Identification and Characterization of a Novel Protein Interacting with Ral-binding Protein 1, a Putative Effector Protein of Ral
1998; Elsevier BV; Volume: 273; Issue: 2 Linguagem: Inglês
10.1074/jbc.273.2.814
ISSN1083-351X
AutoresMasahiro Ikeda, Osamu Ishida, Takao Hinoi, Shosei Kishida, Akira Kikuchi,
Tópico(s)Protein Interaction Studies and Fluorescence Analysis
ResumoRal-binding protein 1 (RalBP1) is a putative effector protein of Ral and exhibits a GTPase activating activity for Rac and CDC42. To clarify the function of RalBP1, we isolated a novel protein that interacts with RalBP1 by yeast two-hybrid screening and designated it POB1 (partner of RalBP1). POB1 consists of 521 amino acids, shares a homology with Eps15, which has been identified as an epidermal growth factor (EGF) receptor substrate, and has two proline-rich motifs. The POB1 mRNA was expressed in cerebrum, cerebellum, lung, kidney, and testis. POB1 interacted with RalBP1 in COS cells and the C-terminal region of POB1 was responsible for this interaction. The binding domain of RalBP1 to POB1 was distinct from its binding domain to Ral. Ral and POB1 simultaneously interacted with RalBP1 in COS cells. The binding of POB1 to RalBP1 did not affect the GTPase activating activity of RalBP1. Furthermore, POB1 bound to Grb2 but not to Nck or Crk. POB1 was tyrosine-phosphorylated in COS cells upon stimulation with EGF and made a complex with EGF receptor. These results suggest that RalBP1 makes a complex with POB1 and that this complex may provide a link between tyrosine kinase, Src homology 3 (SH3)-containing protein, and Ral. Ral-binding protein 1 (RalBP1) is a putative effector protein of Ral and exhibits a GTPase activating activity for Rac and CDC42. To clarify the function of RalBP1, we isolated a novel protein that interacts with RalBP1 by yeast two-hybrid screening and designated it POB1 (partner of RalBP1). POB1 consists of 521 amino acids, shares a homology with Eps15, which has been identified as an epidermal growth factor (EGF) receptor substrate, and has two proline-rich motifs. The POB1 mRNA was expressed in cerebrum, cerebellum, lung, kidney, and testis. POB1 interacted with RalBP1 in COS cells and the C-terminal region of POB1 was responsible for this interaction. The binding domain of RalBP1 to POB1 was distinct from its binding domain to Ral. Ral and POB1 simultaneously interacted with RalBP1 in COS cells. The binding of POB1 to RalBP1 did not affect the GTPase activating activity of RalBP1. Furthermore, POB1 bound to Grb2 but not to Nck or Crk. POB1 was tyrosine-phosphorylated in COS cells upon stimulation with EGF and made a complex with EGF receptor. These results suggest that RalBP1 makes a complex with POB1 and that this complex may provide a link between tyrosine kinase, Src homology 3 (SH3)-containing protein, and Ral. Ral is a member of small G protein 1The abbreviations used are: G protein, GTP-binding protein; RalGDS, Ral GDP dissociation stimulator; GAP, GTPase-activating protein; RalBP1, Ral-binding protein 1; EGF, epidermal growth factor; SH3, Src homology 3; GST, glutathioneS-transferase; HA, hemagglutinin 1; PCR, polymerase chain reaction; GTPγS, guanosine-5′-0-(3-thiotriphosphate); MBP, maltose-binding protein; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; kb, kilobase(s). superfamily and consists of RalA and RalB (1Chardin P. Tavitian A. EMBO J. 1986; 5: 2203-2208Crossref PubMed Scopus (174) Google Scholar, 2Feig L.A. Urano T. Cantor S. Trends Biochem. Sci. 1996; 21: 438-441Abstract Full Text PDF PubMed Scopus (179) Google Scholar). As well as other small G proteins, Ral has the GDP-bound inactive and the GTP-bound active forms. The GDP-bound