Identification and Characterization of Potential Effector Molecules of the Ras-related GTPase Rap2
1999; Elsevier BV; Volume: 274; Issue: 13 Linguagem: Inglês
10.1074/jbc.274.13.8737
ISSN1083-351X
AutoresVanessa Nancy, Rob M.F. Wolthuis, Marie‐France de Tand, Isabelle Janoueix‐Lerosey, Johannes L. Bos, Jean de Gunzburg,
Tópico(s)Cellular transport and secretion
ResumoIn search for effectors of the Ras-related GTPase Rap2, we used the yeast two-hybrid method and identified the C-terminal Ras/Rap interaction domain of the Ral exchange factors (RalGEFs) Ral GDP dissociation stimulator (RalGDS), RalGDS-like (RGL), and RalGDS-like factor (Rlf). These proteins, which also interact with activated Ras and Rap1, are effectors of Ras and mediate the activation of Ral in response to the activation of Ras. Here we show that the full-length RalGEFs interact with the GTP-bound form of Rap2 in the two-hybrid system as well as in vitro. When co-transfected in HeLa cells, an activated Rap2 mutant (Rap2Val-12) but not an inactive protein (Rap2Ala-35) co-immunoprecipitates with RalGDS and Rlf; moreover, Rap2-RalGEF complexes can be isolated from the particulate fraction of transfected cells and were localized by confocal microscopy to the resident compartment of Rap2,i.e. the endoplasmic reticulum. However, the overexpression of activated Rap2 neither leads to the activation of the Ral GTPase via RalGEFs nor inhibits Ras-dependent Ral activation in vivo. Several hypotheses that could explain these results, including compartmentalization of proteins involved in signal transduction, are discussed. Our results suggest that in cells, the interaction of Rap2 with RalGEFs might trigger other cellular responses than activation of the Ral GTPase. In search for effectors of the Ras-related GTPase Rap2, we used the yeast two-hybrid method and identified the C-terminal Ras/Rap interaction domain of the Ral exchange factors (RalGEFs) Ral GDP dissociation stimulator (RalGDS), RalGDS-like (RGL), and RalGDS-like factor (Rlf). These proteins, which also interact with activated Ras and Rap1, are effectors of Ras and mediate the activation of Ral in response to the activation of Ras. Here we show that the full-length RalGEFs interact with the GTP-bound form of Rap2 in the two-hybrid system as well as in vitro. When co-transfected in HeLa cells, an activated Rap2 mutant (Rap2Val-12) but not an inactive protein (Rap2Ala-35) co-immunoprecipitates with RalGDS and Rlf; moreover, Rap2-RalGEF complexes can be isolated from the particulate fraction of transfected cells and were localized by confocal microscopy to the resident compartment of Rap2,i.e. the endoplasmic reticulum. However, the overexpression of activated Rap2 neither leads to the activation of the Ral GTPase via RalGEFs nor inhibits Ras-dependent Ral activation in vivo. Several hypotheses that could explain these results, including compartmentalization of proteins involved in signal transduction, are discussed. Our results suggest that in cells, the interaction of Rap2 with RalGEFs might trigger other cellular responses than activation of the Ral GTPase. Ras proteins are monomeric GTPases that play a pivotal role in the control of cell proliferation; they function as binary switches by cycling between an inactive form bound to GDP and the active GTP-bound state (1Bourne H.R. Sanders D.A. McCormick F. Nature. 