Cloning of a DNA binding protein that is a tyrosine kinase substrate and recognizes an upstream initiator-like sequence in the promoter of the preprodynorphin gene

Abstract

A 90 bp fragment prepared from the promoter region of the rat preprodynorphin gene formed a complex with rat brain nuclear extracts as assessed by gel mobility shift assays. An 8 base pair sequence, CACTCTCC, termed upstream regulatory element (URE), was identified within this fragment as a binding site by DNase 1 footprint analysis and gel mobility shift assays with synthetic oligonucleotides. The URE is a consensus sequence for a transcription initiator (Inr) element although in the preprodynorphin promoter it is located upstream at -208 and overlaps a region conserved between rat and human promoters. A unique 310 amino acid protein (UreB1) that specifically bound the URE was cloned from a rat brain cDNA library using the URE-containing oligonucleotide. Recombinantly expressed, affinity purified UreB1 protein retains specific binding to the URE oligonucleotide. UreB1 contains a tyrosine kinase phosphorylation consensus and binding is enhanced following phosphorylation with the p43v-abl tyrosine kinase. The UreB1 tyrosine phosphoprotein increases transcription in vitro, consistent with a positive transcriptional regulatory function. UreB1 transcripts are well expressed in subsets of neurons in multiple brain areas suggesting that, in addition to regulation of the preprodynorphin gene, it may have a more generalized role in gene transcription.