Oncogenic insertional mutations in the P-loop of Ras are overactive in MAP kinase signaling.

Mutations of Ras with three extra amino acids inserted into the phosphate-binding (P) loop have been investigated both in vitro and in vivo. Such mutants have originally been detected as oncogenes both in the ras and the TC21 genes. Biochemical experiments reveal the molecular basis of their oncogenic potential: the mutants show a strongly attenuated binding affinity for nucleotides, most notably for GDP, leading to a preference for GTP binding. Furthermore, both the intrinsic as well as the GAP-stimulated GTP hydrolysis are drastically diminished. The binding interaction with GAP is reduced, whereas binding to the Ras-binding domain of the downstream effector c-Raf1 is not altered appreciably. Microinjection into PC12 cells shows the mutants to be as potent to induce neurite outgrowth as conventional oncogenic Ras mutants. Unexpectedly, their ability to stimulate the MAP kinase pathway as measured by a reporter gene assay in RK13 cells is much higher than that of the normal oncogenic mutant G12V. This characteristic was attributed to an increased stimulation of c-Raf1 kinase activity by the insertional Ras mutants.