1991; 349: 117-127Crossref PubMed Scopus (2786) Google Scholar). Activation, through the dissociation of bound GDP and subsequent binding of GTP, is catalyzed by GEFs, 1The abbreviations used are:GEF, guanine nucleotide exchange factor; Gpp(NH)p, guanosine imido 5′triphosphate; GST, glutathione S-transferase; GST-RalBD, GST fusion protein containing the Ral binding domain of RLIP76, a Ral effector; HA, hemagglutinin; RalGDS, Ral GDP dissociation stimulator; RBD, Ras binding domain; RGL, RalGDS-like; RID, Ras and Rap interaction domain of RalGEFs; Rlf, RalGDS-like factor; PI3K, phosphatidylinositol 3-OH kinase. such as CDC25/Ras-GRF and Sos (2Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1782) Google Scholar, 3Chardin P. Camonis J.H. Gane N. van Aelst L. Wigler S.J.M. Bar Sagi D. Science. 1993; 260: 1338-1343Crossref PubMed Scopus (674) Google Scholar, 4Shou C. Farnsworth C.L. Neel B.G. Feig L.A. Nature. 1992; 358: 351-354Crossref PubMed Scopus (294) Google Scholar). Return to the inactive state is ensured via stimulation of the low intrinsic GTPase activity of Ras by GTPase-activating proteins, such as p120-GTPase-activating protein, neurofibromin, and GapIP4BP (2Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1782) Google Scholar, 5Cullen P.J. Hsuan J.J. Truong O. Letcher A.J. Jackson T.R. Dawson A.P. Irvine R.F. Nature. 1995; 376: 527-530Crossref PubMed Scopus (287) Google Scholar). In the active GTP-bound state, Ras exerts its biological effects by turning on several effectors that activate downstream pathways. Soluble serine/threonine kinases B-Raf and c-Raf, once activated by Ras-GTP through a mechanism that is not fully understood, trigger a cascade of protein kinases that results in the activation of mitogen-activated protein kinases extracellular signal-regulated kinases 1 and 2 (6Marshall C.J. Curr. Opin. Cell Biol. 1995; 8: 197-204Crossref Scopus (475) Google Scholar). Another target of Ras is the catalytic subunit of phosphatidylinositol 3-OH kinase (PI3K) (7Rodriguez-Viviana P. Warne P. Dhand R. Vanhaesebroeck B. Gout I. Fry M.J. Waterfield M.D. Downward J. Nature. 1994; 370: 527-532Crossref PubMed Scopus (1749) Google Scholar); this pathway leads to the activation of the protein kinase Akt (8Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1896) Google Scholar, 9Franke T.F. Yang S.I. Chan T.O. Datta K. Kazlauskas A. Morrison D.K. Kaplan D. Tsichlis P.N. Cell. 1995; 81: 727-736Abstract Full Text PDF PubMed Scopus (1839) Google Scholar), as well as the activation of Ras-related proteins of the Rho/Rac/Cdc42 family involved in controlling the polymerization state of the actin cytoskeleton, cell adhesion, and gene transcription (10Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5271) Google Scholar, 11RodriguezViciana P. Warne P.H. Khwaja A. Marte B.M. Pappin D. Das P. Waterfield M.D. Ridley A. Downward J. Cell. 1997; 89: 457-467Abstract Full Text Full Text PDF PubMed Scopus (965) Google Scholar). Ras is also able to activate the related GTPase Ral through RalGEFs that constitute direct effectors of Ras (12Urano T. Emkey R. Feig L.A. EMBO J. 1996; 15: 810-816Crossref PubMed Scopus (302) Google Scholar, 13White M.A. Vale T. Camonis J.H. Schaefer E. Wigler M.H. J. Biol. Chem. 1996; 271: 16439-16442Crossref PubMed Scopus (218) Google Scholar, 14Kishida S. Koyama S. Matsubara K. Kishida M. Matsuura Y. Kikuchi A. Oncogene. 1997; 15: 2899-2907Crossref PubMed Scopus (58) Google Scholar, 15Wolthuis R.M.F. Zwartkruis F. Moen T.C. Bos J.L. Curr Biol. 1998; 8: 471-474Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar); three such proteins, namely RalGDS (16Albright C.F. Giddings B.W. Liu J. Vito M. Weinberg R.A. EMBO, J. 1993; 12: 339-347Crossref PubMed Scopus (159) Google Scholar), RGL (17Kikuchi A. Demo S.D. Ye Z.H. Chen Y.W. Williams L. Mol. Cell. Biol. 1994; 14: 7483-7491Crossref PubMed Scopus (248) Google Scholar), and Rlf (18Wolthuis R.M.F. Bauer B. van't Veer L.J. de Vries-Smits A.M.M. Cool R. Spaargaren M. Wittinghofer A. Burgering B.M.T. Bos J.L. Oncogene. 1996; 13: 353-362PubMed Google Scholar) have been extensively characterized, and the isolation of another member of this family has been recently reported (19D'Adamo D.R. Novick S. Kahn J.M. Leonardi P. Pellicer A. Oncogene. 