form of Ral is converted to the GTP-bound form by RalGDS, and inversely the GTP-bound form is changed to the GDP-bound form by RalGAP (3Emkey R. Freedman S. Feig L.A. J. Biol. Chem. 1991; 266: 9703-9706Abstract Full Text PDF PubMed Google Scholar, 4Albright C.F. Giddings B.W. Liu J. Vito M. Weinberg R.A. EMBO J. 1993; 12: 339-347Crossref PubMed Scopus (159) Google Scholar). We and other groups have found that RalGDS is a putative effector protein of Ras (5Kikuchi A. Demo S.D. Ye Z.H. Chen Y.W. Williams L.T. Mol. Cell. Biol. 1994; 14: 7483-7491Crossref PubMed Scopus (244) Google Scholar, 6Hofer F. Fields S. Schneider C. Martin G.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11089-11093Crossref PubMed Scopus (254) Google Scholar, 7Spaargaren M. Bischoff J.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12609-12613Crossref PubMed Scopus (249) Google Scholar). Since RalGDS stimulates the GDP/GTP exchange of Ral (4Albright C.F. Giddings B.W. Liu J. Vito M. Weinberg R.A. EMBO J. 1993; 12: 339-347Crossref PubMed Scopus (159) Google Scholar), it is possible that there is a new signaling pathway from Ras to Ral through RalGDS. Indeed, it has been shown that RalGDS stimulates the GDP/GTP exchange of Ral in a Ras-dependent manner in COS cells and that a dominant negative form of Ral blocks a Ras-dependent transformation in NIH3T3 cells (8Urano T. Emkey R. Feig L.A. EMBO J. 1996; 15: 810-816Crossref PubMed Scopus (300) Google Scholar). It has been also demonstrated that Ral is required for Src- and Ras-dependent activation of phospholipase D and that it regulates the initiation of border cell migration induced by Ras in Drosophila oogenesis (9Jiang H. Luo J.-Q. Urano T. Frankel P. Lu Z. Foster D.A. Feig L.A. Nature. 1995; 378: 409-412Crossref PubMed Scopus (247) Google Scholar, 10Lee T. Feig L.A. Montell D.J. Development (Camb.). 1996; 122: 409-418PubMed Google Scholar). Furthermore, it has been shown that RalGDS and Raf synergistically stimulate cellular proliferation and gene expression (11White M.A. Vale T. Camonis J.H. Schaefer E. Wigler M.H. J. Biol. Chem. 1996; 271: 16439-16442Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 12Okazaki M. Kishida S. Hinoi T. Hasegawa T. Tamada M. Kataoka T. Kikuchi A. Oncogene. 1997; 14: 515-521Crossref PubMed Scopus (56) Google Scholar). These results indicate that RalGDS and Ral act downstream of Ras and mediate Ras functions. However, the mechanism by which Ral regulates cellular functions is not known. One possible clue to clarify the mode of action of Ral is RalBP1 (13Cantor S.B. Urano T. Feig L.A. Mol. Cell. Biol. 1995; 15: 4578-4584Crossref PubMed Scopus (261) Google Scholar, 14Jullien-Flores V. Dorseuil O. Romero F. Letourneur F. Saragosti S. Berger R. Tavitian A. Gacon G. Camonis J.H. J. Biol. Chem. 1995; 270: 22473-22477Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 15Park S.H. Weinberg R.A. Oncogene. 1995; 11: 2349-2355PubMed Google Scholar). RalBP1 has a Ral-binding domain in its C-terminal region and binds to the GTP-bound form of Ral but not to the GDP-bound form. A mutation in the effector loop of Ral impairs its interaction with RalBP1 and RalBP1 inhibits the RalGAP activity for Ral (13Cantor S.B. Urano T. Feig L.A. Mol. Cell. Biol. 1995; 15: 4578-4584Crossref PubMed Scopus (261) Google Scholar, 16Hinoi T. Kishida S. Koyama S. Ikeda M. Matsuura Y. Kikuchi A. J. Biol. Chem. 1996; 271: 19710-19716Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). These results suggest that RalBP1 is an effector protein of Ral. RalBP1 also has a RhoGAP homology domain in its central region and exhibits the GAP activity for Rac1 and CDC42 but not for RhoA (13Cantor S.B. Urano T. Feig L.A. Mol. Cell. Biol. 1995; 15: 4578-4584Crossref PubMed Scopus (261) Google Scholar, 14Jullien-Flores V. Dorseuil O. Romero F. Letourneur F. Saragosti S. Berger R. Tavitian A. Gacon G. Camonis J.H. J. Biol. Chem. 1995; 270: 22473-22477Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 15Park S.H. Weinberg R.A. Oncogene. 