Behre Gerhard, Singh Sheo M, Liu Huaitian, Bortolin Laura T, Christopeit Max, Radomska Hanna S, Rangatia Janki, Hiddemann Wolfgang, Friedman Alan D, Tenen Daniel G
Department of Internal Medicine III, University Hospital Grosshadern, Ludwig-Maximilians-University Munich and GSF-National Research Center for Environment and Health, D-81377 Munich, Germany.
J Biol Chem. 2002 Jul 19;277(29):26293-9. doi: 10.1074/jbc.M202301200. Epub 2002 Apr 26.
The transcription factor C/EBP alpha regulates early steps of normal granulocyte differentiation since mice with a disruption of the C/EBP alpha gene do not express detectable levels of the granulocyte colony-stimulating factor receptor and produce no neutrophils. We have recently shown that C/EBP alpha function is also impaired in acute myeloid leukemias. However, how the transcriptional activity of C/EBP alpha is regulated both in myelopoiesis and leukemogenesis is not fully understood. The current study demonstrates that activated Ras enhances the ability of C/EBP alpha to transactivate the granulocyte colony-stimulating factor receptor promoter and a minimal promoter containing only C/EBP DNA binding sites. Ras signaling activates C/EBP alpha via the transactivation domain because it enhances the transactivation function of a fusion protein containing a Gal4 DNA binding domain and the C/EBP alpha transactivation domain and does not change C/EBP alpha DNA binding. Ras acts on serine 248 of the C/EBP alpha transactivation domain, because it does not enhance the transactivation function of a C/EBP alpha serine 248 to alanine point mutant. Interestingly, serine 248 of C/EBP alpha is a protein kinase C (PKC) consensus site, and a PKC inhibitor blocks the activation of C/EB alpha by Ras. Ras signaling leads to phosphorylation of C/EBP alpha in vivo. Finally, mutation of serine 248 to alanine obviates the ability of C/EBP alpha to induce granulocytic differentiation. These data suggest a model where Ras signaling enhances the activity of C/EBP alpha to induce granulocytic differentiation by phosphorylation of serine 248.
转录因子C/EBPα调节正常粒细胞分化的早期步骤，因为C/EBPα基因缺失的小鼠不表达可检测水平的粒细胞集落刺激因子受体，也不产生中性粒细胞。我们最近发现，C/EBPα的功能在急性髓系白血病中也受到损害。然而，C/EBPα的转录活性在骨髓生成和白血病发生过程中是如何被调节的，目前还不完全清楚。当前的研究表明，激活的Ras增强了C/EBPα反式激活粒细胞集落刺激因子受体启动子和一个仅含C/EBP DNA结合位点的最小启动子的能力。Ras信号通过反式激活结构域激活C/EBPα，因为它增强了含有Gal4 DNA结合结构域和C/EBPα反式激活结构域的融合蛋白的反式激活功能，并且不改变C/EBPα的DNA结合能力。Ras作用于C/EBPα反式激活结构域的丝氨酸248，因为它不增强C/EBPα丝氨酸248突变为丙氨酸的点突变体的反式激活功能。有趣的是，C/EBPα的丝氨酸248是蛋白激酶C（PKC）的共有位点，PKC抑制剂可阻断Ras对C/EBPα的激活。Ras信号在体内导致C/EBPα的磷酸化。最后，丝氨酸248突变为丙氨酸消除了C/EBPα诱导粒细胞分化的能力。这些数据提示了一个模型，即Ras信号通过丝氨酸248的磷酸化增强C/EBPα诱导粒细胞分化的活性。
