A cancer-associated K-RAS mutant that preferentially binds GDP displays gain-of-function and atypical signaling

Abstract

Background: The RAS genes, which are the prototype for the RAS superfamily of small GTPases, encode guanine nucleotide binding proteins that are frequently mutated in cancer. The superfamily has five major branches: RAS, RHO, RAB, RAN, and ARF. Its wild type members function as molecular switches that are active when GTP-bound and inactive when GDP-bound. Previously analyzed cancer-associated point mutants of RAS, such as those affecting codons 12, 13, and 61, and mutants of other superfamily members have followed this paradigm, as their steady-state level of bound GTP is higher than that of the wild type protein. However, it is possible that a less common cancer-associated mutant might be active when bound to GDP. Methods: We searched the Catalog of Somatic Mutations in Cancer (COSMIC) database and the TCGA database in the NCI Genomic Data Commons cancer-associated mutations in the RAS superfamily beyond the commonly mutated RAS codons, conducted biological and biochemical tissue culture studies and in vitro biochemical guanine nucleotide binding studies of a K-RAS mutant identified by this search, and complemented these findings with all-atom molecular dynamics simulations. Results: The database search identified a recurrent cancer-associated K-RAS mutant, M67L, as well as another cancer-associated mutant that affect the same codon in K-RAS and N-RAS and additional point mutants in the analogous codon in 10 other members of the RAS superfamily of GTPases. Detailed analysis of the K-RAS M67L mutant indicated that it has a gain-of-function biologically, but its low GTP level is similar to that of wild type K-RAS. Unlike the GDP-bound wild type, the GDP-bound M67L mutant interacts with and activates at least three RAS effectors: RALGDS, PI3K-p110a, and RAPGEF6. However, it interacts weakly with the prototypic RAS effector, RAF. All-atom molecular dynamics simulations of the mutant, conducted with and without RALGDS, provide structural insight into these characteristics of the GDP-bound mutant. Conclusion: Cancer-associated mutations that are active when GDP-bound occur in K-RAS and probably in other members of the RAS superfamily.