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Dimerization of Substrate Adaptors Can Facilitate Cullin-mediated Ubiquitylation of Proteins by a “Tethering” Mechanism

2006; Elsevier BV; Volume: 281; Issue: 34 Linguagem: Inglês

10.1074/jbc.m601119200

1083-351X

Michael McMahon, Nerys Thomas, Ken Itoh, Masayuki Yamamoto, John D. Hayes,

Coenzyme Q10 studies and effects

The prevalence and mechanistic significance of self-association among substrate adaptors for the Cul-Rbx family of ubiquitin ligases remain unclear. We now report that it is as a homodimer that the substrate adaptor Keap1 interacts with Cul3. The resulting complex facilitates ubiquitylation of the Nrf2 transcription factor but only when this substrate possesses within its Neh2 domain a second cryptic Keap1-binding site, the DLG motif, in addition to its previously described ETGE site. Both motifs recognize overlapping surfaces on Keap1, and the seven lysine residues of Nrf2 that act as ubiquitin acceptors lie between them. Based on these data, we propose a “fixed-ends” model for Nrf2 ubiquitylation in which each binding site becomes tethered to a separate subunit of the Keap1 homodimer. This two-site interaction between Keap1 and Nrf2 constrains the mobility of the target lysine residues in the Neh2 domain, increasing their average concentration in the vicinity of the Rbx-bound ubiquitin-conjugating enzyme, and thus the rate at which the transcription factor is ubiquitylated. We show that self-association is a general feature of Cul3 substrate adaptors and propose that the fixed-ends mechanism is commonly utilized to recruit, orientate, and ubiquitylate substrates upon this family of ubiquitin ligases. The prevalence and mechanistic significance of self-association among substrate adaptors for the Cul-Rbx family of ubiquitin ligases remain unclear. We now report that it is as a homodimer that the substrate adaptor Keap1 interacts with Cul3. The resulting complex facilitates ubiquitylation of the Nrf2 transcription factor but only when this substrate possesses within its Neh2 domain a second cryptic Keap1-binding site, the DLG motif, in addition to its previously described ETGE site. Both motifs recognize overlapping surfaces on Keap1, and the seven lysine residues of Nrf2 that act as ubiquitin acceptors lie between them. Based on these data, we propose a “fixed-ends” model for Nrf2 ubiquitylation in which each binding site becomes tethered to a separate subunit of the Keap1 homodimer. This two-site interaction between Keap1 and Nrf2 constrains the mobility of the target lysine residues in the Neh2 domain, increasing their average concentration in the vicinity of the Rbx-bound ubiquitin-conjugating enzyme, and thus the rate at which the transcription factor is ubiquitylated. We show that self-association is a general feature of Cul3 substrate adaptors and propose that the fixed-ends mechanism is commonly utilized to recruit, orientate, and ubiquitylate substrates upon this family of ubiquitin ligases. Ubiquitylation underpins virtually all biological processes as it is the major mechanism regulating the stability of critical effector molecules in eukaryotic cells. It refers to the formation of an isopeptide bond between the C terminus of ubiquitin and the ϵ-NH2 group of a lysine residue in a target protein. The reaction proceeds in three stages with the final critical step, transfer of activated ubiquitin from an E2 2The abbreviations used are: E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; BCL6, B-cell lymphoma 6; BTB, Broad complex, Tramtrack, and Bric-a-brac; CHX, cycloheximide; Cul, Cullin; ECH, erythroid cell-derived protein with cnc homology (i.e. Gallus gallus Nrf2); HA, hemagglutinin; IVR, intervening region; MEF, mouse embryonic fibroblast; Neh, Nrf2-ECH homology; Nrf, NF-E2 p45-related factor; PLZF, promyelocytic leukemia zinc finger; Rbx, RING box protein; RIPA, radioimmune precipitation assay; Sul, sulforaphane; EGFP, enhanced green fluorescent protein; MBP, maltose-binding protein; GFP, green fluorescent protein. 