Arkadia (RING Finger Protein 111) Mediates Sumoylation-Dependent Stabilization of Nrf2 Through K48-Linked Ubiquitination

Background/Aims: The transcription factor Nrf2 is a master regulator of the antioxidant defense system, protecting cells from oxidative damage. We previously reported that the SUMO-targeted E3 ubiquitin ligase (STUbL), RING finger protein 4 (RNF4) accelerated the degradation rate of Nrf2 in promyelocytic leukemia-nuclear body (PML-NB)-enriched fractions and decreased Nrf2-mediated gene transcription. The mechanisms that regulate Nrf2 nuclear levels are poorly understood. In this study, we aim to explore the role of the second mammalian STUbL, Arkadia/RNF111 on Nrf2. Methods: Arkadia mediated ubiquitination was detected using co-immunoprecipitation assays in which whole cell lysates were immunoprecipated with anti-Nrf2 antibody and Western blotted with anti-hemagglutinin (HA) antibody or anti-Lys-48 ubiquitin-specific antibody. The half-life of Nrf2 was detected in whole cell lysates and promyelocytic leukemia-nuclear body enriched fractions by cycloheximide-chase. Reporter gene assays were performed using the antioxidant response element (ARE)-containing promoter Heme oxygenase-1 (HO-1). Results: We show that Arkadia/RNF111 is able to ubiquitinate Nrf2 resulting in the stabilization of Nrf2. This stabilization was mediated through Lys-48 ubiquitin chains, contrary to traditionally degradative role of Lys-48 ubiquitination, suggesting that Lys-48 ubiquitination of Nrf2 protects Nrf2 from degradation thereby allowing Nrf2-dependent gene transcription. Conclusion: Collectively, these findings highlight a novel mechanism to positively regulate nuclear Nrf2 levels in response to oxidative stress through Arkadia-mediated K48-linked ubiquitination of Nrf2.

[1]  H. Ovaa,et al.  Hybrid Chains: A Collaboration of Ubiquitin and Ubiquitin-Like Modifiers Introducing Cross-Functionality to the Ubiquitin Code , 2020, Frontiers in Chemistry.

[2]  K. Hofmann,et al.  Arkadia/RNF111 is a SUMO-targeted ubiquitin ligase with preference for substrates marked with SUMO1-capped SUMO2/3 chain , 2019, Nature Communications.

[3]  Hongxia Zhu,et al.  MS‐based strategies for identification of protein SUMOylation modification , 2019, Electrophoresis.

[4]  W. Qiu,et al.  Exosomes derived from human umbilical cord mesenchymal stem cells alleviate inflammatory bowel disease in mice through ubiquitination. , 2018, American journal of translational research.

[5]  Nobuhiro Nakamura,et al.  Ubiquitin System , 2018, International journal of molecular sciences.

[6]  W. Birchmeier,et al.  RNF4-Dependent Oncogene Activation by Protein Stabilization , 2016, Cell reports.

[7]  N. Setterblad,et al.  Oxidative stress–induced assembly of PML nuclear bodies controls sumoylation of partner proteins , 2014, The Journal of cell biology.

[8]  R. Dohmen,et al.  SUMO-targeted ubiquitin ligases. , 2014, Biochimica et biophysica acta.

[9]  Sebastian A. Wagner,et al.  RNF111/Arkadia is a SUMO-targeted ubiquitin ligase that facilitates the DNA damage response , 2013, The Journal of cell biology.

[10]  R. Hay.  Decoding the SUMO signal. , 2013, Biochemical Society transactions.

[11]  I. Arinze,et al.  Trafficking of the Transcription Factor Nrf2 to Promyelocytic Leukemia-Nuclear Bodies , 2013, The Journal of Biological Chemistry.

[12]  A. Dejean,et al.  Arkadia, a Novel SUMO-Targeted Ubiquitin Ligase Involved in PML Degradation , 2013, Molecular and Cellular Biology.

