Inducible gene deletion reveals different roles for B-Raf and Raf-1 in B-cell antigen receptor signalling.
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.
Brummer
Tilman
T
Department of Molecular Immunology, Biology III, University of Freiburg and Max-Planck-Institute for Immunobiology, Stuebeweg 51, Germany.
Shaw
Peter E
PE
Reth
Michael
M
Misawa
Yukiko
Y
D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism; D002460:Cell Line; D002645:Chickens; D004268:DNA-Binding Proteins / Q000235:genetics; D004789:Enzyme Activation; D017353:Gene Deletion; D005786:Gene Expression Regulation / Q000502:physiology; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016296:Mutagenesis; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D048493:Proto-Oncogene Proteins B-raf; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics; D019908:Proto-Oncogene Proteins c-raf / Q000235:genetics / Q000502:physiology*; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D014157:Transcription Factors / Q000235:genetics
D004268:DNA-Binding Proteins; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D016760:Proto-Oncogene Proteins c-fos; D011947:Receptors, Antigen, B-Cell; D014157:Transcription Factors; D048493:Proto-Oncogene Proteins B-raf; D019908:Proto-Oncogene Proteins c-raf; D020928:Mitogen-Activated Protein Kinases