Title: Ubiquitination of Keap1, a BTB-Kelch Substrate Adaptor Protein for Cul3, Targets Keap1 for Degradation by a Proteasome-independent Pathway
Abstract: Keap1 is a BTB-Kelch protein that functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex. Keap1 targets its substrate, the Nrf2 transcription factor, for ubiquitination and subsequent degradation by the 26 S proteasome. Inhibition of Keap1-dependent ubiquitination of Nrf2 increases steady-state levels of Nrf2 and enables activation of cytoprotective Nrf2-dependent genes. In this report, we demonstrate that Keap1 and three other BTB-Kelch proteins, including GAN1, ENC1, and Sarcosin, are ubiquitinated by a Cul3-dependent complex. Ubiquitination of Keap1 is markedly increased in cells exposed to quinone-induced oxidative stress, occurs in parallel with inhibition of Keap1-dependent ubiquitination of Nrf2, and results in decreased steady-state levels of Keap1, particularly in cells that are unable to synthesize glutathione. Degradation of Keap1 is independent of the 26 S proteasome, because inhibitors of the 26 S proteasome do not prevent loss of Keap1 following exposure of cells to quinone-induced oxidative stress. Our results suggest that a switch from substrate to substrate adaptor ubiquitination is a critical regulatory step that controls steady-state levels of both BTB-Kelch substrate adaptor proteins and their cognate substrates. Keap1 is a BTB-Kelch protein that functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex. Keap1 targets its substrate, the Nrf2 transcription factor, for ubiquitination and subsequent degradation by the 26 S proteasome. Inhibition of Keap1-dependent ubiquitination of Nrf2 increases steady-state levels of Nrf2 and enables activation of cytoprotective Nrf2-dependent genes. In this report, we demonstrate that Keap1 and three other BTB-Kelch proteins, including GAN1, ENC1, and Sarcosin, are ubiquitinated by a Cul3-dependent complex. Ubiquitination of Keap1 is markedly increased in cells exposed to quinone-induced oxidative stress, occurs in parallel with inhibition of Keap1-dependent ubiquitination of Nrf2, and results in decreased steady-state levels of Keap1, particularly in cells that are unable to synthesize glutathione. Degradation of Keap1 is independent of the 26 S proteasome, because inhibitors of the 26 S proteasome do not prevent loss of Keap1 following exposure of cells to quinone-induced oxidative stress. Our results suggest that a switch from substrate to substrate adaptor ubiquitination is a critical regulatory step that controls steady-state levels of both BTB-Kelch substrate adaptor proteins and their cognate substrates. Oxidative stress results from an imbalance between the production and removal of reactive oxygen species and has been implicated in numerous pathophysiological settings, including cancer, neurodegeneration, aging, and cardiovascular disease (1Ames B.N. Shigenaga M.K. Halliwell B. 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Keap1 is a member of the large BTB-Kelch protein family, more than 40 of which are encoded by the human genome (18Prag S. Adams J.C. BMC Bioinformatics. 2003; 4: 42Crossref PubMed Scopus (129) Google Scholar). Recent reports by several groups have demonstrated that Keap1 functions as a substrate adaptor protein for a Cul3-Rbx1 E3 1The abbreviations used are: E3, ubiquitin-protein isopeptide ligase; E2, ubiquitin carrier protein; HA, hemagglutinin; DTT, dithiothreitol; CBD, C-terminal chitin binding domain; tBHQ, tert-butylhydroquinone; γGCS, γ-glutamyl cysteine synthase. 