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Wilms Tumor Gene on X Chromosome (WTX) Inhibits Degradation of NRF2 Protein through Competitive Binding to KEAP1 Protein*

  • Nathan D. Camp
    Affiliations
    Howard Hughes Medical Institute, Department of Pharmacology, Institute for Stem Cell and Regenerative Medicine, University of Washington School of Medicine, Seattle, Washington 98195
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  • Richard G. James
    Affiliations
    Howard Hughes Medical Institute, Department of Pharmacology, Institute for Stem Cell and Regenerative Medicine, University of Washington School of Medicine, Seattle, Washington 98195
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  • David W. Dawson
    Affiliations
    Department of Pathology and Laboratory Medicine, Jonsson Comprehensive Cancer Center, David Geffen School of Medicine, UCLA, Los Angeles, California 90095-1732
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  • Feng Yan
    Affiliations
    Department of Cell and Developmental Biology, Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599
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  • James M. Davison
    Affiliations
    Department of Cell and Developmental Biology, Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599
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  • Scott A. Houck
    Affiliations
    Department of Cell and Developmental Biology, Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599
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  • Xiaobo Tang
    Affiliations
    Howard Hughes Medical Institute, Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington 98195
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  • Ning Zheng
    Footnotes
    Affiliations
    Howard Hughes Medical Institute, Department of Pharmacology, University of Washington School of Medicine, Seattle, Washington 98195
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  • Michael B. Major
    Correspondence
    To whom correspondence may be addressed: 450 West Dr., Rm. 31-351, Chapel Hill, NC 27599. Tel.: 919-259-2695
    Affiliations
    Department of Cell and Developmental Biology, Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599
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  • Randall T. Moon
    Correspondence
    To whom correspondence may be addressed: 815 Mercer St., Rm. S524, Seattle, WA 98109-358056. Tel.: 206-543-1722
    Footnotes
    Affiliations
    Howard Hughes Medical Institute, Department of Pharmacology, Institute for Stem Cell and Regenerative Medicine, University of Washington School of Medicine, Seattle, Washington 98195
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant T32 GM07270 from USPHS and NRSA (to N. D. C.).
    This article contains supplemental Figs. S1–S3.
    1 Investigator of the Howard Hughes Medical Institute.
Open AccessPublished:January 03, 2012DOI:https://doi.org/10.1074/jbc.M111.316471
      WTX is a tumor suppressor protein that is lost or mutated in up to 30% of cases of Wilms tumor. Among its known functions, WTX interacts with the β-transducin repeat containing family of ubiquitin ligase adaptors and promotes the ubiquitination and degradation of the transcription factor β-catenin, a key control point in the WNT/β-catenin signaling pathway. Here, we report that WTX interacts with a second ubiquitin ligase adaptor, KEAP1, which functions to regulate the ubiquitination of the transcription factor NRF2, a key control point in the antioxidant response. Surprisingly, we find that unlike its ability to promote the ubiquitination of β-catenin, WTX inhibits the ubiquitination of NRF2. WTX and NRF2 compete for binding to KEAP1, and thus loss of WTX leads to rapid ubiquitination and degradation of NRF2 and a reduced response to cytotoxic insult. These results expand our understanding of the molecular mechanisms of WTX and reveal a novel regulatory mechanism governing the antioxidant response.

      Introduction

      FAM123B/WTX/AMER1 (hereafter referred to as WTX) is located on the X chromosome and encodes a tumor suppressor protein that is lost or mutated in up to 30% of the cases of Wilms tumor, the most common pediatric kidney cancer (
      • Rivera M.N.
      • Kim W.J.
      • Wells J.
      • Driscoll D.R.
      • Brannigan B.W.
      • Han M.
      • Kim J.C.
      • Feinberg A.P.
      • Gerald W.L.
      • Vargas S.O.
      • Chin L.
      • Iafrate A.J.
      • Bell D.W.
      • Haber D.A.
      An X chromosome gene, WTX, is commonly inactivated in Wilms tumor.
      ,
      • Perotti D.
      • Gamba B.
      • Sardella M.
