title: Regulation and role of Raf-1/B-Raf heterodimerization.
abstract: 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.
authors.affiliation: Signalling and Proteomics Laboratory, Beatson Institute for Cancer Research, Garscube Estate, Glasgow G61 1BD, United Kingdom.
authors.forename: Linda K
authors.initials: LK
authors.lastname: Rushworth
authors.forename: Alison D
authors.initials: AD
authors.lastname: Hindley
authors.forename: Eric
authors.initials: E
authors.lastname: O'Neill
authors.forename: Walter
authors.initials: W
authors.lastname: Kolch
mesh_terms: 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
chemical_list: 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