A Novel RalGEF-like Protein, RGL3, as a Candidate Effector for Rit and Ras

Abstract

The small GTPase Rit is a close relative of Ras, and constitutively active Rit can induce oncogenic transformation. Although the effector loops of Rit and Ras are highly related, Rit fails to interact with the majority of the known Ras candidate effector proteins, suggesting that novel cellular targets may be responsible for Rit transforming activity. To gain insight into the cellular function of Rit, we searched for Rit-binding proteins by yeast two-hybrid screening. We identified the C-terminal Rit/Ras interaction domain of a protein we have designated RGL3 (RalGEF-like 3) that shares 35% sequence identity with the known Ral guanine nucleotide exchange factors (RalGEFs). RGL3, through a C-terminal 99-amino acid domain, interacted in a GTP- and effector loop-dependent manner with Rit and Ras. Importantly, RGL3 exhibited guanine nucleotide exchange activity toward the small GTPase Ral that was stimulated in vivo by the expression of either activated Rit or Ras. These data suggest that RGL3 functions as an exchange factor for Ral and may serve as a downstream effector for both Rit and Ras. The small GTPase Rit is a close relative of Ras, and constitutively active Rit can induce oncogenic transformation. Although the effector loops of Rit and Ras are highly related, Rit fails to interact with the majority of the known Ras candidate effector proteins, suggesting that novel cellular targets may be responsible for Rit transforming activity. To gain insight into the cellular function of Rit, we searched for Rit-binding proteins by yeast two-hybrid screening. We identified the C-terminal Rit/Ras interaction domain of a protein we have designated RGL3 (RalGEF-like 3) that shares 35% sequence identity with the known Ral guanine nucleotide exchange factors (RalGEFs). RGL3, through a C-terminal 99-amino acid domain, interacted in a GTP- and effector loop-dependent manner with Rit and Ras. Importantly, RGL3 exhibited guanine nucleotide exchange activity toward the small GTPase Ral that was stimulated in vivo by the expression of either activated Rit or Ras. These data suggest that RGL3 functions as an exchange factor for Ral and may serve as a downstream effector for both Rit and Ras. guanine nucleotide exchange factor glutathione S-transferase GTPase-activating protein guanosine 5′-3-O-(thio)triphosphate polyacrylamide gel electrophoresis polymerase chain reaction dithiothreitol influenza hemagglutinin epitope phosphatidylinositol 3-kinase rapid amplification of cDNA ends Rit binding domain bovine serum albumin base pair nickel-nitrilotriacetic acid phosphate-buffered saline Members of the Ras superfamily of monomeric GTPases function as binary molecular switches to control a wide variety of cellular processes including proliferation, differentiation, nuclear transport, cytoskeleton organization, and vesicular transport (1Campbell S.L. Khosravi-Far R. Rossman K.L. Clark G.J. Der C.J. Oncogene. 1998; 17: 1395-1413Crossref PubMed Scopus (919) Google Scholar, 2Macara I.G. Lounsbury K.M. Richards S.A. McKiernan C. Bar-Sagi D. FASEB J. 1996; 10: 625-630Crossref PubMed Scopus (211) Google Scholar, 3Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5216) Google Scholar). They share the ability to cycle between inactive GDP- and active GTP-bound structural states. In the active GTP-bound state Ras-related proteins interact with a variety of cellular targets to elicit their biological effects (4Bourne H.R. Sanders D.A. McCormick F. Nature. 1991; 349: 117-127Crossref PubMed Scopus (2684) Google Scholar). This cycle is tightly controlled in vivo by two classes of regulatory proteins. Activation, through the dissociation of bound GDP and subsequent binding of GTP, is catalyzed by guanine nucleotide exchange factors (GEFs)1 (5Quilliam L.A. Khosravi-Far R. Huff S.Y. Der C.J. BioEssays. 