      • Spreafico F.
      • Terenziani M.
      • Collini P.
      • Pession A.
      • Nantron M.
      • Fossati-Bellani F.
      • Radice P.
      Functional inactivation of the WTX gene is not a frequent event in Wilms' tumors.
      ,
      • Ruteshouser E.C.
      • Robinson S.M.
      • Huff V.
      Wilms tumor genetics. Mutations in WT1, WTX, and CTNNB1 account for only about one-third of tumors.
      ). Recently, germ line mutations in WTX were also discovered in families suffering from osteopathia striata congenital with cranial sclerosis (OSCS),
      The abbreviations used are: OSCS
      osteopathia striata congenital with cranial sclerosis
      APC
      adenomatous polyposis coli
      ARE
      antioxidant-response element
      BTRC
      β-transducin repeat containing
      tBHQ
      tert-butylhydroquinone.
      a debilitating skeletal dysplasia that is often accompanied by developmental abnormalities and fatality in males (
      • Perdu B.
      • de Freitas F.
      • Frints S.G.
      • Schouten M.
      • Schrander-Stumpel C.
      • Barbosa M.
      • Pinto-Basto J.
      • Reis-Lima M.
      • de Vernejoul M.C.
      • Becker K.
      • Freckmann M.L.
      • Keymolen K.
      • Haan E.
      • Savarirayan R.
      • Koenig R.
      • Zabel B.
      • Vanhoenacker F.M.
      • Van Hul W.
      Osteopathia striata with cranial sclerosis owing to WTX gene defect.
      ,
      • Jenkins Z.A.
      • van Kogelenberg M.
      • Morgan T.
      • Jeffs A.
      • Fukuzawa R.
      • Pearl E.
      • Thaller C.
      • Hing A.V.
      • Porteous M.E.
      • Garcia-Miñaur S.
      • Bohring A.
      • Lacombe D.
      • Stewart F.
      • Fiskerstrand T.
      • Bindoff L.
      • Berland S.
      • Adès L.C.
      • Tchan M.
      • David A.
      • Wilson L.C.
      • Hennekam R.C.
      • Donnai D.
      • Mansour S.
      • Cormier-Daire V.
      • Robertson S.P.
      Germ line mutations in WTX cause a sclerosing skeletal dysplasia but do not predispose to tumorigenesis.
      ).
      We previously reported that WTX regulates the stability of β-catenin (
      • Major M.B.
      • Camp N.D.
      • Berndt J.D.
      • Yi X.
      • Goldenberg S.J.
      • Hubbert C.
      • Biechele T.L.
      • Gingras A.C.
      • Zheng N.
      • Maccoss M.J.
      • Angers S.
      • Moon R.T.
      Wilms tumor suppressor WTX negatively regulates WNT/β-catenin signaling.
      ). The regulation of the stability of β-catenin is a key control point of the WNT/β-catenin signaling pathway and the broader protein networks with which it interacts (
      • van Amerongen R.
      • Mikels A.
      • Nusse R.
      Alternative wnt signaling is initiated by distinct receptors.
      ). In the absence of WNT ligand, β-catenin is phosphorylated by a multiprotein complex often called the “destruction complex” and is subsequently recognized by the SCFBTRC (Skp, Cullin, F-box) ubiquitin ligase complex where it is ubiquitinated and targeted for proteasomal degradation (
      • Yost C.
      • Torres M.
      • Miller J.R.
      • Huang E.
      • Kimelman D.
      • Moon R.T.
      The axis-inducing activity, stability, and subcellular distribution of β-catenin is regulated in Xenopus embryos by glycogen synthase kinase 3.
      ,
      • Winston J.T.
      • Strack P.
      • Beer-Romero P.
      • Chu C.Y.
      • Elledge S.J.
      • Harper J.W.
      The SCFβ-TRCP-ubiquitin ligase complex associates specifically with phosphorylated destruction motifs in IκBα and β-catenin and stimulates IκBα ubiquitination in vitro.
      ,
      • Liu C.
      • Kato Y.
      • Zhang Z.
      • Do V.M.
