Abstract
The nuclear factor (NF)-κB transcription factor has essential roles in inflammation and oncogenesis. Its ubiquitous RelA subunit is regulated by several post-translational modifications, including phosphorylation, ubiquitination and acetylation. Ubiquitination promotes the termination of RelA-dependent transcription, but its regulation is incompletely understood. Through mass spectrometry analysis of ubiquitinated RelA, we identified seven lysines that were attached to degradative and non-degradative forms of polyubiquitin. Interestingly, lysines targeted for acetylation were among the residues identified as ubiquitin acceptor sites. Mutation of these particular sites resulted in decreased polyubiquitination. Acetylation and ubiquitination were found to inhibit each other, consistent with their use of overlapping sites. Reconstitution of rela−/− fibroblasts with wild-type and mutant forms of RelA revealed that modifications at these residues can have activating and inhibitory functions depending on the target gene context. Altogether, this study elucidates that ubiquitination and acetylation can modulate each other and regulate nuclear NF-κB function in a gene-specific manner.
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References
Baeuerle PA, Baltimore D . (1988). IκB: a specific inhibitor of the NF-κB transcription factor. Science 242: 540–546.
Bassères DS, Baldwin AS . (2006). Nuclear factor-κB and inhibitor of κB kinase pathways in oncogenic initiation and progression. Oncogene 25: 6817–6830.
Buerki C, Rothgiesser KM, Valovka T, Owen HR, Rehrauer H, Fey M et al. (2008). Functional relevance of novel p300-mediated lysine 314 and 315 acetylation of RelA/p65. Nucleic Acids Res 36: 1665–1680.
Burstein E, Ganesh L, Dick RD, van De Sluis B, Wilkinson JC, Klomp LW et al. (2004). A novel role for XIAP in copper homeostasis through regulation of MURR1. EMBO J 23: 244–254.
Burstein E, Hoberg JE, Wilkinson AS, Rumble JM, Csomos RA, Komarck CM et al. (2005). COMMD proteins: a novel family of structural and functional homologs of MURR1. J Biol Chem 280: 22222–22232.
Chen FE, Huang DB, Chen YQ, Ghosh G . (1998). Crystal structure of p50/p65 heterodimer of transcription factor NF-κB bound to DNA. Nature 391: 410–413.
Chen L, Fischle W, Verdin E, Greene WC . (2001). Duration of nuclear NF-κB action regulated by reversible acetylation. Science 293: 1653–1657.
Chen LF, Mu Y, Greene WC . (2002). Acetylation of RelA at discrete sites regulates distinct nuclear functions of NF-κB. EMBO J 21: 6539–6548.
Chen Z, Hagler J, Palombella VJ, Melandri F, Scherer D, Ballard D et al. (1995). Signal-induced site-specific phosphorylation targets IκBα to the ubiquitin-proteasome pathway. Genes Dev 9: 1586–1597.
Ea CK, Baltimore D . (2009). Regulation of NF-κB activity through lysine monomethylation of p65. Proc Natl Acad Sci USA 106: 18972–18977.
Fan Y, Mao R, Zhao Y, Yu Y, Sun W, Song P et al. (2009). Tumor necrosis factor-α induces RelA degradation via ubiquitination at lysine 195 to prevent excessive nuclear factor-κB activation. J Biol Chem 284: 29290–29297.
Geng H, Wittwer T, Dittrich-Breiholz O, Kracht M, Schmitz ML . (2009). Phosphorylation of NF-κB p65 at Ser468 controls its COMMD1-dependent ubiquitination and target gene-specific proteasomal elimination. EMBO Rep 10: 381–386.
Gerritsen ME, Williams AJ, Neish AS, Moore S, Shi Y, Collins T . (1997). CREB-binding protein/p300 are transcriptional coactivators of p65. Proc Natl Acad Sci USA 94: 2927–2932.
Hayden MS, Ghosh S . (2008). Shared principles in NF-κB signaling. Cell 132: 344–362.
Henkel T, Machleidt T, Alkalay I, Krönke M, Ben-Neriah Y, Baeuerle PA . (1993). Rapid proteolysis of IκB-α is necessary for activation of transcription factor NF-κB. Nature 365: 182–185.
Hoffmann A, Natoli G, Ghosh G . (2006). Transcriptional regulation via the NF-κB signaling module. Oncogene 25: 6706–6716.
Karin M, Greten FR . (2005). NF-κB: linking inflammation and immunity to cancer development and progression. Nat Rev Immunol 5: 749–759.
Karin M . (2006). Nuclear factor-κB in cancer development and progression. Nature 441: 431–436.
Kiernan R, Bres V, Ng RW, Coudart MP, El Messaoudi S, Sardet C et al. (2003). Post-activation turn-off of NF-κB-dependent transcription is regulated by acetylation of p65. J Biol Chem 278: 2758–2766.
Lawrence T, Bebien M, Liu GY, Nizet V, Karin M . (2005). IKKα limits macrophage NF-κB activation and contributes to the resolution of inflammation. Nature 434: 1138–1143.
