Over-expressed, N-terminally truncated BRAF is detected in the nucleus of cells with nuclear phosphorylated MEK and ERK

Abstract

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.

Nuclear Localization of BRAFV600E Is Associated with HMOX-1 Upregulation and Aggressive Behavior of Melanoma Cells

“…In the first study, an exogenous truncated BRAF transfected in NIH3T3 cells was used, and this artificial system cannot be generalized or applied to the endogenous BRAF. In the second study, Shin et al used a non-cancer C2C12 myoblast cell line and found a very tiny fraction of WT BRAF in the nucleus compared to a strong nuclear presence of BRAF V600E [ 34 , 35 ]. Our hypothesis is that WT BRAF activation requires external growth factors, and its localization is totally cytoplasmic.…”