The Keap1 BTB/POZ Dimerization Function Is Required to Sequester Nrf2 in Cytoplasm

Abstract

Transactivation of phase II detoxification enzymes and antioxidant proteins is mediated by the Cap'N′Collar transcription factor, Nrf2, which is sequestered in the cytoplasm by the actin-binding protein Keap1. Mutation of a conserved serine (S104A) within the Keap1 BTB/POZ domain disrupts Keap1 dimerization and eliminates the ability of Keap1 to sequester Nrf2 in the cytoplasm and repress Nrf2 transactivation. Disruption of endogenous Keap1 dimerization using BTB/POZ dominant negative proteins also inhibits the ability of Keap1 to retain Nrf2 in the cytoplasm. Exposure to an electrophilic agent that induces Nrf2 release and nuclear translocation disrupts formation of a Keap1 complex in vivo. Collectively, these data support the conclusion that Keap1 dimerization is required for Nrf2 sequestration and transcriptional repression. Furthermore, exposure to inducing agents disrupts the Keap1 dimerization function and results in Nrf2 release. Transactivation of phase II detoxification enzymes and antioxidant proteins is mediated by the Cap'N′Collar transcription factor, Nrf2, which is sequestered in the cytoplasm by the actin-binding protein Keap1. Mutation of a conserved serine (S104A) within the Keap1 BTB/POZ domain disrupts Keap1 dimerization and eliminates the ability of Keap1 to sequester Nrf2 in the cytoplasm and repress Nrf2 transactivation. Disruption of endogenous Keap1 dimerization using BTB/POZ dominant negative proteins also inhibits the ability of Keap1 to retain Nrf2 in the cytoplasm. Exposure to an electrophilic agent that induces Nrf2 release and nuclear translocation disrupts formation of a Keap1 complex in vivo. Collectively, these data support the conclusion that Keap1 dimerization is required for Nrf2 sequestration and transcriptional repression. Furthermore, exposure to inducing agents disrupts the Keap1 dimerization function and results in Nrf2 release. glutamate cysteine ligase catalytic glutamate cysteine ligase modulatory antioxidant response element electrophile response element pyrrolidinedithiocarbamate kilobase(s) green fluorescent protein extracellular signal-regulated kinase phosphate-buffered saline wild type phenethyl isothiocyanate Cells respond to certain types of oxidative or electrophilic insults by coordinately up-regulating the expression of various cellular protective and antioxidant genes. Among the genes up-regulated are those encoding the phase II detoxification enzymes, such as NAD(P)H quinone reductase (NQO1), glutathione S-transferase, heme oxygenase-1, and epoxide hydrolyase as well as genes involved in cellular redox maintenance, including the catalytic (GCLC)1 and modulatory (GCLM) subunits of glutamate cysteine ligase (GCL). GCL catalyzes the first and rate-limiting step in the de novo synthesis of glutathione, a critical non-protein thiol involved in detoxification of xenobiotics, electrophiles, free radicals, and reactive oxygen species. Coordinate induction of many of the enzymes in this protective battery is transcriptionally mediated through antioxidant response elements (AREs) (also called electrophile response elements (EpREs)) present in the promoters of the respective genes (1Favreau L.V. Pickett C.B. J. Biol. Chem. 1995; 270: 24468-24474Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 2Mulcahy R.T. Wartman M.A. Bailey H.H. Gipp J.J. J. Biol. Chem. 1997; 272: 7445-7454Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar, 3Nguyen T. Rushmore T.H. Pickett C.B. J. Biol. Chem. 1994; 269: 13656-13662Abstract Full Text PDF PubMed Google Scholar, 4Wild A.C. Gipp J.J. Mulcahy T. Biochem. J. 1998; 332: 373-381Crossref PubMed Scopus (101) Google Scholar, 5Talalay P. Fahey J.W. Holtzclaw W.D. Prestera T. Zhang Y. Toxicol. Lett. (Shannon). 1995; 82–83: 173-179Crossref PubMed Scopus (440) Google Scholar). Antioxidant response elements share a common core sequence, 5′-(G/A)TGA(G/C)NNNGC(G/A)-3′, which bears a remarkable resemblance to Maf recognition elements (MAREs), originally identified as binding sites for the Cap'N′Collar protein NF-E2 within the promoter of the β-globin gene (6Igarashi K. Kataoka K. Itoh K. Hayashi N. Nishizawa M. Yamamoto M. Nature. 