Abstract
Inflammation is a complex biological host reaction to tissue damage, infection and trauma. Extensive study of the inflammatory response has led to the identification of several protein kinases that are essential for signaling and could be potential therapeutic targets. The RSK family of kinases has multiple cellular functions. In our study, we found that RSK2 is a mediator for inflammation signaling and interacts with TRAF6. In vitro kinase assay results indicated that RSK2 strongly phosphorylates TRAF6 at serines 46, 47 and 48. Ectopic overexpression of TRAF6 or knocking down RSK2 expression confirmed that RSK2 is a positive regulator of TRAF6 K63 ubiquitination. TRAF6 is also required for RSK2 ubiquitination. TRAF6 bridges the TNF receptor superfamily and intracellular signaling for the induction of proinflammatory cytokines. We developed a colon inflammation model using RSK2 wild type (WT) and knockout (KO) mice. As expected, F4/80 and CD3 infiltration were significantly upregulated in WT mice compared to RSK2 KO mice. Furthermore, inflammation signaling, including Ikkα/β, p38 and JNKs, was dramatically upregulated in WT mice. Colon tissue immunoprecipitation results further confirmed that TRAF6 K63 ubiquitination was lower in RSK2 KO mice. Overall, these results indicate that phosphorylation of TRAF6 (S46, 47, 48) by RSK2 is required for TRAF6 K63 ubiquitination and inflammation signaling.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 50 print issues and online access
$259.00 per year
only $5.18 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Zhang JM, An J. Cytokines, inflammation, and pain. Int Anesthesiol Clin. 2007;45:27–37.
Balkwill F, Mantovani A. Inflammation and cancer: back to Virchow? Lancet . 2001;357:539–45.
Balkwill F, Charles KA, Mantovani A. Smoldering and polarized inflammation in the initiation and promotion of malignant disease. Cancer Cell. 2005;7:211–7.
Terzic J, Grivennikov S, Karin E, Karin M. Inflammation and colon cancer. Gastroenterology . 2010;138:2101–14. e5
Malakhova M, Tereshko V, Lee SY, Yao K, Cho YY, Bode A, et al. Structural basis for activation of the autoinhibitory C-terminal kinase domain of p90 RSK2. Nat Struct & Mol Biol. 2008;15:112–3.
Liu K, Cho YY, Yao K, Nadas J, Kim DJ, Cho EJ, et al. Eriodictyol inhibits RSK2-ATF1 signaling and suppresses EGF-induced neoplastic cell transformation. J Biol Chem. 2011;286:2057–66.
Cho YY, Yao K, Bode AM, Bergen HR 3rd, Madden BJ, Oh SM, et al. RSK2 mediates muscle cell differentiation through regulation of NFAT3. J Biol Chem. 2007;282:8380–92.
Zhu F, Zykova TA, Peng C, Zhang J, Cho YY, Zheng D, et al. Phosphorylation of H2AX at Ser139 and a new phosphorylation site Ser16 by RSK2 decreases H2AX ubiquitination and inhibits cell transformation. Cancer Res. 2011;71:393–403.
Cho YY, Yao K, Kim HG, Kang BS, Zheng D, Bode AM, et al. Ribosomal S6 kinase 2 is a key regulator in tumor promoter induced cell transformation. Cancer Res. 2007;67:8104–12.
Anjum R, Blenis J. The RSK family of kinases: emerging roles in cellular signalling. Nat Rev Mol Cell Biol. 2008;9:747–58.
Broom OJ, Widjaya B, Troelsen J, Olsen J, Nielsen OH. Mitogen activated protein kinases: a role in inflammatory bowel disease? Clin Exp Immunol. 2009;158:272–80.
Wu H, Arron JR. TRAF6, a molecular bridge spanning adaptive immunity, innate immunity and osteoimmunology. Bioessays 2003;25:1096–105.
O’Neill LA, Bowie AG. The family of five: TIR-domain-containing adaptors in Toll-like receptor signalling. Nat Rev Immunol. 2007;7:353–64.
Kawai T, Akira S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol. 2010;11:373–84.
