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.
Chen
Changmin
C
Laboratory for Cell and Molecular Biology, Division of Hematology and Oncology, Beth Israel Deaconess Medical Center, and Department of Medicine, Harvard Medical School, Boston, MA 02215, USA.
Sytkowski
Arthur J
AJ
D000818:Animals; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D004357:Drug Synergism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004921:Erythropoietin / Q000494:pharmacology*; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D051379:Mice; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009025:Morpholines / Q000494:pharmacology; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017467:Receptors, Erythropoietin / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
D002867:Chromones; D004791:Enzyme Inhibitors; D005419:Flavonoids; D009025:Morpholines; D011518:Proto-Oncogene Proteins; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins; D004921:Erythropoietin; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D012694:Serine; D019908:Proto-Oncogene Proteins c-raf; D020929:Mitogen-Activated Protein Kinase Kinases; D017027:Protein Tyrosine Phosphatases; D018631:ras Proteins; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one