      • Yankner B.A.
      • He X.
      β-Trcp couples β-catenin phosphorylation-degradation and regulates Xenopus axis formation.
      ). In the presence of WNT ligand, phosphorylation of β-catenin is attenuated, resulting in the nuclear accumulation of β-catenin and the regulation of transcription. Through proteomic and functional dissection of the WNT/β-catenin signaling pathway, we discovered that WTX associates with β-catenin as well as proteins in the destruction complex, including adenomatous polyposis coli (APC), AXIN1, BTRC (commonly referred to as β-TrCP), and FBXW11 (commonly referred to as β-TrCP2) (
      • Major M.B.
      • Camp N.D.
      • Berndt J.D.
      • Yi X.
      • Goldenberg S.J.
      • Hubbert C.
      • Biechele T.L.
      • Gingras A.C.
      • Zheng N.
      • Maccoss M.J.
      • Angers S.
      • Moon R.T.
      Wilms tumor suppressor WTX negatively regulates WNT/β-catenin signaling.
      ). Although the precise mechanism(s) is unknown, WTX promotes the ubiquitination and degradation of β-catenin.
      In addition to regulating the stability of β-catenin, WTX has also been shown to play a role in regulating WNT signal transduction at the membrane (
      • Tanneberger K.
      • Pfister A.S.
      • Brauburger K.
      • Schneikert J.
      • Hadjihannas M.V.
      • Kriz V.
      • Schulte G.
      • Bryja V.
      • Behrens J.
      Amer1/WTX couples Wnt-induced formation of PtdIns(4,5)P2 to LRP6 phosphorylation.
      ). Furthermore, WTX controls cell-cell adhesion through interactions with APC at the plasma membrane (
      • Grohmann A.
      • Tanneberger K.
      • Alzner A.
      • Schneikert J.
      • Behrens J.
      AMER1 regulates the distribution of the tumor suppressor APC between microtubules and the plasma membrane.
      ) and modulates the activity of the WT1 (Wilms tumor 1) transcription factor in the nucleus (
      • Rivera M.N.
      • Kim W.J.
      • Wells J.
      • Stone A.
      • Burger A.
      • Coffman E.J.
      • Zhang J.
      • Haber D.A.
      The tumor suppressor WTX shuttles to the nucleus and modulates WT1 activity.
      ). Thus, it is possible that the loss of WTX contributes to disease through distinct mechanisms in specific subcellular compartments.
      Our previous analysis of the WTX protein interaction network identified an association between WTX and KEAP1 (Kelch-like ECH-associated protein 1) (
      • Major M.B.
      • Camp N.D.
      • Berndt J.D.
      • Yi X.
      • Goldenberg S.J.
      • Hubbert C.
      • Biechele T.L.
      • Gingras A.C.
      • Zheng N.
      • Maccoss M.J.
      • Angers S.
      • Moon R.T.
      Wilms tumor suppressor WTX negatively regulates WNT/β-catenin signaling.
      ). KEAP1 is a substrate recognition module for the CUL3-based E3 ubiquitin ligase that constitutively ubiquitinates the transcription factor NRF2 (NF-E2-related factor-2; NFE2L2) (
      • Cullinan S.B.
      • Gordan J.D.
      • Jin J.
      • Harper J.W.
      • Diehl J.A.
      The Keap1-BTB protein is an adaptor that bridges Nrf2 to a Cul3-based E3 ligase. Oxidative stress sensing by a Cul3-Keap1 ligase.
      ,
      • Kobayashi A.
      • Kang M.I.
      • Okawa H.
      • Ohtsuji M.
      • Zenke Y.
      • Chiba T.
      • Igarashi K.
      • Yamamoto M.
      Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2.
      ,
      • Zhang D.D.
      • Lo S.C.
      • Cross J.V.
      • Templeton D.J.
      • Hannink M.
      Keap1 is a redox-regulated substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex.
      ). In the presence of cytotoxic stress such as xenobiotics and antioxidants, KEAP1 is inhibited, and NRF2 is no longer targeted for ubiquitination and degradation. NRF2 then accumulates in the nucleus where it regulates transcription of genes involved in the “phase II” antioxidant response (
      • Kensler T.W.
