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TRIM15 and CYLD regulate ERK activation via lysine-63-linked polyubiquitination

Nature Cell Biology volume 23, pages 978–991 (2021)Cite this article

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Abstract

The extracellular-signal-regulated kinases ERK1 and ERK2 (hereafter ERK1/2) represent the foremost mitogenic pathway in mammalian cells, and their dysregulation drives tumorigenesis and confers therapeutic resistance. ERK1/2 are known to be activated by MAPK/ERK kinase (MEK)-mediated phosphorylation. Here, we show that ERK1/2 are also modified by lysine-63 (K63)-linked polyubiquitin chains. We identify the tripartite motif–containing protein TRIM15 as a ubiquitin ligase and the tumour suppressor CYLD as a deubiquitinase of ERK1/2. TRIM15 and CYLD regulate ERK ubiquitination at defined lysine residues through mutually exclusive interactions as well as opposing activities. K63-linked polyubiquitination enhances ERK interaction with and activation by MEK. Downregulation of TRIM15 inhibits the growth of both drug-responsive and drug-resistant melanomas. Moreover, high TRIM15 expression and low CYLD expression are associated with poor prognosis of patients with melanoma. These findings define a role of K63-linked polyubiquitination in the ERK signalling pathway and suggest a potential target for cancer therapy.

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Fig. 1: K63 ubiquitination of ERK1/2 and the identification of TRIM15 as a ubiquitin ligase.
Fig. 2: TRIM15 activates ERK1/2 by conjugating K63 ubiquitin to specific Lys residues.
Fig. 3: TRIM15 interacts with ERK1/2 through conserved domains.
Fig. 4: TRIM15-mediated ubiquitination promotes ERK interaction with and activation by MEK.
Fig. 5: CYLD deubiquitinates and inactivates ERK1/2.
Fig. 6: CYLD associates with ERK and inhibits its interaction with TRIM15 and MEK.
Fig. 7: An oncogenic role for TRIM15 in melanoma.
Fig. 8: TRIM15 and CYLD expression in melanoma cell lines and specimens.

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Data availability

The dataset from these resources that supports the findings of this study is available at OncoLnc (http://www.oncolnc.org), GEPIA (http://gepia.cancer-pku.cn) and cBioPortal (https://www.cbioportal.org). Public melanoma datasets used in this study were deposited at the GEO under the accession numbers GSE3189, GSE7929, GSE61992 and GSE50509. All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

References

  1. Morrison, D. K. MAP kinase pathways. Cold Spring Harb. Perspect. Biol. 4, a011254 (2012).

  2. Shaul, Y. D. & Seger, R. The MEK/ERK cascade: from signaling specificity to diverse functions. Biochim. Biophys. Acta 1773, 1213–1226 (2007).

    Article  CAS  PubMed  Google Scholar 

  3. Lavoie, H., Gagnon, J. & Therrien, M. ERK signalling: a master regulator of cell behaviour, life and fate. Nat. Rev. Mol. Cell Biol. 21, 607–632 (2020).

    Article  CAS  PubMed  Google Scholar 

  4. Vogelstein, B. et al. Cancer genome landscapes. Science 339, 1546–1558 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Johannessen, C. M. et al. COT drives resistance to RAF inhibition through MAP kinase pathway reactivation. Nature 468, 968–972 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Nazarian, R. et al. Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation. Nature 468, 973–977 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Villanueva, J. et al. Acquired resistance to BRAF inhibitors mediated by a RAF kinase switch in melanoma can be overcome by cotargeting MEK and IGF-1R/PI3K. Cancer Cell 18, 683–695 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Samatar, A. A. & Poulikakos, P. I. Targeting RAS-ERK signalling in cancer: promises and challenges. Nat. Rev. Drug Discov. 13, 928–942 (2014).

    Article  CAS  PubMed  Google Scholar 

  9. Ryan, M. B., Der, C. J., Wang-Gillam, A. & Cox, A. D. Targeting RAS-mutant cancers: is ERK the key? Trends Cancer 1, 183–198 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Komander, D. & Rape, M. The ubiquitin code. Annu. Rev. Biochem. 81, 203–229 (2012).

    Article  CAS  PubMed  Google Scholar 

  11. Deng, L. et al. Activation of the IκB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain. Cell 103, 351–361 (2000).

