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.