EXPRESSION AND ACTIVATION OF B-RAF KINASE ISOFORMS IN HUMAN AND MURINE LEUKEMIA-CELL LINES

The B-raf/c-Rmil proto-oncogene belongs to the raf/mil family of serin e/threonine protein kinases. It encodes multiple protein isoforms prev iously shown to be expressed predominantly in neural tissues. We repor t here that B-Raf proteins of 95 and 72 kDa are also expressed in vari ous human and murine hematopoietic cell lines. Their relative level of expression is variable depending on the cell line examined. The highe st level of expression of p95(B-raf) was found in UT-7 cells, a human pluripotent cell line established from a patient with a megakaryoblast ic leukemia. These cells are able to differentiate toward erythroid or myeloid lineage phenotypes in presence of erythropoietin (EPO) or gra nulocyte-macrophage colony-stimulating factor (GM-CSF) respectively. W e show that treatment of UT-7 cells with EPO, GM-CSF or stem cell fact or (SCF) rapidly induces phosphorylation of p95(B-raf) as indicated by a shift of electrophoretic mobility. This increase in phosphorylation is correlated with a three-fold increase of B-Raf kinase activity. B- Raf activation also increases in a dose-dependent manner in response t o EPO and GM-CSF. We also show that both p95(B-raf) and p72(B-raf) can be activated by IL-3 in murine BAF-3 pro-B cells and by anti-CD3 in h uman Jurkat cells, respectively. These observations provide the first evidence that the B-Raf kinase is involved in signal transduction path ways regulating proliferation and differentiation of hematopoietic cel ls of both myeloid and lymphoid lineages.