Identification of residues in the cysteine-rich domain of Raf-1 that control Ras binding and Raf-1 activity.
We have identified mutations in Raf-1 that increase binding to Ras. The mutations were identified making use of three mutant forms of Ras that have reduced Raf-1 binding (Winkler, D. G., Johnson, J. C., Cooper, J. A., and Vojtek, A. B. (1997) J. Biol. Chem. 272, 24402-24409). One mutation in Raf-1, N64L, suppresses the Ras mutant R41Q but not other Ras mutants, suggesting that this mutation structurally complements the Ras R41Q mutation. Missense substitutions of residues 143 and 144 in the Raf-1 cysteine-rich domain were isolated multiple times. These Raf-1 mutants, R143Q, R143W, and K144E, were general suppressors of three different Ras mutants and had increased interaction with non-mutant Ras. Each was slightly activated relative to wild-type Raf-1 in a transformation assay. In addition, two mutants, R143W and K144E, were active when tested for induction of germinal vesicle breakdown in Xenopus oocytes. Interestingly, all three cysteine-rich domain mutations reduced the ability of the Raf-1 N-terminal regulatory region to inhibit Xenopus oocyte germinal vesicle breakdown induced by the C-terminal catalytic region of Raf-1. We propose that a direct or indirect regulatory interaction between the N- and C-terminal regions of Raf-1 is reduced by the R143W, R143Q, and K144E mutations, thereby increasing access to the Ras-binding regions of Raf-1 and increasing Raf-1 activity.
Winkler
D G
DG
Fred Hutchinson Cancer Research Center, Seattle, Washington 98109-1024, USA.
Cutler
R E
RE
Drugan
J K
JK
Campbell
S
S
Morrison
D K
DK
Cooper
J A
JA
D048948:14-3-3 Proteins; D000818:Animals; D002384:Catalysis; D003062:Codon; D003545:Cysteine / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D011506:Proteins / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014446:Tyrosine 3-Monooxygenase; D014981:Xenopus; D015003:Yeasts; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
D048948:14-3-3 Proteins; D003062:Codon; D011506:Proteins; D014446:Tyrosine 3-Monooxygenase; D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins; D003545:Cysteine