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

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.

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.

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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.

Author information

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Authors

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.

Corresponding author

Correspondence to Xiaolu Yang.

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Competing interests

The authors declare no competing interests.

Additional information

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.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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.

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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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