Rapid phosphorylation of Ets-2 accompanies mitogen-activated protein kinase activation and the induction of heparin-binding epidermal growth factor gene expression by oncogenic Raf-1
Rapid phosphorylation of Ets-2 accompanies mitogen-activated protein kinase activation and the induction of heparin-binding epidermal growth factor gene expression by oncogenic Raf-1
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DOI：
10.1128/mcb.17.5.2401
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发表时间：
1997-05-01
影响因子：
5.3
通讯作者：
McMahon, M
中科院分区：
文献类型：
--
作者：
McCarthy, SA;Chen, D;McMahon, M
肝素结合表皮生长因子（HB-EGF）基因转录在由致癌性Pas和Raf转化的NIH 3 T3细胞中被快速激活，并介导在这些细胞中观察到的c-Jun N-末端激酶（JNKs）的自分泌激活。与荧光素酶报告基因连接的鼠HB-EGF基因的启动子的1.7-kb片段在Delta Raf-1：ER（致癌人Raf 1的条件活性形式）活化后被强烈诱导。Delta Raf-1：ER激活启动子需要一个复合AP-1/Ets转录因子结合位点，位于翻译起始位点上游bp-974和-988之间。在体内基因组足迹表明，该复合AP-1/Ets元件的基础水平的占用增加后，三角洲Raf-1：ER激活。Ets-2和促分裂原活化蛋白（MAP）激酶表达载体的共转染强烈增强了HB-EGF启动子对Delta Raf-1：ER的激活。增强激活需要p44 MAP激酶催化活性和Ets-2的Pointed结构域中的苏氨酸72。生物化学测定证明了p42和P43 MAP激酶磷酸化苏氨酸72上的Ets-2的能力。重要的是，在完整细胞中，Ets-2在该残基上的磷酸化动力学密切反映了p42和p34 MAP激酶的激活以及在Delta Raf-1：ER激活后观察到的HB-EGF基因转录的开始。这些数据坚定地确立了Ets-2作为Raf-MEK-MAP激酶信号通路的直接靶点，并强烈暗示Ets-2参与HB-EGF基因表达的调节。
Heparin-binding epidermal growth factor (HB-EGF) gene transcription is rapidly activated in NIH 3T3 cells transformed by oncogenic Pas and Raf and mediates the autocrine activation of the c-Jun N-terminal kinases (JNKs) observed in these cells. A 1.7-kb fragment of the promoter of the murine HB-EGF gene linked to a luciferase reporter was strongly induced following activation of Delta Raf-1:ER, a conditionally active form of oncogenic human Raf l. Promoter activation by Delta Raf-1:ER required a composite AP-1/Ets transcription factor binding site located between bp -974 and -988 upstream of the translation initiation site. In vivo genomic footprinting indicated that the basal level of occupancy of this composite AP-1/Ets element increased following Delta Raf-1:ER activation. Cotransfection of Ets-2 and pll mitogen-activated protein (MAP) kinase expression vectors strongly potentiated HB-EGF promoter activation in response to Delta Raf-1:ER. Potentiated activation required both p44 MAP kinase catalytic activity and threonine 72 in the Pointed domain of Ets-2. Biochemical assays demonstrated the ability of the p42 and pll MAP kinases to phosphorylate Ets-2 on threonine 72. Importantly, in intact cells, the kinetics of phosphorylation of Ets-2 on this residue closely mirror the activation of the p42 and p34 MAP kinases and the observed onset of HB-EGF gene transcription following Delta Raf-1:ER activation. These data firmly establish Ets-2 as a direct target of the Raf-MEK-MAP kinase signaling pathway and strongly implicate Ets-2 in the regulation of HB-EGF gene expression.