2The abbreviations used are: E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; BCL6, B-cell lymphoma 6; BTB, Broad complex, Tramtrack, and Bric-a-brac; CHX, cycloheximide; Cul, Cullin; ECH, erythroid cell-derived protein with cnc homology (i.e. Gallus gallus Nrf2); HA, hemagglutinin; IVR, intervening region; MEF, mouse embryonic fibroblast; Neh, Nrf2-ECH homology; Nrf, NF-E2 p45-related factor; PLZF, promyelocytic leukemia zinc finger; Rbx, RING box protein; RIPA, radioimmune precipitation assay; Sul, sulforaphane; EGFP, enhanced green fluorescent protein; MBP, maltose-binding protein; GFP, green fluorescent protein. ubiquitin-conjugating enzyme to an acceptor lysine, facilitated by E3 ubiquitin ligases (1Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2887) Google Scholar). Modular complexes based around at least four Cullin-RING box (Cul-Rbx) holoenzymes constitute the largest family of E3 ligases identified to date. These holoenzymes, Cul1-Rbx1, Cul2-Rbx1, Cul3-Rbx1, and Cul5-Rbx2, differ in the nature of the substrate adaptors they bind (2Cardozo T. Pagano M. Nat. Rev. Mol. Cell Biol. 2004; 5: 739-751Crossref PubMed Scopus (870) Google Scholar, 3Willems A.R. Schwab M. Tyers M. Biochim. Biophys. Acta. 2004; 1695: 133-170Crossref PubMed Scopus (377) Google Scholar, 4Petroski M.D. Deshaies R.J. Nat. Rev. Mol. Cell Biol. 2005; 6: 9-20Crossref PubMed Scopus (1651) Google Scholar, 5Kamura T. Maenaka K. Kotoshiba S. Matsumoto M. Kohda D. Conaway R.C. Conaway J.W. Nakayama K.I. Genes Dev. 2004; 18: 3055-3065Crossref PubMed Scopus (363) Google Scholar). For example, Cul1-Rbx1 recruits protein dimers comprising the S-phase kinase-associated protein 1 (Skp1) bound to the eponymous domain found in over 40 F-box proteins (2Cardozo T. Pagano M. Nat. Rev. Mol. Cell Biol. 2004; 5: 739-751Crossref PubMed Scopus (870) Google Scholar). The resulting E3 ligase is termed SCFF-box (Skp1, Cul1, and F-box, with the specific F-box protein identified in supercript). Cul3-Rbx1 recruits Broad complex, Tramtrack, and Bric-a-brac (BTB) proteins to generate a large family of ligases that, by analogy with SCF ligases, are referred to as BC3BBTB ubiquitin ligases (6Pintard L. Willems A. Peter M. EMBO J. 2004; 23: 1681-1687Crossref PubMed Scopus (297) Google Scholar, 7Xu L. Wei Y. Reboul J. Vaglio P. Shin T.H. Vidal M. Elledge S.J. Harper J.W. Nature. 2003; 425: 316-321Crossref PubMed Scopus (383) Google Scholar, 8Geyer R. Wee S. Anderson S. Yates J. Wolf D.A. Mol. Cell. 2003; 12: 783-790Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 9Furukawa M. He Y.J. Borchers C. Xiong Y. Nat. Cell Biol. 2003; 5: 1001-1007Crossref PubMed Scopus (332) Google Scholar). 3Various nomenclatures have been proposed to indicate the composition of Cullin-based protein complexes. We have utilized that described in Ref. 3Willems A.R. Schwab M. Tyers M. Biochim. Biophys. Acta. 2004; 1695: 133-170Crossref PubMed Scopus (377) Google Scholar to which the reader is referred for further details. 