      • Wakabayashi N.
      • Biswal S.
      Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway.
      ,
      • Hayes J.D.
      • McMahon M.
      NRF2 and KEAP1 mutations. Permanent activation of an adaptive response in cancer.
      ,
      • Venugopal R.
      • Jaiswal A.K.
      Nrf1 and Nrf2 positively and c-Fos and Fra1 negatively regulate the human antioxidant response element-mediated expression of NAD(P)H:quinone oxidoreductase1 gene.
      ).
      Although many studies have reported that small molecules and stressors regulate NRF2 stability and function, relatively little is known about regulation of KEAP1 and NRF2 through protein-protein interactions. Here, we report that WTX directly binds to KEAP1. Using gain-of-function and loss-of-function approaches, we found that WTX stabilizes NRF2 and positively regulates its transcriptional activity. We identified an ETGE motif within the KEAP1 interacting domain of WTX that is nearly identical to that of NRF2. We show that this motif is not only required for the interaction of WTX with KEAP1 but is also required for the ability of WTX to regulate NRF2 stability and activity. Our observations support a model whereby WTX competes with NRF2 for binding to KEAP1, thereby promoting the NRF2-mediated antioxidant response.

      DISCUSSION

      The WTX gene encodes a tumor suppressor protein; it is located on the X chromosome and is somatically lost or mutated in 7–30% of cases of Wilms tumor (
      • Rivera M.N.
      • Kim W.J.
      • Wells J.
      • Driscoll D.R.
      • Brannigan B.W.
      • Han M.
      • Kim J.C.
      • Feinberg A.P.
      • Gerald W.L.
      • Vargas S.O.
      • Chin L.
      • Iafrate A.J.
      • Bell D.W.
      • Haber D.A.
      An X chromosome gene, WTX, is commonly inactivated in Wilms tumor.
      ,
      • Perotti D.
      • Gamba B.
      • Sardella M.
      • Spreafico F.
      • Terenziani M.
      • Collini P.
      • Pession A.
      • Nantron M.
      • Fossati-Bellani F.
      • Radice P.
      Functional inactivation of the WTX gene is not a frequent event in Wilms' tumors.
      ,
      • Ruteshouser E.C.
      • Robinson S.M.
      • Huff V.
      Wilms tumor genetics. Mutations in WT1, WTX, and CTNNB1 account for only about one-third of tumors.
      ). Germ line mutations in WTX give rise to OSCS, a debilitating and fatal disease that largely affects the skeletal system (
      • Perdu B.
      • de Freitas F.
      • Frints S.G.
      • Schouten M.
      • Schrander-Stumpel C.
      • Barbosa M.
      • Pinto-Basto J.
      • Reis-Lima M.
      • de Vernejoul M.C.
      • Becker K.
      • Freckmann M.L.
      • Keymolen K.
      • Haan E.
      • Savarirayan R.
      • Koenig R.
      • Zabel B.
      • Vanhoenacker F.M.
      • Van Hul W.
      Osteopathia striata with cranial sclerosis owing to WTX gene defect.
      ,
      • Jenkins Z.A.
      • van Kogelenberg M.
      • Morgan T.
      • Jeffs A.
      • Fukuzawa R.
      • Pearl E.
      • Thaller C.
      • Hing A.V.
      • Porteous M.E.
      • Garcia-Miñaur S.
      • Bohring A.
      • Lacombe D.
      • Stewart F.
      • Fiskerstrand T.
      • Bindoff L.
      • Berland S.
      • Adès L.C.
      • Tchan M.
      • David A.
      • Wilson L.C.
      • Hennekam R.C.
      • Donnai D.
      • Mansour S.
      • Cormier-Daire V.
      • Robertson S.P.
      Germ line mutations in WTX cause a sclerosing skeletal dysplasia but do not predispose to tumorigenesis.