1994; 367: 568-572Crossref PubMed Scopus (404) Google Scholar, 7Kotkow K.J. Orkin S.H. Mol. Cell. Biol. 1995; 15: 4640-4647Crossref PubMed Scopus (148) Google Scholar). When dimerized with certain b-zip transcription factors, members of the Cap'N′Collar transcription factor family (NF-E2, Nrf1, Nrf2) are able to bind and transactivate ARE or ARE-like sequences. Although members of the small Maf family of proteins (Maf F, Maf G, and Maf K) as well as members of the Jun family (c-Jun, JunD) have been implicated as potential binding partners for Cap'N′Collar proteins, specific dimerization partners for Nrf2 in the context of AREs have not been clearly defined (8–21). Overexpression of Nrf2 up-regulates expression of ARE-containing genes, demonstrating that Nrf2 is a major determinant in the ARE-mediated induction of genes encoding several of the Phase II detoxification enzymes and both GCL subunits. Constitutive and induced expression of several ARE-containing genes, including glutathioneS-transferase, NQO1, GCLC, and GCLM are reduced in Nrf2 knock-out mice (22Aoki Y. Sato H. Nishimura N. Takahashi S. Itoh K. Yamamoto M. Toxicol. Appl. Pharmacol. 2001; 173: 154-160Crossref PubMed Scopus (277) Google Scholar, 23Chan K. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12731-12736Crossref PubMed Scopus (530) Google Scholar, 24Chan J.Y. Kwong M. Biochim. Biophys. Acta. 2000; 1517: 19-26Crossref PubMed Scopus (272) Google Scholar, 25Chan K. Han X.D. Kan Y.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4611-4616Crossref PubMed Scopus (665) Google Scholar, 26Cho H.Y. Jedlicka A.E. Reddy S.P. Kensler T.W. Yamamoto M. Zhang L.Y. Kleeberger S.R. Am. J. Respir. Cell Mol. Biol. 2002; 26: 175-182Crossref PubMed Scopus (597) Google Scholar, 27Enomoto A. Itoh K. Nagayoshi E. Haruta J. Kimura T. O'Connor T. Harada T. Yamamoto M. Toxicol. Sci. 2001; 59: 169-177Crossref PubMed Scopus (659) Google Scholar, 28Hayes J.D. Chanas S.A. Henderson C.J. McMahon M. Sun C. Moffat G.J. Wolf C.R. Yamamoto M. Biochem. Soc. Trans. 2000; 28: 33-41Crossref PubMed Scopus (291) Google Scholar, 29McMahon 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). Nrf2 null mice are more susceptible to the carcinogenic effects of benzyo(a)pyrene than are wild-type mice and are less responsive to the chemoprotective effects of Oltipraz, a chemopreventive agent that functions in part via ARE induction (30Ramos-Gomez M. Kwak M.K. Dolan P.M. Itoh K. Yamamoto M. Talalay P. Kensler T.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3410-3415Crossref PubMed Scopus (1011) Google Scholar). By virtue of its central role in the transactivation of detoxification genes involved in cellular responses to oxidative or electrophilic stresses, Nrf2 activation is a critical target for cancer chemoprevention and other protective strategies (31Kwak M.K. Egner P.A. Dolan P.M. Ramos-Gomez M. Groopman J.D. Itoh K. Yamamoto M. Kensler T.W. Mutat. Res. 2001; 480–481: 305-315Crossref PubMed Scopus (228) Google Scholar, 32Kensler T.W. Curphey T.J. Maxiutenko Y. Roebuck B.D. Drug Metabol. Drug Interact. 2000; 17: 3-22Crossref PubMed Scopus (122) Google Scholar). Attempts to identify Nrf2 binding partners by yeast two-hybrid screens resulted in the identification of Keap1, a homologue of theDrosophila Kelch protein (33Itoh K. Wakabayashi N. Katoh Y. Ishii T. Igarashi K. Engel J.D. Yamamoto M. Genes Dev. 1999; 13: 76-86Crossref PubMed Scopus (2917) Google Scholar). Subsequent experiments demonstrated that Keap1 normally sequesters Nrf2 in the cytoplasm. After an oxidative/electrophilic stress or stimulus, Nrf2 is liberated from Keap1, whereupon it translocates to the nucleus and, in association with dimerization partners, binds ARE sequences to induce expression of the battery of genes that contribute to the protective response (33Itoh K. Wakabayashi N. Katoh Y. Ishii T. Igarashi K. Engel J.D. Yamamoto M. Genes Dev. 1999; 13: 76-86Crossref PubMed Scopus (2917) Google Scholar). Although the nature of the molecular event(s) that signals dissociation of the Keap1·Nrf2 complex is unknown, it has been hypothesized that thiol modulation of one or more reactive cysteine(s) in Keap1 might result in a conformational change that releases Nrf2 (33Itoh K. Wakabayashi N. Katoh Y. Ishii T. Igarashi K. Engel J.D. Yamamoto M. Genes Dev. 1999; 13: 76-86Crossref PubMed Scopus (2917) Google Scholar). Alternatively, other post-translational mechanisms such as phosphorylation of Nrf2 or Keap1 by kinases activated by oxidative or electrophilic stress might trigger Nrf2 release from Keap1. Members of the Kelch family, including Keap1, contain an N-terminal BTB/POZ domain and a series of 4–7 C-terminal Kelch repeats (34Albagli O. Dhordain P. Deweindt C. Lecocq G. Leprince D. Cell Growth Differ. 