1995; 17: 395-404Crossref PubMed Scopus (193) Google Scholar). Return to the inactive state is stimulated by GTPase-activating proteins that promote rapid hydrolysis (6Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1756) Google Scholar), thus completing the cycle. The prototypic Ras proteins, Ha-, Ki, and N-Ras, play a pivotal role in the control of cellular growth and differentiation through their interaction with a variety of cellular effectors that in turn activate downstream signaling pathways (1Campbell S.L. Khosravi-Far R. Rossman K.L. Clark G.J. Der C.J. Oncogene. 1998; 17: 1395-1413Crossref PubMed Scopus (919) Google Scholar, 7Malumbres M. Pellicer A. Front. Biosci. 1998; 3: 887-912Crossref PubMed Scopus (12) Google Scholar, 8Marshall C.J. Curr. Opin. Cell Biol. 1996; 8: 197-204Crossref PubMed Scopus (473) Google Scholar). The best characterized of the Ras effectors are the Raf family of serine-threonine kinases including Raf1, B-Raf, and A-Raf (9Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2282) Google Scholar). Once activated by Ras-GTP, through a mechanism that is incompletely understood, Raf kinases trigger a cascade of protein kinases that results in the activation of extracellular signal-regulated kinases 1 and 2 (10Marshall M. Mol. Reprod. Dev. 1995; 42: 493-499Crossref PubMed Scopus (86) Google Scholar). Ras-mediated extracellular signal-regulated kinase activation leads to the stimulation of various transcription factors that regulate the expression of genes involved in proliferation and oncogenesis (9Robinson M.J. Cobb M.H. Curr. Opin. Cell Biol. 1997; 9: 180-186Crossref PubMed Scopus (2282) Google Scholar, 11Treisman R. Curr. Opin. Genet. & Dev. 1994; 4: 96-101Crossref PubMed Scopus (620) Google Scholar,12Treisman R. Curr. Opin. Cell Biol. 1996; 8: 205-215Crossref PubMed Scopus (1163) Google Scholar). Whereas the Raf/extracellular signal-regulated kinase pathway is a central downstream signaling pathway activated by Ras, recent studies have established that Raf-independent pathways are also required for Ras function. Due in large part to yeast two-hybrid screening analysis, an expanding number of potential Ras effectors have been identified (reviewed in Ref. 1Campbell S.L. Khosravi-Far R. Rossman K.L. Clark G.J. Der C.J. Oncogene. 1998; 17: 1395-1413Crossref PubMed Scopus (919) Google Scholar). Although these proteins do not share obvious sequence similarity, they each bind Ras in a manner that requires an intact effector region and is GTP-dependent. Evidence has been presented recently that in addition to the Raf kinases, Ras can activate RalGEF proteins, which serve as guanine nucleotide exchange factors for the Ras-like GTPase Ral (1Campbell S.L. Khosravi-Far R. Rossman K.L. Clark G.J. Der C.J. Oncogene. 1998; 17: 1395-1413Crossref PubMed Scopus (919) Google Scholar, 13Bos J.L. EMBO J. 1998; 17: 6776-6782Crossref PubMed Scopus (287) Google Scholar), and phosphatidylinositol 3-kinase (PI-3K) (14Rodriguez-Viciana P. Warne P.H. Dhand R. Vanhaesebroeck B. Gout I. Fry M.J. Waterfield M.D. Downward J. Nature. 1994; 370: 527-532Crossref PubMed Scopus (1726) Google Scholar), which leads to the activation of both Rho family GTPases (3Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5216) Google Scholar) and the protein kinase AKT (15Franke T.F. Yang S.I. Chan T.O. Datta K. Kazlauskas A. Morrison D.K. Kaplan D.R. Tsichlis P.N. Cell. 1995; 81: 727-736Abstract Full Text PDF PubMed Scopus (1826) Google Scholar, 16Burgering B.M. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1878) Google Scholar). Studies with Ras effector loop mutants suggest that Ras-mediated cellular transformation requires the activation of at least two of these three known cellular pathways (Raf, RalGDSs, and PI-3K) (1Campbell S.L. Khosravi-Far R. Rossman K.L. Clark G.J. Der C.J. Oncogene. 1998; 17: 1395-1413Crossref PubMed Scopus (919) Google Scholar). Therefore, Ras exerts its cellular functions through the activation of numerous effector pathways. We recently identified two novel Ras-related GTPases, Rit and Rin (17Shao H. Kadono-Okuda K. Finlin B.S. Andres D.A. Arch. Biochem. Biophys. 1999; 371: 207-219Crossref PubMed Scopus (59) Google Scholar), that share ∼50% sequence identity with Ras. We found that stable expression of constitutively active Rit (Rit79L), but not activated Rin, induces strong growth transformation to NIH3T3 cells. 