3Various nomenclatures have been proposed to indicate the composition of Cullin-based protein complexes. We have utilized that described in Ref. 3Willems A.R. Schwab M. Tyers M. Biochim. Biophys. Acta. 2004; 1695: 133-170Crossref PubMed Scopus (377) Google Scholar to which the reader is referred for further details.Structural similarities exist among substrate adaptors. For example, Skp1 and BTB proteins all utilize BTB folds to interact with Cul proteins (3Willems A.R. Schwab M. Tyers M. Biochim. Biophys. Acta. 2004; 1695: 133-170Crossref PubMed Scopus (377) Google Scholar). Additionally, the domains utilized to recruit substrates can adopt analogous super-secondary structures; whereas F-box proteins frequently use WD40 domains to recruit substrates and BTB proteins commonly exploit Kelch-repeat domains for this purpose, both domains fold to give six-bladed β-propeller structures (3Willems A.R. Schwab M. Tyers M. Biochim. Biophys. Acta. 2004; 1695: 133-170Crossref PubMed Scopus (377) Google Scholar, 10Stogios P.J. Prive G.G. Trends Biochem. Sci. 2004; 29: 634-637Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 11Adams J. Kelso R. Cooley L. Trends Cell Biol. 2000; 10: 17-24Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar). In conjunction with the seminal crystallographic study on the structure of the SCFF-box of Skp2 complex (12Zheng N. Schulman B.A. Song L. Miller J.J. Jeffrey P.D. Wang P. Chu C. Koepp D.M. Elledge S.J. Pagano M. Conaway R.C. Conaway J.W. Harper J.W. Pavletich N.P. Nature. 2002; 416: 703-709Crossref PubMed Scopus (1151) Google Scholar), these common features among adaptors suggest that probably all Cul-Rbx ligases function by recruiting and juxtaposing an E2 enzyme, via a C-terminal Rbx protein, and a substrate, via an N-terminal adaptor.The above model, in which a single substrate adaptor interacts with an E3 holoenzyme, may be incomplete as two examples exist supporting the idea that self-association of F-box proteins is required for ubiquitylation of some substrates by Cul1-Rbx1. First, association of two F-box proteins, Pop1p and Pop2p, is necessary for polyubiquitylation of Rum1p in fission yeast (13Wolf D.A. McKeon F. Jackson P.K. Curr. Biol. 1999; 9: 373-376Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 14Seibert V. Prohl C. Schoultz I. Rhee E. Lopez R. Abderazzaq K. Zhou C. Wolf D.A. BMC Biochem. 2002; 3: 22Crossref PubMed Scopus (31) Google Scholar). Second, two F-box proteins, β-transducin repeat-containing protein 1 (βTrCP1) and βTrCP2, form hetero- and homo-oligomers with each other, but only the homo-oligomers could target phosphorylated IκBα for ubiquitylation (15Suzuki H. Chiba T. Suzuki T. Fujita T. Ikenoue T. Omata M. Furuichi K. Shikama H. Tanaka K. J. Biol. Chem. 2000; 275: 2877-2884Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). The various rationales proposed to explain the necessity for adaptor self-association (discussed in Ref. 3Willems A.R. Schwab M. Tyers M. Biochim. Biophys. Acta. 2004; 1695: 133-170Crossref PubMed Scopus (377) Google Scholar) remain unsupported by evidence, and it is not clear whether these findings are idiosyncratic or of general significance. In particular, an analogous role for BTB protein dimers in substrate ubiquitylation by Cul3-Rbx1 has not been reported. Yet the BTB adaptor protein Keap1 (Kelch-like ECH-associated protein 1) homodimerizes in bacteria (16Dinkova-Kostova A.T. Holtzclaw W.D. Wakabayashi N. Biochemistry. 2005; 44: 6889-6899Crossref PubMed Scopus (176) Google Scholar, 17Wakabayashi N. Dinkova-Kostova A.T. Holtzclaw W.D. Kang M.I. Kobayashi A. Yamamoto M. Kensler T.W. Talalay P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2040-2045Crossref PubMed Scopus (809) Google Scholar). Additionally, evidence from the BTB domains of both B-cell lymphoma 6 (BCL6) and promyelocytic leukemia zinc finger (PLZF) transcription factors (18Ahmad K.F. Engel C.K. Prive G.