The RafC1 cysteine-rich domain contains multiple distinct regulatory epitopes which control Ras-dependent Raf activation.
Activation of c-Raf-1 (referred to as Raf) by Ras is a pivotal step in mitogenic signaling. Raf activation is initiated by binding of Ras to the regulatory N terminus of Raf. While Ras binding to residues 51 to 131 is well understood, the role of the RafC1 cysteine-rich domain comprising residues 139 to 184 has remained elusive. To resolve the function of the RafC1 domain, we have performed an exhaustive surface scanning mutagenesis. In our study, we defined a high-resolution map of multiple distinct functional epitopes within RafC1 that are required for both negative control of the kinase and the positive function of the protein. Activating mutations in three different epitopes enhanced Ras-dependent Raf activation, while only some of these mutations markedly increased Raf basal activity. One contiguous inhibitory epitope consisting of S177, T182, and M183 clearly contributed to Ras-Raf binding energy and represents the putative Ras binding site of the RafC1 domain. The effects of all RafC1 mutations on Ras binding and Raf activation were independent of Ras lipid modification. The inhibitory mutation L160A is localized to a position analogous to the phorbol ester binding site in the protein kinase C C1 domain, suggesting a function in cofactor binding. Complete inhibition of Ras-dependent Raf activation was achieved by combining mutations K144A and L160A, which clearly demonstrates an absolute requirement for correct RafC1 function in Ras-dependent Raf activation.
Abteilung Strukturelle Biologie, Max-Planck-Institut für Molekulare Physiologie, Dortmund, Germany.
M
M
Daub
J
J
Jöckel
T
T
Quack
C K
CK
Weber
F
F
Schmitz
U R
UR
Rapp
A
A
Wittinghofer
C
C
Block
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry*; D004789:Enzyme Activation / Q000235:genetics; D000939:Epitopes / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D050356:Lipid Metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D011485:Protein Binding / Q000502:physiology; D011493:Protein Kinase C / Q000737:chemistry; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry*; D011817:Rabbits; D015398:Signal Transduction / Q000502:physiology; D015533:Transcriptional Activation / Q000502:physiology; D018631:ras Proteins / Q000502:physiology*
D000939:Epitopes; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D018631:ras Proteins; D003545:Cysteine