1995; 11: 2349-2355PubMed Google Scholar). Recently we have found that Ral localized to the membrane through its post-translational modification induces translocation of RalBP1 from the cytosol to the membrane and that the post-translational modifications of Rac1 and CDC42 enhance the GAP activity of RalBP1 (17Matsubara K. Hinoi T. Koyama S. Kikuchi A. FEBS Lett. 1997; 410: 169-174Crossref PubMed Scopus (29) Google Scholar). Therefore, RalBP1 may link Ral to Rac or CDC42 on the membrane. However, there is no evidence obtained so far that RalBP1 regulates the activities of Rac and CDC42 in intact cells. It is not known whether RalBP1 has additional activities. To gain more insight into the action of RalBP1, we sought to isolate proteins which interact with RalBP1 by yeast two-hybrid screening. We describe here the isolation of a novel RalBP1-interacting protein, which is designated POB1 (partner of RalBP1). POB1 shares a homology with Eps15, which has been identified as an EGF receptor substrate (18Fazioli F. Minichiello L. Matoskova B. Wong W.T. Di Fiore P.P. Mol. Cell. Biol. 1993; 13: 5814-5828Crossref PubMed Scopus (238) Google Scholar), and has two proline-rich motifs. POB1 and Ral simultaneously interact with RalBP1 on the different sites and these proteins make a ternary complex. Furthermore, POB1 binds to Grb2, is tyrosine-phosphorylated in COS cells in response to EGF, and makes a complex with EGF receptor. These results suggest that RalBP1 and POB1 may provide a link between tyrosine kinase, SH3-containing protein, and Ral. Yeast strain L40, plasmid vectors for two-hybrid screening, pGAD10-derived human brain cDNA library, and pEF-BOS were kindly supplied from Drs. Y. Takai, K. Tanaka, and S. Nagata (Osaka University, Suita, Japan) (19Mizushima S. Nagata S. Nucleic Acids Res. 1990; 18: 5322Crossref PubMed Scopus (1502) Google Scholar, 20Shimizu K. Kawabe H. Minami S. Honda T. Takaishi K. Shirataki H. Takai Y. J. Biol. Chem. 1996; 271: 27013-27017Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). λgt10 human brain cDNA library was from Dr. K. Kaibuchi (Nara Institute Science and Technology, Ikoma, Japan) (21Kikuchi A. Kaibuchi K. Hori Y. Nonaka H. Sakoda T. Kawamura M. Mizuno T. Takai Y. Oncogene. 1992; 7: 289-293PubMed Google Scholar). pGEX/Grb2 was a generous gift from Drs. H. Miki and T. Takenawa (Institute of Medical Science, University of Tokyo, Tokyo, Japan) (22Miki H. Miura K. Matuoka K. Nakata T. Hirokawa N. Orita S. Kaibuchi K. Takai Y. Takenawa T. J. Biol. Chem. 1994; 269: 5489-5492Abstract Full Text PDF PubMed Google Scholar, 23Watanabe K. Fukuchi T. Hosoya H. Shirasawa T. Matuoka K. Miki H. Takenawa T. J. Biol. Chem. 1995; 270: 13733-13739Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). pBSSK/Nck and pGFP/Crk were generous gifts from Drs. H. Hanafusa (Rockefeller University, New York) and M. Matsuda (National Institute of Health, Tokyo, Japan) (24Tanaka S. Hattori S. Kurata T. Nagashima K. Fukui Y. Nakamura S. Matsuda M. Mol. Cell. Biol. 1993; 13: 4409-4415Crossref PubMed Scopus (97) Google Scholar), respectively. The anti-GST and Ral rabbit polyclonal antibodies were made by a routine method and supplied from Dr. M. Nakata (Sumitomo Electric Industries, Yokohama, Japan). The anti-HA antibody 12CA5 was kindly provided by Dr. Q. Hu (Chiron Corp., Emeryville, CA). The anti-Myc antibody was prepared from 9E10 