Ras interaction with two distinct binding domains in Raf-1 may be required for Ras transformation.
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).
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599, USA.
J K
JK
Drugan
R
R
Khosravi-Far
M A
MA
White
C J
CJ
Der
Y J
YJ
Sung
Y W
YW
Hwang
S L
SL
Campbell
D016475:3T3 Cells; D000818:Animals; D002471:Cell Transformation, Neoplastic; D003545:Cysteine / Q000378:metabolism; D051379:Mice; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D014364:Tryptophan / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism*
D011518:Proto-Oncogene Proteins; D014364:Tryptophan; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins; D003545:Cysteine