1The abbreviations used are: E3, ubiquitin-protein isopeptide ligase; E2, ubiquitin carrier protein; HA, hemagglutinin; DTT, dithiothreitol; CBD, C-terminal chitin binding domain; tBHQ, tert-butylhydroquinone; γGCS, γ-glutamyl cysteine synthase. ubiquitin ligase complex (19Cullinan S.B. Gordan J.D. Jin J. Harper J.W. Diehl J.A. Mol. Cell. Biol. 2004; 24: 8477-8486Crossref PubMed Scopus (743) Google Scholar, 20Kobayashi 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 (1564) Google Scholar, 21Zhang D.D. Lo S.C. Cross J.V. Templeton D.J. Hannink M. Mol. Cell. Biol. 2004; 24: 10941-10953Crossref PubMed Scopus (929) Google Scholar, 22Furukawa M. Xiong Y. Mol. Cell. Biol. 2005; 25: 162-171Crossref PubMed Scopus (568) Google Scholar). The N-terminal BTB domain and central linker region of Keap1 bind Cul3, whereas the C-terminal Kelch domain of Keap1 binds Nrf2 via residues located within loops that extend out from the bottom of the Kelch domain (20Kobayashi 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 (1564) Google Scholar, 21Zhang D.D. Lo S.C. Cross J.V. Templeton D.J. Hannink M. Mol. Cell. Biol. 2004; 24: 10941-10953Crossref PubMed Scopus (929) Google Scholar, 23Li X. Zhang D. Hannink M. Beamer L.J. J. Biol. Chem. 2004; 279: 54750-54758Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Under conditions of homeostatic cell growth, Keap1 brings Nrf2 into the Cul3-Rbx1 complex and enables ubiquitin conjugation onto specific lysine residues located within the N-terminal Neh2 domain of Nrf2 (21Zhang D.D. Lo S.C. Cross J.V. Templeton D.J. Hannink M. Mol. Cell. Biol. 2004; 24: 10941-10953Crossref PubMed Scopus (929) Google Scholar). However, following exposure of cells to a wide variety of chemical inducers of Nrf2-dependent transcription, Keap1-dependent ubiquitination of Nrf2 is blocked, enabling Nrf2 to accumulate in the nucleus and activate expression of Nrf2-dependent genes (21Zhang D.D. Lo S.C. Cross J.V. Templeton D.J. Hannink M. Mol. Cell. Biol. 2004; 24: 10941-10953Crossref PubMed Scopus (929) Google Scholar, 24Zhang D.D. Hannink M. Mol. Cell. Biol. 2003; 23: 8137-8151Crossref PubMed Scopus (1069) Google Scholar). The molecular definition of Keap1 as a substrate adaptor protein for Cul3 provides a conceptual framework for understanding how Keap1-dependent ubiquitination of Nrf2 is regulated. In general, the six cullin proteins encoded by the human genome function as scaffold proteins that bring together a substrate protein and a ubiquitin-charged E2 ubiquitin conjugation (Ubc) protein (25Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2857) Google Scholar, 26Deshaies R.J. Annu. Rev. Cell Dev. Biol. 1999; 15: 435-467Crossref PubMed Scopus (1078) Google Scholar). The E2 protein does not associate directly with the cullin protein but is brought into the complex by the cullin-associated Rbx1 protein. Likewise, the substrate does not typically associate directly with the cullin protein but is brought into the complex by a substrate adaptor protein. Once the entire E3 ubiquitin ligase complex is assembled, the ubiquitin molecule is transferred from a conserved cysteine residue in the Ubc protein to one or more lysine residues in the substrate protein. Cullin-based E3 ubiquitin ligases typically catalyze the addition of a multiubiquitin chain onto the substrate protein and thereby target the substrate protein for proteasome-mediated degradation. Cullin-based E3 ubiquitin ligase complexes are dynamic complexes that undergo cycles of assembly and disassembly (27Cope G.A. Deshaies R.J. Cell. 2003; 114: 663-671Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 28Wolf D.A. Zhou C. Wee S. Nat. Cell Biol. 2003; 5: 1029-1033Crossref PubMed Scopus (161) Google Scholar). The ability of cullin-based E3 ubiquitin ligase complexes to undergo facile substrate