    Article  CAS  PubMed  Google Scholar 

  12. Wang, C. et al. TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature 412, 346–351 (2001).

    Article  CAS  PubMed  Google Scholar 

  13. Yang, W. L. et al. The E3 ligase TRAF6 regulates Akt ubiquitination and activation. Science 325, 1134–1138 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Chan, C. H. et al. The Skp2-SCF E3 ligase regulates Akt ubiquitination, glycolysis, herceptin sensitivity, and tumorigenesis. Cell 149, 1098–1111 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Chen, Z. J. & Sun, L. J. Nonproteolytic functions of ubiquitin in cell signaling. Mol. Cell 33, 275–286 (2009).

    Article  CAS  PubMed  Google Scholar 

  16. Kulathu, Y. & Komander, D. Atypical ubiquitylation—the unexplored world of polyubiquitin beyond Lys48 and Lys63 linkages. Nat. Rev. Mol. Cell Biol. 13, 508–523 (2012).

    Article  CAS  PubMed  Google Scholar 

  17. Yau, R. & Rape, M. The increasing complexity of the ubiquitin code. Nat. Cell Biol. 18, 579–586 (2016).

    Article  CAS  PubMed  Google Scholar 

  18. Ozato, K., Shin, D. M., Chang, T. H. & Morse, H. C. III. TRIM family proteins and their emerging roles in innate immunity. Nat. Rev. Immunol. 8, 849–860 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Hatakeyama, S. TRIM proteins and cancer. Nat. Rev. Cancer 11, 792–804 (2011).

    Article  CAS  PubMed  Google Scholar 

  20. Guo, L. et al. A cellular system that degrades misfolded proteins and protects against neurodegeneration. Mol. Cell 55, 15–30 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Chen, L., Zhu, G., Johns, E. M. & Yang, X. TRIM11 activates the proteasome and promotes overall protein degradation by regulating USP14. Nat. Commun. 9, 1223 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Chen, L. et al. Enhanced degradation of misfolded proteins promotes tumorigenesis. Cell Rep. 18, 3143–3154 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Liu, Y. et al. TRIM25 promotes the cell survival and growth of hepatocellular carcinoma through targeting Keap1-Nrf2 pathway. Nat. Commun. 11, 348 (2020).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Zhu, G. et al. TRIM11 prevents and reverses protein aggregation and rescues a mouse model of Parkinson’s disease. Cell Rep. 33, 108418 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Pertel, T. et al. TRIM5 is an innate immune sensor for the retrovirus capsid lattice. Nature 472, 361–365 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Li, Q. et al. Tripartite motif 8 (TRIM8) modulates TNFα- and IL-1β-triggered NF-κB activation by targeting TAK1 for K63-linked polyubiquitination. Proc. Natl Acad. Sci. USA 108, 19341–19346 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. McEwan, W. A. et al. Intracellular antibody-bound pathogens stimulate immune signaling via the Fc receptor TRIM21. Nat. Immunol. 14, 327–336 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Gack, M. U. et al. TRIM25 RING-finger E3 ubiquitin ligase is essential for RIG-I-mediated antiviral activity. Nature 446, 916–920 (2007).

    Article  CAS  PubMed  Google Scholar 

  29. Lopez-Bergami, P. et al. Rewired ERK-JNK signaling pathways in melanoma. Cancer Cell 11, 447–460 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. McGill, G. G. et al. Bcl2 regulation by the melanocyte master regulator Mitf modulates lineage survival and melanoma cell viability. Cell 109, 707–718 (2002).

    Article  CAS  PubMed  Google Scholar 

  31. Kim, Y. J., Tsang, T., Anderson, G. R., Posimo, J. M. & Brady, D. C. Inhibition of BCL2 family members increases the efficacy of copper chelation in BRAF(V600E)-driven melanoma. Cancer Res. 80, 1387–1400 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Chapman, P. B. et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N. Engl. J. Med. 364, 2507–2516 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Goetz, E. M., Ghandi, M., Treacy, D. J., Wagle, N. & Garraway, L. A. ERK mutations confer resistance to mitogen-activated protein kinase pathway inhibitors. Cancer Res. 74, 7079–7089 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Bardwell, L. & Thorner, J. A conserved motif at the amino termini of MEKs might mediate high-affinity interaction with the cognate MAPKs. Trends Biochem. Sci. 21, 373–374 (1996).

