Fig. 6

a The role of serine phosphorylation was examined using STF-Luc assay in an RNF43(R127P) mutant background. Luciferase activity in mock-transfected cells was set to 1 (mean ± sd). Schematic of RNF43 mutants used in Figs. 5, 6, Supplementary Fig. 7, 9 is shown. Independent values of each sample are shown as red circles. Asterisks indicate significant differences (P < 0.05, one-way ANOVA, n = 3 biologically independent samples) from RNF43(R127P) cells. b Colony-forming activity was evaluated following expression of RNF43 phospho-mutant forms in Cle-H3 cells via soft agar assay and volume of colonies was estimated. Scale bars, 100 μm. Asterisks indicate significant differences from RNF43(R127P) tumour. c Tumour growth was examined in nude mice with Cle-H3 cells following the expression of RNF43 phospho-mutant forms at 5 wks after Cle-H3 injection and tumour weight was measured. Images for all of the tumours are shown. Scale bar, 1 cm. NT indicates no tumour observed. Bar graphs and error bars in (b, c) represents mean ± sem of biologically independent samples. Red circles indicate individual values of each sample. The P values for the indicated comparisons were determined by one-way ANOVA (P < 0.05). n = 98–134 (b), n = 6–12 (c) biologically independent samples. Asterisks or ND indicates significant or no significant difference in indicated comparisons, respectively. d Ubiquitination of Fzd5 by RNF43 phospho-mutants was examined with bafilomycin A₁ by immunoprecipitation (IP)-IB experiments. e Schematic of molecular mechanism and biological role of RNF43 phosphorylation in Wnt signalling and multi-step tumorigenesis. Wild-type RNF43 is activated by serine phosphorylation. Oncogenic RNF43 with R127P extracellular mutation is reverted to a functional tumour suppressor via the introduction of phosphomimetic mutation.