Regulation and role of Raf-1/B-Raf heterodimerization.
The Ras-Raf-MEK-extracellular signal-regulated kinase (ERK) pathway participates in the control of many fundamental cellular processes including proliferation, survival, and differentiation. The pathway is deregulated in up to 30% of human cancers, often due to mutations in Ras and the B-Raf isoform. Raf-1 and B-Raf can form heterodimers, and this may be important for cellular transformation. Here, we have analyzed the biochemical and biological properties of Raf-1/B-Raf heterodimers. Isolated Raf-1/B-Raf heterodimers possessed a highly increased kinase activity compared to the respective homodimers or monomers. Heterodimers between wild-type Raf-1 and B-Raf mutants with low or no kinase activity still displayed elevated kinase activity, as did heterodimers between wild-type B-Raf and kinase-negative Raf-1. In contrast, heterodimers containing both kinase-negative Raf-1 and kinase-negative B-Raf were completely inactive, suggesting that the kinase activity of the heterodimer specifically originates from Raf and that either kinase-competent Raf isoform is sufficient to confer high catalytic activity to the heterodimer. In cell lines, Raf-1/B-Raf heterodimers were found at low levels. Heterodimerization was enhanced by 14-3-3 proteins and by mitogens independently of ERK. However, ERK-induced phosphorylation of B-Raf on T753 promoted the disassembly of Raf heterodimers, and the mutation of T753 prolonged growth factor-induced heterodimerization. The B-Raf T753A mutant enhanced differentiation of PC12 cells, which was previously shown to be dependent on sustained ERK signaling. Fine mapping of the interaction sites by peptide arrays suggested a complex mode of interaction involving multiple contact sites with a main Raf-1 binding site in B-Raf encompassing T753. In summary, our data suggest that Raf-1/B-Raf heterodimerization occurs as part of the physiological activation process and that the heterodimer has distinct biochemical properties that may be important for the regulation of some biological processes.
Rushworth
Linda K
LK
Signalling and Proteomics Laboratory, Beatson Institute for Cancer Research, Garscube Estate, Glasgow G61 1BD, United Kingdom.
Hindley
Alison D
AD
O'Neill
Eric
E
Kolch
Walter
W
D048948:14-3-3 Proteins / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D020930:MAP Kinase Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D046912:Multiprotein Complexes; D009154:Mutation; D020932:Nerve Growth Factor / Q000494:pharmacology; D016716:PC12 Cells / Q000187:drug effects; D010766:Phosphorylation; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000378:metabolism*; D051381:Rats
D048948:14-3-3 Proteins; D004791:Enzyme Inhibitors; D046912:Multiprotein Complexes; D020932:Nerve Growth Factor; C482123:Braf protein, rat; D048493:Proto-Oncogene Proteins B-raf; D019908:Proto-Oncogene Proteins c-raf; D048049:Extracellular Signal-Regulated MAP Kinases; D020930:MAP Kinase Kinase Kinases