adaptor exchange is a critical functional property, because each cullin protein supports the ability of a large number of substrate adaptor proteins to target their substrates for degradation. Several mechanisms have been suggested to account for facile exchange of substrate adaptor proteins by cullin-based E3 ubiquitin ligase complexes. In yeast, several well characterized F-box-containing substrate adaptor proteins, including Grr1, cdc4p, and Met30, all of which function as substrate adaptors for Cul1, are labile proteins that are subject to auto-ubiquitination by the same Cul1-Rbx1 complex that directs ubiquitination of their substrates (29Galan J.M. Peter M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9124-9129Crossref PubMed Scopus (225) Google Scholar, 30Zhou P. Howley P.M. Mol. Cell. 1998; 2: 571-580Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Because F-box proteins play critical roles in the cell cycle and in the response of cells to environmental conditions, rapid degradation of a specific substrate adaptor protein may enable timely accumulation of its substrate protein. Rapid degradation of a substrate adaptor protein will also minimize competition for limiting amounts of the cullin-Rbx1 complex and provides a mechanism for substrate adaptor exchange (27Cope G.A. Deshaies R.J. Cell. 2003; 114: 663-671Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). In mammalian cells, which contain numerous substrate adaptor proteins that specifically utilize individual cullin proteins, only two F-box proteins, Skp2 and βTrCP, are known to be regulated at the level of ubiquitination and proteasome-mediated degradation (31Li Y. Gazdoiu S. Pan Z.Q. Fuchs S.Y. J. Biol. Chem. 2004; 279: 11074-11080Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 32Bashir T. Dorrello N.V. Amador V. Guardavaccaro D. Pagano M. Nature. 2004; 428: 190-193Crossref PubMed Scopus (398) Google Scholar, 33Wei W. Ayad N.G. Wan Y. Zhang G.J. Kirschner M.W. Kaelin Jr., W.G. Nature. 2004; 428: 194-198Crossref PubMed Scopus (389) Google Scholar, 34Wirbelauer C. Sutterluty H. Blondel M. Gstaiger M. Peter M. Reymond F. Krek W. EMBO J. 2000; 19: 5362-5375Crossref PubMed Scopus (150) Google Scholar). Additional mechanisms have been uncovered that contribute to substrate adaptor exchange in mammalian cells. For example, the CAND1 protein has been proposed to displace a substrate adaptor protein from Cul1 following deneddylation of the Cul1 protein by the COP9 signalosome (28Wolf D.A. Zhou C. Wee S. Nat. Cell Biol. 2003; 5: 1029-1033Crossref PubMed Scopus (161) Google Scholar, 35Liu J. Furukawa M. Matsumoto T. Xiong Y. Mol. Cell. 2002; 10: 1511-1518Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 36Zheng J. Yang X. Harrell J.M. Ryzhikov S. Shim E.H. Lykke-Andersen K. Wei N. Sun H. Kobayashi R. Zhang H. Mol. Cell. 2002; 10: 1519-1526Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). In this report, we demonstrate that Keap1 is ubiquitinated in vivo and in vitro by the same Cul3-Rbx1 complex that ubiquitinates its substrate, Nrf2. Other members of the BTB-Kelch protein family are also able to assemble into functional E3 ubiquitin ligase complexes with Cul3 that support ubiquitination of the respective BTB-Kelch protein, providing evidence that the ability to function as a substrate adaptor protein for Cul3 is a conserved property of this large family of proteins. Quinone-induced oxidative stress enhances ubiquitination of Keap1 and decreases steady-state levels of Keap1, resulting in a corresponding increase in steady-state levels of Nrf2. However, in contrast to Nrf2 degradation, which is blocked by inhibitors of the 26 S proteasome, proteasome inhibitors