cells. RalBP1 cDNA was synthesized by reverse-transcriptase PCR as described (16Hinoi T. Kishida S. Koyama S. Ikeda M. Matsuura Y. Kikuchi A. J. Biol. Chem. 1996; 271: 19710-19716Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). [α-32P]dCTP, [35S]GTPγS, and [γ-32P]GTP were purchased from Amersham Inc. (Buckinghamshire, United Kingdom). The anti-phosphotyrosine and EGF receptor antibodies were from ICN Biomedicals, Inc. (Costa Mesa, CA) and Transduction Laboratories (Lexington, KY), respectively. EGF was purchased from Life Technologies, Inc. Other materials were from commercial sources. To construct pBTM116HA/RalBP1, pUC19/RalBP1 (16Hinoi T. Kishida S. Koyama S. Ikeda M. Matsuura Y. Kikuchi A. J. Biol. Chem. 1996; 271: 19710-19716Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) was digested with BamHI, and the 1.9-kb fragment encoding full-length RalBP1 was inserted into theBamHI cut pBTM116HA. To construct pCGN/POB1 and pCGN/POB1-(1–374) (amino acids 1–374), the 1.6- and 1.1-kb fragments encoding full-length POB1 and POB1-(1–374) with PmaCI andEcoRI sites were synthesized by PCR. These fragments were digested with PmaCI and EcoRI, blunted with Klenow fragment, and inserted into the SmaI cut pCGN. To construct pCGN/POB1-(375–521), pGAD10/POB1-(375–521) was digested with BglII and inserted into the BamHI cut pCGN. pBJ-Myc/RalBP1, pMAL/RalBP1, and pMAL/RalBP1-(364–647) were constructed as described (16Hinoi T. Kishida S. Koyama S. Ikeda M. Matsuura Y. Kikuchi A. J. Biol. Chem. 1996; 271: 19710-19716Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 17Matsubara K. Hinoi T. Koyama S. Kikuchi A. FEBS Lett. 1997; 410: 169-174Crossref PubMed Scopus (29) Google Scholar). To construct pMAL/RalBP1-(1–210), pUC19/RalBP1 was digested with BamHI and PvuII. The 0.6-kb fragment encoding RalBP1-(1–210) with BamHI andPvuII sites was inserted into pMAL-c2, which was digested with XbaI, blunted with Klenow fragment, and digested withBamHI. pMAL-c2 was digested with EcoRI, blunted with Klenow fragment, and self-ligated to generate pMAL/ΔEcoRI. To construct pMAL/RalBP1-(210–415), the 0.6-kb fragment encoding RalBP1-(210–415) with BamHI sites synthesized by PCR was digested with BamHI and inserted into the BamHI cut pMAL/ΔEcoRI. To construct pMAL/RalBP1-(391–499) and pMAL/RalBP1-(500–647), the 0.3- and 0.44-kb fragments encoding RalBP1-(391–499) and RalBP1-(500–647) withBamHI sites were synthesized by PCR and digested withBamHI, then inserted into the BamHI cut pMAL-c2. To construct pCGN/RalBP1, pBJ-Myc/RalBP1 was digested withXbaI and BamHI, and the 1.9-kb fragment encoding full-length RalBP1 was inserted into the XbaI andBamHI cut pCGN. To generate pUC19-Myc, pBJ-Myc was digested with XbaI and EcoRI and the fragment encoding Myc epitope was inserted into the EcoRI-XbaI cut pUC19. To construct pUC19-Myc/RalBG23V, the 0.6-kb fragment encoding RalBG23V with XbaI sites synthesized by PCR was digested with XbaI and inserted into theXbaI cut pUC19-Myc. To construct pEF-BOS-Myc/RalBG23V, pUC19-Myc/RalBG23V was digested with KpnI and HindIII and blunted with T4 DNA polymerase. The 0.7-kb fragment encoding Myc-RalBG23V was inserted into pEF-BOS, which was digested with XbaI and blunted with Klenow fragment. To construct pGEX/POB1-(375–521), pGAD10/POB1-(375–521) was digested withNdeI, blunted with Klenow fragment, and digested withBamHI. The 0.6-kb fragment encoding POB1-(375–521) was inserted into the SmaI and BamHI cut pGEX2T. To construct pGEX/Nck, pBSSK/Nck was digested with BamHI and the 1.1-kb fragment encoding Nck was inserted into the BamHI cut pGEX2T. To construct pGEX/Crk, pGFP/Crk was digested withBamHI and the 0.9-kb fragment encoding Crk was inserted into the BamHI cut pGEX2T. pCGN/RalBG23V, pCGN/RalBS28N, pGEX/RalB, and pV-IKS/Rac1 were constructed as described (17Matsubara K. Hinoi T. Koyama S. Kikuchi A. FEBS Lett. 1997; 410: 169-174Crossref PubMed Scopus (29) Google Scholar, 25Ikeda M. Koyama S. Okazaki M. Dohi K. Kikuchi A. FEBS Lett. 1995; 375: 37-40Crossref PubMed Scopus (28) Google Scholar, 26Murai H. Ikeda M. Kishida S. 