1997; 14: 1295-1305Crossref PubMed Scopus (49) Google Scholar). Elegant genetic experiments have shown that in most cellular systems, the complementary action of at least two of these three pathways (Raf, PI3K, and RalGEFs) is necessary for Ras to transform murine fibroblasts in culture (11RodriguezViciana P. Warne P.H. Khwaja A. Marte B.M. Pappin D. Das P. Waterfield M.D. Ridley A. Downward J. Cell. 1997; 89: 457-467Abstract Full Text Full Text PDF PubMed Scopus (965) Google Scholar, 13White M.A. Vale T. Camonis J.H. Schaefer E. Wigler M.H. J. Biol. Chem. 1996; 271: 16439-16442Crossref PubMed Scopus (218) Google Scholar, 20Joneson T. White M.A. Wigler M.H. Bar-Sagi D. Science. 1996; 271: 810-812Crossref PubMed Scopus (359) Google Scholar, 21Khosravi-Far R. White M.A. Westwick J.K. Solski P. Chrzanowska-Wodnicka Van Aelst L. Wigler M.H. Der C.J. Mol. Cell. Biol. 1996; 16: 3923-3933Crossref PubMed Scopus (330) Google Scholar, 22White M.A. Nicolette C. Minden A. Polverino A. van Aelst L. Karin M. Wigler M.H. Cell. 1995; 80: 533-541Abstract Full Text PDF PubMed Scopus (629) Google Scholar). Other Ras effectors, such as the ζ isoform of protein kinase C (23Diaz-Meco M.T. Lozano J. Municio M.M. Berra E. Frutos S. Sanz L. Moscat J. J. Biol. Chem. 1994; 269: 31706-31710Abstract Full Text PDF PubMed Google Scholar), Rin (24Han L. Colicelli J. Mol. Cell. Biol. 1995; 15: 1318-1323Crossref PubMed Google Scholar), and AF-6/Canoe (25Kuriyama M. Harada N. Kuroda S. Yamamoto T. Nakafuku M. Iwamatsu A. Yamamoto D. Prasad R. Croce C. Canaani E. Laibuchi K. J. Biol. Chem. 1996; 271: 607-610Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar) have been identified; their involvement in Ras action has not yet been documented. The Rap group of Ras-related proteins is composed of the two closely related Rap1A and Rap1B (which are 95% identical) and the two 90% identical Rap2A and Rap2B proteins (26Pizon V. Lerosey I. Chardin P. Tavitian A. Nucleic Acids Res. 1988; 16: 7719Crossref PubMed Scopus (114) Google Scholar, 27Pizon V. Chardin P. Lerosey I. Olofsson B. Tavitian A. Oncogene. 1988; 3: 201-204PubMed Google Scholar, 28Ohmstede C.-A. Farrel F.X. Reep B.R. Clemetson K.J. Lapetina E.G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6527-6531Crossref PubMed Scopus (63) Google Scholar); overall, Rap1 and Rap2 share 60% identity. They constitute the closest Ras relatives because they share more than 50% overall identity with Ras proteins and exhibit very similar three-dimensional structures (29Nassar N. Horn G. Herrmann C. Scherer A. McCormick F. Wittinghofer Nature. 1995; 375: 554-560Crossref PubMed Scopus (565) Google Scholar, 30Cherfils J. Menetrey J. LeBras G. LeBras G. Janoueix Lerosey I. de Gunzburg J. Garel J.R. Auzat I. EMBO J. 1997; 16: 5582-5591Crossref PubMed Scopus (62) Google Scholar). Moreover, Rap1 contains the same effector domain as Ras, which has prompted speculations that Rap proteins may behave as Ras antagonists. Indeed, the Krev-1 gene encoding Rap1A had been isolated on the basis that its overexpression was able to revert the phenotype of Ras-transformed NIH 3T3 fibroblasts (31Kitayama H. Sugimoto Y. Matsuzaki T. Ikawa Y. Noda M. Cell. 1989; 56: 77-84Abstract Full Text PDF PubMed Scopus (826) Google Scholar); since then, several laboratories have provided evidence that overexpression of Rap1 can indeed interfere with Ras function. Molecular basis for such findings resides in the fact that Rap1 is capable of binding with affinities similar to or in some cases even higher than Ras with Ras effectors, such as Raf-1 kinase, PI3K, and RalGEFs (7Rodriguez-Viviana P. Warne P. Dhand R. Vanhaesebroeck B. Gout I. Fry M.J. Waterfield M.D. Downward J. Nature. 