MECHANISM OF TRANSCRIPTIONAL ACTIVATION BY THE HUMAN ETS FAMILY PROTEIN IN VITRO

用抗人ERG-2蛋白的特异性抗体， 来自KG 1细胞（人前髓细胞系）的ERG-2蛋白已经被研究。 鉴定和纯化。 最佳结合序列G/CCaGGAAG， 从随机寡核苷酸中筛选出ERG-2的纯化片段 蛋白质，类似于对人ETS-1和 果蝇E74蛋白。 因此，这些结果证实了ETS家族 蛋白代表一类DNA结合蛋白， 识别富含嘌呤的核心序列GGAA 的一些因素 稳定ERG-2 DNA复合物似乎存在于粗核中 KG 1细胞的提取物。 这种推定的ERG-2结合因子和 正在研究其相互作用的性质。 研究发现ERG-2蛋白的磷酸化涉及一个过程。 与ETS-1所述不同的信号转导途径 和ETS-2蛋白质。 在ERG-2蛋白中， ETS-1和ETS-2中不存在的酪氨酸磷酸化也 被识别。 ERG-2蛋白的作用机制 磷酸化正在被详细研究，以了解其作为一种 转录因子 为了表征ETS家族蛋白作为转录因子的特性， 激活剂，体外转录系统与部分纯化的 来自HeLa细胞的蛋白质，其被ERG-2特异性激活， ETS-1蛋白，已经开发。 使用纯化的成分，该计划 主要研究：1）ETS蛋白和基本转录因子是如何与 能够相互作用以激活转录; 2）ETS如何 蛋白质和其它转录激活因子或其它调节因子 能够合作并激活基因转录， 细胞刺激; 3）ETS蛋白的磷酸化如何能够 来影响这些蛋白质间的相互作用。

With the specific antibody against human ERG-2 protein, a 52 kDa human ERG-2 protein from KG1 cells (human premyeloid cell line) has been identified and purified. The optimal binding sequence, G/CCaGGAAG, which was selected from random oligo-nucleotides with the purified ERG-2 protein, was similar to the one determined for the human ETS-1 and Drosophila E74 proteins. Thus, these results confirm that the ETS family proteins represent a class of DNA-binding proteins that specifically recognize the purine-rich core sequence, GGAA. Some factors which stabilize the ERG-2 DNA complex appear to be present in the crude nuclear extract of KG1 cells. This putative ERG-2 binding factor(s) and the nature of its interaction is being investigated. It has been found that phosphorylation of the ERG-2 protein involves a different signal transduction pathway from that described for the ETS-1 and ETS-2 proteins. In ERG-2 protein, the consensus sequences for tyrosine phosphorylation that are not present in ETS-1 and ETS-2 have also been identified. The mechanism and function of ERG-2 protein phosphorylation is being studied in detail to understand its role as a transcription factor. In order to characterize the ETS family proteins as transcriptional activators, an in vitro transcription system with the partially-purified proteins from HeLa cells, which were specifically activated with ERG-2 and ETS-1 proteins, has been developed. Using purified components, the plan is to study: 1) how the ETS proteins and basic transcriptional factors are able to interact with each other to activate transcription; 2) how the ETS proteins and other transcriptional activators or other regulatory factors are able to cooperate and activate gene transcription in response to cellular stimuli; 3) how the phosphorylation of the ETS proteins are able to effect these protein-protein interactions.