    Article  CAS  PubMed  Google Scholar 

  35. Tanoue, T. & Nishida, E. Molecular recognitions in the MAP kinase cascades. Cell Signal. 15, 455–462 (2003).

    Article  CAS  PubMed  Google Scholar 

  36. Tanoue, T., Adachi, M., Moriguchi, T. & Nishida, E. A conserved docking motif in MAP kinases common to substrates, activators and regulators. Nat. Cell Biol. 2, 110–116 (2000).

    Article  CAS  PubMed  Google Scholar 

  37. Kinoshita, T. et al. Crystal structure of human mono-phosphorylated ERK1 at Tyr204. Biochem. Biophys. Res. Commun. 377, 1123–1127 (2008).

    Article  CAS  PubMed  Google Scholar 

  38. Taylor, S. S. & Kornev, A. P. Protein kinases: evolution of dynamic regulatory proteins. Trends Biochem. Sci. 36, 65–77 (2011).

    Article  CAS  PubMed  Google Scholar 

  39. Roskoski, R. Jr. ERK1/2 MAP kinases: structure, function, and regulation. Pharmacol. Res. 66, 105–143 (2012).

    Article  CAS  PubMed  Google Scholar 

  40. Alessi, D. R. et al. Identification of the sites in MAP kinase kinase-1 phosphorylated by p74raf-1. EMBO J. 13, 1610–1619 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Mevissen, T. E. T. & Komander, D. Mechanisms of deubiquitinase specificity and regulation. Annu. Rev. Biochem. 86, 159–192 (2017).

    Article  CAS  PubMed  Google Scholar 

  42. Clague, M. J., Urbe, S. & Komander, D. Breaking the chains: deubiquitylating enzyme specificity begets function. Nat. Rev. Mol. Cell Biol. 20, 338–352 (2019).

    Article  CAS  PubMed  Google Scholar 

  43. Lu, Z., Xu, S., Joazeiro, C., Cobb, M. H. & Hunter, T. The PHD domain of MEKK1 acts as an E3 ubiquitin ligase and mediates ubiquitination and degradation of ERK1/2. Mol. Cell 9, 945–956 (2002).

    Article  CAS  PubMed  Google Scholar 

  44. Zhang, Y. et al. Nitration-induced ubiquitination and degradation control quality of ERK1. Biochem. J. 476, 1911–1926 (2019).

    Article  CAS  PubMed  Google Scholar 

  45. Bignell, G. R. et al. Identification of the familial cylindromatosis tumour-suppressor gene. Nat. Genet. 25, 160–165 (2000).

    Article  CAS  PubMed  Google Scholar 

  46. Sun, S. C. CYLD: a tumor suppressor deubiquitinase regulating NF-κB activation and diverse biological processes. Cell Death Differ. 17, 25–34 (2010).

    Article  CAS  PubMed  Google Scholar 

  47. Massoumi, R. et al. Down-regulation of CYLD expression by snail promotes tumor progression in malignant melanoma. J. Exp. Med. 206, 221–232 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Trompouki, E. et al. CYLD is a deubiquitinating enzyme that negatively regulates NF-κB activation by TNFR family members. Nature 424, 793–796 (2003).

    Article  CAS  PubMed  Google Scholar 

  49. Brummelkamp, T. R., Nijman, S. M., Dirac, A. M. & Bernards, R. Loss of the cylindromatosis tumour suppressor inhibits apoptosis by activating NF-κB. Nature 424, 797–801 (2003).

    Article  CAS  PubMed  Google Scholar 

  50. Kovalenko, A. et al. The tumour suppressor CYLD negatively regulates NF-κB signalling by deubiquitination. Nature 424, 801–805 (2003).

    Article  CAS  PubMed  Google Scholar 

  51. Levin-Salomon, V., Kogan, K., Ahn, N. G., Livnah, O. & Engelberg, D. Isolation of intrinsically active (MEK-independent) variants of the ERK family of mitogen-activated protein (MAP) kinases. J. Biol. Chem. 283, 34500–34510 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Davies, H. et al. Mutations of the BRAF gene in human cancer. Nature 417, 949–954 (2002).