1995; 6: 1193-1198PubMed Google Scholar, 35Soltysik-Espanola M. Rogers R.A. Jiang S. Kim T.A. Gaedigk R. White R.A. Avraham H. Avraham S. Mol. Biol. Cell. 1999; 10: 2361-2375Crossref PubMed Scopus (81) Google Scholar, 36Xue F. Cooley L. Cell. 1993; 72: 681-693Abstract Full Text PDF PubMed Scopus (377) Google Scholar, 37Kim I.F. E. 1999; PubMed Scopus Google Scholar). BTB/POZ protein Kelch repeats have been implicated in binding to the and the formation of M. Rogers R.A. Jiang S. Kim T.A. Gaedigk R. White R.A. Avraham H. Avraham S. Mol. Biol. Cell. 1999; 10: 2361-2375Crossref PubMed Scopus (81) Google Scholar, 36Xue F. Cooley L. Cell. 1993; 72: 681-693Abstract Full Text PDF PubMed Scopus (377) Google Scholar, 37Kim I.F. E. 1999; PubMed Scopus Google Scholar, Cooley L. J. Cell Biol. 1997; PubMed Scopus Google Scholar, S. A. M.A. J. 1997; 17: PubMed Google Scholar, R. Genes Dev. 1994; PubMed Scopus Google Scholar). of BTB/POZ from various proteins with Keap1 the of a conserved serine to Mutation of the in the transcription factor the of the BTB/POZ domain is critical to protein function Engel Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). a of this conserved serine in Keap1 to the role of the BTB/POZ domain in Keap1 of to sequester Nrf2 in the and of Nrf2 transactivation of a vivo. Keap1 a molecular complex in the to this Exposure of to the pyrrolidinedithiocarbamate formation of the molecular Collectively, the that the BTB/POZ dimerization function of Keap1 is required for Nrf2 sequestration and that of endogenous Keap1 in is involved in Nrf2 release. for Keap1 by Yamamoto and Itoh of (33Itoh K. Wakabayashi N. Katoh Y. Ishii T. Igarashi K. Engel J.D. Yamamoto M. Genes Dev. 1999; 13: 76-86Crossref PubMed Scopus (2917) Google Scholar). by with and the sites of of the that of promoter from the GCLM gene of the gene has been Mulcahy R.T. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). for Nrf2 by Itoh and to A.C. Mulcahy R.T. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). by of a of the in the of and proteins transcription sequences and for in and by and the by first with by of the and the the in Keap1 by or with and the to and from the and and from in with and using the and as Mulcahy R.T. Biochem. Biophys. Res. 1999; PubMed Scopus Google Scholar, A.C. Mulcahy R.T. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). in and using a as by the expression to expression of of in and results are as with and with for in for in fluorescent and with a fluorescent with isothiocyanate and with and as with by for in for with a of in for in and for After the with to the as in the in and with a with using the from as in the the of and are by and as with of and of or Keap1 and in by with an by with in and in nuclear protein and to using a with for with a of in the After with the for an and with and with a and as with of and of or of Keap1 and in a and with and and for to by the of protein for with for the of protein the of and to and for using a of in and with as and with of and of as in the for the of of After been with for of protein and for an with and in for to a of proteins in the of and in the and by the of of the the of and with of are called in and and the and by as with or to or for Cells in and of the in and by using the and of the in with and and for by using the proteins not by in in and by using the for the as conserved serine in the Keap1 domain to an and the ability of this to repress Nrf2 transactivation of a in an ARE to for Nrf2 binding and gene induction Mulcahy R.T. Biochem. Biophys. Res. 1999; PubMed Scopus Google Scholar, A.C. Mulcahy R.T. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Mulcahy R.T. