Extended Data Fig. 7: The RAS-PP1C interface favours MRAS while N/KRAS compensate for MRAS loss.

a. RAS and RAS-related proteins sequence alignment. Amino acid sequences of human RAS-related proteins MRAS Q71R, RRAS, RRAS2 and aligned with KRAS, NRAS and HRAS. Identically conserved residues are shaded red. The site of the Q71R mutation in the present structure is shaded yellow. Secondary structure elements of MRAS are indicated above the alignment. Symbols above the alignment indicate residues that in the holoenzyme complex lie in the interface with SHOC2 (yellow stars) or PP1Cα (blue diamonds). b. SHOC2, PP1Cα, and KRAS (GppNHp) were mixed in a 1:1:1.2 molar ratio and subjected to size-exclusion chromatography using a Superdex 200 Increase 10/300 GL column. In the absence of MRAS, the ternary complex formed with KRAS (left). SHOC2, PP1Cα, MRAS and KRAS were combined (right) in a 1:1:1.2:1.2 molar ratio, respectively, before SEC analysis. MRAS and KRAS were both charged with non-hydrolyzable GTP analogue GppNHp prior to combining with SHOC2 and PP1C. The resulting UV280 absorbance trace is shown in the top panel, and Coomassie-stained SDS-PAGE gel of the corresponding fractions is shown in the lower panel. Note that the ternary complex assembles with MRAS, while KRAS and excess MRAS elutes as expected for the unbound GTPases. The data are representative of two independent experiments. c. A scatter plot with the dependency score of RAF1 on the x axis and the dependency score of SHOC2 on the y axis. The dashed lines indicate a dependency score of zero (no dependency). A highly negative dependency score implies that a given cell line is highly dependent on that gene. Cell lines dependent on both SHOC2 and RAF1 are indicated in the lower left of the scatter plot. d. Western Blot analysis of MiaPaca2 parental cells or SHOC2 KO cells treated with trametinib (10 nM) for 24 h and siRNA targeting MRAS (100 nM) for 48 h. Densitometry quantification of pCRAF(S259)/CRAF and pERK/ERK levels from Western Blot analysis normalized to trametinib treated parental cells. Samples were derived from the same experiment and blots were processed in parallel. Representative images shown are from two independent experiments.