Modulation of kinase activity and oncogenic properties by alternative splicing reveals a novel regulatory mechanism for B-Raf.
Members of the raf oncogene family encode serine/threonine protein kinases, 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 activation, regulation, and kinase activity. In particular, B-Raf was shown to display higher MEK kinase activity than Raf-1. By using both two-hybrid analysis and coimmunoprecipitation experiments, we demonstrate here that B-Raf also markedly differs from Raf-1 by a higher affinity for MEK. We previously reported that the B-raf gene encodes multiple protein isoforms resulting from complex alternative splicing of two exons (exons 8b and 10) located upstream of B-Raf kinase domain. In the present study, we show that these naturally occurring modifications within the protein sequence markedly modulate both the biochemical and oncogenic 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 presence of exon 8b sequences seems to have opposite effects. Therefore, alternative splicing represents a novel regulatory mechanism for a protein of the Raf family.
Papin
C
C
Unité Mixte de Recherche 146 du CNRS, Institut Curie, Centre Universitaire, Laboratoire 110, 91405 Orsay Cédex, France.
Denouel-Galy
A
A
Laugier
D
D
Calothy
G
G
Eychène
A
A
D016475:3T3 Cells; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D002455:Cell Division; D002478:Cells, Cultured; D002642:Chick Embryo; D004789:Enzyme Activation; D005091:Exons; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D048728:MAP Kinase Kinase Kinase 1; D051379:Mice; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D009857:Oncogenes; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012160:Retina; D014162:Transfection
D007527:Isoenzymes; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D048728:MAP Kinase Kinase Kinase 1; C482576:MAP3K1 protein, human; C482578:Map3k1 protein, mouse