Elucidation of binding determinants and functional consequences of Ras/Raf-cysteine-rich domain interactions.
Raf-1 is a critical downstream target of Ras and contains two distinct domains that bind Ras. The first Ras-binding site (RBS1) in Raf-1 has been shown to be essential for Ras-mediated translocation of Raf-1 to the plasma membrane, whereas the second site, in the Raf-1 cysteine-rich domain (Raf-CRD), has been implicated in regulating Raf kinase activity. While recognition elements that promote Ras.RBS1 complex formation have been characterized, relatively little is known about Ras/Raf-CRD interactions. In this study, we have characterized interactions important for Ras binding to the Raf-CRD. Reconciling conflicting reports, we found that these interactions are essentially independent of the guanine nucleotide bound state, but instead, are enhanced by post-translational modification of Ras. Specifically, our findings indicate that Ras farnesylation is sufficient for stable association of Ras with the Raf-CRD. Furthermore, we have also identified a Raf-CRD variant that is impaired specifically in its interactions with Ras. NMR data also suggests that residues proximal to this mutation site on the Raf-CRD form contacts with Ras. This Raf-CRD mutant impairs the ability of Ras to activate Raf kinase, thereby providing additional support that Ras interactions with the Raf-CRD are important for Ras-mediated activation of Raf-1.
Williams
J G
JG
Department of Biochemistry and Biophysics, Department of Pharmacology, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Drugan
J K
JK
Yi
G S
GS
Clark
G J
GJ
Der
C J
CJ
Campbell
S L
SL
D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D003545:Cysteine; D011485:Protein Binding; D011487:Protein Conformation; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry* / Q000378:metabolism; D018631:ras Proteins / Q000737:chemistry* / Q000378:metabolism
D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins; D003545:Cysteine