Chen Changmin, Sytkowski Arthur J
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.
Blood. 2004 Jul 1;104(1):73-80. doi: 10.1182/blood-2003-04-1340. Epub 2004 Mar 16.
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.
促红细胞生成素（EPO）受体的激活会引发一系列信号转导事件。我们曾报道，在BaF3-EpoR细胞中，EPO通过两条途径上调c-myc的表达——一条是作用于转录起始的磷脂酰肌醇3激酶（PI3K）途径，另一条是影响延伸的Raf-1-丝裂原活化蛋白激酶（MAPK）途径。我们现在发现，EPO可诱导Raf-1在丝氨酸338位点及羧基末端结构域发生磷酸化，导致其电泳迁移率改变（过度磷酸化）。重要的是，MEK 1抑制剂PD98059仅阻断Raf-1的过度磷酸化，而不影响丝氨酸338位点的磷酸化。对Raf-1过度磷酸化的这种抑制导致Raf-1激酶活性增加以及MEK磷酸化增加，这表明Raf-1的过度磷酸化抑制了其MEK激酶活性。缺失Raf-1与Ras相互作用所涉及的前184个氨基酸，对EPO诱导的磷酸化没有影响。引入显性负性N17Ras或GAP对EPO诱导的Raf-1激酶活性及ELK激活没有影响。N17Ras无法抑制另一种细胞系——劳舍尔小鼠红白血病细胞系中的ELK激活，该细胞系内源性表达EPO受体并对该激素产生分化反应。这些结果表明，在这些细胞中存在一种不依赖Ras的Raf-1和MEK激活机制。