      ). As the discovery of WTX and its mutations in human disease is relatively new, the molecular function(s) and developmental or homeostatic consequences of its loss is only beginning to be unraveled. Compelling data from numerous research groups have described functions for WTX in controlling WNT/β-catenin signaling, cell-cell adhesion, apoptosis, and transcription (
      • Rivera M.N.
      • Kim W.J.
      • Wells J.
      • Driscoll D.R.
      • Brannigan B.W.
      • Han M.
      • Kim J.C.
      • Feinberg A.P.
      • Gerald W.L.
      • Vargas S.O.
      • Chin L.
      • Iafrate A.J.
      • Bell D.W.
      • Haber D.A.
      An X chromosome gene, WTX, is commonly inactivated in Wilms tumor.
      ,
      • Major M.B.
      • Camp N.D.
      • Berndt J.D.
      • Yi X.
      • Goldenberg S.J.
      • Hubbert C.
      • Biechele T.L.
      • Gingras A.C.
      • Zheng N.
      • Maccoss M.J.
      • Angers S.
      • Moon R.T.
      Wilms tumor suppressor WTX negatively regulates WNT/β-catenin signaling.
      ,
      • Tanneberger K.
      • Pfister A.S.
      • Brauburger K.
      • Schneikert J.
      • Hadjihannas M.V.
      • Kriz V.
      • Schulte G.
      • Bryja V.
      • Behrens J.
      Amer1/WTX couples Wnt-induced formation of PtdIns(4,5)P2 to LRP6 phosphorylation.
      ,
      • Grohmann A.
      • Tanneberger K.
      • Alzner A.
      • Schneikert J.
      • Behrens J.
      AMER1 regulates the distribution of the tumor suppressor APC between microtubules and the plasma membrane.
      ,
      • Rivera M.N.
      • Kim W.J.
      • Wells J.
      • Stone A.
      • Burger A.
      • Coffman E.J.
      • Zhang J.
      • Haber D.A.
      The tumor suppressor WTX shuttles to the nucleus and modulates WT1 activity.
      ). Recently, characterization of Wtx deletion in mice revealed that Wtx regulates mesenchymal progenitor cell fate specification in part through β-catenin (
      • Moisan A.
      • Rivera M.N.
      • Lotinun S.
      • Akhavanfard S.
      • Coffman E.J.
      • Cook E.B.
      • Stoykova S.
      • Mukherjee S.
      • Schoonmaker J.A.
      • Burger A.
      • Kim W.J.
      • Kronenberg H.M.
      • Baron R.
      • Haber D.A.
      • Bardeesy N.
      The WTX tumor suppressor regulates mesenchymal progenitor cell fate specification.
      ).
      Here, we expand on the knowledge of the molecular mechanisms of action of WTX with the observation that WTX regulates the steady-state levels of NRF2 and NRF2-dependent transcription by competing with NRF2 for binding to KEAP1. KEAP1 forms a homodimer and interacts with CUL3 through its BTB domain (
      • Ogura T.
      • Tong K.I.
      • Mio K.
      • Maruyama Y.
      • Kurokawa H.
      • Sato C.
      • Yamamoto M.
      Keap1 is a forked-stem dimer structure with two large spheres enclosing the intervening double glycine repeat and C-terminal domains.
      ,
      • Zipper L.M.
      • Mulcahy R.T.
      The Keap1 BTB/POZ dimerization function is required to sequester Nrf2 in cytoplasm.
      ) and interacts with the N-terminal Neh2 domain of a single NRF2 molecule through its KELCH repeats. The Neh2 domain of NRF2 contains two highly conserved regions, one bearing an LXXQDXDLG (DLG) motif (
      • McMahon M.
      • Thomas N.
      • Itoh K.
      • Yamamoto M.
      • Hayes J.D.
      Redox-regulated turnover of Nrf2 is determined by at least two separate protein domains, the redox-sensitive Neh2 degron and the redox-insensitive Neh6 degron.
      ,
      • Katoh Y.
      • Iida K.
      • Kang M.I.
      • Kobayashi A.
      • Mizukami M.
      • Tong K.I.
      • McMahon M.
      • Hayes J.D.
      • Itoh K.
      • Yamamoto M.