2E. V. Rusyn, E. R. Reynolds, H. Shao, T. M. Grana, T. O. Chan, D. A. Andres, A. D. Cox (2000)Oncogene in press.2E. V. Rusyn, E. R. Reynolds, H. Shao, T. M. Grana, T. O. Chan, D. A. Andres, A. D. Cox (2000)Oncogene in press. Rit79L-transformed cells proliferate in low serum, form colonies in soft agar, and form tumors in nude mice similar to that of the analogous Ha-Ras61L oncogenic mutant. Furthermore, Rit79L stimulates transcription from reporter constructs controlled by minimal promoters containing recognition sites for SRF, NF-κB, Elk, and Jun. However, no activation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, or p38, or of PI-3K/Akt/PKB kinases was observed. The high degree of amino acid conservation between the effector loops of Ras and Rit when combined with the ability of activated Rit to transform NIH3T3 cells raised the possibility that Rit-dependent cellular transformation might be mediated in part by the activation of known Ras effector proteins. However, a combination of biochemical and yeast two-hybrid studies suggest that Rit may interact with only a limited subset of the known Ras-binding proteins, including Ral exchange factors and AF-6 (17Shao H. Kadono-Okuda K. Finlin B.S. Andres D.A. Arch. Biochem. Biophys. 1999; 371: 207-219Crossref PubMed Scopus (59) Google Scholar). Taken together these results suggest that Rit might use unique signaling pathways to regulate cellular proliferation and transformation. In order to elucidate the signal transduction pathway(s) utilized by Rit, the yeast two-hybrid system was used to isolate Rit-binding proteins. This screen identified clones encoding a new member of the growing family of RalGEFs that we have termed RGL3 (RalGDS-like 3). In this study, we show that RGL3 interacts with GTP-bound Ras and Rit in an effector loop-dependent manner. In addition, evidence is presented demonstrating that RGL3 is a Ral exchange factor whose in vivo GEF activity is stimulated by GTP-bound Rit and Ras. Expression of activated Rit in HEK293 cells caused an increase in GTP-bound Ral levels and provides the first evidence for a potential link between the Rit and Ral signal transduction pathways. Therefore, the results of this study demonstrate that RGL3 is an exchange factor for Ral GTPases that may represent a downstream binding target for both the Rit and Ras GTPases. The mouse embryo two-hybrid cDNA library (18Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1660) Google Scholar) in pVP16 (LEU2, amp r) was obtained from Dr. Michael White (University of Texas Southwestern Medical Center, Dallas). The yeast two-hybrid vectors pGBDC1-3 (TRP1,amp r), pGADC1-3 (LEU2,amp r), and the Saccharomyces cerevisiae strain PJ69-4A was provided by Dr. Philip James (University of Wisconsin, Madison) (19James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar). For library screening, the yeast reporter stain PJ69-4A transformed with a bait plasmid that expressed a fusion between the GAL4 DNA binding domain and wild type Rit deleted of its C-terminal 18 amino acids was grown in synthetic minimal media lacking tryptophan. Preliminary studies indicated that a Gal4-Rit fusion bearing a C-terminal deletion in Rit was more efficiently imported to the nucleus (data not shown). Yeast cells were made competent and transformed with 1 mg of the mouse-cDNA library in the presence of sheared carrier DNA as described (17Shao H. Kadono-Okuda K. Finlin B.S. Andres D.A. Arch. Biochem. Biophys. 1999; 371: 207-219Crossref PubMed Scopus (59) Google Scholar). After recovery, approximately 1 × 107 primary transformants were selected for growth at 30 °C for 15 days on synthetic media lacking tryptophan, leucine, adenine, and histidine and containing 2 mm 3-aminotriazole. Surviving colonies were streaked onto synthetic minimal media lacking tryptophan and leucine and containing 80 mg/liter 5-bromo-4-chloro-3-indolyl-β-d-galactoside (X-Gal) to test for β-galactosidase expression. We obtained three independent groups (including four independent isolates of RGL3-RBD). The pVP16 plasmids containing putative Rit-interacting cDNAs were rescued from yeast cells and transformed into Escherichia coli strain HB101. Those cDNAs that exhibited a Rit-dependent genotype upon retransformation were characterized further. For two-hybrid assays, competent PJ69-4A yeast were co-transformed with pVP16-RGL3-RBD and wild type or mutant small GTP-binding proteins as described previously (17Shao H. Kadono-Okuda K. Finlin B.S. Andres D.A. Arch. Biochem. Biophys. 