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. Author manuscript; available in PMC: 2022 Mar 8.
Published in final edited form as: Nat Cell Biol. 2021 Sep 8;23(9):978–991. doi: 10.1038/s41556-021-00732-8

Fig. 1 |. K63 ubiquitination of ERK1/2 and identification of TRIM15 as a ubiquitin ligase.

Fig. 1 |

a, b, K63-ubiquitination of ERK1/2 and Flag-ERK1 correlates with their activation following IGF1 stimulation. Serum-starved SK-MEL-28 cells (a), or control and TRIM15-knockout HEK293T expressing Flag-ERK1 (b), were treated with or without IGF1 (100 ng/ml IGF1) as indicated. Cell lysates were made in SDS-containing buffer, diluted, and immunoprecipitated with control IgG, anti-ERK1/2 antibody (a), or anti-Flag antibody (b) (d-IP). d-IP samples and whole cell lysates (WCL) were analyzed by Western blot.

c, K63-ubiquitination of Flag-ERK1 correlates with its activation following EGF stimulation. A549 cells transfected with Flag-ERK1 were serum-starved and treated with or without EGF (50 ng/ml) for 4 h. Cell lysates were subjected to d-IP with anti-Flag antibody. d-IP samples and whole cell lysates (WCL) were analyzed by Western blot.

d, e, TRIM15 mediates ERK1/2 ubiquitination. HEK293T cells were transfected with Flag-ERK1, HA-Ub, and increasing amounts of HA-TRIM15 as indicated (d), or with Flag-ERK1, Flag-ERK2, TRIM15-YFP, or TRIM15ΔRB-YFP, together with HA-Ub together (e). d-IP samples and WCL were analyzed by Western blot.

f, g, TRIM15 is a K63-linkage specific ubiquitin ligase for ERK1/2. HEK293T cells were transfected with Flag-ERK1, HA-TRIM15, and wild-type (WT) or mutant ubiquitin as indicated. d-IP samples and WCL were analyzed by Western blot.

h, Proteasome blockage does not alter ERK1 ubiquitination or abundance. HEK293T cells were transfected with Flag-ERK1, HA-TRIM15, and ubiquitin and treated with MG132 (10 μM) as indicated. d-IP samples and WCL were analyzed by Western blot.

i-k, TRIM15 promotes ubiquitination of endogenous ERK1/2. G361 cells transfected with control or TRIM15 expression plasmid (i), A375 cells transduced with control (Ctrl) or TRIM15 shRNA lentiviral vector (j), and SK-MEL-28 cells transfected with negative control (NC) or TRIM15 siRNA and treated with or without IGF1 (100 ng/ml) for 16 h (k) were analyzed for ERK1/2 ubiquitination (i-k) and phosphorylation (k).

Assays in panels a-k have been performed two times with similar results.