Extended Data Fig. 4: TRIM15 activates ERK1/2 by ubiquitinating them on specific Lys residues.
From: TRIM15 and CYLD regulate ERK activation via lysine-63-linked polyubiquitination
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.