[13]  A. Cuadrado,et al.  Nrf2 is controlled by two distinct β-TrCP recognition motifs in its Neh6 domain, one of which can be modulated by GSK-3 activity. , 2012, Oncogene.

[14]  T. Hunter,et al.  Poly-Small Ubiquitin-like Modifier (PolySUMO)-binding Proteins Identified through a String Search* , 2012, The Journal of Biological Chemistry.

[15]  M. Rapé,et al.  The Ubiquitin Code , 2012, Annual review of biochemistry.

[16]  R. Hay,et al.  SUMO-targeted ubiquitin E3 ligase RNF4 is required for the response of human cells to DNA damage. , 2012, Genes & development.

[17]  P. Rada,et al.  SCF/β-TrCP Promotes Glycogen Synthase Kinase 3-Dependent Degradation of the Nrf2 Transcription Factor in a Keap1-Independent Manner , 2011, Molecular and Cellular Biology.

[18]  Jianqi Yang,et al.  Acetylation-Deacetylation of the Transcription Factor Nrf2 (Nuclear Factor Erythroid 2-related Factor 2) Regulates Its Transcriptional Activity and Nucleocytoplasmic Localization* , 2010, The Journal of Biological Chemistry.

[19]  R. Hay,et al.  Arsenic-Induced SUMO-Dependent Recruitment of RNF4 into PML Nuclear Bodies , 2010, Molecular biology of the cell.

[20]  H. de Thé,et al.  PML nuclear bodies. , 2010, Cold Spring Harbor perspectives in biology.

[21]  Donna D. Zhang,et al.  Phosphorylation of Nrf2 at Multiple Sites by MAP Kinases Has a Limited Contribution in Modulating the Nrf2-Dependent Antioxidant Response , 2009, PloS one.

[22]  C. B. Pickett,et al.  The Nrf2-Antioxidant Response Element Signaling Pathway and Its Activation by Oxidative Stress* , 2009, Journal of Biological Chemistry.

[23]  Donna D. Zhang,et al.  Acetylation of Nrf2 by p300/CBP Augments Promoter-Specific DNA Binding of Nrf2 during the Antioxidant Response , 2009, Molecular and Cellular Biology.

[24]  R. Dohmen,et al.  Arsenic trioxide stimulates SUMO‐2/3 modification leading to RNF4‐dependent proteolytic targeting of PML , 2008, FEBS letters.

[25]  John A Tainer,et al.  A SIM-ultaneous role for SUMO and ubiquitin. , 2008, Trends in biochemical sciences.

[26]  M. Lei,et al.  Arsenic degrades PML or PML–RARα through a SUMO-triggered RNF4/ubiquitin-mediated pathway , 2008, Nature Cell Biology.

[27]  M. Tatham,et al.  RNF4 is a poly-SUMO-specific E3 ubiquitin ligase required for arsenic-induced PML degradation , 2008, Nature Cell Biology.

[28]  X. He,et al.  Phosphorylation of Nrf2 in the transcription activation domain by casein kinase 2 (CK2) is critical for the nuclear translocation and transcription activation function of Nrf2 in IMR‐32 neuroblastoma cells , 2008, Journal of biochemical and molecular toxicology.

[29]  Rosa Bernardi,et al.  Structure, dynamics and functions of promyelocytic leukaemia nuclear bodies , 2007, Nature Reviews Molecular Cell Biology.

[30]  Erica S. Johnson,et al.  Ubiquitin-dependent Proteolytic Control of SUMO Conjugates* , 2007, Journal of Biological Chemistry.

[31]  John A Tainer,et al.  SUMO‐targeted ubiquitin ligases in genome stability , 2007, The EMBO journal.

[32]  Konstantinos J. Mavrakis,et al.  Arkadia Induces Degradation of SnoN and c-Ski to Enhance Transforming Growth Factor-β Signaling* , 2007, Journal of Biological Chemistry.

[33]  Shyam Biswal,et al.  Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway. , 2007, Annual review of pharmacology and toxicology.

