Figure 3: K-Ras insertion mutants have reduced intrinsic GTPase activity and are insensitive to GAP stimulation.
From: KRAS insertion mutations are oncogenic and exhibit distinct functional properties
(a) Phosphorimaging of α-P32 nucleotides eluted from indicated recombinant wild-type K-Ras (WT), K-RasG12D (D12), K-RasE62_A66dup (5AA), or K-RasG60_A66dup (7AA) proteins and separated by thin layer chromatography. Labelling, elution, and electrophoresis were performed in duplicate. (b) Quantification of the data shown in panel a indicating the percentage of GTP-bound Ras after 2 hr incubation. (c) Intrinsic GTP hydrolysis of recombinant K-Ras proteins. Scintillation counting of phosphates liberated from K-Ras proteins loaded with γ-P32 GTP at indicated time points. Best fit linear regressions are shown. Data are average±s.d. of duplicate reactions and are representative of three independent experiments. (d) Scintillation counting of free phosphates released by K-Ras proteins that were loaded with γ-P32 GTP and incubated with the indicated concentrations of a recombinant peptide encoding the GAP domain of p120 GAP. Data are average±s.d. of duplicate reactions and representative of three independent experiments.