Li M, Luo J, Brooks CL, Gu W . (2002). Acetylation of p53 inhibits its ubiquitination by Mdm2. J Biol Chem 277: 50607–50611.
Lois C, Hong EJ, Pease S, Brown EJ, Baltimore D . (2002). Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 295: 868–872.
Maine GN, Gluck N, Zaidi IW, Burstein E . (2009). Bimolecular affinity purification (BAP): tandem affinity purification using two protein baits. Cold Spring Harb Protoc (10.1101/pdb.prot5318).
Maine GN, Li H, Zaidi IW, Basrur V, Elenitoba-Johnson KS, Burstein E . (2010). A bimolecular affinity purification method under denaturing conditions for rapid isolation of a ubiquitinated protein for mass spectrometry analysis. Nat Protoc 5: 1447–1459.
Maine GN, Mao X, Komarck CM, Burstein E . (2007). COMMD1 promotes the ubiquitination of NF-κB subunits through a Cullin-containing ubiquitin ligase. EMBO J 26: 436–447.
Mao X, Gluck N, Li D, Maine GN, Li H, Zaidi IW et al. (2009). GCN5 is a required cofactor for a ubiquitin ligase that targets NF-κB/RelA. Genes Dev 23: 849–861.
Moreno R, Sobotzik JM, Schultz C, Schmitz ML . (2010). Specification of the NF-κB transcriptional response by p65 phosphorylation and TNF-induced nuclear translocation of IKKɛ. Nucleic Acids Res 38: 6029–6044.
Natoli G, Chiocca S . (2008). Nuclear ubiquitin ligases, NF-κB degradation, and the control of inflammation. Sci Signal 1: pe1.
Rothgiesser KM, Fey M, Hottiger MO . (2010). Acetylation of p65 at lysine 314 is important for late NF-κB-dependent gene expression. BMC Genomics 11: 22.
Ryo A, Suizu F, Yoshida Y, Perrem K, Liou YC, Wulf G et al. (2003). Regulation of NF-κB signaling by Pin1-dependent prolyl isomerization and ubiquitin-mediated proteolysis of p65/RelA. Mol Cell 12: 1413–1426.
Saccani S, Marazzi I, Beg AA, Natoli G . (2004). Degradation of promoter-bound p65/RelA is essential for the prompt termination of the nuclear factor κB response. J Exp Med 200: 107–113.
Sakurai H, Chiba H, Miyoshi H, Sugita T, Toriumi W . (1999). IκB kinases phosphorylate NF-κB p65 subunit on serine 536 in the transactivation domain. J Biol Chem 274: 30353–30356.
Silverman N, Maniatis T . (2001). NF-κB signaling pathways in mammalian and insect innate immunity. Genes Dev 15: 2321–2342.
Tanaka T, Grusby MJ, Kaisho T . (2007). PDLIM2-mediated termination of transcription factor NF-κB activation by intranuclear sequestration and degradation of the p65 subunit. Nat Immunol 8: 584–591.
Wright CW, Duckett CS . (2009). The aryl hydrocarbon nuclear translocator alters CD30-mediated NF-κB-dependent transcription. Science 323: 251–255.
Xu M, Skaug B, Zeng W, Chen ZJ . (2009). A ubiquitin replacement strategy in human cells reveals distinct mechanisms of IKK activation by TNFα and IL-1β. Mol Cell 36: 302–314.
Yang XD, Huang B, Li M, Lamb A, Kelleher NL, Chen LF . (2009). Negative regulation of NF-κB action by Set9-mediated lysine methylation of the RelA subunit. EMBO J 28: 1055–1066.
Yang XD, Tajkhorshid E, Chen LF . (2010). Functional interplay between acetylation and methylation of the RelA subunit of NF-κB. Mol Cell Biol 30: 2170–2180.
Zhong H, May MJ, Jimi E, Ghosh S . (2002). The phosphorylation status of nuclear NF-κB determines its association with CBP/p300 or HDAC-1. Mol Cell 9: 625–636.
Zhong H, Voll RE, Ghosh S . (1998). Phosphorylation of NF-κB p65 by PKA stimulates transcriptional activity by promoting a novel bivalent interaction with the coactivator CBP/p300. Mol Cell 1: 661–671.
Acknowledgements
We are grateful to Zhijian ‘James’ Chen, Maria S Soengas and Marty W Mayo for providing cell lines that were utilized in these studies. We also thank Doris Newel and Oliver Dittrich for their technical assistance with the microarray experiments. This work was supported by an NIH R01 DK073639, a CCFA Senior Research Award and a UTSW DOCS Award to EB. The work of MLS and MK is supported by grants from the DFG.
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Li, H., Wittwer, T., Weber, A. et al. Regulation of NF-κB activity by competition between RelA acetylation and ubiquitination. Oncogene 31, 611–623 (2012). https://doi.org/10.1038/onc.2011.253
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DOI: https://doi.org/10.1038/onc.2011.253
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