Fig. 5: KRAS recruits TRIM21 to regulate DUSP6 stability.
a Schematic of the immunoprecipitation followed by mass spectrometry (IP-MS) experiment employed to identify binding partners of FLAG-KRASG12V. 3 biological replicates from each condition were pooled for mass spectrometry. Linear plot shows the enriched proteins. b IP western blots showing the interaction between HA-KRASG12V and endogenous TRIM21 and DUSP6 in 293 T cells stably expressing vector or HA-KRASG12V treated with DMSO, trametinib, or ulixertinib for 16 h. The bar graph includes quantification of three independent experiments. Data was presented as mean ± SEM. P-values were calculated using one-way ANOVA followed by Dunnet’s multiple comparisons test. c Western blots, and quantification of serial endogenous DUSP6 protein levels in 293 T cells expressing empty vector or GFP-tagged TRIM21 following treatment with CHX (10 µg/mL) for the indicated durations. d IP experiment showing differences in K48-polyubiquitination of FLAG-tagged DUSP6 in 293 T cells transfected with the vector, wild-type, or enzymatically inactive (C54Y) TRIM21. e IP experiment showing differences in K48-polyubiquitination of FLAG-tagged DUSP6 with/without TRIM21 co-expression following 16 h treatment with DMSO, ulixertinib, or trametinib in 293 T cells. f Western blots showing changes in p-HER2, p-ERK1/2, and DUSP6 in Pa01c and HPAC cells stably expressing a scramble shRNA or two different shRNAs against TRIM21. b was conducted three times, (c–f) were conducted two times, and one set of data for each was shown. g 2-dimensional colony formation assay showing the colony-forming ability of the indicated Pa01c and HPAC cells co-treated with DMSO or afatinib for 2 weeks. h GSEA plots showing the enrichment of ERBB2 and KRAS signatures in TRIM21-silenced HPAC cells subjected to bulk RNA sequencing. i proposed model of the mechanism by which KRAS/MAPK inhibitors dissociate TRIM21 from DUSP6 and causes the latter to be destabilized, leading to sustained phospho-activation of HER2. Phospho-activated HER2 may in turn promotes DUSP6 degradation while simultaneously activate other survival cascades such as the PI3K-AKT pathway. Source data are provided in Source Data file.
