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Mol. Cell. Biol. doi:10.1128/MCB.01265-06
Copyright (c) 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Differential regulation of B-Raf isoforms by phosphorylation and auto-inhibitory mechanisms

Institut Curie, Centre de Recherche, Orsay F-91405 France; CNRS, UMR 146, Orsay F-91405 France

^* To whom correspondence should be addressed. Email: Alain.Eychene{at}curie.u-psud.fr.

	Abstract

The B-Raf proto-oncogene encodes several isoforms resulting from alternative splicing in the hinge region upstream of the kinase domain. The presence of exon 8b in the B2-Raf_8b isoform and exon 9b in the B3-Raf_9b isoform differentially regulates B-Raf by decreasing and increasing MEK activating and oncogenic activities, respectively. Using different cell systems, we investigated here the molecular basis of this regulation. We show that exons 8b and 9b interfere with the ability of B-Raf N-terminal region to interact with and inhibit the C-terminal kinase domain, thus modulating the auto-inhibition mechanism in an opposite manner. Exons 8b and 9b are flanked by two residues reported to down-regulate B-Raf activity upon phosphorylation. S365A mutation increased the activity of all B-Raf isoforms, but the effect was more pronounced on B2-Raf_8b. This was correlated to the high level of S365 phosphorylation in this isoform, whereas the B3-Raf_9b isoform was poorly phosphorylated on this residue. In contrast, S429 was equally phosphorylated in all B-Raf isoforms, but S429A mutation activated B2-Raf_8b, whereas it inhibited B3-Raf_9b. These results indicate that phosphorylation on both S365 and S429 participate in the differential regulation of B-Raf isoforms through distinct mechanisms. Finally, we show that auto-inhibition and phosphorylation represent independent but convergent mechanisms accounting for B-Raf regulation by alternative splicing.