      Evolutionary conserved N-terminal domain of Nrf2 is essential for the Keap1-mediated degradation of the protein by proteasome.
      ) and the other bearing a DXETGE motif (
      • Kobayashi M.
      • Itoh K.
      • Suzuki T.
      • Osanai H.
      • Nishikawa K.
      • Katoh Y.
      • Takagi Y.
      • Yamamoto M.
      Identification of the interactive interface and phylogenic conservation of the Nrf2-Keap1 system.
      ). These motifs interact with a separate KEAP1 molecule in the KEAP1 homodimer, although the DXETGE motif binds with ∼100-fold higher affinity (
      • Tong K.I.
      • Katoh Y.
      • Kusunoki H.
      • Itoh K.
      • Tanaka T.
      • Yamamoto M.
      Keap1 recruits Neh2 through binding to ETGE and DLG motifs. Characterization of the two-site molecular recognition model.
      ). WTX contains a similar SPETGE motif that can directly inhibit the interaction between NRF2 and the KELCH repeats of KEAP1 in vitro. As WTX does not contain a DLG motif similar to NRF2, it is possible that NRF2 can still interact with a KEAP1 dimer through this low affinity interaction motif. We hypothesize that WTX enhances NRF2 steady-state levels by disrupting the conformation of the E3 ubiquitin ligase complex, resulting in lower ubiquitination of NRF2. In support of our findings, expression of the NRF2 target genes NQO1 and HMOX1 was significantly enhanced in HEK293T cells overexpressing a fragment of WTX containing the KEAP1 interaction domain (
      • Kim M.K.
      • Min D.J.
      • Rabin M.
      • Licht J.D.
      Functional characterization of Wilms tumor-suppressor WTX and tumor-associated mutants.
      ).
      In addition to the ETGE motif, we determined that serine 286 is also required for the ability of WTX to interact with KEAP1 and regulate NRF2. Interestingly, mutation of serine 286 to either glutamic or aspartic acid enhanced the functional effects of WTX. Although these mutations may only present conformational changes in WTX that make it more suitable for binding to KEAP1, both glutamic and aspartic acid resemble phosphorylated serine. Thus, phosphorylation of WTX at serine 286 may increase its affinity for KEAP1. Furthermore, we found that WTX is phosphorylated in vivo, and KEAP1 interacts with endogenous WTX that is phosphorylated at serine 286, raising the intriguing possibility that the interaction between WTX and KEAP1 is regulated by a yet to be identified kinase(s).
      Under normal conditions, NRF2 is constitutively ubiquitinated through its association with KEAP1 (
      • Cullinan S.B.
      • Gordan J.D.
      • Jin J.
      • Harper J.W.
      • Diehl J.A.
      The Keap1-BTB protein is an adaptor that bridges Nrf2 to a Cul3-based E3 ligase. Oxidative stress sensing by a Cul3-Keap1 ligase.
      ,
      • Kobayashi A.
      • Kang M.I.
      • Okawa H.
      • Ohtsuji M.
      • Zenke Y.
      • Chiba T.
      • Igarashi K.
      • Yamamoto M.
      Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2.
      ,
      • Zhang D.D.
      • Lo S.C.
      • Cross J.V.
      • Templeton D.J.
      • Hannink M.
      Keap1 is a redox-regulated substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex.
      ). In the presence of oxidative stress, cysteine residues in KEAP1 are modified resulting in a conformational change that disrupts the ubiquitination of NRF2, and NRF2 accumulates in the nucleus where it regulates gene transcription (
      • Zhang D.D.
      • Lo S.C.
      • Cross J.V.
      • Templeton D.J.
      • Hannink M.
      Keap1 is a redox-regulated substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex.
      ,
      • Yamamoto T.
      • Suzuki T.
      • Kobayashi A.
      • Wakabayashi J.
      • Maher J.
      • Motohashi H.
      • Yamamoto M.
      Physiological significance of reactive cysteine residues of Keap1 in determining Nrf2 activity.