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). Yeast strain L40 (MAT a trp1 leu2 his3 ade2 LYS2::lexA-HIS3 URA3::lexA-lacZ) was used as a host for the two-hybrid screening (27Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1663) Google Scholar). Yeast cells were grown on rich medium (YAPD) containing 2% glucose, 2% Bacto-peptone, 1% Bacto-yeast extracts, and 0.002% adenine sulfate. Yeast transformations were performed by the lithium acetate method. Transformants were selected on SD medium containing 2% glucose, 0.67% yeast nitrogen base without amino acids, and appropriate amino acids, which were supplemented to SD medium when required. A strain L40 carrying pBTM116HA/RalBP1 was transformed with a human brain cDNA library constructed in pGAD10. pBTM116HA/RalBP1 directs the expression of a fusion between the DNA-binding domain of LexA and the entire RalBP1 from an ADH promoter. Approximately 6 × 106 transformants were screened for the growth on SD plate medium lacking tryptophan, leucine, and histidine as evidenced by transactivation of a LexA-HIS3reporter gene and histidine prototrophy. His+ colonies were scored for β-galactosidase activity. Plasmids harboring cDNAs were recovered from positive colonies and introduced by electroporation into E. coli HB101 on the M9 plate lacking leucine. HB101 isleuB −, and this defect can be complemented by the LEU2 gene in the library plasmid. Then library plasmids were recovered from HB101 and transformed again into L40 containing pBTM116HA/RalBP1. The nucleotide sequences of plasmid DNAs, which conferred the LacZ+ phenotype on L40 containing pBTM116HA/RalBP1, were determined. The clone identified by the yeast two-hybrid method contained a region that interacted with RalBP1 and the 3′-end but did not contain the 5′-end of an open reading frame. To obtain full-length cDNA, the clone was labeled with random primers and [α-32P]dCTP and used to screen λgt10 human brain cDNA library. However, the initial screening did not identify the 5′-end of the gene. To obtain more 5′ sequence information, the 5′-most 650 nucleotides of the largest clone was labeled with [α-32P]dCTP to screen the same library, and a number of positive clones were isolated. All clones, collectively spanning 3.2 kb, were sequenced using double-strand templates and Thermo Sequenase premixed cycle sequencing kit (Amersham, Buckinghamshire, UK) and Hitachi DNA sequencer SQ-5500 (Hitachi Ltd., Tokyo, Japan). To construct full-length POB1 cDNA clone, the cDNA fragment containing POB1 codons 10–521 (amino acid numbers) from the initial screening was subcloned into pUC19. The cDNA fragment containing POB1 codons 1–253 (amino acid numbers) from the second screening was jointed to this sequence to obtain pUC19/POB1. Total RNA was extracted from various rat tissues as described (28Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63190) Google Scholar). Northern blot analysis was performed as described (29Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982Google Scholar). Twenty μg of total RNA was then resolved by agarose gel electrophoresis and transferred to nitrocellulose filters. A 1.6-kb cDNA probe corresponding to full-length POB1 was labeled with random primers and [α-32P]dCTP and hybridized to the membrane. The membrane was washed and exposed to