CLK3 is frequently mutated and activated in human CCA. (Ai) Schematic presentation of somatic mutations in CLK3 in 100 CCA patients. (Aii and Aiii) Sanger sequencing to identify CLK3 mutations in 100 (ii) and 75 (iii) human CCAs. Arrows indicate the location of the mutation in the tumor, but not in the tumor tissue. (B) IP kinase assay. Briefly, CLK3 was immunoprecipitated with anti-HA antibodies from HEK293 cells expressing WT-CLK3 or its mutants. The immunoprecipitated proteins mixed with the SRSF1 protein (a known substrate of CLK3) synthetic peptide and [γ-³²P] ATP. The phosphocellulose paper assay was used to measure kinase activity. The results were normalized to 1.0 for WT-CLK3. (C) The effects of human CCA-associated CLK3 mutants on the proliferation and colony formation of HCCC9810 cells. (D) The impact of human CCA-associated CLK3 mutants on HCCC9810 cell wound healing and invasion. (E) Heatmap showing WT-CLK3 and its mutants in CCA patients on purine metabolism in HCCC9810 cells. (F–H) The effects of WT-CLK3 and its mutants in CCA patients on purine intermediates in HCCC9810 cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data are representative of three independent experiments with similar results (Bii and E) or are from three independent experiments (Bi, C, D, and F–H; mean ± SEM). P values were calculated using unpaired Student’s t test (Ci) or one-way ANOVA (Bi, Cii, D, and F–H). AMP, adenosine monophosphate; GMP, guanosine monophosphate; IMP, inosine monophosphate; TUBA, alpha tubulin.