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Expression and activation of B-Raf kinase isoforms in human and murine leukemia cell lines
A Eychène1, I Dusanter-Fourt, J V Barnier
1Unité de Recherche Associée 1443 du CNRS, Institut Curie, Centre Universitaire, Orsay, France.
Abstract:
The B-raf/c-Rmil proto-oncogene belongs to the raf/mil family of serine/threonine protein kinases. It encodes multiple protein isoforms previously shown to be expressed predominantly in neural tissues. We report here that B-Raf proteins of 95 and 72 kDa are also expressed in various human and murine hematopoietic cell lines. Their relative level of expression is variable depending on the cell line examined. The highest level of expression of p95B-raf was found in UT-7 cells, a human pluripotent cell line established from a patient with a megakaryoblastic leukemia. These cells are able to differentiate toward erythroid or myeloid lineage phenotypes in presence of erythropoietin (EPO) or granulocyte-macrophage colony-stimulating factor (GM-CSF) respectively. We show that treatment of UT-7 cells with EPO, GM-CSF or stem cell factor (SCF) rapidly induces phosphorylation of p95B-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 to EPO and GM-CSF. We also show that both p95B-raf and p72B-raf can be activated by IL-3 in murine BAF-3 pro-B cells and by anti-CD3 in human Jurkat cells, respectively. These observations provide the first evidence that the B-Raf kinase is involved in signal transduction pathways regulating proliferation and differentiation of hematopoietic cells of both myeloid and lymphoid lineages.
Insights
The B-Raf kinase, previously found in neural tissues, is also present in hematopoietic cells. This study demonstrates its activation by growth factors, suggesting a role in blood cell proliferation and differentiation.
Area of Science:
- Molecular Biology
- Cell Biology
- Hematology
Background:
- The B-raf proto-oncogene encodes serine/threonine protein kinases.
- B-Raf isoforms were predominantly found in neural tissues.
- The role of B-Raf in hematopoietic cells was previously unknown.
Purpose of the Study:
- To investigate the expression and activation of B-Raf proteins in human and murine hematopoietic cell lines.
- To determine the involvement of B-Raf in hematopoietic cell signaling pathways.
Main Methods:
- Western blotting to detect B-Raf protein isoforms (p95B-raf and p72B-raf).
- Electrophoretic mobility shift assays to assess B-Raf phosphorylation.
- Kinase assays to measure B-Raf activity.
- Treatment of cell lines (UT-7, BAF-3, Jurkat) with cytokines (EPO, GM-CSF, SCF, IL-3) and anti-CD3 antibody.
Main Results:
- B-Raf proteins (p95 and 72 kDa) are expressed in various hematopoietic cell lines.
- Growth factors (EPO, GM-CSF, SCF, IL-3) and anti-CD3 rapidly induce B-Raf phosphorylation and increase its kinase activity.
- B-Raf activation is dose-dependent in response to EPO and GM-CSF.
- Highest p95B-raf expression was observed in UT-7 megakaryoblastic leukemia cells.
Conclusions:
- B-Raf kinase is expressed and activated in hematopoietic cells.
- B-Raf is involved in signal transduction pathways regulating the proliferation and differentiation of myeloid and lymphoid lineages.
- These findings highlight a novel role for B-Raf in hematopoiesis.