1994; 370: 527-532Crossref PubMed Scopus (1749) Google Scholar, 18Wolthuis R.M.F. Bauer B. van't Veer L.J. de Vries-Smits A.M.M. Cool R. Spaargaren M. Wittinghofer A. Burgering B.M.T. Bos J.L. Oncogene. 1996; 13: 353-362PubMed Google Scholar, 32Herrmann C. Horn G. Spaargaren M. Wittinghofer A. J. Biol. Chem. 1996; 271: 6794-6800Abstract Full Text PDF PubMed Scopus (305) Google Scholar). Yet the physiological role of Rap1 appears to vary according to the biological model studied. Rap1 seems to promote cAMP-dependent as well as NGF-stimulated B-Raf and mitogen-activated protein kinase activation in PC12 cells (33Vossler M.R. Yao H. York R.D. Pan M.G. Rim C.S. Stork P.J.S. Cell. 1997; 89: 73-82Abstract Full Text Full Text PDF PubMed Scopus (950) Google Scholar, 34York R.D. Yao H. Dillon T. Ellig C.L. Eckert S.P. McCleskey E.W. Stork P.J.S. Nature. 1998; 392: 622-626Crossref PubMed Scopus (764) Google Scholar). Conversely, it has also been shown to participate in the maintenance of T cell anergy by acting as a negative regulator of T cell receptor (TCR)-mediated interleukin-2 gene transcription (35Boussiotis V.A. Freeman G.J. Berezovskaya A. Barber D.L. Nadler L.M. Science. 1997; 278: 124-127Crossref PubMed Scopus (396) Google Scholar) and to inhibit Raf kinase by forming a catalytically inactive complex in quiescent Chinese hamster ovary cells that is reversed upon insulin stimulation (71Okada S. Matsuda M. Anafi M. Pawson T. Pessin J.E. EMBO J. 1998; 17: 2554-2565Crossref PubMed Scopus (82) Google Scholar). It is also possible that the function of Rap1 is independent of regulating Ras signaling, because activation of endogenous Rap1 by extracellular signals fails to interfere with Ras effector signaling in fibroblasts (72Zwartkruis F.J.T. Wolthuis R.M.F. Nabben N. Franke B. Bos J.L. EMBO J. 1998; 17: 5905-5912Crossref PubMed Scopus (191) Google Scholar). In contrast with Rap1, no function has yet been attributed to Rap2. Although it also contains the same effector domain as Ras, except for a single substitution of a serine to phenylalanine at position 39 (a similar substitution in Ras only moderately affects its transforming potential), its overexpression does not antagonize Ras signaling (36Jimenez B. Pizon V. Lerosey I. Béranger F. Tavitian A. de Gunzburg J. Int. J. Cancer. 1991; 49: 471-479Crossref PubMed Scopus (27) Google Scholar). In an effort to uncover the function of Rap2, we searched for potential effectors by using the yeast two-hybrid system. This enabled us to identify a novel protein, RPIP8, that specifically interacts with Rap2 and is principally expressed in brain (37Janoueix-Lerosey I. Pacheva E. de Tand M.F. Tavitian A. de Gunzburg J. Eur. J. Biochem. 1998; 252: 290-298Crossref PubMed Scopus (43) Google Scholar). As described in this paper, we also isolated partial cDNAs encoding the C-terminal region of the RalGEFs RalGDS, RGL, and Rlf. These three related proteins, which constitute effectors of Ras, are capable of inducing activation of the Ras-related Ral GTPase, i.e. nucleotide exchange leading to the formation of active Ral-GTP complexes (12Urano T. Emkey R. Feig L.A. EMBO J. 1996; 15: 810-816Crossref PubMed Scopus (302) Google Scholar, 38Wolthuis R.M.F. deRuiter N.D. Cool R.H. Bos J.L. EMBO J. 1997; 16: 6748-6761Crossref PubMed Scopus (145) Google Scholar, 39Murai H. Ikeda M. Ishida O. Okazaki-Kishida M. Matsuura Y. Kikuchi A. J. Biol. Chem. 1997; 272: 10483-10490Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Although Ras and Rap1 can both interact with RalGDS and Rlf in cells, only Ras is capable of inducing activation of the GTPase Ral in vivo(12Urano T. Emkey R. Feig L.A. EMBO J. 1996; 15: 810-816Crossref PubMed Scopus (302) Google Scholar, 14Kishida S. Koyama S. Matsubara K. Kishida M. Matsuura Y. Kikuchi A. Oncogene. 