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，在52 kDa的人 KG1细胞(人早幼粒细胞系)中的Erg-2蛋白已被 鉴定和提纯。最优结合序列G/CCaGGAAG，它 与纯化的ERG-2一起从随机寡核苷酸中选出 蛋白质，与为人类ETS-1确定的相似 果蝇E74蛋白。因此，这些结果证实了ETS家族 蛋白质代表一类DNA结合蛋白，它能特异性地 识别富含嘌呤的核心序列GGAA。一些因素，其中 稳定ERG-2 DNA复合体似乎存在于粗核中 KG1细胞提取液。这个推测的ERG-2结合因子(S)和 其相互作用的性质正在调查中。 已发现ERG-2蛋白的磷酸化涉及一种 与ETS-1所描述的信号转导途径不同 和Ets-2蛋白。在ERG-2蛋白中， Ets-1和Ets-2中没有的酪氨酸磷酸化也 已被确认身份。ERG-2蛋白的机制和功能 正在对磷酸化进行详细的研究，以了解它作为一种 转录因子。 为了鉴定ETS家族蛋白的转录功能 激活剂，一种部分纯化的体外转录系统 HeLa细胞中被ERG-2和ERG-2特异性激活的蛋白质 ETS-1蛋白已被开发出来。使用提纯的组件，该计划 是为了研究：1)ETS蛋白和基本转录因子是如何 能够相互作用以激活转录；2)ETS如何 蛋白质和其他转录激活物或其他调节因子 能够合作并激活基因转录以响应 细胞刺激；3)ETS蛋白的磷酸化是如何 以实现这些蛋白质之间的相互作用。

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.