[34]  T. Hunter,et al.  Conserved function of RNF4 family proteins in eukaryotes: targeting a ubiquitin ligase to SUMOylated proteins. , 2007, Nature Reviews Molecular Cell Biology.

[35]  Pier Paolo Pandolfi,et al.  The mechanisms of PML-nuclear body formation. , 2006, Molecular cell.

[36]  X. He,et al.  Arsenic Induces NAD(P)H-quinone Oxidoreductase I by Disrupting the Nrf2·Keap1·Cul3 Complex and Recruiting Nrf2·Maf to the Antioxidant Response Element Enhancer* , 2006, Journal of Biological Chemistry.

[37]  Peter O'Hare,et al.  Comparison of the SUMO1 and ubiquitin conjugation pathways during the inhibition of proteasome activity with evidence of SUMO1 recycling. , 2005, The Biochemical journal.

[38]  C. B. Pickett,et al.  Nrf2 Controls Constitutive and Inducible Expression of ARE-driven Genes through a Dynamic Pathway Involving Nucleocytoplasmic Shuttling by Keap1* , 2005, Journal of Biological Chemistry.

[39]  Makoto Kobayashi,et al.  Molecular mechanisms activating the Nrf2-Keap1 pathway of antioxidant gene regulation. , 2005, Antioxidants & redox signaling.

[40]  Yue Xiong,et al.  BTB Protein Keap1 Targets Antioxidant Transcription Factor Nrf2 for Ubiquitination by the Cullin 3-Roc1 Ligase , 2005, Molecular and Cellular Biology.

[41]  D. Fushman,et al.  Polyubiquitin chains: polymeric protein signals. , 2004, Current opinion in chemical biology.

[42]  Mark Hannink,et al.  Keap1 Is a Redox-Regulated Substrate Adaptor Protein for a Cul3-Dependent Ubiquitin Ligase Complex , 2004, Molecular and Cellular Biology.

[43]  Masayuki Yamamoto,et al.  Nrf2-Keap1 defines a physiologically important stress response mechanism. , 2004, Trends in molecular medicine.

[44]  Masayuki Yamamoto,et al.  Oxidative Stress Sensor Keap1 Functions as an Adaptor for Cul3-Based E3 Ligase To Regulate Proteasomal Degradation of Nrf2 , 2004, Molecular and Cellular Biology.

[45]  Ken Itoh,et al.  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* , 2004, Journal of Biological Chemistry.

[46]  Takeshi Imamura,et al.  Arkadia amplifies TGF‐β superfamily signalling through degradation of Smad7 , 2003 .

[47]  C. B. Pickett,et al.  Regulatory mechanisms controlling gene expression mediated by the antioxidant response element. , 2003, Annual review of pharmacology and toxicology.

[48]  Randal J. Kaufman,et al.  Nrf2 Is a Direct PERK Substrate and Effector of PERK-Dependent Cell Survival , 2003, Molecular and Cellular Biology.

[49]  James Douglas Engel,et al.  Integration and diversity of the regulatory network composed of Maf and CNC families of transcription factors. , 2002, Gene.

[50]  R. Arkell,et al.  Induction of the mammalian node requires Arkadia function in the extraembryonic lineages , 2001, Nature.

[51]  P. Pandolfi,et al.  Role of SUMO-1-modified PML in nuclear body formation. , 2000, Blood.

[52]  E. Yeh,et al.  Pml Is Critical for Nd10 Formation and Recruits the Pml-Interacting Protein Daxx to This Nuclear Structure When Modified by Sumo-1 , 1999, The Journal of cell biology.

[53]  P. Freemont,et al.  SUMO-1 modification of the acute promyelocytic leukaemia protein PML: implications for nuclear localisation. , 1999, Journal of cell science.

[54]  J. D. Engel,et al.  Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain. , 1999, Genes & development.

[55]  T. Sternsdorf,et al.  Evidence for Covalent Modification of the Nuclear Dot–associated Proteins PML and Sp100 by PIC1/SUMO-1 , 1997, The Journal of cell biology.