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). of the and resulted in an in expression with with and an of a wild-type Keap1 expression with reduced Nrf2 transactivation of the by When the with Nrf2, expression of the gene from that in with Nrf2 and wild-type Keap1 and Keap1 proteins to that the with the not in or protein the Nrf2 the of a protein in with for wild-type or Keap1. in the nucleus in with the cytoplasm in with and wild-type Keap1 with the fluorescent to that in with that the protein to sequester Nrf2 in the cytoplasm. also by of nuclear from with of in the nucleus of with wild-type Keap1 with the results of the with and the in the nucleus with that in the of with of the Keap1·Nrf2 by experiments using from with and wild-type or Keap1. of the to the of Keap1 not the function of the wild-type protein in of its ability to bind and sequester Nrf2 not Cell with a and for the of Keap1 by to the Keap1 protein in from the of the Keap1 reduced in with the to that in wild-type Keap1 for that of in of Keap1 in with the Keap1 expression by also with to in to the that wild-type and the protein with the that the not the Keap1 and serine to in Keap1 is conserved in BTB/POZ identified to A. S. A. H. J.D. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar, H. R. Cancer Res. 1999; 59: Google Scholar). the BTB/POZ domain has been to an dimerization (34Albagli O. Dhordain P. Deweindt C. Lecocq G. Leprince D. Cell Growth Differ. 1995; 6: 1193-1198PubMed Google Scholar, R. Genes Dev. 1994; PubMed Scopus Google hypothesized that the with the in the dimerization function of the Keap1 BTB/POZ this the ability of Keap1 to using wild-type and Keap1 proteins in and Keap1 proteins of and protein and Keap1 proteins with the Keap1 in the and Keap1 proteins and with the the of Keap1 protein in from of the proteins is of an association and Keap1 When a of and proteins with the both and Keap1 proteins in the and and with the results that the the of Keap1 dimerization and wild-type wild-type Keap1 protein the proteins and this not by of the to Keap1. the complex in Keap1 that of the BTB/POZ domain complex dimerization and of Nrf2 sequestration with that the ability of Keap1 to might an element in Nrf2 Keap1 dimerization might involved in Nrf2 in with expression to an agent to induce Nrf2 nuclear and ARE transactivation A.C. Mulcahy R.T. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, Mulcahy R.T. Biochem. Biophys. Res. 2000; PubMed Scopus Google Scholar). After the and for and a in from the of the complex in to the in the complex formation not a in the of this Exposure to phenethyl isothiocyanate also the of the Keap1 complex not oxidative/electrophilic agents are of Keap1 dimerization in vivo. the formation of the complex with the of formation in of the protein by a of other the of in by an to for the the the protein from and by with the Keap1 in from the in the from than that in from that exposure Keap1 the complex less in the demonstrated that induction of GCLM activation of both and protein kinase to Nrf2 binding the ARE Mulcahy R.T. Biochem. Biophys. Res. 2000; PubMed Scopus Google Scholar). the of both and in the formation and of the Keap1 complex in with wild-type Keap1. with the or the of and the formation or of the complex by as Although activation of the complex exposure exposure of to to Nrf2 binding to the GCLM ARE Mulcahy R.T. Biochem. Biophys. Res. 2000; PubMed Scopus Google not Keap1 complex by that dissociation of the Keap1 complex in via an Collectively, the data that Keap1 dimerization is required for Keap1·Nrf2 of the dimerization potential of endogenous Keap1 in to result in Nrf2 and expression of the this Keap1 proteins by the C-terminal Kelch from the and proteins to and in BTB/POZ proteins with an molecular of to BTB/POZ with the the proteins to in for protein with the of a expression of the gene as (33Itoh K. Wakabayashi N. Katoh Y. Ishii T. Igarashi K. Engel J.D. Yamamoto M. Genes Dev. 1999; 13: 76-86Crossref PubMed Scopus (2917) Google Scholar). of the expression resulted in a in gene of the release of endogenous Nrf2 function as dominant expression not by of the that Nrf2, a of the Cap'N′Collar family of transcription factors, a central role in the expression and induction of a battery of detoxification and antioxidant enzymes, including NQO1, glutathione S-transferase, and J.Y. Kwong M. Biochim. Biophys. Acta. 2000; 1517: 19-26Crossref PubMed Scopus (272) Google Scholar, 28Hayes J.D. Chanas S.A. Henderson C.J. McMahon M. Sun C. Moffat G.J. Wolf C.R. Yamamoto M. Biochem. Soc. Trans. 