1999; 371: 207-219Crossref PubMed Scopus (59) Google Scholar). The plasmids containing Ras-like small GTP-binding proteins (pGBT9-Ha-Ras, pGBT9-Rap1A, pGBT9-Rap1B, and pGBT9-RalA) were kindly provided by Dr. Gilbert White (University of North Carolina, Chapel Hill) (20Peterson S.N. Trabalzini L. Brtva T.R. Fischer T. Altschuler D.L. Martelli P. Lapetina E.G. Der C.J. White G.C., II J. Biol. Chem. 1996; 271: 29903-29908Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Transformants were plated on minimal synthetic medium lacking tryptophan and leucine. Colonies were then streaked on plates containing 2 mm 3-aminotriazole and lacking tryptophan, leucine, adenine, and histidine to assay for growth. β-Galactosidase activity was determined using the Luminescent β-Galactosidase Detection Kit II (CLONTECH). The ability of pGBT9-RalA to interact with pVP16-RalBD was confirmed using this system (data not shown). PCR was performed on pVP16-RGL3-Rit binding domain (RBD) to isolate the 99-amino acid C-terminal Rit-binding domain containing 5′-XbaI and 3′-HindIII restriction sites. The product was subcloned to the corresponding sites of pGEX-KG to create pGEX-KG-RGL3-RBD. The 288-bp cDNA insert was excised from this vector and radiolabeled with [α-32P]dCTP using a Nick Translation labeling kit (Life Technologies, Inc.). Sixty six RGL3 cDNAs were isolated from a total of ∼5 × 105 plaque-forming units of a mouse kidney library (21Finlin B.S. Andres D.A. J. Biol. Chem. 1997; 272: 21982-21988Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The size of each cDNA insert was determined by restriction mapping, and selected clones were analyzed by DNA sequencing using T7, SP6, and specific internal primers. Of the initial positives, none was found to be full-length at the 5′ end. To obtain the extreme 5′ end of the RGL3 cDNA clone, 5′-RACE was carried out using the SMART RACE cDNA amplification kit (CLONTECH) according to the manufacturer's instructions. First-strand synthesis was primed with a specific oligonucleotide corresponding to a sequence located near the 5′ end of the longest RGL3 cDNA (5′ CAC AGT CCT CCG CAA ACT C 3′). Poly(A)+ mRNA was purified from mouse kidney using Straight As mRNA Isolation System (Novagen), and an RGL3-specific oligonucleotide (5′ GTC TGC TCT CCC TTA GCC TCC TTC AAG 3′) was used in the 5′-RACE PCR. Two independently isolated RGL3 5′-RACE products were sequenced and found to encode identical nucleotide sequences. To construct a full RGL3 coding region, nucleotides 1–402 of 5′-RACE RGL3 were amplified by PCR to contain a 5′-EcoRI site and ligated to nucleotide 403–2129 of the largest library cDNA clone through a unique internal BglII site in the bacterial expression vector pGEX-KG. The resulting plasmid, pGEX-KG-RGL3WT, was characterized by restriction mapping, and the entire coding region was sequenced on both strands. Bacterial expression and yeast two-hybrid vectors for wild type and mutant Rit have been described previously (17Shao H. Kadono-Okuda K. Finlin B.S. Andres D.A. Arch. Biochem. Biophys. 1999; 371: 207-219Crossref PubMed Scopus (59) Google Scholar). Mammalian expression constructs were prepared by polymerase chain reaction amplification of the desired fragment containing flanking BglII restriction sites. The products were subcloned to the BamHI site of pKH3 (22Mattingly R.R. Sorisky A. Brann M.R. Macara I.G. Mol. Cell. Biol. 1994; 14: 7943-7952Crossref PubMed Scopus (84) Google Scholar), and each plasmid was verified by DNA sequencing. This allowed expression of Rit bearing three copies of the influenza hemagglutinin (HA) epitope tag at the N terminus. To express full-length constitutively activated Ras in HEK293 cells, pCGN-RasQ61L (Dr. Adrienne Cox, University of North Carolina at Chapell Hill) was digested with BamHI, and the released DNA fragment was inserted into the corresponding site in pKH3. RGL3 