do not block degradation of Keap1. Keap1 ubiquitination and proteasome-independent degradation of Keap1 following exposure to quinone-induced oxidative stress is markedly enhanced in glutathione-deficient cells. The isothiocyanate sulforaphane, a well characterized cancer-preventive inducer of Nrf2-dependent transcription, inhibits Keap1-dependent ubiquitination of Nrf2 but does not induce Keap1 ubiquitination, indicating that Keap1 differentially responds to inducers of Nrf2-dependent transcription. Our results indicate that ubiquitination targets BTB-Kelch proteins for degradation by a proteasome-independent pathway and suggest that a switch from substrate to substrate adaptor ubiquitination is a critical regulatory step that controls steady-state levels of both BTB-Kelch substrate adaptor proteins and their cognate substrates. Construction of Recombinant DNA Molecules—Plasmids expressing wild type Keap1, Nrf2, Cul3, Cul3DN, Rbx1, and HA-Ub proteins have been previously described (21Zhang D.D. Lo S.C. Cross J.V. Templeton D.J. Hannink M. Mol. Cell. Biol. 2004; 24: 10941-10953Crossref PubMed Scopus (929) Google Scholar). cDNA clones of GAN1, ENC1, and sarcosin were purchased from ATCC (American Type Culture Collection). The CBD-tagged versions of GAN1, ENC1, and sarcosin were generated by insertion of a PCR-generated DNA fragment encoding the chitin binding domain of Bacillus circulans chitinase A1 gene upstream of the stop codon. The integrity of all of the plasmids used in this study was confirmed by sequence analysis. Cell Culture and Transfections—COS-1 and MDA-MB-231 cells were purchased from ATCC. Cells were maintained in either Dulbecco's modified Eagle's medium or Eagle's minimal essential medium in the presence of 10% fetal bovine serum. GCS-2 cells were grown in knockout Dulbecco's modified Eagle's medium supplemented with 15% ES-cell qualified bovine serum, 2.5 mm glutathione, and 100 μm β-mercaptoethanol. Transfections were performed with Lipofectamine Plus (Invitrogen) according to the manufacturer's instructions. Antibodies, Immunoprecipitation, and Immunoblot Analysis—The anti-Keap1 antibody has been described previously (24Zhang D.D. Hannink M. Mol. Cell. Biol. 2003; 23: 8137-8151Crossref PubMed Scopus (1069) Google Scholar). Antibodies against Nrf2 and Myc (Santa Cruz Biotechnology), ubiquitin (Sigma), chitin binding domain (New England Biolabs), and HA (Covance) were purchased from commercial sources. For detection of protein expression in total cell lysates, cells were lysed in sample buffer (50 mm Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 100 mm DTT, 0.1% bromphenol blue) at 48 h post-transfection. For immunoprecipitation assays, cells were lysed in radioimmune precipitation assay buffer (10 mm sodium phosphate, pH 8.0, 150 mm NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing 1 mm DTT, 1 mm phenylmethylsulfonyl fluoride, and protease inhibitor mixture (Sigma). Cell lysates were pre-cleared with protein A beads and incubated with 2 μg of affinity-purified antibodies for 2 h at 4 °C, followed by incubation at 4 °C with protein A-agarose beads for 2 h. Immunoprecipitated complexes were washed four times with RIPA buffer and eluted in sample buffer by boiling for 4 min, electrophoresed through SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and subjected to immunoblot analysis. In Vivo Ubiquitination—For detection of ubiquitinated proteins in vivo, cells were transfected with expression vectors for HA-ubiquitin and the indicated proteins. Expression vectors for HA-Cul3 and Myc-Rbx1 were included in some of experiments, as indicated. Cells were rapidly lysed by boiling in a buffer containing 2% SDS, 