The B-raf gene is mapped to human chromosome 7q33-36. Analysis of genomic B-raf clones identified a B-raf pseudogene in addition to the active gene. The B-raf pseudogene is also located on chromosome 7 near the active gene. B-raf is the only raf gene for which alternate size transcripts have been observed. Recently, an isolated cDNA clone, that has a 5-prime extension, relative to the previously characterized B-raf sequence, corresponds well with the second form of the B-Raf kinase, a 95 kiloDalton phosphoprotein that is predominantly expressed in PC12 (pheochromocytoma) cells. Like Raf-1, B-Raf coordinates the signalling pathways of several growth factors. Both differentiation factors and mitogens induced B-raf kinase activity. Since B-raf was predominantly expressed in neural tissues, we wanted to address whether B-raf would play a role in nerve growth factor (NGF)-induced differentiation. This work was done in PC12 cells, which are extensively used as an in vitro model system to study the mechanism of growth factor-induced differentiation and proliferation. In PC12 cells, NGF rapidly stimulated both serine and threonine phosphorylation in vivo and autophosphorylation activity in vitro of the B-raf protein. The major in vitro autophosphorylation site was identified as threonine 372 in the CR2 domain. Induction of B-Raf kinase activity in NGF-treated PC12 cells required expression of kinase-active trk receptors. The exact position of B-Raf kinase in the various kinase cascades initiated at the receptor remains to be elucidated. Recent experiments with a dominant-negative mutant of Ras M17 indicates that B-Raf acts downstream of both Trk substrate (PLC-gamma) and Ras in NGF-treated PC12 cells. B-Raf inhibition experiments with B-Raf-specific monoclonal antibodies, anti-sense expression vectors, and B-raf-specific dominant-negative mutants in PC12 cells should allow us to investigate the role of B-Raf in neuronal differention.
