Persistent activation of mitogen-activated protein kinases p42 and p44 and ets-2 phosphorylation in response to colony-stimulating Factor 1/c-fms Signaling
Persistent activation of mitogen-activated protein kinases p42 and p44 and ets-2 phosphorylation in response to colony-stimulating Factor 1/c-fms Signaling
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DOI：
10.1128/mcb.18.9.5148
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发表时间：
1998-09-01
影响因子：
5.3
通讯作者：
Ostrowski, MC
中科院分区：
文献类型：
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作者：
Fowles, LF;Martin, ML;Ostrowski, MC
特异性识别ets-2中磷酸苏氨酸72的抗体用于确定内源性ets-2响应集落刺激因子1（CSF-1）/c-fms信号传导的磷酸化状态。在原代巨噬细胞（正常表达c-fms的细胞）和工程化表达人c-fms的成纤维细胞中检测到ets-2的磷酸化。在前者细胞中，ets-2是CSF-1即刻早期反应基因，在2 ~ 4 h后检测到磷酸化ets-2，与ets-2蛋白的表达一致。在成纤维细胞中，ets-2组成型表达，并迅速成为响应于CSF-1磷酸化。在这两种细胞系统中，ets-2磷酸化是持久的，最大的磷酸化检测8至2 1小时后，CSF-1刺激，并与CSF-1靶尿激酶纤溶酶原激活剂（uPA）基因的激活。使用重组ets-2蛋白作为底物的激酶测定表明，有丝分裂原活化蛋白（MAP）激酶p42和p44在两种细胞类型中响应于CSF-1被组成性激活。免疫耗竭实验和MAP激酶激酶抑制剂PD 98059的使用表明，这两种MAP激酶是响应CSF-1/c-fms信号传导而激活的主要ets-2激酶。在巨噬细胞系RAW 264中，raf激酶的条件表达诱导ets-2表达和磷酸化，以及uPA mRNA表达。瞬时测定将ets/AP-1应答元件映射为基础和CSF-1刺激的uPA报告基因活性的关键。这些结果表明CSF-1对raf/MAP激酶通路的持续激活对于巨噬细胞ets-2表达和翻译后激活都是必要的。
An antibody that specifically recognized phosphothreonine 72 in ets-2 was used to determine the phosphorylation status of endogenous ets-2 in response to colony-stimulating factor 1 (CSF-1)/c-fms signaling. Phosphorylation of ets-2 was detected in primary macrophages, cells that normally express c-fms, and in fibroblasts engineered to express human c-fms. In the former cells, ets-2 was a CSF-1 immediate early response gene, and phosphorylated ets-2 was detected after 2 to 4 h, coincident with expression of ets-2 protein. In fibroblasts, ets-2 was constitutively expressed and rapidly became phosphorylated in response to CSF-1. In both cell systems, ets-2 phosphorylation was persistent, with maximal phosphorylation detected 8 to 2 1 h after CSF-1 stimulation, and was correlated with activation of the CSF-1 target urokinase plasminogen activator (uPA) gene. Kinase assays that used recombinant ets-2 protein as a substrate demonstrated that mitogen-activated protein (MAP) kinases p42 and p44 were constitutively activated in both cell types in response to CSF-1. Immune depletion experiments and the use of the MAP kinase kinase inhibitor PD98059 indicate that these two MAP kinases are the major ets-2 kinases activated in response to CSF-1/c-fms signaling. In the macrophage cell line RAW264, conditional expression of raf kinase induced ets-2 expression and phosphorylation, as well as uPA mRNA expression. Transient assays mapped ets/AP-1 response elements as critical for basal and CSF-1 stimulated uPA reporter gene activity. These results indicate that persistent activation of the raf/MAP kinase pathway by CSF-1 is necessary for both ets-2 expression and posttranslational activation in macrophages.