150 mm NaCl, 10 mm Tris-HCl, and 1 mm DTT. This rapid lysis procedure inactivates cellular ubiquitin hydrolases and therefore preserves ubiquitin-protein conjugates present in cells prior to lysis. Protein-protein interactions, including association of Nrf2 with Keap1, are also disrupted by this lysis procedure. For immunoprecipitation, these lysates were diluted 5-fold in buffer lacking SDS and incubated with anti-Keap1, anti-Nrf2, or anti-CBD antibodies accordingly. Immunoprecipitated proteins were analyzed by immunoblot with antibodies directed against the HA epitope. In Vitro Ubiquitination—For ubiquitination of the BTB-Kelch proteins in vitro, COS-1 cells were transfected with expression vectors for the individual CBD-tagged BTB-Kelch protein, HA-Cul3, and Myc-Rbx1. The transfected cells were lysed in buffer B (15 mm Tris-HCL (pH 7.4), 500 mm NaCl, and 0.25% Nonidet P-40) containing 1 mm DTT, 1 mm phenylmethylsulfonyl fluoride, and protease inhibitor mixture. The lysates were pre-cleared with protein A beads prior to incubation with chitin beads (New England Biolabs) for 4 h at 4 °C. Chitin beads were washed twice with buffer B, twice with buffer A (25 mm Tris-HCl (pH 7.5), 10% (v/v) glycerol, 1 mm EDTA, 0.01% Nonidet P-40 and 0.1 m NaCl), and twice with reaction buffer (50 mm Tris-HCl (pH 7.5), 5 mm MgCl2, 2 mm NaF, and 0.6 mm DTT). The pellets were incubated with ubiquitin (300 pmol), E1 (2 pmol), E2-UbcH5a (10 pmol), and ATP (2 mm) in 1× reaction buffer in a total volume of 30 μl for 1 h at 37 °C. Ubiquitin, E1, and E2-UbcH5a were purchased from Boston Biochem. The chitin beads were centrifuged at 3000 × g, resuspended in 2% SDS, 150 mm NaCl, 10 mm Tris-HCl (pH 8.0) and 1 mm DTT and boiled for 5 min to release bound proteins, inactivate any contaminating ubiquitin hydrolases, and disrupt protein-protein interactions. The supernatant was diluted 5-fold with buffer lacking SDS prior to immunoprecipitation with anti-CBD antibodies. Immunoprecipitated proteins were subjected to immunoblot analysis with anti-ubiquitin antibodies. Assembly into a Functional Ubiquitin Ligase Complex Is a Conserved Property of BTB-Kelch Proteins—The major function of substrate adaptor proteins is to target specific substrate proteins for ubiquitination. However, ubiquitination of substrate adaptor proteins may also play an important role in cellular physiology. For example, auto-ubiquitination of several yeast F-box proteins is responsible for their rapid turnover during progression through the cell cycle (29Galan J.M. Peter M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9124-9129Crossref PubMed Scopus (225) Google Scholar, 30Zhou P. Howley P.M. Mol. Cell. 1998; 2: 571-580Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). To examine the possibility that Keap1 is ubiquitinated by a Cul3-Rbx1 complex in vitro, the Keap1-Cul3-Rbx1 complex was purified from COS-1 cells transfected with expression vectors for Keap1, Cul3, and Rbx1. To facilitate purification of Keap1 and Keap1-associated proteins, an expression vector encoding a Keap1 protein containing a C-terminal chitin binding domain (CBD) was utilized in this experiment. Proteins that bound to the chitin beads were incubated with purified E1, E2-UbcH5a, ubiquitin, and ATP. Subsequently, anti-Keap1 immunoprecipitates were prepared under strongly denaturing conditions and subjected to immunoblot analysis using anti-ubiquitin antibodies (Fig. 1A, top panel). Keap1-ubiquitin conjugates were readily observed in the presence of both Cul3 and Rbx1 (Fig. 1A, top panel, lane 5), and required the addition of ubiquitin, E1, and E2-UbcH5a to the in vitro reaction (Fig. 2C, top panel, lanes 2–4). Immunoblot