Biochemical and functional characterization of germ line KRAS mutations

Abstract

Germ line missense mutations in HRAS and KRAS and in genes encoding molecules that function up- or downstream of Ras in cellular signaling networks cause a group of related developmental disorders that includes Costello syndrome, Noonan syndrome, and cardiofaciocutaneous syndrome. We performed detailed biochemical and functional studies of three mutant K-Ras proteins (P34R, D153V, and F156L) found in individuals with Noonan syndrome and cardiofaciocutaneous syndrome. Mutant K-Ras proteins demonstrate a range of gain-of-function effects in different cell types, and biochemical analysis supports the idea that the intrinsic Ras guanosine nucleotide triphosphatase (GTPase) activity, the responsiveness of these proteins to GTPase-activating proteins, and guanine nucleotide dissociation all regulate developmental programs in vivo.

FIG. 1.

Biochemical analysis of WT and mutant K-Ras proteins. (A) Schematic representation of K-Ras4B showing the distribution of the amino acid substitutions encoded by germ line mutations found for developmental disorders (above) and the three amino acids that are commonly altered by cancer-associated somatic mutations (below). The P-loop, switch I (Sw I), and switch II (Sw II) domains are conserved among all Ras isoforms (H-Ras, N-Ras, K-Ras4A, and K-Ras4B). These isoforms vary considerably in the hypervariable region. The germ line substitutions characterized in this study are shown in red. (B) Intrinsic GTP hydrolysis measured as the number of cpm released over time. (C and D) GTP hydrolysis stimulated by various concentrations of the GRD of neurofibromin (C) or p120 GAP (D). GTP hydrolysis was measured after 8 min. (E and F) Dissociation of bound GTP (E) and GDP (F) from WT and mutant K-Ras proteins over time.

FIG. 2.

Functional characterization of P34R, D153V, and F156L K-Ras proteins. (A) Ras signaling in transiently transfected COS-7 cells under basal growth conditions or after 6 h in 0.1% fetal bovine serum (starvation). (B) CFU-GM colony formation of fetal liver cells expressing WT or mutant K-Ras proteins over a range of GM-CSF concentrations. (C) BFU-E colony formation of fetal liver cells expressing WT and mutant K-Ras proteins over a range of erythropoietin (EPO) concentrations. Data show number of colonies per 200,000 GFP-positive fetal liver cells. (D) Phosphorylation of signaling proteins downstream of Ras in macrophage progenitors expressing WT or mutant K-Ras proteins. The data shown in panels A to D are representative of at least three independent experiments.