Fig. 5. Interactions at the RAF1-CRD interface with KRAS are required for full RAF1 kinase activity.

a FLAG-RAF1 WT and mutants for kinase assays were immunoprecipitated with FLAG antibody from 293T cells and assessed for binding to Strep2-KRAS. b Kinase activity of purified RAF1 mutants from cells co-expressing KRAS Q61L (shown in panel 5a). c FLAG-RAF1 WT and Strep2-KRAS mutants were co-expressed and isolated as in a. KRAS mutations were introduced into the constitutively active KRAS-Q61L background and “-” denotes Q61L with no additional mutations. d Kinase activity of purified RAF1 co-expressed with KRAS mutants in the constitutively active Q61L background (shown in c). e Sequence alignment of residues present in the switch I, and interswitch region of human RAS isoforms and members of RAS subfamily. Fully conserved residues are highlighted in black. The switch and inter-switch regions are indicated above the alignment. KRAS residues that are involved at the KRAS-CRD and KRAS-RBD interfaces are highlighted above the alignment using ovals and triangles, respectively. The red boxes and cyan color highlight a lack of conservation in the interswitch region among RAS isoforms and MRAS as well as other members of the RAS subfamily. f FLAG-KRAS WT or mutant, or constitutively active MRAS-Q71L immune precipitates were probed for endogenous RAF1 interaction and pathway activation was measured in lysates. “HTE” denotes substitution of inter-switch residues ⁴³QVV⁴⁵ (KRAS) to HTE (MRAS), and “HTE-NQWAI” was changed from ⁴³QVV⁴⁵-⁴⁸GETCL⁵² (KRAS) to HTE-NQWAI (MRAS). Representative of n = 3 independent experiments. g The KRAS-CRD interaction interface, with CRD shown in electrostatic surface representation and the KRAS residues that participate at the interface shown in stick representation. Structural superposition of MRAS (PDB 1X1S) with KRAS shows residue E55 from MRAS clashes with CRD. Bar graphs represent mean phosphorylation ± SD and data points for three independent experiments, except R89L where n = 2. Source data are provided as a Source Data file.