Mammalian Raf-1 is activated by mutations that restore Raf signaling in Drosophila

An interaction with the Ras proto‐oncogene product is a requirement for Raf‐1 activation in many signaling cascades. The significance of this interaction is demonstrated by the fact that a mutation preventing the Ras–Raf interaction severely impairs the function of both mammalian (Raf‐1) and Drosophila (D‐Raf) Raf proteins. In D‐Raf, however, dominant intragenic mutations have been identified that suppress the effect of the Ras‐binding site (RBS) mutation. To address the mechanism by which these mutations restore Raf signaling, we have introduced the suppressor mutations into the analogous residues of mammalian Raf‐1. Here, we show that rather than compensating for the RBS mutation by restoring the Ras–Raf‐1 interaction, the suppressor mutations increase the enzymatic and biological activity of Raf‐1, allowing Raf‐1 to signal in the absence of Ras binding. Surprisingly, we find that while one of the suppressor mutations (P181L) increases the basal kinase activity of Raf‐1, it also abolishes the ability of wild‐type Raf‐1 to become activated by Ras. This mutation occurs in the cysteine‐rich domain (CRD) of Raf‐1 and demonstrates the importance of this region for a productive Ras–Raf interaction. Finally, we present evidence that the most activating suppressor mutation (G498S) increases Raf‐1 activity by introducing a novel phosphorylation site into the L12 activation loop of the Raf‐1 kinase domain.