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12123-12128Crossref PubMed Scopus (242) Google Scholar, 19Ahmad K.F. Melnick A. Lax S. Bouchard D. Liu J. Kiang C.L. Mayer S. Takahashi S. Licht J.D. Prive G.G. Mol. Cell. 2003; 12: 1551-1564Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar) suggests dimerization of BTB domains may be obligatory (20Melnick A. Ahmad K.F. Arai S. Polinger A. Ball H. Borden K.L. Carlile G.W. Prive G.G. Licht J.D. Mol. Cell. Biol. 2000; 20: 6550-6567Crossref PubMed Scopus (151) Google Scholar).Keap1 recruits the antioxidant NF-E2 p45-related factor 2 (Nrf2) transcription factor to the BC3B holoenzyme (21Kobayashi A. Kang M.I. Okawa H. Ohtsuji M. Zenke Y. Chiba T. Igarashi K. Yamamoto M. Mol. Cell. Biol. 2004; 24: 7130-7139Crossref PubMed Scopus (1595) Google Scholar, 22Cullinan S.B. Gordan J.D. Jin J.O. Harper J.W. Diehl J.A. Mol. Cell. Biol. 2004; 24: 8477-8486Crossref PubMed Scopus (750) Google Scholar, 23Furukawa M. Xiong Y. Mol. Cell. 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The ability of BC3BKeap1 to ubiquitylate the factor is inhibited by oxidative modification of specific cysteine residues in Keap1 (17Wakabayashi N. Dinkova-Kostova A.T. Holtzclaw W.D. Kang M.I. Kobayashi A. Yamamoto M. Kensler T.W. Talalay P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2040-2045Crossref PubMed Scopus (809) Google Scholar, 27Nguyen T. Sherratt P.J. Huang H.C. Yang C.S. Pickett C.B. J. Biol. Chem. 2003; 278: 4536-4541Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar, 28Stewart D. Killeen E. Naquin R. Alam S. Alam J. J. Biol. Chem. 2003; 278: 2396-2402Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar, 29Zhang D.D. Hannink M. Mol. Cell. Biol. 2003; 23: 8137-8151Crossref PubMed Scopus (1085) Google Scholar, 30Hong F. Freeman M.L. Liebler D.C. Chem. Res. Toxicol. 2005; 18: 1917-1926Crossref PubMed Scopus (333) Google Scholar, 31Hong F. Sekhar K.R. Freeman M.L. Liebler D.C. J. Biol. Chem. 2005; 280: 31768-31775Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar), and this allows rapid repletion of Nrf2 protein levels in stressed cells. As Nrf2 promotes the transcription of genes whose products promote reductive chemistries, Keap1 provides the cell with a negative feedback control loop that plays a pivotal role in maintaining redox homeostasis (32Ishii T. Itoh K. Takahashi S. Sato H. Yanagawa T. Katoh Y. Bannai S. Yamamoto M. J. Biol. Chem. 2000; 275: 16023-16029Abstract Full Text Full Text PDF PubMed Scopus (1221) Google Scholar, 33McMahon M. Itoh K. Yamamoto M. Chanas S.A. Henderson C.J. McLellan L.I. Wolf C.R. Cavin C. Hayes J.D. Cancer Res. 2001; 61: 3299-3307PubMed Google Scholar, 34Lee J.M. Johnson J.A. J. Biochem. Mol. Biol. 2004; 37: 139-143Crossref PubMed Google Scholar, 35Hayes J.D. McMahon M. Mol. Cell. 2006; 21: 732-734Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar).We now report that Keap1 in mammalian cells exists as a dimeric protein and that it interacts with Cul3-Rbx1 in this oligomeric form. Additionally, we demonstrate that ubiquitylation of Nrf2 by BC3BKeap1/Keap1 4This nomenclature has been chosen to convey the essentially dimeric nature of the substrate adaptor. 