      ). In addition to our findings, several recent studies have also revealed that NRF2 signaling is regulated through protein-protein interactions. The cyclin-dependent kinase inhibitor p21, a p53-regulated gene with pro-survival properties, was recently shown to bind the DLG motif of NRF2 and inhibit its interaction with KEAP1, resulting in elevated NRF2 levels under both basal and chemically induced conditions (
      • Chen W.
      • Sun Z.
      • Wang X.J.
      • Jiang T.
      • Huang Z.
      • Fang D.
      • Zhang D.D.
      Direct interaction between Nrf2 and p21(Cip1/WAF1) up-regulates the Nrf2-mediated antioxidant response.
      ). Another recent study identified p62 as a novel interactor of KEAP1 (
      • Komatsu M.
      • Kurokawa H.
      • Waguri S.
      • Taguchi K.
      • Kobayashi A.
      • Ichimura Y.
      • Sou Y.S.
      • Ueno I.
      • Sakamoto A.
      • Tong K.I.
      • Kim M.
      • Nishito Y.
      • Iemura S.
      • Natsume T.
      • Ueno T.
      • Kominami E.
      • Motohashi H.
      • Tanaka K.
      • Yamamoto M.
      The selective autophagy substrate p62 activates the stress-responsive transcription factor Nrf2 through inactivation of Keap1.
      ). During autophagy, p62 directs ubiquitinated proteins to degradation by the lysosome. By binding to KEAP1 through an ETGE-like motif similar to that of NRF2 and WTX, p62 targets KEAP1 for autophagic degradation, thus contributing to the stabilization of NRF2. Combined with WTX, these examples highlight the complex regulation of NRF2 degradation and its importance in both homeostasis and response to cytotoxic stress.
      Whereas WTX inhibits the ubiquitination of NRF2, we previously reported that WTX promotes the ubiquitination of β-catenin by the SCFBTRC ubiquitin ligase complex. To our knowledge, WTX is the first described protein to interact with two E3 ubiquitin ligase adaptors and have opposite regulatory effects on their respective substrates. How is this possible? WTX interacts with the KELCH repeats that form the β-propeller fold of KEAP1 and likely inhibits the formation of a functional CUL3-KEAP1-NRF2 complex. Through a separable domain, WTX interacts with both BTRC and β-catenin, suggesting that it binds to an intact SCFBTRC-substrate complex (
      • Major M.B.
      • Camp N.D.
      • Berndt J.D.
      • Yi X.
      • Goldenberg S.J.
      • Hubbert C.
      • Biechele T.L.
      • Gingras A.C.
      • Zheng N.
      • Maccoss M.J.
      • Angers S.
      • Moon R.T.
      Wilms tumor suppressor WTX negatively regulates WNT/β-catenin signaling.
      ). BTRC also employs a β-propeller fold for substrate capture (
      • Wu G.
      • Xu G.
      • Schulman B.A.
      • Jeffrey P.D.
      • Harper J.W.
      • Pavletich N.P.
      Structure of a β-TrCP1-Skp1-β-catenin complex. Destruction motif binding and lysine specificity of the SCF(β-TrCP1) ubiquitin ligase.
      ), but it is unlikely that WTX interacts with the β-propeller fold of BTRC in a similar fashion as KEAP1 as this would inhibit the formation of a functional CUL1-SKP1-BTRC-β-catenin complex and result in elevated β-catenin levels. Interestingly, a recent study demonstrated that NRF2 is phosphorylated by GSK3β in the central Neh6 domain (DSGIS, residues 334–338), creating an SCFBTRC destruction motif similar to that of β-catenin (
      • Rada P.
      • Rojo A.I.
      • Chowdhry S.
      • McMahon M.
      • Hayes J.D.
      • Cuadrado A.
      SCF/β-TrCP promotes glycogen synthase kinase 3-dependent degradation of the Nrf2 transcription factor in a Keap1-independent manner.
      ). This phosphorylated form of NRF2 is recognized by BTRC. Additionally, KEAP1 and BTRC have been identified in the same complex by mass spectrometry (
      • Sowa M.E.
      • Bennett E.J.
      • Gygi S.P.
      • Harper J.W.
