Figure 3
From: Functional characterisation of a novel class of in-frame insertion variants of KRAS and HRAS
VMOS RAS variants are insensitive to GEFs. (A–F) Nucleotide exchange rates in the absence or presence of RasGRP1 (150 nM) or SOS (1.5 μM) of wild type (A,E) and variant RAS (B–D,F). For variant RAS an additional control was included to which EDTA was added at the indicated point in time. (G) RasGRP1 (150 nM) catalysed nucleotide exchange activity on wild type KRAS (200 nM) in the presence of various concentrations of the KRAS case1 variant. (H) SOS (150 nM) catalysed nucleotide exchange activity on wild type KRAS (200 nM) in the presence of variant RAS (20 μM) as indicated. Variant RAS was used as obtained from the protein purification and therefore in part loaded with GDP and in part with GTP. The portion of GTP loaded RAS was 64% (case 1), 71% (case 2), 39% (case 1), and 77% (case 4). Note: The reduced ability of the case 2 variant cannot be attributed to lower GTP loading. (I) SOS catalysed nucleotide exchange was measured as in (H) in the presence of various concentration of the case 1 KRAS variant. The decay of the fluorescence signal was fitted to a single exponential functions and the obtained rate constants (kobs) were plotted against the concentration of the KRAS variant.
