Extended Data Fig. 9: Mapping of missense mutations on the structures of the ALK—ALKAL2 and LTK—ALKAL1 complexes.
From: Structural basis of cytokine-mediated activation of ALK family receptors
a, Mapping of most frequent SNPs (GnomAD) to the ALKAL2—ALKTG complex shown in top view. SNPs also found in COSMIC database (https://cancer.sanger.ac.uk/cosmic) are also indicated on the bottom view. Mutations further characterized in this study are coloured red. Inset shows the detailed position of R753 in the ALKTG—ALKAL2 complex. b, Mapping of most frequent SNPs (GnomAD) to the ALKAL1—LTKTG complex shown in top view. SNPs also found in COSMIC database (https://cancer.sanger.ac.uk/cosmic) are also indicated on the bottom view. c, Western blot analysis of the expression levels of ALKWT and the ALKR753Q and ALKF856S mutants and their ERK phosphorylation (left). On the right-side western blot analysis of phosphorylated ALKWT, ALKR753Q and ALKF856S is shown. Representative results from three biologically independent experiments with similar results. Uncropped western blot scans are provided in source data. d, Sanger sequencing of cDNA showing WT or mutant ALK expression in isogenic Ba/F3 cells. e, Cell proliferation of ALK expressing Ba/F3 cells treated with 10 nM ALKAL2 with and without addition of crizotinib for ALK R753Q and F856S mutants. Data for EV and WT ALK are repeated from Extended Data Fig. 3 for direct comparison. Crizotinib is also able to inhibit ALKAL2 induced proliferation for mutant ALK, indicating ALK dependent signalling. (n = 3 biologically independent experiments; mean ± s.d.; two-way ANOVA with Tukey’s multiple comparison test compared with DMSO control). f, Proliferation of Ba/F3 cells expressing ALK carrying the R753Q or F856S mutation upon stimulation by a concentration series of ALKAL2, ALKAL1 or ALKAL1FL at indicated concentration (n=3 biologically independent experiments; mean ± s.d.; two-way ANOVA with Tukey’s multiple comparison test compared with ALKWT. ALKAL-induced cell growth relative to that of cells cultured with IL-3 is shown in a heatmap representation. EV and ALKWT controls are included for comparison. *P < 0.05, **P < 0.01 and ***P < 0.001. Exact P-values are provided in source data).
