Autophosphorylation on S614 inhibits the activity and the transforming potential of BRAF

The BRAF proto-oncogene serine/threonine-protein kinase, known as BRAF, belongs to the RAF kinase family. It regulates the MAPK/ERK signalling pathway affecting several cellular processes such as growth, survival, differentiation, and cellular transformation. BRAF is mutated in similar to 8% of all human cancers with the V600E mutation constituting similar to 90% of mutations. Here, we have used quantitative mass spectrometry to map and compare phosphorylation site patterns between BRAF and BRAF V600E. We identified sites that are shared as well as several quantitative differences in phosphorylation abundance. The highest difference is phosphorylation of 5614 in the activation loop which is similar to 5fold enhanced in BRAF V600E. Mutation of 5614 increases the kinase activity of both BRAF and BRAF V600E and the transforming ability of BRAF V600E. The phosphorylation of 5614 is mitogen inducible and the result of autophosphorylation. These data suggest that phosphorylation at this site is inhibitory, and part of the physiological shut-down mechanism of BRAF signalling. (C) 2016 Elsevier Inc. All rights reserved.