MODULATION OF KINASE-ACTIVITY AND ONCOGENIC PROPERTIES BY ALTERNATIVESPLICING REVEALS A NOVEL REGULATORY MECHANISM FOR B-RAF

Members of the raf oncogene family encode serine/ threonine protein ki nases, which activate the mitogen-activated protein kinase kinase MEKs (MAPK or ERK kinases) through direct interaction and phosphorylation. Several recent studies have revealed interesting differences between two members of this family, Raf-1 and B-Raf, regarding their activatio n, regulation, and kinase activity. In particular, B-Raf was shown to display higher MEK kinase activity than Raf-1. By using both two-hybri d analysis and coimmunoprecipitation experiments, we demonstrate here that B-Raf also markedly differs from Raf-1 by a higher affinity for M EK. We previously reported that the B-raf gene encodes multiple protei n isoforms resulting from complex alternative splicing of two exons (e xons 8b and 10) located upstream of E-Raf kinase domain. In the presen t study, we show that these naturally occurring modifications within t he protein sequence markedly modulate both the biochemical and oncogen ic properties of B-Raf. The presence of exon 10 sequences enhances the affinity for MEK, the basal kinase activity, as well as the mitogenic and transforming properties of full-length B-Raf, whereas the presenc e of exon 8b sequences seems to have opposite effects. Therefore, alte rnative splicing represents a novel regulatory mechanism for a protein of the Raf family.