analysis of anti-Keap1 immunoprecipitates confirmed the presence of equivalent levels of Keap1-CBD in all reactions (Fig. 1A, bottom panel).Fig. 2A, MDA-MB-231 cells were transfected with expression vectors for HA-ubiquitin, Keap1, and HA-Cul3 and increasing amounts of an expression vector for Rbx1. Cells were lysed under denaturing conditions and subjected to immunoblot analysis with Keap1 antibodies (bottom panel). Equivalent aliquots of each lysates were subjected to immunoprecipitation with anti-Keap1 antibodies. Anti-Keap1 immunoprecipitates were analyzed by immunoblot with anti-HA antibodies (top panel). B, 293T cells were transfected with expression vectors for HA-ubiquitin and Keap1. Total lysates (left panel) or anti-HA immunoprecipitates (right panel) were analyzed by immunoblot with anti-Keap1 antibodies. The samples were analyzed in parallel on adjacent lanes. The left panel was exposed for a shorter time than the right panel. C, MDA-MB-231 cells were transfected with expression vectors for HA-ubiquitin and Keap1 and the expression vector for either Cul3 or Cul3DN. Cells were either left untreated or treated with 10 μm MG132 for 4 h prior to lysis. Equivalent aliquots of each lysates were analyzed by immunoblot with anti-Keap1 antibodies (bottom panel) or subjected to immunoprecipitation with anti-Keap1 antibodies. Anti-Keap1 immunoprecipitates were analyzed by immunoblot with anti-HA antibodies (top panel). D, cell lysates from MDA-MB-231 cells transfected with expression vectors for Keap1 and the indicated wild-type or mutant HA-ubiquitin proteins were immunoprecipitated with anti-Keap1 antibodies. The immunoprecipitated proteins were analyzed by immunoblot with anti-HA antibodies.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Keap1 is one of more than 40 BTB-Kelch proteins that are encoded by the human genome (18Prag S. Adams J.C. BMC Bioinformatics. 2003; 4: 42Crossref PubMed Scopus (129) Google Scholar, 38Stogios P.J. Prive G.G. Trends Biochem. Sci. 2004; 29: 634-637Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). The members of the BTB-Kelch family share both a common domain organization and ∼25% sequence identity, when compared on a one-to-one basis with each other. The biochemical functions of this family of proteins are, in general, not known, although the ability of Keap1 to function as a substrate adaptor protein suggests a common function for these proteins. To determine if other BTB-Kelch proteins may function as substrate adaptor proteins for Cul3, the ability of several different BTB-Kelch proteins to associate with Cul3 and Rbx1 to form a functional ubiquitin ligase complex was determined. The three BTB-Kelch proteins used for these experiments, GAN1, ENC1, and sarcosin, were selected because of their known or suspected roles in cancer and neurodegenerative diseases (39Bomont P. Cavalier L. Blondeau F. Ben Hamida C. Belal S. Tazir M. Demir E. Topaloglu H. Korinthenberg R. Tuysuz B. Landrieu P. Hentati F. Koenig M. Nat. Genet. 2000; 26: 370-374Crossref PubMed Scopus (307) Google Scholar, 40Cullen V.C. Brownlees J. Banner S. Anderton B.H. Leigh P.N. Shaw C.E. Miller C.C. Neuroreport. 2004; 15: 873-876Crossref PubMed Scopus (14) Google Scholar, 41Liang X.-Q. Avraham H.K. Jiang S. Avraham S. Oncogene. 2004; 23: 5890-5990Crossref PubMed Scopus (39) Google Scholar, 42Fujita M. Furukawa Y. Tsunoda T. Tanaka T. Ogawa M. Nakamura Y. Cancer Res. 2001; 61: 7722-7726PubMed Google Scholar, 43Spence H.J. Johnston I. Ewart K. Buchanan S.J. Fitzgerald U. Ozanne B.W. Oncogene. 