4This nomenclature has been chosen to convey the essentially dimeric nature of the substrate adaptor. requires a second previously unrecognized Keap1-binding site, the DLG motif, to be present in its Neh2 domain (Fig. 1A). Based on these findings, we present a “fixed-ends” model for recruitment and orientation of Nrf2 upon BC3BKeap1/Keap1. The model accounts for the target lysine specificity displayed by the E3 complex and also provides a generally applicable rationale for substrate adaptor dimerization.MATERIALS AND METHODSPlasmids—The pcDNA3.1/V5mNrf2, pCG-GAL(HA)mNeh2, pcDNA3.1/V5HisCmKeap1, pcDNA3.1/mKeap1, and pHisUb constructs have been described previously (36McMahon M. Itoh K. Yamamoto M. Hayes J.D. J. Biol. Chem. 2003; 278: 21592-21600Abstract Full Text Full Text PDF PubMed Scopus (854) Google Scholar). A plasmid encoding mKeap1 fused at its N terminus with EGFP, pEGFP-mKeap1, was generated by PCR of the mKeap1 coding region using oligonucleotides bearing mismatches that introduced XhoI and EcoRI sites (forward primer, 5′-GAGGAACTGTGTCTCGAGATCCGGAACCCCATG-3′ and reverse primer, 5′-GTTGTCAGTGCTCAGGAATTCCAAGTGCTTCAGCAG-3′), and following restriction the product was ligated into pEGFP-C3 (Clontech). The pET15bmKeap1 plasmid encoding a hexahistidine-mKeap1 fusion protein was generated by PCR amplification of the mKeap1 coding sequence with the following primer pair: 5′-CTTGTCATCCGGAACCATATGCAGCCCGAACCC-3′ and 5′-CTGTTGTCAGTGCTCGAGTATTCCAAGTGCTTC-3′. The product was digested with NdeI and XhoI and ligated into NdeI/XhoI-digested pET15b (Novagen). The pMal-mKeap1Δ1-307, encoding a maltose-binding protein (MBP) fused to the Kelch-repeat domain of mKeap1, was generated by PCR amplification of the mKeap1 Kelch-repeat sequence with the following primer pair: 5′-CTACCTGGTGCAGATATTCGAATTCCTCACGCTGCACAAGCCC-3′ and 5′-CTGTCCTGTTGTCAGTCTGCAGGTATTCCAAGTGCTTCAG-3′. The product was digested with EcoRI and PstI and ligated into EcoRI/PstI-digested pMal-2cX (New England Biolabs). An expression construct for hexahistidine-Xpress-mCul3 protein, pcDNA4/HisMaxBmCul3, was generated by PCR of the mCul3 coding region in IMAGE clone 3981140 using oligonucleotides bearing mismatches introducing EcoRI and XhoI sites (forward primer, 5′-GGCCGAGCACGAATTCGAATCTGAGCAAAGGC-3′ and reverse primer, 5′-CTCTCAGAGGGACTCGAGGCTTGATCTGAG-3′), and following restriction the product was ligated into pcDNA4/HisMaxB (Invitrogen). Plasmids for expression of both Myc-tagged and HA-tagged RhoBTB2 (provided by Dr Christopher Carpenter, Harvard Medical School) and Myc-tagged and FLAG-tagged SPOP (provided by Dr Maarten van Lohuizen, The Netherlands Cancer Institute) have been described previously (37Wilkins A. Ping Q. Carpenter C.L. Genes Dev. 2004; 18: 856-861Crossref PubMed Scopus (103) Google Scholar, 38Hernandez-Munoz I. Lund A.H. van der Stoop P. Boutsma E. Muijrers I. Verhoeven E. Nusinow D.A. Panning B. Marahrens Y. van Lohuizen M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 7635-7640Crossref PubMed Scopus (246) Google Scholar). For transfection, the pCMVβgal vector (Clontech) was used as an internal control. Constructs expressing mutated forms of the above proteins were generated by site-directed mutagenesis using the GeneEditor™ kit (Promega).Bacterial Expression and Purification of Protein—To purify recombinant hexahistidine-mKeap1, the pET15bmKeap1 plasmid was transformed into Escherichia coli BL21(DE3)pLysS (Novagen) and selected on LB agar containing 100 μg/ml ampicillin and 34 μg/ml chloramphenicol. One colony was used to inoculate 200 ml of LB broth containing 500 μg/ml ampicillin and 34 μg/ml chloramphenicol, and the culture was grown at 37 °C to an A600 of 0.4. Expression of hexahistidine-mKeap1 protein was induced by addition of isopropylβ-d-thiogalactopyranoside to a final concentration of 1 mm, and after a further 2 h at 30 °C the