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.
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.
Neurobiology and Anesthesiology Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892.
J
J
Gu
K
K
Ren
R
R
Dubner
M J
MJ
Iadarola
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D004399:Dynorphins / Q000235:genetics*; D004926:Escherichia coli; D015870:Gene Expression; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism; D010525:Peripheral Nerves / Q000378:metabolism*; D010750:Phosphoproteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D016548:Prosencephalon / Q000378:metabolism*; D011498:Protein Precursors / Q000235:genetics*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D014158:Transcription, Genetic; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases
D004268:DNA-Binding Proteins; D004399:Dynorphins; D010750:Phosphoproteins; D011498:Protein Precursors; D011505:Protein-Tyrosine Kinases; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; D044767:Ubiquitin-Protein Ligases; C036448:pre-prodynorphin; C502830:HUWE1 protein, human; C000610971:Huwe1 protein, rat