1997; 15: 2899-2907Crossref PubMed Scopus (58) Google Scholar). By themselves, RalGEFs exhibit little biological activity, only slightly stimulating transcription from the c-fospromoter; however, upon co-expression with activated Raf, RalGDS greatly synergizes to activate c-fos promoter activity, as well as cell proliferation and morphological transformation (13White M.A. Vale T. Camonis J.H. Schaefer E. Wigler M.H. J. Biol. Chem. 1996; 271: 16439-16442Crossref PubMed Scopus (218) Google Scholar, 40Okazaki M. Kishida S. Hinoi T. Hasegawa T. Tamada M. Kataoka T. Kikuchi A. Oncogene. 1997; 14: 515-521Crossref PubMed Scopus (56) Google Scholar). Moreover, targeting Rlf to the plasma membrane constitutively activates the protein, which is then able to stimulate gene induction and cell growth (38Wolthuis R.M.F. deRuiter N.D. Cool R.H. Bos J.L. EMBO J. 1997; 16: 6748-6761Crossref PubMed Scopus (145) Google Scholar). RalGEFs exhibit considerable homology among each other in their 130 most C-terminal residues, which constitute the Ras and Rap1 interaction domain (RID) (18Wolthuis R.M.F. Bauer B. van't Veer L.J. de Vries-Smits A.M.M. Cool R. Spaargaren M. Wittinghofer A. Burgering B.M.T. Bos J.L. Oncogene. 1996; 13: 353-362PubMed Google Scholar). They contain a conserved central domain homologous to the RasGEF CDC25 that is responsible for their exchange factor activity toward Ral as well as their stimulating effects on cell growth and gene induction (16Albright C.F. Giddings B.W. Liu J. Vito M. Weinberg R.A. EMBO, J. 1993; 12: 339-347Crossref PubMed Scopus (159) Google Scholar, 38Wolthuis R.M.F. deRuiter N.D. Cool R.H. Bos J.L. EMBO J. 1997; 16: 6748-6761Crossref PubMed Scopus (145) Google Scholar). In this study, we show that Rap2 binds to full-length RalGEFs in vitro as well as in vivo. This interaction only occurs with active Rap2; Rap2-RalGEF complexes are found in the particulate fraction of transfected cells, and active Rap2 is capable of recruiting RalGDS and Rlf to its resident compartment, the endoplasmic reticulum, suggesting that RalGEFs may indeed constitute effectors of Rap2 function. However, ectopic expression of activated Rap2 does not lead to the activation of the GTPase Ral, nor does it interfere with the ability of Ras to activate Ral. These results suggest that RalGEFs could also serve a function other than activating Ral in cells and that this novel function could be regulated by their interaction with the GTPase Rap2. Screening of a mouse brain cDNA library with the first 168 residues of Rap2A containing a Gly-12 to Val substitution fused to the C terminus of the bacterial LexA protein has already been described (37Janoueix-Lerosey I. Pacheva E. de Tand M.F. Tavitian A. de Gunzburg J. Eur. J. Biochem. 1998; 252: 290-298Crossref PubMed Scopus (43) Google Scholar). The coding sequence for the full-length Rap2B protein was obtained by reverse transcription-polymerase chain reaction from total RNA of the human pro-erythrocyte line HEL (a generous gift from Dr. Dominique Dumesnil) using oligonucleotides 5′-ACT GGG ATC CAC CAT GAG AGA GTA CAA AGT GGT GGTG-3′ and 5′-CCC TCG TCG ACG GAC TAC GCC GCG TAG TTC ATC TGC CGC AC-3′ as forward and reverse amplimers respectively and Pfu polymerase (Stratagene); the resulting product was digested with BamHI and SalI and cloned into pGBT10 (3Chardin P. Camonis J.H. Gane N. van Aelst L. Wigler S.J.M. Bar Sagi D. Science. 