bacteria were harvested, resuspended in 5 ml of binding buffer (20 mm Tris-Cl, pH 7.9, 5 mm imidazole, 0.5 mm NaCl, and 0.01% (v/v) Nonidet P-40), lysed by addition of 1 mg of lysozyme, and sonicated. Hexahistidine-mKeap1 inclusion bodies were harvested by centrifugation (10,000 × g, 15 min, 4 °C), and washed with two sequential volumes of 20 mm Tris-Cl, pH 7.5, 10 mm EDTA, and 1% (v/v) Triton X-100. The final pellet was solubilized in Laemmli sample buffer. The purity of the hexahistidine-mKeap1 (>95%) and concentration were determined by SDS-PAGE followed by Coomassie staining. To purify MBP-mKeap1Δ1-307, pMal-mKeap1Δ1-307 was transformed into E. coli BL21(DE3)pLysS, and MBP-mKeap1Δ1-307 was induced as described above. Post-induction, the bacteria were resuspended in 5 ml of Column buffer (20 mm Tris, pH 7.6, 200 mm NaCl, 1 mm EDTA), lysed by addition of 1 mg of lysozyme, and sonicated. The lysate was clarified by centrifugation (10,000 × g, 15 min, 4 °C), sterile-filtered, and passed through an amylose column (New England Biolabs). After extensively washing the column, the MBP fusion protein was eluted in Column buffer supplemented with 10 mm maltose. Fractions containing protein were dialyzed against 2 volumes of 10 mm HEPES, pH 7.6, 150 mm NaCl, and 0.01% (v/v) Nonidet P-40, and stored at -80 °C.Cell Culture, Transfections, and Chemical Challenge—COS1 and RL34 cells were maintained as described previously (36McMahon M. Itoh K. Yamamoto M. Hayes J.D. J. Biol. Chem. 2003; 278: 21592-21600Abstract Full Text Full Text PDF PubMed Scopus (854) Google Scholar). Keap1-/- mouse embryonic fibroblast (MEF) cells and their wild-type counterparts (39Wakabayashi N. Itoh K. Wakabayashi J. Motohashi H. Noda S. Takahashi S. Imakado S. Kotsuji T. Otsuka F. Roop D.R. Harada T. Engel J.D. Yamamoto M. Nat. Genet. 2003; 35: 238-245Crossref PubMed Scopus (676) Google Scholar) were cultured as described previously (40Nioi P. McMahon M. Itoh K. Yamamoto M. Hayes J.D. Biochem. J. 2003; 374: 337-348Crossref PubMed Scopus (389) Google Scholar). Cells were seeded into 60-mm tissue culture dishes and were either 90% confluent (COS1) or 30% confluent (keap1-/- MEF) when transfected ∼18 h later with constructs using Lipofectamine 2000 (Invitrogen). Cells were challenged with either 10 μm cycloheximide (CHX; Calbiochem) or sulforaphane (Sul; LKT Laboratories) at least 40 h after plating.Whole-cell Extracts, in Vivo Ubiquitylation Assay, Immunoprecipitation, and Immunoblots—For immunoblots, whole-cell lysates were prepared by scraping cell monolayers into ice-cold radioimmune precipitation assay (RIPA) buffer (50 mm Tris-Cl, pH 7.4, 150 mm NaCl, 1% (v/v) Nonidet P-40, 0.5% (w/v) deoxycholate, 0.1% (w/v) SDS). Lysates were clarified by centrifugation (16,000 × g, 15 min, 4 °C). The in vivo ubiquitylation assay was carried out as described previously (36McMahon M. Itoh K. Yamamoto M. Hayes J.D. J. Biol. Chem. 2003; 278: 21592-21600Abstract Full Text Full Text PDF PubMed Scopus (854) Google Scholar). To immunoprecipitate EGFP-, V5-, or Myc-tagged proteins, clarified whole-cell lysates were prepared as described above, except that deoxycholate and SDS were omitted from the RIPA buffer. On occasion, clarified lysates that contained different ectopic proteins were mixed together. 