Differential Regulation of B-Raf Isoforms by Phosphorylation and AutoinhibitoryMechanisms

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 the B-Raf N-terminal region to interact with and inhibit the C-terminal kinase domain, thus modulating the autoinhibition mechanism in an opposite manner. Exons 8b and 9b are flanked by two residues reported to down-regulate B-Raf activity upon phosphorylation. The S365A mutation increased the activity of all B-Raf isoforms, but the effect on B2-Raf_8b was more pronounced. 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 the 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 autoinhibition and phosphorylation represent independent but convergent mechanisms accounting for B-Raf regulation by alternative splicing.

We thank Brian Rudkin for helpful advice on PC12 cells. We also thank Carole Burns, Andrew Doedens, Celio Pouponnot, and Nathalie Rocques for comments on the manuscript.

This work was funded by the Centre National de la Recherche Scientifique, by the Institut Curie, and by grants from the Ligue Nationale Contre le Cancer (Comité de l'Essonne) and INCA (melanoma network). I.H. was supported by fellowships from the Ligue Nationale Contre le Cancer and the Association pour la Recherche sur le Cancer. A.E. and S.D. are INSERM investigators.