1993; 260: 1338-1343Crossref PubMed Scopus (674) Google Scholar) restricted with the same enzymes. The absence of mutations was verified by DNA sequencing. This construct was transformed into Saccharomyces cerevisiaestrain HF7c (MATa, ura3–52, his3–200, lys2–801, ade2–101, trp1–901, leu2–3, 112, gal4–452, gal80–538, LYS2::GAL1-HIS3, URA3::(GAL17-mers) 3 -CYCI-lacZ) and used to screen a cDNA library from human Jurkat cells as described (41Benichou S. Bomsel M. Bodeus M. Durand H. Doute M. Letourneur F. Camonis J.H. Benarous R. J. Biol. Chem. 1994; 269: 30073-30076Abstract Full Text PDF PubMed Google Scholar). Interactions between identified partners were performed by mating with Ras family proteins expressed as fusions with the DNA binding domain of GAL4 in HF7c yeast, and putative effectors fused to the activation domain of GAL4 in yeast of the strain Y187 (MATα, ura3–52, his3, ade2–101, trp1–901, leu2–3, 112, met - , gal4Δ, gal80Δ, URA3::GAL1-lacZ). Interaction was assessed by the capacity of diploid conjugants to grow in the absence of histidine and to express β-galactosidase activity, with similar results. pGAD-RalGDS was a generous gift from Dr. Michael White (13White M.A. Vale T. Camonis J.H. Schaefer E. Wigler M.H. J. Biol. Chem. 1996; 271: 16439-16442Crossref PubMed Scopus (218) Google Scholar), pGAD-Raf was from L. van Aelst (42Van Aelst L. Barr M. Marcus S. Polverino A. Wigler M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6213-6217Crossref PubMed Scopus (514) Google Scholar), pGAD-B-Raf was from A. Eychène (43Papin C. Denouel A. Calothy G. Eychène A. Oncogene. 1996; 12: 2213-2221PubMed Google Scholar), and pPC86-Rlf was described previously (18Wolthuis R.M.F. Bauer B. van't Veer L.J. de Vries-Smits A.M.M. Cool R. Spaargaren M. Wittinghofer A. Burgering B.M.T. Bos J.L. Oncogene. 1996; 13: 353-362PubMed Google Scholar). Ras family proteins were expressed as GST fusions, purified on glutathione-Sepharose beads, and loaded with GDP or Gpp(NH)p prior to binding reactions as described previously (37Janoueix-Lerosey I. Pacheva E. de Tand M.F. Tavitian A. de Gunzburg J. Eur. J. Biochem. 1998; 252: 290-298Crossref PubMed Scopus (43) Google Scholar). Potential partners were transcribed and translated in vitro in the presence of [35S]methionine in a coupled reticulocyte lysate using the bacteriophage T7 RNA polymerase (TNT, Promega) from templates obtained as follows. The RIDs of RalGEFs (obtained from the two-hybrid screens described in this study) were amplified with polymerase chain reaction with Pfu polymerase and subcloned at the 5′ EcoRI site of the pGEMMyc4 vector (a generous gift from Harald Stenmark and Marino Zerial) in frame with a Myc epitope and downstream of the bacteriophage T7 promoter. The 5′ and 3′ oligonucleotide primers used to amplify these sequences were as follows: for RalGDS, 5′-TGTG GAA TTC GCC TCC ACC ACG CCC GTG GCT GCC-3′ and 5′-CCTTG CTC GAG TCA GAA GAT GCC CTT GGC AAA TCTT-3′; for RGL, 5′-TGTG GAA TTC AAC AAT CCT AAA ATC CAC AAG CGC-3′ and 5′-CCTTG CTC GAG TCA GAG GGT GAT CTT GCT GTG CCT-3′; and for Rlf, 5′-TGTC GAA TTC TCC CCT AGG CCT TCT CGG GGT-3′ and 5′-GTCT GTC GACTCAG AAC AGT GCC CGTGC-3′. The RBD of c-Raf, obtained as a GST fusion from A. Wittinghofer (44Herrmann C. Martin G.A. Wittinghofer A. J. Biol. Chem. 1995; 270: 2901-2905Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar), was excised with BamHI andEcoRI, subcloned into the pMalC2 vector (New England Biolabs), amplified with oligonucleotides homologous to vector sequences providing a 3′ XhoI site, and cloned as above in pGEMMyc4. The absence of mutations in all clones was assessed by DNA sequencing. RPIP8 was transcribed and translated from pBS-RPIP8 as described (37Janoueix-Lerosey I. Pacheva E. de Tand M.F. Tavitian A. de Gunzburg J. Eur. J. Biochem. 