    Article  CAS  PubMed  Google Scholar 

  53. Flaherty, K. T. et al. Inhibition of mutated, activated BRAF in metastatic melanoma. N. Engl. J. Med. 363, 809–819 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Bollag, G. et al. Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma. Nature 467, 596–599 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Planchard, D. et al. Dabrafenib plus trametinib in patients with previously treated BRAF(V600E)-mutant metastatic non-small cell lung cancer: an open-label, multicentre phase 2 trial. Lancet Oncol. 17, 984–993 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Long, G. V. et al. Increased MAPK reactivation in early resistance to dabrafenib/trametinib combination therapy of BRAF-mutant metastatic melanoma. Nat. Commun. 5, 5694 (2014).

    Article  CAS  PubMed  Google Scholar 

  57. Rizos, H. et al. BRAF inhibitor resistance mechanisms in metastatic melanoma: spectrum and clinical impact. Clin. Cancer Res. 20, 1965–1977 (2014).

    Article  CAS  PubMed  Google Scholar 

  58. Krepler, C. et al. A comprehensive patient-derived xenograft collection representing the heterogeneity of melanoma. Cell Rep. 21, 1953–1967 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Xu, L. et al. Gene expression changes in an animal melanoma model correlate with aggressiveness of human melanoma metastases. Mol. Cancer Res. 6, 760–769 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Talantov, D. et al. Novel genes associated with malignant melanoma but not benign melanocytic lesions. Clin. Cancer Res. 11, 7234–7242 (2005).

    Article  CAS  PubMed  Google Scholar 

  61. Uchil, P. D. et al. TRIM15 is a focal adhesion protein that regulates focal adhesion disassembly. J. Cell Sci. 127, 3928–3942 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Yang, W. L. et al. Cycles of ubiquitination and deubiquitination critically regulate growth factor-mediated activation of Akt signaling. Sci. Signal. 6, ra3 (2013).

    PubMed  Google Scholar 

  63. Tang, J. et al. Critical role for Daxx in regulating Mdm2. Nat. Cell Biol. 8, 855–862 (2006).

    Article  CAS  PubMed  Google Scholar 

  64. Jiang, P. et al. p53 regulates biosynthesis through direct inactivation of glucose-6-phosphate dehydrogenase. Nat. Cell Biol. 13, 310–316 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank Y. Zhao, J. L. Riley, S. Eblen, A. Catling, I. Asangani, W. Mothes and P. Uchil for plasmids and/or cell lines, and W. Wu, H. Li, X. Su, Y. Xu, R. Wang, S. Ghaisas, D. Harischandra and K. Lv for technical assistance. This research was supported by grants from NIH (R01CA182675, R01CA184867, R01CA235760, and R01CA243520) to X.Y.

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Authors and Affiliations

  1. Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Guixin Zhu & Xiaolu Yang

  2. Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Guixin Zhu & Xiaolu Yang

  3. Molecular and Cellular Oncogenesis Program, Melanoma Research Center, The Wistar Institute, Philadelphia, PA, USA

    Meenhard Herlyn

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  1. Guixin Zhu
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  2. Meenhard Herlyn
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Contributions

G.Z. and X.Y. planned this project, analysed data and wrote the manuscript. G.Z. designed and conducted experiments. X.Y. supervised the study and acquired funding. M.H. helped with the PDX experiment.

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Correspondence to Xiaolu Yang.

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Peer review information Nature Cell Biology thanks Tony Hunter, Ze’ev Ronai and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Identification of TRIM15 as a ubiquitin ligase for ERK.

a, Screening of human TRIM proteins for ERK1 ubiquitination. HEK293T cells were transfected with Flag-ERK1 and each of the first eighteen human TRIMs. 24 h after transfection, cell lysates were made in SDS-containing buffer, diluted, and immunoprecipitated (denaturing IP or d-IP) with anti-Flag antibody. Immunoprecipitates were analyzed for ERK1 ubiquitination by Western blot using ubiquitin antibody and for sample loading by Ponceau S staining. HC, heavy chain. n.s., non-specific. b, TRIM15, but not TRIM17, promotes ERK1 ubiquitination. HEK293T cells were transfected with TRIM15 or TRIM17, together with Flag-ERK1 and HA-Ub. Flag-ERK1 was immunoprecipitated. d-IP samples and whole cell lysates (WCL) were analyzed by Western blot. c, HEK293T cells transfected with Flag-ERK1, HA-TRIM15, and wild-type (WT) or mutant ubiquitin proteins as indicated were analyzed for Flag-ERK1 ubiquitination with both anti-ubiquitin (Ub) and anti-HA antibodies and for protein expression. d, HEK293T cells transfected with Flag-ERK1, HA-Ub, HA-TRIM15, and Myc-TRAF2 as indicated were analyzed for Flag-ERK1 ubiquitination and protein expression.

