Ras interaction with two distinct binding domains in Raf-1 may be required for Ras transformation

• Published in: The Journal of biological chemistry 1996-01, Vol.271 (1), p.233-237
• Main Authors: Drugan, J K, Khosravi-Far, R, White, M A, Der, C J, Sung, Y J, Hwang, Y W, Campbell, S L
• Format: Article
• Language: English
• Subjects: 3T3 Cells; Animals; Cell Transformation, Neoplastic; Cysteine - metabolism; Mice; Protein Binding; Protein-Serine-Threonine Kinases - metabolism; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins c-raf; ras Proteins - metabolism; Tryptophan - metabolism
• ISSN: 0021-9258
• DOI: 10.1074/jbc.271.1.233

Although Raf-1 is a critical Ras effector target, how Ras mediates Raf-1 activation remains unresolved. Raf-1 residues 55-131 define a Ras-binding domain essential for Raf-1 activation. Therefore, our identification of a second Ras-binding site in the Raf-1 cysteine-rich domain (residues 139-184) was unexpected and suggested a more complex role for Ras in Raf-1 activation. Both Ras recognition domains preferentially associate with Ras-GTP. Therefore, mutations that impair Ras activity by perturbing regions that distinguish Ras-GDP from Ras-GTP (switch I and II) may disrupt interactions with either Raf-1-binding domain. We observed that mutations of Ras that impaired Ras transformation by perturbing its switch I (T35A and E37G) or switch II (G60A and Y64W) domain preferentially diminished binding to Raf-1-(55-131) or the Raf-1 cysteine-rich domain, respectively. Thus, these Ras-binding domains recognize distinct Ras-GTP determinants, and both may be essential for Ras transforming activity. Finally, since Ha-Ras T35A and E37G mutations prevent Ras interaction with full-length Raf-1, we suggest that Raf-Cys is a cryptic binding site that is unmasked upon Ras interaction with Raf-1-(55-131).