RNA expression profile (RNA-Seq) in mouse colonic tumors expressing endogenous K-Ras-WT, K-Ras-G12D, or K-RAS-G12D/K104Q
Ras proteins are GTPases that regulate a wide range of cellular processes. The activity of Ras is dependent on its nucleotide-binding status, which is modulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Previously, we demonstrated that mutation of lysine 104 to glutamine (K104Q) attenuates the transforming capacity of oncogenic K-Ras by interrupting GEF induced nucleotide exchange. To assess the effect of this mutation in vivo, we used CRISPR/Cas9 to generate mouse models carrying the K104Q point mutation in wild-type and conditional K- RasLSL-G12D alleles. Homozygous animals for K104Q were viable, fertile, and arose at Mendelian frequency, indicating that K104Q is not a complete loss of function mutation. Consistent with our previous findings from in vitro studies, however, the oncogenic activity of K-RasG12D was significantly attenuated by mutation at K104. These data demonstrate that lysine at position 104 is critical for the full oncogenic activity of mutant K-Ras and suggest that modification at K104, for example acetylation, may also regulate its activity. Using biochemical and structural analysis, we found that the G12D and K104Q mutations cooperate to suppress GEF-mediated nucleotide exchange, explaining the preferential effect of K104Q on oncogenic K-Ras. Furthermore, we found that K104 site forms allosteric network with three residues (M72, R73 and G75) on ?2 helix at switch II. Intriguingly, point mutation at glutamine 75 to alanine (G75A) showed strong negative regulatory effect on K-RasG12D. Collectively, modulating these sites may provide a unique therapeutic approach in cancer expressing mutant K-Ras. Overall design: RNA-Seq of colonic tumor from three mouse models, Fabp-Cre/+ (Control), Fabp-Cre/+; K-Ras-G12D/+ (Experimental), and Fabp-Cre/+; K-Ras-G12D-K104Q/+ (Experimental). Colonic tumor from the control model expressed K-Ras-WT. Colonic tumor from the experimental model expressed oncogenic K-Ras-G12D or K-Ras-G12D-K104Q/+.