC-1024; Bio-Rad). FLAG-RA-GEF-1 was purified from Sf9 cells as described above. GST-fused protein kinase A regulatory subunit Iα (GST-PKA-RIα) was prepared as described previously (24Liao 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). The cAMP binding assay was performed essentially as described previously (24Liao 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), with minor modifications. FLAG-RA-GEF-1 (1 µg) immobilized on anti-FLAG M2 resin or GST-PKA-RIα (0.5 µg) immobilized on glutathione-agarose resin was incubated in a 100-µl reaction mixture containing 10 mmpotassium phosphate, pH 6.8, 150 mm NaCl, 1 mmEDTA, 100 µg/ml bovine serum albumin, 25 mmβ-mercaptoethanol, and 500 nm [2,8-3H]cAMP (10,000 cpm/pmol) at 25 °C for 90 min with gentle shaking. After extensive washing, bound proteins were eluted with 0.2% SDS and counted for 3H label. The cGMP binding assay was carried out in a similar manner, except that [8-3H]cGMP (5,000 cpm/pmol) replaced cAMP. RA-GEF-1 acts as a GEF for Rap1 and Rap2 (24Liao 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, 25de 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, 26Ohtsuka 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, 27Pham 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), and its RA domain efficiently associates with Rap1A·GTP (24Liao 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) but not with Rap2A·GTP (data not shown) in vitro. However, the role of the RA domain in the regulation of GEF activity remained unknown. To clarify this point, we first tried to identify a mutation in the RA domain that abolishes the binding activity. Arg-20 of RalGDS is critical for the association between RalGDS-RID and Rap1, as indicated by the finding that the R20A mutation of RalGDS completely eliminated the binding activity (6Huang L. Weng X. Hofer F. Martin G.S. Kim S.H. Nat. Struct. Biol. 1997; 4: 609-615Crossref PubMed Scopus (66) Google Scholar). Although this Arg residue is conserved in the RA domain of RA-GEF-1 (Arg-611), RA-GEF-1 carrying the R611A mutation still retained a residual binding activity toward Rap1A·GTP (data not shown). Subsequently, we deleted 21 N-terminal amino acids (amino acids 606–626) of the RA domain by oligonucleotide-directed mutagenesis (Fig. 1A), and the association of MBP-RAWT and MBP-RAMUT with GDP-bound and GTP-bound forms of Rap1A was examined (Fig.1 B). Whereas MBP-RAWT associated with Rap1Ain vitro in a GTP-dependent fashion, as we described previously (24Liao 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), MBP-RAMUT exhibited no detectable binding to Rap1A. To examine the effect of the RA domain mutation on GEF activity, we assayed the GEF activities of full-length wild-type RA-GEF-1 and its RA domain mutant (RA-GEF-1ΔRA). Both proteins were expressed with a FLAG tag in Sf9 cells and purified to near homogeneity by using anti-FLAG M2 resin. As shown in Fig.2A, both of these proteins exhibited virtually the same enzymatic activity to stimulate GDP release from Rap1A. Therefore, the internal deletion in the RA domain does not interfere with the intrinsic activity of the GEF domain. It has been reported that binding of a ligand to the regulatory domain of certain GEFs, such as Epac/cAMP-GEF (21de 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 (1634) Google Scholar, 22Kawasaki 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 (1179) Google Scholar) and Asef (28Kawasaki Y. Senda T. Ishidate T. Koyama R. Morishita T. Iwayama Y. Higuchi O. Akiyama T. Science. 2000; 289: 1194-1197Crossref PubMed Scopus (299) Google Scholar), modulates GEF activity. To examine the possibility of ligand induction of GEF activity in vitro, FLAG-RA-GEF-1 and FLAG-RA-GEF-1ΔRA were incubated with guanosine 5′-O-(3-thiotriphosphate)-loaded Rap1A, and reaction mixtures were subjected to in vitro GEF assays. FLAG-RA-GEF-1 and FLAG-RA-GEF-1ΔRA preincubated with or without guanosine 5′-O-(3-thiotriphosphate)-loaded Rap1A exhibited no significant differences in their GEF activity toward Rap1A (data not shown). GEF activities of RA-GEF-1 and RA-GEF-1ΔRA in vivo were examined by pull-down assays for Rap1A·GTP. RA-GEF-1 and RA-GEF-1ΔRA were expressed as FLAG-tagged proteins in COS-7 cells in combination with HA-tagged wild-type Rap1A (HA-Rap1AWT). Cell lysates prepared from transfectants were incubated with immobilized GST-RalGDS-RID, and bound HA-Rap1AWT·GTP was quantitated by immunoblotting (Fig. 2 B). Coexpression of FLAG-RA-GEF-1 caused a 6-fold increase in the amounts of the GTP-bound form of HA-Rap1AWT, which were almost equivalent to those of constitutively active HA-Rap1A (HA-Rap1AV12). In contrast, the GTP-bound Rap1A level in FLAG-RA-GEF-1ΔRA-expressing cells was ∼3-fold lower than that in cells expressing FLAG-RA-GEF-1. We next examined B-Raf activation following expression of RA-GEF

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