Zhao W, Wang L, Zhang M, Yuan C, Gao C. E3 ubiquitin ligase tripartite motif 38 negatively regulates TLR-mediated immune responses by proteasomal degradation of TNF receptor-associated factor 6 in macrophages. J Immunol. 2012;188:2567–74.
Adhikari A, Xu M, Chen ZJ. Ubiquitin-mediated activation of TAK1 and IKK. Oncogene . 2007;26:3214–26.
Chen ZJ. Ubiquitination in signaling to and activation of IKK. Immunol Rev. 2012;246:95–106.
Huang G, Shi LZ, Chi H. Regulation of JNK and p38 MAPK in the immune system: signal integration, propagation and termination. Cytokine . 2009;48:161–9.
Lamothe B, Besse A, Campos AD, Webster WK, Wu H, Darnay BG. Site-specific Lys-63-linked tumor necrosis factor receptor-associated factor 6 auto-ubiquitination is a critical determinant of I kappa B kinase activation. J Biol Chem. 2007;282:4102–12.
Wertz IE, Dixit VM. Signaling to NF-kappaB: regulation by ubiquitination. Cold Spring Harb Perspect Biol. 2010;2:a003350.
Liu A, Gong P, Hyun SW, Wang KZ, Cates EA, Perkins D, et al. TRAF6 protein couples Toll-like receptor 4 signaling to Src family kinase activation and opening of paracellular pathway in human lung microvascular endothelia. J Biol Chem. 2012;287:16132–45.
Cho YY, Yao K, Pugliese A, Malakhova ML, Bode AM, Dong Z. A regulatory mechanism for RSK2 NH(2)-terminal kinase activity. Cancer Res. 2009;69:4398–406.
Zhang X, Zhang J, Zhang L, van Dam H, ten Dijke P. UBE2O negatively regulates TRAF6-mediated NF-kappaB activation by inhibiting TRAF6 polyubiquitination. Cell Res. 2013;23:366–77.
Jiao S, Zhang Z, Li C, Huang M, Shi Z, Wang Y, et al. The kinase MST4 limits inflammatory responses through direct phosphorylation of the adaptor TRAF6. Nat Immunol. 2015;16:246–57.
Mukhopadhyay D, Riezman H. Proteasome-independent functions of ubiquitin in endocytosis and signaling. Science. 2007;315:201–5.
Yin Q, Lin SC, Lamothe B, Lu M, Lo YC, Hura G, et al. E2 interaction and dimerization in the crystal structure of TRAF6. Nat Struct & Mol Biol. 2009;16:658–66.
Yang WL, Wang J, Chan CH, Lee SW, Campos AD, Lamothe B, et al. The E3 ligase TRAF6 regulates Akt ubiquitination and activation. Science. 2009;325:1134–8.
Bekes M, Salvesen GS. The CULt of caspase-8 ubiquitination. Cell. 2009;137:604–6.
Wu ZH, Wong ET, Shi Y, Niu J, Chen Z, Miyamoto S, et al. ATM- and NEMO-dependent ELKS ubiquitination coordinates TAK1-mediated IKK activation in response to genotoxic stress. Mol Cell. 2010;40:75–86.
Hinz M, Stilmann M, Arslan SC, Khanna KK, Dittmar G, Scheidereit C. A cytoplasmic ATM-TRAF6-cIAP1 module links nuclear DNA damage signaling to ubiquitin-mediated NF-kappaB activation. Mol Cell. 2010;40:63–74.
De Robertis M, Massi E, Poeta ML, Carotti S, Morini S, Cecchetelli L, et al. The AOM/DSS murine model for the study of colon carcinogenesis: From pathways to diagnosis and therapy studies. J Carcinog. 2011;10:9.
Lin HH, Faunce DE, Stacey M, Terajewicz A, Nakamura T, Zhang-Hoover J, et al. The macrophage F4/80 receptor is required for the induction of antigen-specific efferent regulatory T cells in peripheral tolerance. J Exp Med. 2005;201:1615–25.
Wang Y, Becker D, Vass T, White J, Marrack P, Kappler JW. A conserved CXXC motif in CD3epsilon is critical for T cell development and TCR signaling. PLoS Biol. 2009;7:e1000253.
