CLK3-dependent phosphorylation of USP13 at Y708 promotes CCA progression by activating c-Myc–mediated purine synthesis. (A) Knockdown of CLK3 blocked the interaction of USP13 with c-Myc in HuCCT1 cells treated as indicated with MG132. (B) HCCC9810 cells were transfected with His-WT-USP13 or USP13-Y708F or together with Flag-CLK3 and treated with MG132. Co-IP was performed using His antibody. (C) Proximity ligation assay indicating the binding of USP13 to c-Myc in HCCC9810 cells. Bar, 50 µm. (D) Computational molecular docking to analyze the molecular mechanism by which USP13-Y708 phosphorylation was involved in its interaction with c-Myc. Based on the prediction in ZDOCK and Pymol software, Y708 of USP13 lay in the interface between c-Myc and USP13, and Y708 phosphorylation greatly enhanced the binding affinity of USP13 to c-Myc by increasing hydrophilic forces. (E) HEK293T cells were transfected with WT-USP13 or phospho mutants and then treated using CHX. Western blotting was performed as indicated. (F) HEK293T cells were treated with WT-USP13 or phospho-deficient or phosphomimetic mutant with HA-tagged ubiquitin and MG132. Western blotting was performed to determine c-Myc ubiquitination. (G) ChIP assays from HCCC9810 cells treated as indicated. (H) Quantitative RT-PCR in samples treated as indicated. (I and J) The impacts of WT-USP13 and its phosphorylation mutants on CCA growth and metastasis using mice model. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data are representative of three independent experiments with similar results (A–C, E, and F) or are from three independent experiments (G–J; mean ± SEM). P values were calculated using unpaired Student’s t test (I) or one-way ANOVA (G, H, and J). KD, knockdown; PLA, proximity ligation assay; TUBA, alpha tubulin.