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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.

SUBMITTER: Rushworth LK

PROVIDER: S-EPMC1430271 | biostudies-literature | 2006 Mar

REPOSITORIES: biostudies-literature

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Regulation and role of Raf-1/B-Raf heterodimerization.

Rushworth Linda K LK Hindley Alison D AD O'Neill Eric E Kolch Walter W

Molecular and cellular biology 20060301 6

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 R ...[more]

PMID: 16508002

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Project description:The dimerization of RAF kinases represents a key event in their activation cycle and in RAS/ERK pathway activation. Genetic, biochemical and structural approaches provided key insights into this process defining RAF signaling output and the clinical efficacy of RAF inhibitors (RAFi). However, methods reporting the dynamics of RAF dimerization in living cells and in real time are still in their infancy. Recently, split luciferase systems have been developed for the detection of protein-protein-interactions (PPIs), incl. proof-of-concept studies demonstrating the heterodimerization of the BRAF and RAF1 isoforms. Due to their small size, the Nanoluc luciferase moieties LgBiT and SmBiT, which reconstitute a light emitting holoenzyme upon fusion partner promoted interaction, appear as well-suited to study RAF dimerization. Here, we provide an extensive analysis of the suitability of the Nanoluc system to study the homo- and heterodimerization of BRAF, RAF1 and the related KSR1 pseudokinase. We show that KRASG12V promotes the homo- and heterodimerization of BRAF, while considerable KSR1 homo- and KSR1/BRAF heterodimerization already occurs in the absence of this active GTPase and requires a salt bridge between the CC-SAM domain of KSR1 and the BRAF-specific region. We demonstrate that loss-of-function mutations impairing key steps of the RAF activation cycle can be used as calibrators to gauge the dynamics of heterodimerization. This approach identified the RAS-binding domains and the C-terminal 14-3-3 binding motifs as particularly critical for the reconstitution of RAF mediated LgBiT/SmBiT reconstitution, while the dimer interface was less important for dimerization but essential for downstream signaling. We show for the first time that BRAFV600E, the most common BRAF oncoprotein whose dimerization status is controversially portrayed in the literature, forms homodimers in living cells more efficiently than its wildtype counterpart. Of note, Nanoluc activity reconstituted by BRAFV600E homodimers is highly sensitive to the paradox-breaking RAFi PLX8394, indicating a dynamic and specific PPI. We report the effects of eleven ERK pathway inhibitors on RAF dimerization, incl. third-generation compounds that are less-defined in terms of their dimer promoting abilities. We identify Naporafenib as a potent and long-lasting dimerizer and show that the split Nanoluc approach discriminates between type I, I1/2 and II RAFi. Video Abstract.

Dataset Information

Regulation and role of Raf-1/B-Raf heterodimerization.

Publications

Regulation and role of Raf-1/B-Raf heterodimerization.

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