Kodak X-Omat film. MBP fused to RalBP1 (MBP-RalBP1), MBP-RalBP1-(364–647), GST fused to RalB (GST-RalB), and GST-Grb2 were purified from E. coli as described (16Hinoi T. Kishida S. Koyama S. Ikeda M. Matsuura Y. Kikuchi A. J. Biol. Chem. 1996; 271: 19710-19716Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 17Matsubara K. Hinoi T. Koyama S. Kikuchi A. FEBS Lett. 1997; 410: 169-174Crossref PubMed Scopus (29) Google Scholar, 22Miki H. Miura K. Matuoka K. Nakata T. Hirokawa N. Orita S. Kaibuchi K. Takai Y. Takenawa T. J. Biol. Chem. 1994; 269: 5489-5492Abstract Full Text PDF PubMed Google Scholar, 23Watanabe K. Fukuchi T. Hosoya H. Shirasawa T. Matuoka K. Miki H. Takenawa T. J. Biol. Chem. 1995; 270: 13733-13739Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). TheE. coli expressing MBP-RalBP1-(1–210), MBP-RalBP1-(210–415), MBP-RalBP1-(391–499), MBP-RalBP1-(500–647), GST-POB1-(375–521), GST-Crk, and GST-Nck were grown at 37 °C to an absorbance of 0.5 (absorbance at 600 nm), then isopropyl-β-d-thiogalactopyranoside was added at a final concentration of 0.3 mm. After further incubation was carried out for 2 h at 37 °C, the expressed proteins were purified in accordance with the manufacturer's instructions. Approximately 70 amino acids from pMAL-c2 were attached to the C terminus of RalBP1-(1–210), RalBP1-(210–415), and RalBP1-(391–499). The post-translationally modified form of Rac1 was purified fromSpodoptera frugiperda 9 cells as described (17Matsubara K. Hinoi T. Koyama S. Kikuchi A. FEBS Lett. 1997; 410: 169-174Crossref PubMed Scopus (29) Google Scholar, 26Murai H. Ikeda M. Kishida S. 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) COS cells (10-cm-diameter plate) transfected with pCGN-, pBJ-, and pEF-BOS-derived plasmids were lysed in 0.5 ml of lysis buffer (20 mm Tris/HCl (pH 7.5), 1% nonidet P-40, 137 mmNaCl, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, and 10 μg/ml leupeptin) as described (30Kikuchi A. Williams L.T. J. Biol. Chem. 1996; 271: 588-594Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). POB1 and its deletion mutants were tagged with HA epitope at their N termini. RalBP1 was tagged with Myc epitope at its N terminus. The lysates (620 μg of protein) were immunoprecipitated with the anti-Myc or HA antibody. The precipitates were washed once with 20 mm Tris/HCl (pH 7.5), 1% nonidet P-40, 137 mmNaCl, and 10% glycerol, twice with 100 mm Tris/HCl (pH 7.5) and 0.5 m LiCl, and once with 10 mmTris/HCl (pH 7.5). The precipitates were subjected to SDS-polyacrylamide gel electrophoresis (31Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207231) Google Scholar), transferred to nitrocellulose filters, and probed with the anti-HA or Myc antibody as described (16Hinoi T. Kishida S. Koyama S. Ikeda M. Matsuura Y. Kikuchi A. J. Biol. Chem. 1996; 271: 19710-19716Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 26Murai H. Ikeda M. Kishida S. 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, 30Kikuchi A. Williams L.T. J. Biol. Chem. 1996; 271: 588-594Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Where specified, the lysates expressing RalBP1 and POB1 with RalG23V or RalS28N were used. Various MBP fused to RalBP1 deletion mutants (20 pmol each) were incubated with GST-POB1-(375–521) (44 pmol) or the GTPγS-bound form of GST-Ral (44 pmol) in 40 μl of reaction mixture (20 mm Tris/HCl (pH 7.5), 1 mm dithiothreitol, and 0.05% CHAPS) for 1 h at 4 °C. The GTPγS-bound form of Ral was made as described (16Hinoi T. Kishida S. Koyama S. Ikeda M. Matsuura Y. Kikuchi A. J. Biol. Chem. 1996; 271: 19710-19716Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The amylose resin was added to this mixture and further