1 μg of either rabbit anti-GFP, goat anti-V5, or mouse anti-c-Myc (clone 9E10) (all from Abcam) was added to each clarified lysate and mixed by end-over-end tumbling at 4 °C for 2 h. At this point, immunocomplexes were gathered with protein G-Sepharose 4B (Sigma) at 4 °C for 15 min. Material that remained bound to the resin after washing with modified RIPA buffer was eluted in 50 μl of Laemmli reducing sample buffer. Biochemical analyses were carried out by standard methods (36McMahon M. Itoh K. Yamamoto M. Hayes J.D. J. Biol. Chem. 2003; 278: 21592-21600Abstract Full Text Full Text PDF PubMed Scopus (854) Google Scholar). Antibodies used included mouse anti-V5 and mouse anti-Xpress (Invitrogen), goat anti-hKeap1 (E20; Santa Cruz Biotechnology), mouse anti-HA, (Roche Applied Science), rabbit anti-GFP, goat anti-c-Myc, and rabbit anti-FLAG (the latter three were all from Abcam). During this study, a rabbit anti-mKeap1 antiserum was generated by immunization of a female New Zealand White rabbit with bacterially expressed MBP-mKeap1Δ1-307 protein and was used at a dilution of 1:5000.Gel Filtration—Confluent monolayers of cells in 60-mm dishes were lysed by scraping them into 300 μl of ice-cold 50 mm sodium phosphate, pH 7.2, 150 mm NaCl, containing 1% (v/v) Tween 20. The clarified lysate was applied to a Superdex-200 10/300 GL 10 × 300-mm column (Amersham Biosciences) and was eluted at 0.25 ml/min with lysis buffer. Fractions of 250 μl were collected.RESULTS AND DISCUSSIONKeap1 Protein Exists as a Stable Dimer, but Not as a Monomer, in Mammalian Cells—To determine whether Keap1 oligomerizes in mammalian cells, we co-expressed EGFP- and V5-tagged forms of the protein in COS1 cells. After 24 h, whole-cell lysates were prepared from each dish of cells. EGFP-tagged mKeap1 was immunoprecipitated from these lysates and probed for the presence of V5-tagged adaptor by immunoblotting. The V5-tagged protein specifically co-immunoprecipitated with its EGFP-tagged counterpart (Fig. 2A, cf. lanes 1 and 2 with lanes 7 and 8), indicating that Keap1 self-associates. These data also show that the interaction of the adaptor protein with itself is unaffected by treatment of COS1 cells with doses of the oxidative stressor Sul that are sufficient to inhibit BC3BKeap1 activity (36McMahon M. Itoh K. Yamamoto M. Hayes J.D. J. Biol. Chem. 2003; 278: 21592-21600Abstract Full Text Full Text PDF PubMed Scopus (854) Google Scholar, 41Itoh K. Wakabayashi N. Katoh Y. Ishii T. O'Connor T. Yamamoto M. Genes Cells. 2003; 8: 379-391Crossref PubMed Scopus (655) Google Scholar).FIGURE 2mKeap1 exists as a homodimer. A, the indicated proteins were expressed heterologously in COS1 cells. Duplicate dishes of cells were treated with 0, 15, or 90 μm Sul for 2 h before whole-cell lysates were prepared, EGFP-mKeap1 immunoprecipitated (IP), and both whole-cell lysates (input) and IP fractions blotted with mouse anti-V5 and rabbit anti-GFP. WB, Western blot. B, a whole-cell lysate was prepared from COS1 cells expressing mKeap1 and size-fractionated on a Superdex-200 column. Fractions of 0.25 ml were collected and blotted with goat anti-hKeap1, either as combined aliquots from eight consecutive fractions (panel i), or individually (panel ii). The column was calibrated using thyroglobulin (669 kDa, elution volume of 9.15 ml), apoferritin (445 kDa, elution volume of 9.26 ml), catalase (202 kDa, elution volume of 10.42 ml), β-amylase (200 kDa, elution volume of 10.75 ml), alcohol dehydrogenase (150 kDa, elution volume of 11.98), albumin (63.5 kDa, elution volume of 13.47 ml), carbonic anhydrase (29 kDa, elution volume of 16.53 ml), chymotrypsinogen (20.4 kDa, elution volume of 16.68 ml), and ribonuclease A (15.6 kDa, elution volume of 17.32 ml) as size standards. The graph of log Mw versus Kav for these nine proteins is depicted in panel iii, where Kav is defined as (Ve - Vo)/(Vt - Vo). Ve indicates the elution volume for protein of interest; Vo indicates the void volume (7.7 ml); Vt indicates the total column volume (24 ml).