Erythropoietin regulation of Raf-1 and MEK: evidence for a ras-independent mechanism

Stimulation of the erythropoietin (EPO) receptor triggers a cascade of signaling events. We reported that EPO upregulates c-myc expression through 2 pathways in BaF3-EpoR cells-a phosphatidylinositol 3-kinase (PI3K) pathway operating on transcriptional initiation and a Raf-1-mitogen-activated protein kinase (MAPK) pathway affecting elongation. We now show that EPO induces phosphorylation of Raf-1 at serine 338 and within the carboxy-terminal domain, resulting in an electrophoretic mobility change (hyperphosphorylation). Importantly, MEK 1 inhibitor PD98059 blocked only the hyperphosphorylation of Raf-1 but not the phosphorylation at serine 338. This inhibition of Raf-1 hyperphosphorylation resulted in increased kinase activity of Raf-1 and increased phosphorylation of MEK, suggesting that the hyperphosphorylation of Raf-1 inhibits its MEK kinase activity. Deletion of the first 184 amino acids of Raf-1, which are involved in its interaction with Ras, had no effect on EPO-induced phosphorylation. Introducing the dominant-negative N17Ras or GAP had no effect on EPO-induced kinase activity of Raf-1 and ELK activation. N17Ras failed to inhibit ELK activation in another cell line-Rauscher murine erythroleukemia-which expresses the EPO receptor endogenously and differentiates in response to the hormone. These results indicate the presence of a Ras-independent mechanism for Raf-1 and MEK activation in these cells. (C) 2004 by The American Society of Hematology.