Source data

Extended Data Fig. 2 Expression of TRIM15 in melanoma cells and its role in ERK activation.

a, Expression of TRIM15 and CYLD, and phosphorylation of ERK1/2 and ELK-1, in melanoma cell lines. b, Tumor cell lines used in the current work and the status of BRAF, NRAS, and KRAS mutations. WM3960 was a cell line established from patient-derived xenografts (PDX) tumors58. c, HEK293T cells transfected with Flag-ERK1 and/or HA-TRIM15 were analyzed for Flag-ERK1 phosphorylation by anti-Flag d-IP, followed by Western blot. d, e, ERK1/2 activation in HT29 cells (which harbors BRAFV600E) stably expressing control (shCtrl) or one of the two independent TRIM15 shRNAs (#1 and #2) (d), and in parental and TRIM15-knockout SK-MEL-173 cells (e). f, g, Levels of cyclin D1 and BCL2 proteins in A375 cells transfected with control (Ctrl) or TRIM15 siRNA (f), or treated with vehicle (DMSO) or trametinib (2 μM) for 24 h.

Source data

Extended Data Fig. 3 Mass-spectrometry analysis of ERK1 for potential ubiquitination sites.

Flag-ERK1 protein co-expressed with HA-TRIM15 in HEK293T cells (Flag-ERK1Ub) were purified and analyzed by mass spectrometry. Shown are ubiquitination sites of ERK1 (a), and mass spectrum of peptides surrounding K168 (b) and K302 (c). Ubiquitination at K302 was detected when a relatively small amount of Flag-ERK1Ub protein was used, while ubiquitination at K168 was detected only when a relatively large amount of Flag ERK1Ub protein was used, suggesting that ubiquitination at K302 was more abundant than that at K168.

Extended Data Fig. 4 TRIM15 activates ERK1/2 by ubiquitinating them on specific Lys residues.

a, b, HEK293T cells transfected with wild-type or mutant Flag-ERK1, HA-Ub, and TRIM15-YFP were analyzed for Flag-ERK1 ubiquitination. c, Alignment of human ERK1 (total 379 aa) and mouse ERK2 (total 360 aa) sequences around the ubiquitination sites, which are indicated in red color. d, D14 (left) and SK-MEL-28 (right) cells transduced with control lentiviral vector (Vector) or lentiviral vector expressing Flag-ERK1 or Flag-ERK12KR were analyzed for activation of Flag-ERK1/ERK12KR and phosphorylation of ELK-1. e, Flag-ERK1 or Flag-ERK12KR expressed in HEK293T cells were immunoprecipitated (IP) with anti-Flag mAb (M2) beads and analyzed for phosphorylation. f, A375 cells transduced with control lentiviral vector or lentiviral vector expressing Flag-ERK1, Flag-ERK12KR, Flag-ERK2, or Flag-ERK22KR were treated with or without 2 μM PLX4032 (PLX) for 24 h. Bright field images of cells are shown. Scale bar, 100 μm. g, Flag-tagged ERK1, ERK2, and TRIM15 proteins purified from HEK293T cells, and His-Ub and His-Ub K63R purchased from a commercial source, were analyzed by SDS-PAGE and Coomassie blue staining. h, i, Ribbon diagram of human ERK1 structure (PDB ID: 2ZOQ) around K302 (h) and K168 (i), analyzed by PyMOL 2.0. Two α-helix domains (αF and αH), key residues, activation loop, and catalytic loop are indicated. j, A375 cells stably expressing Flag-ERK1 and Flag-ERK1K302R were analyzed for activation of Flag-ERK1/ERK1K302R and endogenous ERK1/2.