2000; 19: 1266-1276Crossref PubMed Scopus (47) Google Scholar). To facilitate purification and detection of these BTB-Kelch proteins, fusion proteins containing a CBD were constructed. Expression vectors for the CBD-tagged BTB-Kelch proteins were transfected into COS-1 cells alone or with expression vectors for HA-Cul3 and Myc-Rbx1. Cell lysates were subjected to affinity purification using chitin beads. Neither Cul3 nor Rbx1 purified with the chitin beads in the absence of a CBD-tagged BTB-Kelch protein (Fig. 1B). Each of these BTB-Kelch proteins was able to associate with both Cul3 and Rbx1, as determined by immunoblot analysis of the purified complexes using anti-HA or anti-Myc antibodies (Fig. 1C, middle and bottom panels, respectively). The ability of the complexes formed between the respective BTB-Kelch proteins, Cul3 and Rbx1, to support ubiquitin conjugation onto the BTB-Kelch protein was determined. The chitin beads containing the purified complexes were incubated with E1, E2-UbcH5a, ubiquitin, and ATP. Subsequently, anti-CBD immunoprecipitates prepared under strongly denaturing conditions were subjected to immunoblot analysis with anti-ubiquitin antibodies. Ubiquitin conjugation, onto each of the BTB-Kelch proteins that was dependent upon the addition of E1 into the in vitro reaction, was observed (Fig. 1D). The presence of ubiquitin conjugation onto Keap1 and GAN1 in the absence of co-expressed Cul3 and Rbx1 proteins presumably represents co-purification of endogenous Cul3 and Rbx1 proteins with these two BTB-Kelch proteins (Fig. 1D, lanes 3 and 6). Keap1 Is Ubiquitinated by a Cul3-Rbx1 Complex in Vivo—To determine if Keap1 is ubiquitinated in vivo, MDA-MB-231 cells were co-transfected with expression vectors for HA-ubiquitin, HA-Cul3, and Keap1 and increasing amounts of the expression vector for Myc-Rbx1. Cells were lysed under denaturing conditions and subjected to immunoprecipitation with Keap1 antibodies. The presence of ubiquitin-conjugated Keap1 proteins was assessed by immunoblot analysis of anti-Keap1 immunoprecipitates with anti-HA antibodies. Ubiquitin conjugation onto ectopically expressed Keap1 was readily observed and was increased by coexpression of Cul3 (Fig. 2A, top panel, lanes 1–3). Low levels of co-expressed Rbx1 slightly increased the levels of ubiquitin conjugation onto Keap1 (Fig. 2A, top panel, lane 4). Increased Rbx1 expression did not further increase Keap1 ubiquitination, but markedly decreased steady-state levels of Keap1 in a dose-dependent manner (Fig. 2A, lanes 5–8). To confirm that Keap1 is ubiquitinated in vivo, cell lysates from 293T cells transfected with expression vectors for Keap1 and HA-ubiquitin were immunoprecipitated with anti-HA antibodies. The presence of Keap1 in anti-HA immunoprecipitates was confirmed by immunoblot analysis with anti-Keap1 antibodies (Fig. 2B). To define the role of Cul3 in ubiquitination of Keap1 in vivo, we determined the ability of a dominant-negative Cul3 protein lacking the C-terminal Rbx1 binding domain to block Keap1 ubiquitination. Cell lysates were collected from MDA-MB-231 cells transfected with expression vectors for Keap1 and either wild-type or dominant-negative Cul3. As expected, co-expression of Keap1 with the wild-type Cul3 protein increased ubiquitination onto Keap1 (Fig. 2C, top panel, lanes 3–6). Co-expression of Keap1 with the dominant-negative Cul3 protein decreased levels of ubiquitin-conjugated Keap1 (Fig. 2C, top panel, lanes 7 and 8). Treatment of the transfected cells with MG132 prior to collection of cell lysates enhanced ubiquitin conjugation onto Keap1 (Fig. 2C, top panel, compare even and odd lanes) but did not alter steady-state levels of Keap1 (Fig. 2C, bottom panel, compare even and odd lanes). Taken together, these results indicate that ubiquitination of Keap1 is mediated by