Ricciotti E, FitzGerald GA. Prostaglandins and inflammation. Arterioscler Thromb Vasc Biol. 2011;31:986–1000.
Swatek KN, Komander D. Ubiquitin modifications. Cell Res. 2016;26:399–422.
Kulathu Y, Komander D. Atypical ubiquitylation - the unexplored world of polyubiquitin beyond Lys48 and Lys63 linkages. Nat Rev Mol Cell Biol. 2012;13:508–23.
Zinngrebe J, Montinaro A, Peltzer N, Walczak H. Ubiquitin in the immune system. EMBO Rep. 2014;15:28–45.
Corn JE, Vucic D. Ubiquitin in inflammation: the right linkage makes all the difference. Nat Struct Mol Biol. 2014;21:297–300.
Richards SA, Dreisbach VC, Murphy LO, Blenis J. Characterization of regulatory events associated with membrane targeting of p90 ribosomal S6 kinase 1. Mol Cell Biol. 2001;21:7470–80.
Yang WL, Wu CY, Wu J, Lin HK. Regulation of Akt signaling activation by ubiquitination. Cell Cycle. 2010;9:487–97.
Pickart CM. Mechanisms underlying ubiquitination. Annu Rev Biochem. 2001;70:503–33.
Pearce LR, Komander D, Alessi DR. The nuts and bolts of AGC protein kinases. Nat Rev Mol Cell Biol. 2010;11:9–22.
Newton AC. Regulation of the ABC kinases by phosphorylation: protein kinase C as a paradigm. Biochem J. 2003;370:361–71.
Hunter T. The age of crosstalk: phosphorylation, ubiquitination, and beyond. Mol Cell. 2007;28:730–8.
Zhou F, Zhang X, van Dam H, Ten Dijke P, Huang H, Zhang L. Ubiquitin-specific protease 4 mitigates Toll-like/interleukin-1 receptor signaling and regulates innate immune activation. J Biol Chem. 2012;287:11002–10.
Choo YS, Zhang Z. Detection of protein ubiquitination. J Vis Exp.2009;30:1293
Davidson LA, Callaway ES, Kim E, Weeks BR, Fan YY, Allred CD, et al. Targeted deletion of p53 in Lgr5-expressing intestinal stem cells promotes colon tumorigenesis in a preclinical model of colitis-associated cancer. Cancer Res. 2015;75:5392–7.
Acknowledgements
We thank Simin Zhao and Todd Schuster for supporting experiments, Nicki Brickman and Dr. Tia Rai for assistance in submitting our manuscript (The Hormel Institute, University of Minnesota). This work was funded by The Hormel Foundation, National Institutes of Health grants CA166011, CA187027, CA196639 and NSF of Henan Province China No. 162300410337.
Author contributions:
KY, SL, and ZD study conception and research design; KY, SL, DL, HY, CP, JR, TL, HC, GJ, ZZ, YH, and WM performed the research; KY, SL, and DL analysis and interpretation of the data; KY, AMB, and ZD wrote the paper.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Rights and permissions
About this article
Cite this article
Yao, K., Lee, SY., Peng, C. et al. RSK2 is required for TRAF6 phosphorylation-mediated colon inflammation. Oncogene 37, 3501–3513 (2018). https://doi.org/10.1038/s41388-018-0167-6
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41388-018-0167-6
This article is cited by
-
CD147 mediates epidermal malignant transformation through the RSK2/AP-1 pathway
Journal of Experimental & Clinical Cancer Research (2022)
-
Combination of RSK inhibitor LJH-685 and FLT3 inhibitor FF-10101 promoted apoptosis and proliferation inhibition of AML cell lines
Cellular Oncology (2022)
-
LncRNA NEAT1 accelerates renal mesangial cell injury via modulating the miR-146b/TRAF6/NF-κB axis in lupus nephritis
Cell and Tissue Research (2020)
-
Emerging roles of Lys63-linked polyubiquitination in neuronal excitatory postsynapses
Archives of Pharmacal Research (2019)