2000; 28: 33-41Crossref PubMed Scopus (291) Google Scholar, 29McMahon 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, M.K. Itoh K. Yamamoto M. Kensler T.W. Mol. 2001; PubMed Google Scholar). Furthermore, mice have been demonstrated to more susceptible to certain and are to chemopreventive Nrf2 as an factor and the Nrf2 activation as a potential target for chemoprevention strategies J.D. Chanas S.A. Henderson C.J. McMahon M. Sun C. Moffat G.J. Wolf C.R. Yamamoto M. Biochem. Soc. Trans. 2000; 28: 33-41Crossref PubMed Scopus (291) Google Scholar, M. Kwak M.K. Dolan P.M. Itoh K. Yamamoto M. Talalay P. Kensler T.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3410-3415Crossref PubMed Scopus (1011) Google Scholar, M.K. Egner P.A. Dolan P.M. Ramos-Gomez M. Groopman J.D. Itoh K. Yamamoto M. Kensler T.W. Mutat. Res. 2001; 480–481: 305-315Crossref PubMed Scopus (228) Google Scholar, 32Kensler T.W. Curphey T.J. Maxiutenko Y. Roebuck B.D. Drug Metabol. Drug Interact. 2000; 17: 3-22Crossref PubMed Scopus (122) Google Scholar). Although Nrf2 in gene expression is well the and involved in Nrf2 are as (33Itoh K. Wakabayashi N. Katoh Y. Ishii T. Igarashi K. Engel J.D. Yamamoto M. Genes Dev. 1999; 13: 76-86Crossref PubMed Scopus (2917) Google demonstrated that Nrf2 is normally from the nucleus by association with the Keap1. response to or electrophiles, Nrf2 is liberated from Keap1, whereupon it translocates to the with an b-zip and binds to ARE sequences in the promoter of responsive genes. the of Nrf2 in the activation of gene expression exposure to oxidative or electrophilic a of the nature and of the Keap1·Nrf2 an the Kelch Keap1 to a family of proteins a BTB/POZ domain the and Kelch repeats the J. R. Cooley L. Cell Biol. 2000; 10: Full Text Full Text PDF PubMed Scopus Google Scholar). Kelch repeats are involved in binding to M. Rogers R.A. Jiang S. Kim T.A. Gaedigk R. White R.A. Avraham H. Avraham S. Mol. Biol. Cell. 1999; 10: 2361-2375Crossref PubMed Scopus (81) Google Scholar, 36Xue F. Cooley L. Cell. 1993; 72: 681-693Abstract Full Text PDF PubMed Scopus (377) Google Scholar, C. E. E. J. R. 2000; PubMed Scopus Google and other the BTB/POZ domain dimerization as well as the formation of (34Albagli O. Dhordain P. Deweindt C. Lecocq G. Leprince D. Cell Growth Differ. 1995; 6: 1193-1198PubMed Google Scholar, Cooley L. J. Cell Biol. 1997; PubMed Scopus Google Scholar, R. Genes Dev. 1994; PubMed Scopus Google Scholar, E. A. S. J. F. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). Itoh (33Itoh K. Wakabayashi N. Katoh Y. Ishii T. Igarashi K. Engel J.D. Yamamoto M. Genes Dev. 1999; 13: 76-86Crossref PubMed Scopus (2917) Google that Keap1 and Nrf2 via the Kelch and of the proteins, the Kelch of the Keap1 protein is also for the complex to the specific molecular of the association have not been to serine to in the Keap1 protein is conserved in proteins from both the family and members of the transcription factor family Engel Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar, A. S. A. H. J.D. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar, H. R. Cancer Res. 1999; 59: Google Scholar). Mutation of this which is within the core of the BTB/POZ has been to BTB/POZ dimerization Mutation of the serine to an in the transcription factor protein that the BTB/POZ domain an role in A. S. A. H. J.D. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar). a the in the Keap1 BTB/POZ domain (S104A) to the role of BTB/POZ domain in Keap1 protein to repress Nrf2 activation of the not sequester Nrf2 in the cytoplasm and and a with Nrf2 in these data that of this within the BTB/POZ domain reduced the ability of the Keap1 to with Nrf2, that the of the BTB/POZ domain of Keap1 a critical role in Keap1·Nrf2 also the of the Keap1 by wild-type Keap1 and with the the of to sequester Nrf2 not to a association with have that Keap1 proteins the Kelch domain not with the not that the Kelch of Keap1 is for the Keap1·Nrf2 complex to the as results are in with M. J. C. L. K. T. Cell 2002; PubMed Scopus Google demonstrated that Keap1 with and in using this an Kelch domain Kelch of Kelch repeats binding M. J. C. L. K. T. Cell 2002; PubMed Scopus Google Scholar). BTB/POZ domain is an dimerization that (34Albagli O. Dhordain P. Deweindt C. Lecocq G. Leprince D. Cell Growth Differ. 