incubation was performed. After 30 min, the resin was precipitated by centrifugation. The precipitates were probed with the anti-GST antibody. The lysates (465 μg of protein) of COS cells expressing HA-POB1 were incubated with 1 μm GST-Grb2, GST-Nck, GST-Crk, or GST in 170 μl of the lysis buffer for 1 h at 4 °C. GST fused to proteins was precipitated with glutathione-Sepharose 4B. The precipitates were probed with the anti-HA and GST antibodies. COS cells expressing HA-POB1 with or without Myc-RalBP1 were serum-starved for 24 h before stimulation with 100 ng/ml EGF for 15 min. The lysates (620 μg of protein) were prepared as described above except that 5 mm sodium orthovanadate and 50 mmβ-glycerophosphate were added to the lysis buffer and immunoprecipitated with the anti-HA antibody. The precipitates were probed with the anti-HA, Myc, phosphotyrosine, or EGF receptor antibody. The assay for the interaction of the [35S]GTPγS-bound form of Ral with immobilized MBP-RalBP1 in the presence of GST-POB1-(375–521) was carried out as described (17Matsubara K. Hinoi T. Koyama S. Kikuchi A. FEBS Lett. 1997; 410: 169-174Crossref PubMed Scopus (29) Google Scholar). The GAP activity of RalBP1 for the post-translationally modified Rac1 was measured in the presence of GST-POB1-(375–521) as described (17Matsubara K. Hinoi T. Koyama S. Kikuchi A. FEBS Lett. 1997; 410: 169-174Crossref PubMed Scopus (29) Google Scholar). Protein concentrations were determined using bovine serum albumin as a standard (32Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (216440) Google Scholar). To identify proteins that physically interact with RalBP1, we conducted a human brain cDNA library screening with the yeast two-hybrid method. From 6 × 106 initial transformants, we identified five positive clones (His+ and LacZ+) and the library plasmids were recovered from the clones. Among these five plasmids, three clones were found to confer both the His+and LacZ+ phenotypes on L40 containing pBTM116HA/RalBP1. The cDNA inserts from these three clones were 1 kb, and all encoded a single sequence containing an open reading frame of 147 amino acids and the consensus sequence for a stop codon. To identify full-length cDNA of this RalBP1-interacting protein, a human brain cDNA library was screened using the cDNA isolated by the yeast two-hybrid method and a number of overlapping positive clones were isolated. The overlapping clones spanned a distance of 3.2 kb and contained an uninterrupted open reading frame of 1563 base pairs, encoding a predicted protein of 521 amino acids with a calculated M r of 57,901 (Fig.1 A). We designated this protein POB1 for partner of RalBP1. The first ATG was preceded by stop codons in all three reading frames and the 5′-noncoding region had a high percentage of GC base pairs (72%). The second amino acid was also Met. The neighboring sequence of the second ATG was consistent with the translation initiation start proposed by Kozak (33Kozak M. Nucleic Acids Res. 1987; 15: 8125-8148Crossref PubMed Scopus (4168) Google Scholar) better than that of the first ATG, but we could not determine which is the first Met. Data bases were searched for related proteins. Besides short human EST sequences (GenBank™ accession numbers; R90985, H44097, T72336, T72376,R88729, H44028, W38942, G27820, N89827, and AA191588), no protein closely related to POB1 was identified. However, the central region of POB1, residues 126–227, shares 37% amino acid identity with residues 111–211 of Eps15, which has been identified as an EGF receptor substrate (18Fazioli F. Minichiello L. Matoskova B. Wong W.T. Di Fiore P.P. Mol. Cell. Biol. 1993; 13: 5814-5828Crossref PubMed Scopus (238) Google