Over-expressed, N-terminally truncated BRAF is detected in the nucleus of cells with nuclear phosphorylated MEK and ERK
Fiona Hey,
Catherine Andreadi,
Catherine Noble,
Bipin Patel,
Hong Jin,
Tamihiro Kamata,
Kees Straatman,
Jinli Luo,
Kathryn Balmanno,
David T.W. Jones,
V. Peter Collins,
Simon J. Cook,
Christopher J. Caunt,
Catrin Pritchard
Affiliations
Fiona Hey
Department of Molecular Cell Biology, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK
Catherine Andreadi
Leicester Cancer Research Centre, Clinical Sciences Building, University of Leicester, Leicester Royal Infirmary, Leicester LE2 7LX, UK
Catherine Noble
Department of Molecular Cell Biology, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK
Bipin Patel
Department of Molecular Cell Biology, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK
Hong Jin
Department of Molecular Cell Biology, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK
Tamihiro Kamata
Leicester Cancer Research Centre, Clinical Sciences Building, University of Leicester, Leicester Royal Infirmary, Leicester LE2 7LX, UK
Kees Straatman
Core Biotechnology Services, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK
Jinli Luo
Leicester Cancer Research Centre, Clinical Sciences Building, University of Leicester, Leicester Royal Infirmary, Leicester LE2 7LX, UK
Kathryn Balmanno
Signalling Laboratory, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK
David T.W. Jones
Department of Pathology, Division of Molecular Histopathology, University of Cambridge, Cambridge CB2 0QQ, UK
V. Peter Collins
Department of Pathology, Division of Molecular Histopathology, University of Cambridge, Cambridge CB2 0QQ, UK
Simon J. Cook
Signalling Laboratory, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK
Christopher J. Caunt
Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK
Catrin Pritchard
Leicester Cancer Research Centre, Clinical Sciences Building, University of Leicester, Leicester Royal Infirmary, Leicester LE2 7LX, UK; Corresponding author.
BRAF is a cytoplasmic protein kinase, which activates the MEK-ERK signalling pathway. Deregulation of the pathway is associated with the presence of BRAF mutations in human cancer, the most common being V600EBRAF, although structural rearrangements, which remove N-terminal regulatory sequences, have also been reported. RAF-MEK-ERK signalling is normally thought to occur in the cytoplasm of the cell. However, in an investigation of BRAF localisation using fluorescence microscopy combined with subcellular fractionation of Green Fluorescent Protein (GFP)-tagged proteins expressed in NIH3T3 cells, surprisingly, we detected N-terminally truncated BRAF (ΔBRAF) in both nuclear and cytoplasmic compartments. In contrast, ΔCRAF and full-length, wild-type BRAF (WTBRAF) were detected at lower levels in the nucleus while full-length V600EBRAF was virtually excluded from this compartment. Similar results were obtained using ΔBRAF tagged with the hormone-binding domain of the oestrogen receptor (hbER) and with the KIAA1549-ΔBRAF translocation mutant found in human pilocytic astrocytomas. Here we show that GFP-ΔBRAF nuclear translocation does not involve a canonical Nuclear Localisation Signal (NLS), but is suppressed by N-terminal sequences. Nuclear GFP-ΔBRAF retains MEK/ERK activating potential and is associated with the accumulation of phosphorylated MEK and ERK in the nucleus. In contrast, full-length GFP-WTBRAF and GFP-V600EBRAF are associated with the accumulation of phosphorylated ERK but not phosphorylated MEK in the nucleus. These data have implications for cancers bearing single nucleotide variants or N-terminal deleted structural variants of BRAF.
