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.
Cutler
R E
RE
Molecular Basis of Carcinogenesis Laboratory, ABL-Basic Research Program, National Cancer Institute, Frederick Cancer Research and Development Center, MD 21702, USA.
Morrison
D K
DK
D048948:14-3-3 Proteins; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D001665:Binding Sites; D002460:Cell Line; D004330:Drosophila / Q000235:genetics / Q000502:physiology; D004789:Enzyme Activation; D005998:Glycine / Q000378:metabolism; D006801:Humans; D007668:Kidney; D008540:Meiosis; D015689:Oncogene Protein p21(ras) / Q000235:genetics / Q000378:metabolism*; D009865:Oocytes; D010768:Phosphoserine / Q000032:analysis; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D015398:Signal Transduction / Q000502:physiology*; D013489:Suppression, Genetic / Q000502:physiology*; D014446:Tyrosine 3-Monooxygenase; D014981:Xenopus
D048948:14-3-3 Proteins; C000715470:MAS1 protein, human; D011506:Proteins; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D010768:Phosphoserine; D001224:Aspartic Acid; D014446:Tyrosine 3-Monooxygenase; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D015689:Oncogene Protein p21(ras); D005998:Glycine