1998; 252: 290-298Crossref PubMed Scopus (43) Google Scholar). Full-length RalGDS and RGL coding sequences were excised from pCEP4RalGDS and pCEP4RGL, respectively (generous gifts from M. White (13White M.A. Vale T. Camonis J.H. Schaefer E. Wigler M.H. J. Biol. Chem. 1996; 271: 16439-16442Crossref PubMed Scopus (218) Google Scholar)) with BamHI and inserted into theBamHI site of pGEMMyc4; full-length Rlf was excised from pPC86-Rlf with SalI and NotI, blunted with Klenow at its 3′ extremity, and inserted into the 5′ SalI and 3′PstI (blunted with T4 DNA polymerase) sites of pGEMMyc4. For binding experiments, 10 μl of glutathione-Sepharose 4B beads bound to 250 ng of GST, GST-Rap2A, GST-Ha-Ras, GST-Rap1A, and GST-RalA proteins were washed three times in ice-cold exchange buffer (25 mm Tris-HCl, pH 7.5, 2 mm EDTA, and 5 mm dithiothreitol) and incubated for 30 min at 37 °C in 20 μl of exchange buffer containing 150 mm of Gpp(NH)p or GDP. The beads were then diluted in 180 μl of interaction buffer (25 mm Tris-HCl, pH 7.5, 1 mm EDTA, 5 mm MgCl2, and 5 mm dithiothreitol) containing 200 μm of the appropriate nucleotide and incubated for 3 h at 4 °C with 1 μl of in vitrotranslated [35S]methionine-labeled potential effector. Beads were washed four times with 1 ml of interaction buffer and then boiled in SDS gel sample buffer to recover bound proteins. Samples were analyzed by SDS-polyacrylamide gel electrophoresis on 10% polyacrylamide gels; after staining with Coomassie Blue to detect the GST and GST fusion proteins, gels were treated with Amplify (Amersham Pharmacia Biotech), dried, and exposed to film. The cDNAs encoding Ras and Rap2 GTPases as well as RalGEFs were subcloned into mammalian expression vectors under the control of the cytomegalovirus promoter as follows. The coding sequences for Rap2 proteins carrying a Gly to Val substitution at position 12 (Rap2Val-12) and a Thr to Ala substitution at position 35 (Rap2Ala-35) (45Lerosey I. Chardin P. de Gunzburg J. Tavitian A. J. Biol. Chem. 1991; 266: 4315-4321Abstract Full Text PDF PubMed Google Scholar) were amplified by polymerase chain reaction with Pfu using primers 5′-GTGT GGA TCC ACC ATG CGC GAG TAC AAA GTG GTG GTG-3′ and 5′-TCTT CTC GAGC CTA TTG TAT GTT ACA TGC AGA ACA-3′ and inserted into the BamHI and XhoI sites of pcDNA3 (Invitrogen). Full-length RalGDS and RGL sequences excised as indicated above were inserted into the BamHI site of a pRK5 vector engineered to encode an N-terminal Myc epitope fused in frame to the N terminus of the protein of interest (a generous gift from Dr. Alan Hall). HeLa cells were co-transfected with 4 μg of Rap2 and 8 μg of RalGDS or Rlf expression constructs (or the relevant empty vector) per 8.5-cm dish with calcium phosphate; 40 h after transfection, cells were washed twice with phosphate-buffered saline and processed as follows. For experiments performed with total cell extracts, cells were lysed on ice in 25 mm Hepes buffer, pH 7.5, containing 0.1m NaCl, 1% Nonidet P-40, and protease inhibitors, and debris were removed by a 15-min centrifugation at 14,000 ×g. In order to prepare membranes, cells were swollen on ice in hypotonic buffer (25 mm Hepes, pH 7.5, containing protease inhibitors) and lysed by 100 strokes of the tight-fitted pestle of a Dounce homogeneizer. The postnuclear supernatant obtained after centrifugation at 1000 × g for 3 min was submitted to further centrifugation at 45,000 rpm for 30 min in a Beckman TLA 45 rotor; membranes were resuspended in hypotonic buffer containing 0.1 m NaCl and recentrifuged as described above. They were solubilized in the same buffer containing 1% Nonidet P-40 (30 min on ice), and insoluble material was eliminated by a final centrifugation as described above. Solubilized extracts were precleared with protein A-Sepharose and immunoprecipitated with 5 μg of anti-Myc 9E10 antibody (Boehringer Mannheim) followed by protein A-Sepharose as described previously (46de Gunzburg J. Riehl R. Weinberg R.