was expressed using a modified mammalian expression vector. As a first step in constructing this plasmid, the 3× HA tag region of pKH3 was released by XbaI/EcoRI digestion and subcloned to NheI/EcoRI digested pcDNA3.1+Zeo to create pcDNA3.1+Zeo3HA. The BamHI/XhoI fragment of pBluescript-II-SK(+) (Stratagene), containing the polylinker, was then subcloned to the vector to create pcDNA3.1+Zeo3HAa. Finally, the BamHI/HindIII fragment of pcDNA3.1+Zeo3HAa was replaced by the corresponding polylinker region of pGEX-KG to generate pcDNA3.1+Zeo3HAb. The entire open reading frame of RGL3 cDNA was excised from pGEX-KG-RGL3WT by digestion with EcoRI/HindIII and subcloned to the corresponding sites of pcDNA3.1+Zeo3HAb, yielding pcDNA3.1+Zeo3HAb-RGL3. A fragment that encodes amino acids 1–507 of RGL3 (removing the RGL3-RBD) was prepared by PCR amplification of the desired fragment containing 5′-EcoRI and 3′-HindIII sites and a new 3′ stop codon. The product was subcloned to the EcoRI/HindIII sites of pcDNA3.1+Zeo3HAb to generate pcDNA3.1+Zeo3HAb-RGL3-ΔRBD. To produce recombinant RGL3-ΔRBD protein, the RGL3-ΔRBD PCR product was subcloned into the vectors pTrcHisA (Invitrogen) and pGEX-KG. To express His6 and Myc epitope-tagged RGL3 protein, the desired product was generated by PCR (5′-EcoRI and 3′-HindIII sites) and subcloned into pcDNA(−)MycHisA (Invitrogen) to generate pcDNA-MycHisA-RGL3. To produce recombinant His6-RGL3RBD protein, pGEX-KG-RGL3-RBD was digested withBamHI/HindIII, and the excised RGL3-RBD coding DNA was inserted into pET32a (Novagen)BamHI/HindIII sites to create pET32aRGL3RBD. All constructs were analyzed by restriction mapping, and all PCR products were fully sequenced. A single-stranded cDNA probe corresponding to mouse RGL3 (amino acids 613–709) was radiolabeled with [α-32P]dCTP by nick translation and used to probe a mouse multiple tissue Northern blot (CLONTECH). The probe was used at a concentration of 2 × 106cpm/ml in Rapid-hyb buffer (Amersham Pharmacia Biotech), according to manufacturer's instructions. After washing, the blot was exposed to Kodak X-Omat AR film for the indicated time. Recombinant GST-hRitC-Δ (bearing a short C-terminal deletion), GST-HaRas, GST-RGL3RBD, GST-RGL3ΔRBD, GST-RheB, GST-mRinC-Δ, GST-Rap1A, GST-TC21, GST-R-Ras, and GST-RalBD fusion proteins were purified by glutathione-agarose affinity column as described previously (17Shao H. Kadono-Okuda K. Finlin B.S. Andres D.A. Arch. Biochem. Biophys. 1999; 371: 207-219Crossref PubMed Scopus (59) Google Scholar). Recombinant GST fusion proteins were dialyzed (20 mmTris-Cl, pH 7.5, 100 mm NaCl, 1 mm DTT, 1 mm MgCl2, and 20% glycerol) for 12 h at 4 °C and stored in multiple aliquots at −70 °C. Recombinant His6-RitC-Δ, His6-Ha-Ras, His6-RalA, His6-Rab1A, His6-RGL3RBD, and His6-RGL3ΔRBD proteins were expressed and purified as described (17Shao H. Kadono-Okuda K. Finlin B.S. Andres D.A. Arch. Biochem. Biophys. 1999; 371: 207-219Crossref PubMed Scopus (59) Google Scholar). Protein concentrations were determined by the Bradford assay (Bio-Rad), using bovine serum albumin as a standard. Anti-Rit polyclonal antibody was produced by immunizing rabbits with purified GST-hRitC-Δ fusion protein (see above). GST-hRitC-Δ fusion protein (750 μg) was mixed with Freund's complete adjuvant and injected subcutaneously into the back of a female New Zealand White rabbit, age 6 months. Before the first injection, preimmune serum was obtained from the rabbit. Four additional injections were given at 4-week intervals using 250 μg of GST-hRitC-Δ mixed with Freund's incomplete adjuvant. Bleeds were taken 10 days after each injection. Recombinant His6-RitC-Δ and His6-Ha-Ras proteins were loaded with [35S]GTPγS or [3H]GDP as 10 of His6-RitC-Δ or His6-Ha-Ras in a total of were loaded with either 10 or [3H]GDP in buffer that either 1 Rit, mm pH 7.5, 100 mm NaCl, 1 mm MgCl2, 1 mm DTT, and 1 or 10 mm pH 7.5, 100 mm NaCl, mm MgCl2, 1 mm 1 mm DTT, and 1 at 30 °C for 30 was then to the reaction to a concentration of 10 A was from each and the of guanine nucleotide binding was determined by rapid using as described previously (17Shao H. Kadono-Okuda K. Finlin B.S. Andres D.A. Arch. Biochem. Biophys. 