Source data

Extended Data Fig. 5 TRIM15 interacts with ERK1/2 and promotes their association with MEK.

a, Interaction of endogenous TRIM15 and ERK1/2 in A375 cells was analyzed by co-immunoprecipitation (co-IP) assay with control IgG and anti-ERK1/2 antibody. b, Direct TRIM15-ERK1 interaction in vitro and its dependence on the ERK1 CD domain. Purified Flag-TRIM15 protein was incubated with purified GST, GST-ERK1, or GST-ERK12DN immobilized on beads. Pulldown samples and 5% of input TRIM15 were analyzed by Western blot (top) and Ponceau S staining (bottom). c, d, Ubiquitination of ERK1 does not affect its binding to ATP. Flag-ERK1 (c, d), Flag-ERK1Ub (c), and Flag-ERK1K302R (d) were analyzed for interaction with the ATP derivatives 6AH-ATP and AP-ATP conjugated to agarose beads in a pulldown assay. e, f, Ubiquitination of ERK1 does not affect its binding to ELK-1. Flag-ERK1 (e, f), Flag-ERK1Ub (e), and Flag-ERK1K302R (f) were analyzed for interaction with GST and GST-ELK-1 bound to glutathione resins. g, TRIM15 does not affect ERK1-ELK-1 binding in cells. Flag-ERK1 was expressed alone or together with HA-TRIM15 in HEK293T cells. The interaction of Flag-ERK1 with endogenous EKL-1 was analyzed by co-IP. h, Knockdown of TRIM15 decreases ERK1/2-MEK1/2 association. Interaction of endogenous ERK1/2 with MEK1/2 in A375 cells stably expressing control or TRIM15 shRNA were analyzed by co-IP.

Source data

Extended Data Fig. 6 CYLD inhibits ERK1/2 activity and their interaction with MEK1/2.

a, Knockout Cyld in MEFs increases ERK1/2 activity. Cyld+/+ MEFs and Cyld-/- MEFs were analyzed for ERK1 and ELK-1 phosphorylation and CYLD expression by Western blot. b, Interaction of endogenous CYLD and ERK1/2 in A375 cells was analyzed by co-IP with anti-ERK1/2 antibody. c, CYLD4A has weakened ability to inactivate ERK1/2. A375 cells were infected with empty pCDH (EV), pCDH-CYLD, or pCDH-CYLD4A lentiviral vector. Cell lysates were examined for ERK1/2 activation and CYLD/CYLD4A expression. d, CYLD4A is still able to deubiquitinate TRAF2. Myc-TRAF2 and HA-Ub were expressed in the presence or absence of Flag-CYLD4A and Flag-CYLDCA in HEK293T cells. Ubiquitination of Myc-TRAF2 was examined by d-IP with anti-Myc antibody. e, CYLD shows reduced interaction with ERK12DN. Flag-CYLD was incubated with immobilized GST, GST-ERK1, or GST-ERK12DN. The pulldown samples and input were analyzed by SDS-PAGE followed by Western blot and/or Ponceau S staining. f, Knockdown of CYLD increases ERK1-TRIM15 interaction. HEK293T cells were treated with NC or CYLD siRNA, and transfected with HA-TRIM15 alone or together with Flag-ERK1. Interaction between TRIM15 and ERK1 was assayed by co-IP with anti-Flag antibody. g, Knockout of CYLD increases TRIM15-ERK1/2 interaction. Cyld+/+ and Cyld-/- MEFs were analyzed for TRIM15-ERK1/2 interaction using co-IP with anti-ERK1/2 antibody. h, Interaction of Flag-ERK1 with endogenous MEK1 in HEK293T cells stably expressing shCtrl or shCYLD.

Source data

Extended Data Fig. 7 Distinct signaling specificities of CYLD and TRIM15.

a, Frequency of CYLD mutations in melanoma (cBioPortal). b, Schematic paragraph showing melanoma-derived point mutants. CAP-GLY, cytoskeleton-associated protein (CAP)-glycine-rich (GLY) domain; USP, ubiquitin-specific protease. c, In vitro deubiquitination of Di-Ub by CYLD or CYLD mutants. Di-Ub was incubated with purified CYLD or CYLD mutants, and reaction mixtures were analyzed by Western blot (top) and Ponceau S staining (bottom). d, In vitro deubiquitination of Flag-ERK1-Ub by CYLD or CYLD mutants. Ubiquitinated Flag-ERK1 protein (Flag-ERK1-Ub) was treated with CYLD or the indicated CYLD mutants. De-ubiquitination was analyzed by Western blot with anti-ERK antibody. e, NF-κB reporter assays show the effect of wild-type (WT) and mutant CYLD proteins on TRAF2-induced NF-κB signaling in HEK293T cells. Data are Mean ± SD (n = 3 biologically independent samples). **** P < 0.0001, One-way ANOVA followed by Tukey’s multiple comparisons test. f, NF-κB reporter assay show no effect of TRIM15 on NF-κB signaling in HEK293T cells. Data are Mean ± SD (n = 3 biologically independent samples). g, Immunoblot of whole cell lysates from A375 cells treated with PLX4032 (2 μM) or trametinib (1 μM) for 24 h. h, Immunoblot of total cell lysates from A375 cells expressing control or TRIM15 shRNA.