Scholar) (Fig. 1, B and C). The C-terminal region has two proline-rich motifs, PPTPPPRP (amino acids 338–345) and PPPPALPPRP (amino acids 374–383), and a putative coiled-coil structure (Fig. 1 B). By Northern blot analysis, a single band was strongly detected in RNA of rat cerebrum, cerebellum, lung, and testis, and weakly in kidney, but not in heart, thymus, liver, spleen, or adrenal gland (Fig.2). This tissue distribution of POB1 was different from that of RalBP1, which is ubiquitously expressed in rat various tissues (13Cantor S.B. Urano T. Feig L.A. Mol. Cell. Biol. 1995; 15: 4578-4584Crossref PubMed Scopus (261) Google Scholar, 15Park S.H. Weinberg R.A. Oncogene. 1995; 11: 2349-2355PubMed Google Scholar). To examine whether POB1 interacts with RalBP1 in intact cells, we coexpressed HA-POB1 with Myc-RalBP1 in COS cells (Fig.3 A, lanes 1–3). The lower bands, which were seen under POB1, might be its degradation products. When the lysates coexpressing HA-POB1 with Myc-RalBP1 were immunoprecipitated with the anti-Myc antibody, both HA-POB1 and Myc-RalBP1 were detected in the RalBP1 immune complex (Fig. 3 B, lanes 1–3). Similarly when the same lysates were immunoprecipitated with the anti-HA antibody, both HA-POB1 and Myc-RalBP1 were detected in the POB1 immune complex (Fig. 3 B, lane 6). Neither HA-POB1 nor Myc-RalBP1 was immunoprecipitated from the lysates coexpressing HA-POB1 and Myc-RalBP1 with non-immune immunoglobulin (data not shown). These results indicate that POB1 interacts with RalBP1 in intact cells. By the yeast two-hybrid screening, POB1-(375–521) was identified as a RalBP1-binding domain. We examined whether this domain is essential for the binding of POB1 to RalBP1. Myc-RalBP1 was coexpressed with HA-POB1-(1–374) or HA-POB1-(375–521) in COS cells (Fig. 3 A, lanes 4 and5). When the lysates coexpressing Myc-RalBP1 with HA-POB1-(1–374) or HA-POB1-(375–521) were immunoprecipitated with the anti-Myc antibody, HA-POB1-(375–521), but not HA-POB1-(1–374), was detected in the RalBP1 immune complex (Fig. 3 B, lanes 4 and5). Similarly when the same lysates were immunoprecipitated with the anti-HA antibody, Myc-RalBP1 was detected in the POB1-(375–521) immune complex but not in the POB1-(1–374) immune complex (Fig. 3 B, lanes 7 and 8). These results indicate that POB1-(375–521) is necessary and sufficient for the binding of POB1 to RalBP1. To examine which region of RalBP1 interacts with POB1, various RalBP1 deletion mutants were purified as MBP fused to proteins and POB1-(375–521) was purified as a GST fused to protein from E. coli (Fig.4 A). After MBP-RalBP1 deletion mutants were incubated with GST-POB1-(375–521), the amylose resin was added. The resin was precipitated by centrifugation, and the precipitates were probed with the anti-GST antibody. POB1-(375–521) interacted with full-length RalBP1, RalBP1-(364–647), and RalBP1-(500–647) but not with RalBP1-(1–210), RalBP1-(210–415), or RalBP1-(391–499) (Fig. 4 B). These results indicate that the binding of POB1 to RalBP1 is direct and that the C-terminal region of RalBP1 has the POB1-binding domain. It has been shown that the GTP-bound form of Ral binds to the C-terminal region of RalBP1 (RalBP1-(364–647)) (13Cantor S.B. Urano T. Feig L.A. Mol. Cell. Biol. 1995; 15: 4578-4584Crossref PubMed Scopus (261) Google Scholar, 14Jullien-Flores V. Dorseuil O. Romero F. Letourneur F. Saragosti S. Berger R. Tavitian A. Gacon G. Camonis J.H. J. Biol. Chem. 1995; 270: 22473-22477Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 15Park S.H. Weinberg R.A. Oncogene. 1995; 11: 2349-2355PubMed Google Scholar). To examine whether Ral and POB1 bind to the same
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