1999; 371: 207-219Crossref PubMed Scopus (59) Google Scholar). For the in binding the indicated concentrations of His6-RitC-Δ or His6-Ha-Ras with either [35S]GTPγS or [3H]GDP were for 1 h at 4 °C with in of binding buffer (20 mmTris-Cl, pH 7.5, mm NaCl, mm and containing 30 of glutathione-agarose with either 2 of GST or The were in a for 15 and the was The were with 1 of binding and the bound was by For each of His6-RitC-Δ or the specific binding was determined by bound to GST from bound by the fusion To test the interaction of RGL3-RBD with a of Ras GTPases, μg of recombinant GTPase fusion protein or GST was with [35S]GTPγS or [3H]GDP in a total of 80 of buffer that mm pH 7.5, 100 mm NaCl, mm 1 and 1 at °C for 10 was to the reaction to a concentration of 10 mm and at °C for 15 of the reaction was from each reaction to the of guanine nucleotide binding as described The of the reaction was with μg of His6-RGL3RBD and of for 1 h at 4 °C with in of binding The were and and the bound as described full-length RGL3 was generated by in transcription and translation in the presence of using the System Kit according to the of the with as The plasmid was by fragment from and to the corresponding sites of For binding recombinant GST or a of fusion proteins were each with either or GDP as described reaction of the in RGL3 protein into of binding buffer mmTris-Cl, pH 7.5, mm NaCl, 10 containing of either or GDP together with 10 μg of GST-RheB, or GST and of glutathione-agarose After for 2 h at 4 °C with the were by and four with 1 of binding The bound proteins were by the in 15 of buffer containing mm for 10 on were analyzed by gel electrophoresis on polyacrylamide The were with to GST and GST fusion proteins, with (Amersham Pharmacia Biotech), and exposed to HEK293 cells were in modified medium containing bovine serum, and plated h to at of HEK293 cells were with either 10 μg of or and 10 μg using the as described previously (21Finlin B.S. Andres D.A. J. Biol. Chem. 1997; 272: 21982-21988Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, D.A. H. Finlin B.S. Arch. Biochem. Biophys. 1997; PubMed Scopus Google Scholar). were h after by in phosphate-buffered saline and in buffer pH 7.5, 10 mm MgCl2, 1 1 mm 1 1 1 The was for 30 and by 10 in a on The was at 4 °C for at × and the was to a This was then at 4 °C for 15 at 105 × to 105 × and 105 The 105 × was in buffer 100 mm NaCl, 10 mm MgCl2, 1 mm DTT, 1 1 1 1 pH and on for 30 and was by at 4 °C for at 12 × μg) were in of binding buffer (20 mm 250 mm NaCl, 1 10 GTP, mm pH and with of a of for 2 h at 4 °C with to of The were then in a for 10 at × and the was The were then three with binding buffer after which bound proteins were released by in buffer and to gel electrophoresis on two polyacrylamide The presence of RGL3 in the was by with antibody and by (Amersham Pharmacia Biotech), and proteins were by with the The ability of His6-RGL3ΔRBD isolated RGL3 to bind Ras-like GTPases was determined as described previously (17Shao H. Kadono-Okuda K. Finlin B.S. Andres D.A. Arch. Biochem. Biophys. 1999; 371: 207-219Crossref PubMed Scopus (59) Google Scholar). containing μg of GST or fusion protein were at 30 °C for 10 in of buffer (20 mm mm NaCl, 10 1 pH to bound The were three with buffer and in 100 of buffer containing μg of His6-RGL3ΔRBD at 4 °C for 2 were four with and bound proteins were with 15 of buffer mm glutathione in and by gel electrophoresis on polyacrylamide to was by with tag antibody and (Amersham Pharmacia The nucleotide of binding to Ral proteins was with the interaction of the domain from the known containing bound GST or were in buffer for 10 at °C to the bound The of were then by and in the buffer or in Ral exchange buffer (20 mm pH 7.5, mm NaCl, 10 mm 1 mm containing either 1 mm or at °C for 30 to nucleotide the were and in of binding buffer (20 mm pH 7.5, mm NaCl, 1 mm containing the concentrations of and nucleotide or To this was 100 of or After at 4 °C for 2 were three with to the and nucleotide The were in of at °C for to bound protein, and by gel electrophoresis on and were by using tag antibody and by GDP dissociation from recombinant Ral and in