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4007-4011Crossref PubMed Scopus (38) Google Scholar); the presence of Rap2 in immune complexes was revealed by Western blotting with affinity-purified polyclonal anti-Rap2 antibodies (47Béranger F. Tavitian A. de Gunzburg J. Oncogene. 1991; 6: 1835-1842PubMed Google Scholar) and visualized by ECL (Amersham Pharmacia Biotech). 5 × 106 exponentially growing HeLa cells were electroporated in a final volume of 200 μl of culture medium supplemented with 15 mm Hepes buffer, pH 7.5, at 960 microfarads and 240 V in the 0.4-cm electrode gap cuvettes of a Bio-Rad Gene Pulser with 6 μg of each expression construct for Myc-tagged RalGDS or Rlf, and Ras or Rap2 GTPase as indicated. After electroporation, cells were washed once and plated in four 35-mm dishes containing glass coverslips. 24 h later, they were washed twice with phosphate-buffered saline, fixed for 6 min at −20 °C in methanol, and simultaneously incubated with affinity-purified anti-Rap2 or anti-Ha-Ras (Santa-Cruz, catalog no. sc-520) and 9E10 anti-Myc (1 μg/ml, Boehringer Mannheim) antibodies as described previously (47Béranger F. Tavitian A. de Gunzburg J. Oncogene. 1991; 6: 1835-1842PubMed Google Scholar). Complexes were stained with fluorescein isothiocyanate-coupled anti-mouse and tetramethylrhodamine isothiocyanate-coupled anti-rabbit antibodies (Jackson ImmunoResearch Laboratories, Inc.) and visualized with a Leica scanning laser confocal microscope. In order to assess whether Rap2 could activate Ral via RalGDS or Rlf, COS-7 cells grown in 5-cm dishes were transfected with 1.5 μg of pMT2-HA-Ral together with 2 μg of expression vector for RasVal-12 or Rap2Val-12 and/or 1 μg of pcDNA3-Rlf or pcDNA-Myc-RalGDS (a generous gift from Thomas Linnemann and Alfred Wittinghoger) as indicated. After metabolic labeling of cells with [32P]orthophosphate, HA-Ral bound nucleotides were analyzed and quantitated as described (38Wolthuis R.M.F. deRuiter N.D. Cool R.H. Bos J.L. EMBO J. 1997; 16: 6748-6761Crossref PubMed Scopus (145) Google Scholar). Ras-dependent Ral activation in the response to insulin, was measured in A14 cells, which are NIH 3T3 fibroblasts expressing the human insulin receptor (48de Vries-Smits A.M.M. Burgering B.M.T. Leevers S.J. Marshall C.J. Bos J.L. Nature. 1992; 357: 602-604Crossref PubMed Scopus (315) Google Scholar), grown in 5-cm dishes, and transfected with 1 μg of pMT2HA-Ral together with 1 μg of pcDNA3 or 1 μg of pcDNA3-Rap2-Val-12. After serum starvation for 16 h, the cells were stimulated for 5 min with 1 μm of insulin and lysed. Ral-GTP levels were determined by trapping the active complex on beads covered with GST-RalBD fusion proteins (GST fused to the Ral binding domain of the Ral effector RLIP76 (49Wolthuis R.M.F. Franke B. vanTriest M. Bauer B. Cool R.H. Camonis J.H. Akkerman J.W.N. Bos J.L. Mol. Cell. Biol. 1998; 18: 2486-2491Crossref PubMed Scopus (130) Google Scholar)) as described (15Wolthuis R.M.F. Zwartkruis F. Moen T.C. Bos J.L. Curr Biol. 1998; 8: 471-474Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Rap2 and Ral proteins were detected on Western blots using monoclonal antibodies (Transduction Laboratories). In order to search for potential effectors of Rap2, through which it may exert its biological effects, we performed two independent screens using the two-hybrid method in the yeast S. cerevisiae. We screened a mouse brain cDNA library with residues 1–168 of Rap2A carrying a Gly to Val substitution at position 12 (Rap2AVal-12) fused to the C terminus of the DNA binding domain of the bacterial transcription activator LexA as a bait, and a human Jurkat T lymphoma cDNA library with the full-length Rap2B protein fused to the C terminus of the DNA binding domain of
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