title: Regulation of BRAF protein stability by a negative feedback loop involving the MEK-ERK pathway but not the FBXW7 tumour suppressor.
abstract: The (V600E)BRAF oncogenic mutation is detected in a wide range of human cancers and induces hyperactivation of the downstream MEK-ERK signalling cascade. Although output of the BRAF-MEK-ERK pathway is regulated by feed-forward RAF activity, feedback control also plays an important role. One such feedback pathway has been identified in Caenorhabditis elegans and involves ERK-mediated phosphorylation of BRAF within a CDC4 phosphodegron (CPD), targeting BRAF for degradation via CDC4 (also known as FBXW7), a component of the SKP1/CUL1/F-box (SCF) E3 ubiquitin ligase complex. Here we investigate this pathway in mammalian cells. Short-term expression of autochthonous (V600E)BRAF in mouse embryonic fibroblasts (MEFs) leads to down-regulation of BRAF protein levels in a proteasome-dependent manner and (V600E)BRAF has a reduced half-life compared to (WT)BRAF in HEK293(T) cells. These effects were reversed by treatment with the MEK inhibitor PD184352. We have identified the equivalent CPD at residues 400-405 in human BRAF and have found that mutation of ERK phosphorylation sites at residues T401 and S405 in (V600E)BRAF increases the half-life of the protein. While BRAF and FBXW7 co-immunoprecipitated, the overexpression of FBXW7 did not influence the half-life of either (WT)BRAF or (V600E)BRAF. Furthermore, disruption of the substrate-binding site of mouse FBXW7 using the R482Q mutation did not affect the interaction with BRAF and the expression levels of (WT)BRAF and (V600E)BRAF were not altered in MEFs derived from mice with the homozygous knockin (R482Q)FBXW7 mutation. Overall these data confirm the existence of a negative feedback pathway by which BRAF protein stability is regulated by ERK. However, unlike the situation in C. elegans, FBXW7 does not play a unique role in mediating subsequent BRAF degradation.
authors.affiliation: Department of Molecular Cell Biology, University of Leicester, University Road, Leicester LE1 7RH, UK.
authors.forename: Maria Aguilar
authors.initials: MA
authors.lastname: Hernandez
authors.affiliation: Department of Molecular Cell Biology, University of Leicester, University Road, Leicester LE1 7RH, UK.
authors.forename: Bipin
authors.initials: B
authors.lastname: Patel
authors.affiliation: Department of Molecular Cell Biology, University of Leicester, University Road, Leicester LE1 7RH, UK.
authors.forename: Fiona
authors.initials: F
authors.lastname: Hey
authors.affiliation: Department of Molecular Cell Biology, University of Leicester, University Road, Leicester LE1 7RH, UK.
authors.forename: Susan
authors.initials: S
authors.lastname: Giblett
authors.affiliation: Gastrointestinal Stem Cell Biology Laboratory, Wellcome Trust Centre for Human Genetics, Oxford University, Roosevelt Drive, Oxford OX3 7BN, UK.
authors.forename: Hayley
authors.initials: H
authors.lastname: Davis
authors.affiliation: Department of Molecular Cell Biology, University of Leicester, University Road, Leicester LE1 7RH, UK; Department of Cancer Studies, University of Leicester, University Road, Leicester LE1 7RH, UK. Electronic address: cap8@le.ac.uk.
authors.forename: Catrin
authors.initials: C
authors.lastname: Pritchard
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D016922:Cellular Senescence; D015536:Down-Regulation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D044783:F-Box Proteins / Q000502:physiology*; D000073979:F-Box-WD Repeat-Containing Protein 7; D025461:Feedback, Physiological; D057809:HEK293 Cells; D006801:Humans; D020935:MAP Kinase Signaling System; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D009154:Mutation; D055550:Protein Stability; D048493:Proto-Oncogene Proteins B-raf / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000502:physiology; D044767:Ubiquitin-Protein Ligases / Q000502:physiology*
chemical_list: D044783:F-Box Proteins; D000073979:F-Box-WD Repeat-Containing Protein 7; C513272:Fbxw7 protein, mouse; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; C482120:Braf protein, mouse; D048493:Proto-Oncogene Proteins B-raf; D048049:Extracellular Signal-Regulated MAP Kinases; D020929:Mitogen-Activated Protein Kinase Kinases
keywords: BRAF; ERK; FBXW7; Feedback; Protein stability