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Inspection of the data in Fig. 2A revealed that the proportion of mKeap1 in the immunoprecipitate that contained a V5 tag was similar to that in the corresponding whole-cell lysate (Fig. 2A, lanes 1 and 2). This is consistent with self-association of the adaptor proceeding to completion. In agreement with this, when we size-fractionated lysates from homeostatic COS1 cells expressing untagged mKeap1, the protein eluted as a single peak (Fig. 2B (i) and (ii)), with an estimated mass of 147-162 kDa, a value that is approximately twice the theoretical molecular weight of a Keap1 polypeptide (69.5 kDa). These data reveal that overexpressed Keap1 exists solely as a stable dimer under steady-state conditions.Self-association is not an artifact of overexpression as endogenous Keap1 also exhibits this property. To unequivocally identify the endogenous protein, we used two immunological reagents that interact with separate domains of the adaptor. Goat anti-hKeap1 is available commercially and was raised to a peptide within the BTB domain of the adaptor protein. In addition, we generated an antiserum against the Kelch-repeat domain of mKeap1. Both of these reagents recognized hexahistidine-mKeap1 standards (Fig. 3, A and B, lanes 1-3). They also bound to ectopic untagged and EGFP-tagged forms of mKeap1 (Fig. 3, A and B, lanes 5 and 6). Critically, only one endogenous protein in COS1 lysates was recognized by both reagents, and it co-migrated with untagged mKeap1 (Fig. 3, A and B, lane 4). This protein, which we identify as Keap1, was present at approximately only 10 ppm of extracted COS1 protein. In an immortalized rat liver epithelial cell line, RL34, and MEFs, the adaptor is present in even lower amounts being below the limit of detection of our assay (approximately 2 ppm of extracted protein) (Fig. 3, A and B, lanes 7-9). Endogenous Keap1 eluted as a single peak from a Superdex-200 column with proteins of 141-155 kDa (Fig. 3C), indicating that it exists solely as a dimer.FIGURE 3Endogenous Keap1 is a dimer in COS1 cells. Portions of bacterially expressed and purified hexahistidine-mKeap1 (4000, 400, and 40 pg in lanes 1-3, respectively), 10 μg of protein from COS1 cells (lane 4), COS1 cells heterologously expressing untagged mKeap1 or EGFP-mKeap1 (lanes 5 and 6, respectively), RL34 cells (lane 7), wild type (wt) MEF cells (lane 8) and Keap1-/- MEF cells (lane 9) were electrophoresed through 8% polyacrylamide gels and blotted with goat anti-hKeap1 (A) or rabbit anti-mKeap1 (B). WB, Western blot. C, a whole-cell lysate was prepared from COS1 cells and size-fractionated on a Superdex-200 column. Starting from elution volume 7.36 ml and ending at elution volume 16.36 ml (i.e. proteins from >1000 to ∼35 kDa), 36 fractions of 0.25 ml were collected and blotted with rabbit anti-mKeap1. Bands representing Keap1 are indicated by an asterisk.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Dimeric Keap1 Associates with Cul3—The above experiments suggest it is dimeric and not monomeric Keap1 that interacts with Cul3. We developed a co-immunoprecipitation-based assay to further investigate this issue.First, we confirmed that Xpress-tagged mCul3 co-immunoprecipitated with V5-tagged mKeap1 (Fig. 4, cf. lanes 1 and 2, and lanes 3 and 4). Next, we generated a mutant form of Keap1 that was unable to bind Cul3. To do this, we created a model of the Keap1 BTB homodimer structure based on those of BCL6 and PLZF (supplemental Fig. 1,