1995; 6: 1193-1198PubMed Google Scholar, R. Genes Dev. 1994; PubMed Scopus Google Scholar, E. A. S. J. F. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, T. Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). that wild-type Keap1 in the Keap1 to or with a wild-type Keap1 in the wild-type the in the proteins the that the within the BTB/POZ domain the ability of the protein to complex Keap1 protein of from wild-type Keap1 or in from the in the of this in in with the dimerization of proteins in the of this Keap1 complex also reduced in with or not agents that are to result in Nrf2 release from Keap1 and induction of ARE-containing genes A.C. Mulcahy R.T. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, R. R. S. Drug 2001; PubMed Scopus Google Scholar). also resulted in the of a a of in the of that induced a conformational change of the its in the to the that dimerization is involved in Nrf2 sequestration in the cytoplasm and that in Keap1 dimerization potential by or exposure to an inducing agent is involved in Nrf2 release. that the wild-type BTB/POZ protein of in a dominant negative to Nrf2 release by with endogenous Keap1 proteins and endogenous Keap1 the protein the to also the conclusion that Keap1 dimerization a critical role in Nrf2 sequestration and release in vivo. of the Keap1·Nrf2 data from is in the with Keap1 binds to the via the Kelch repeats in the of the protein and with a Keap1 through respective BTB/POZ Nrf2 is in the cytoplasm through its domain and Kelch of the dimerized Keap1 of the Keap1·Nrf2 complex is Keap1 dimerization via the BTB/POZ of the Keap1·Nrf2 association is as is as in in is to one by and Cooley Cooley L. J. Cell Biol. 1997; PubMed Scopus Google to the role of the Kelch protein in the of dimerization Kelch to via respective Kelch that Nrf2 formation of a Keap1 complex that Nrf2 to the as in other molecular also with the that Keap1 dimerization a required common dimerization of BTB/POZ is to that Engel Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar, H. R. Cancer Res. 1999; 59: Google it is also that proteins might contribute to formation of the complex data not such a the BTB/POZ dimerization function of the Keap1 protein is a for complex formation and Nrf2 response to inducing agents Nrf2 is from Keap1 and is free to to the of the complex or and the ability of BTB/POZ proteins to Nrf2 release in that of Keap1 dimerization the trigger for Nrf2 release. the nature of the that are as that of and protein kinases and hypothesized that an or phosphorylation mediated Keap1 complex and Nrf2 release. experiments that Keap1 complex in through an and an or phosphorylation as the Mulcahy R.T. Biochem. Biophys. Res. 2000; PubMed Scopus Google Scholar). kinases such as protein kinase T. Pickett C.B. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google and J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, Kim Mol. Pharmacol. 2000; PubMed Scopus Google have also been implicated in Nrf2 the that kinases activated by exposure the for Keap1 complex and Nrf2 this to to Keap1 complex exposure is that Keap1 a conformational change from thiol of one or more of its reactive support of this M.A. Talalay P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: PubMed Scopus Google data a the of various agents and with the data that oxidative/electrophilic of specific in Keap1 the that results in Keap1 complex and Nrf2 release. the experiments to that the dimerization function with the BTB/POZ domain of Keap1 is required for sequestration of Nrf2 in the cytoplasm and that dissociation of Keap1 dimerization Nrf2 release. Although many to these a for the Keap1·Nrf2 a association of critical to the expression of many detoxification genes and to strategies for cancer for with the and Itoh and Yamamoto for Keap1