      Defining the human deubiquitinating enzyme interaction landscape.
      ). This raises the possibility that multiple E3 ubiquitin ligase complexes consisting of a KEAP1 homodimer, a BTRC homodimer, or a KEAP1/BTRC heterodimer regulate the ubiquitination of NRF2. As WTX interacts with BTRC and KEAP1 through separable domains, we hypothesize that WTX coordinates the adaptors in the E3 ubiquitin ligase complexes, resulting in variable ubiquitination of substrates.
      Although most commonly lost through gene-encompassing deletions, a small percentage of WTX mutations identified in Wilms tumor yield single amino acid substitutions and truncated proteins. Conversely, the majority of WTX mutations identified in OSCS yield truncated proteins. Aligning the location of these mutations with the WTX protein interaction domains suggests relationships of these diseases with specific binding interfaces. The KEAP1 interaction domain lies N-terminal to the domains that bind β-catenin/BTRC/APC and WT1 and remains intact in 9 out of the 20 reported mutation products. Of note, we determined that the K292N substitution inhibited the association of WTX and KEAP1. The β-catenin/BTRC/APC interacting domains encompass residues 280–839. Of the 20 reported mutations, 11 are predicted to alter these binding activities. As the C terminus of WTX binds WT1, this interaction is lost in all truncation products derived from mutations in WTX. These correlations suggest that whereas WT1 is likely central to WTX-associated diseases, the WTX-KEAP1 and WTX-β-catenin/BTRC/APC functional relationships may contribute to a subset of Wilms tumor and OSCS, perhaps accounting for variability in disease onset or progression.
      The observation that WTX regulates the NRF2-mediated antioxidant response supports further study of its role in disease beyond Wilms tumor and OSCS. One of the best-studied diseases with altered KEAP1/NRF2 activity is non-small cell lung cancer, where mutations in KEAP1 lead to constitutive NRF2-mediated transcription. Consequently, cultured lung cancer cell lines with constitutive NRF2 activity are resistant to cell death induced by etoposide (
      • Singh A.
      • Misra V.
      • Thimmulappa R.K.
      • Lee H.
      • Ames S.
      • Hoque M.O.
      • Herman J.G.
      • Baylin S.B.
      • Sidransky D.
      • Gabrielson E.
      • Brock M.V.
      • Biswal S.
      Dysfunctional KEAP1-NRF2 interaction in non-small cell lung cancer.
      ). Similarly, silencing NRF2 restores resistance to chemotherapeutics in KEAP1 mutant cells (
      • Homma S.
      • Ishii Y.
      • Morishima Y.
      • Yamadori T.
      • Matsuno Y.
      • Haraguchi N.
      • Kikuchi N.
      • Satoh H.
      • Sakamoto T.
      • Hizawa N.
      • Itoh K.
      • Yamamoto M.
      Nrf2 enhances cell proliferation and resistance to anticancer drugs in human lung cancer.
      ). We find that silencing WTX sensitizes HEK293T cells to death induced by etoposide, similar to loss of NRF2. Interestingly, although WTX expression is diminished in adult mouse brain and kidney compared with embryonic tissues, expression levels remain high in the lung, suggesting it may contribute to lung cell homeostasis in the adult (
      • Rivera M.N.
      • Kim W.J.
      • Wells J.
      • Driscoll D.R.
      • Brannigan B.W.
      • Han M.
      • Kim J.C.
      • Feinberg A.P.
      • Gerald W.L.
      • Vargas S.O.
      • Chin L.
      • Iafrate A.J.
      • Bell D.W.
      • Haber D.A.
      An X chromosome gene, WTX, is commonly inactivated in Wilms tumor.
      ). Coupled with the established roles of WTX in Wilms tumor and KEAP1/NRF2 in lung cancer, our data support future investigations into a functional role for WTX in lung cancer and other diseases associated with aberrant NRF2 activity.

      Acknowledgments

      We thank Priscila Siesser for unpublished data, Jeffrey Johnson and Seth Goldenberg for reagents, and other members of the Moon laboratory and Major laboratory for helpful discussions.

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