Fig. 4: Canonical RAS isoforms can form active holophosphatases.

a, SPR-derived affinity and cooperativity immobilizing NRAS–GppNHp, NRAS(Q61R)–GTP, KRAS–GppNHp or KRAS(Q61R)–GTP with the indicated analytes. α = K_D^SHOC2/K_D^SHOC2–PP1C. Affinity values over 10,000 nM are highlighted in red. b, In vitro dephosphorylation of autoinhibited and active-state BRAF–MEK1–14-3-3 complexes by the SHOC2 holophosphatase. Purified full-length BRAF complexes in the autoinhibited state (left) or active dimeric state (right) were incubated with lambda phosphatase (PPase), PP1Cα or ternary SHOC2 complexes, and were blotted with phospho-specific antibodies for BRAF phosphorylated at Ser365 and Ser729. Equivalent loading of BRAF complexes is shown by Coomassie staining. Phosphorylated Ser365 is selectively dephosphorylated relative to Ser729 in the active dimer, whereas both are relatively protected in the autoinhibited (14-3-3-bound) state. Experiments were conducted twice with similar results. c, The relationship between the knockdown of SHOC2 and MRAS, HRAS, NRAS and KRAS. The dependency scores of each RAS gene and SHOC2 are shown on the x and y axes, respectively. Dashed lines indicate a dependency score of zero. A highly negative dependency score implies that a given cell line is highly dependent on that gene. Cell lines dependent on both SHOC2 and RAS are indicated at the bottom left. Right, the calculated Pearson correlation coefficient (y axis) applied to each mutation group (group of cell lines containing the associated mutation). A higher positive value indicates a stronger positive relationship: the dependency score of SHOC2 decreases/increases in the same lines as the dependency scores of the RAS genes. The n values above each bar show the number of cell lines in each mutation group. d, Immunoblot analysis of MiaPaca2 parental cells (Par), SHOC2-KO (KO) and stable cell lines reconstituted with SHOC2 mutants after 10 nM trametinib (Tram) treatment for 1 h or 24 h (+). Densitometry quantification (percentage variation) of pCRAF/CRAF and pERK/ERK levels from the immunoblot analysis normalized to untreated MiaPaca2 parental cells. The samples were derived from the same experiment and blots were processed in parallel. The images are representative of two independent experiments.