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.
