B-RAF PROTEIN KINASE--STRUCTURE, EXPRESSION AND ACTIVATION IN VIVO

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Three isotypes of Raf kinases have been identified to date in man: raf-1, A-raf and B-raf. B-raf has the highest levels in fetal brain and adult cerebrum. In addition to the 10- and 13-kilobase (kb) transcripts common to B-raf-expressing tissues, alternate-size transcripts of 2.6 and 4.5 kb were found in testes. Previous work established a partial B-raf sequence. To obtain a full-length cDNA, a 42mer oligonucleotide unique to B-raf was used to screen a human testes cDNA library. A 2.3-kb clone was obtained that contains an open reading frame of 1950 base pairs, presumably encoding full-length B-raf protein. Sequence analysis indicates three conserved regions, CR1, CR2 and CR3, typical of raf protein kinases. In the CR1 region, which encompasses the putative zinc finger region, B-raf shows a 75% homology to A-raf and 65% to raf-1. The serine/threonine-rich CR2 region has a 47.6% homology with both A-raf and raf-1, and CR3 has a 76.4% homology with A-raf and 79% homology with raf-1, which corresponds to the kinase domain. B-raf protein has been expressed in the E. coli T7 expression system. The B-raf fusion protein reacts with antibodies specific for B-raf raised against the C-terminal peptide and to monoclonal antibodies which map into the kinase domain (near a tripeptide conserved sequence motif for all Src family protein kinase sites) which are predicted to react with all raf family members. Consistent with the RNA expression data, the predicted 72.5-kiloDalton B-raf protein is expressed in mouse brain.