Inducible gene deletion reveals different roles for B-Raf and Raf-1 in B-cell antigen receptor signalling

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Abstract
Engagement of the B‐cell antigen receptor (BCR) leads to activation of the Raf–MEK–ERK pathway and Raf kinases play an important role in the modulation of ERK activity. B lymphocytes express two Raf isoforms, Raf‐1 and B‐Raf. Using an inducible deletion system in DT40 cells, the contribution of Raf‐1 and B‐Raf to BCR signalling was dissected. Loss of Raf‐1 has no effect on BCR‐mediated ERK activation, whereas B‐Raf‐deficient DT40 cells display a reduced basal ERK activity as well as a shortened BCR‐mediated ERK activation. The Raf‐1/B‐Raf double deficient DT40 cells show an almost complete block both in ERK activation and in the induction of the immediate early gene products c‐Fos and Egr‐1. In contrast, BCR‐mediated activation of nuclear factor of activated T cells (NFAT) relies predominantly on B‐Raf. Furthermore, complementation of Raf‐1/B‐Raf double deficient cells with various Raf mutants demonstrates a requirement for Ras‐GTP binding in BCR‐mediated activation of both Raf isoforms and also reveals the important role of the S259 residue for the regulation of Raf‐1. Our study shows that BCR‐mediated ERK activation involves a cooperation of both B‐Raf and Raf‐1, which are activated specifically in a temporally distinct manner.