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.

Macrophage migration inhibitory factor interactions with glutathione and S-hexylglutathione.

Macrophage migration inhibitory factor (MIF) has been reported to interact with glutathione and S-hexylglutathione and to possess glutathione S-transferase activity. However, contrary to these reports, a recent NMR study concluded that MIF shows no affinity for glutathione. Re-examination of the glutathione-MIF interactions indicates that the reported increase in fluorescence upon addition of glutathione is because of pH-induced unfolding of the protein and not to any direct interactions. Circular dichroism shows that MIF remains folded from pH 4.5-7.5 but is 50% unfolded at pH 2.9 +/- 0.2. The reported increase in fluorescence can be achieved by acid titration. Under strongly buffered conditions, no fluorescence change is observed upon addition of glutathione. In contrast to the results with glutathione, MIF binds S-hexylglutathione with a Kd of 2.5 +/- 0.6 mM. Using NMR spectroscopy, a binding site which clusters around the N-terminal proline was identified. These data indicate that the binding site for S-hexylglutathione is the same as the catalytic site for the dopachrome tautomerase activity of MIF. Consequently, the binding of S-hexylglutathione as well as hexanethiol inhibits this catalytic activity.