Source data

Extended Data Fig. 8 TRIM15 is critical for the survival of melanoma cells.

a, b, A375 cells expressing control or TRIM15 shRNA were grown on adherent plates. Shown are representative images (a) and relative number (b) of colonies. Data are Mean ± SD (n = 3 biologically independent samples). *** P < 0.001, two-tailed Student’s t-test. c, A375 cells expressing control shRNA, or expressing TRIM15 shRNA and transfected with EV, TRIM15, or TRIM15ΔRB, were grown on adherent plates. Relative numbers of colonies are shown as Mean ± SD (n = 3 biologically independent samples). *** P < 0.001, n.s., no significance, one-way ANOVA test followed by Tukey’s post hoc test. d, Adherent colony formation by control or TRIM15-knockdown A375 cells that were transfected with siCtrl or siCYLD. e, Soft agar colony formation by A375 cells stably expressing shCtrl or shTRIM15 and transfected with or without TRIM15 or TRIM15ΔRB. f, Soft agar colony formation of SK-MEL-28 cells stably expressing control or TRIM15 shRNA, or expressing TRIM15 shRNA and transfected with Flag-ERK1R84S. g, Control or TRIM15-knockdown A375 cells grew on adherent plates for 6 days in the presence of indicated concentration of PLX4032, and stained with Crystal violet. h, Weights of mice that underwent the indicated treatment for two weeks. Data were shown as Mean ± SD (n = 5 biologically independent animals). i, Relative survival of A375 and A375R cells treated with increasing concentrations of PLX4032. Data were shown as Mean ± SD (n = 4 biologically independent samples). j, k, A375R cells expressing control or TRIM15 shRNA were grown on adherent plates and stained with crystal violet. Shown are representative images (j) and relative colony number (k). Data are Mean ± SD (n = 3 biologically independent samples). * P < 0.05, two-tailed Student’s t-test.

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Extended Data Fig. 9 TRIM15 expression in treated and untreated melanoma specimens, and correlation of high TRIM15 and low CYLD expression with poor prognosis of melanoma patients.

a, TRIM15 expression in eleven melanoma samples that initially responded but subsequently progressed on the combined treatment of the BRAF inhibitor dabrafenib and the MEK inhibitor trametinib (treated), and nine matched pre-treatment tumor samples (untreated), based on the microarray dataset (GSE61992)56. The minimum, 25% percentile, median, 75% percentile, maxima are -9.271, -8.848, -8.118, -6.084, and -4.756, respectively, for untreated group; and -9.734, -7.84, -5.619, -4.041, and 3.549, respectively, for treated group. b, c, Average TRIM15 expression in 38 tumors that were treated the BRAF inhibitor dabrafenib or vemurafenib but subsequently progressed (treated) and 21 matched pretreatment tumors (untreated) (b), and TRIM15 expression in 21 matched treated and untreated samples (c). Note that TRIM15 was highly upregulated in two samples (#6 and #21). The results are from the microarray dataset (GSE50509)57. For b, the minimum, 25% percentile, median, 75% percentile, maxima for are -5.492, 1.99, 3.811, 9.627, and 13.59, respectively, for untreated group; and -4.804, 1.927, 4.814, 11.56, and 35.71, respectively, for treated group. d, Frequency of TRIM15 alterations in cancers (cBioPortal). e, CYLD mRNA expression in normal skin and SKCM. Results were analyzed on GEPIA website. **** P < 0.0001, two-sided Student’s t-test. f, Poor survival of melanoma patients with low CYLD expression. Results were analyzed on GEPIA website.

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Zhu, G., Herlyn, M. & Yang, X. TRIM15 and CYLD regulate ERK activation via lysine-63-linked polyubiquitination. Nat Cell Biol 23, 978–991 (2021). https://doi.org/10.1038/s41556-021-00732-8

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