Tyrosine phosphorylation regulates the DNA binding activity of a nuclear factor 1-like repressor protein.
We have previously identified an ubiquitous repressor binding site that binds a nuclear factor 1 (NF-1)-like transcription factor designated BEF-1. The DNA binding activity of BEF-1, a 98-kDa protein, is increased by the oncoproteins of adenovirus, the early region 1a proteins (E1a), which results in the induction of further repression. Using the prototype repressor sequence, first identified in the enhancer of the human polyoma virus BKV-P2 we have shown that phosphorylation of BEF-1 is required for its DNA binding activity. We demonstrate here that the inhibition of DNA binding by BEF-1 dephosphorylated with potato acid phosphatase or calf intestinal alkaline phosphatase was reversed by sodium orthovanadate, a specific inhibitor of phosphotyrosyl-protein phosphatases. In addition, BEF-1 binding activity, but not the binding of related factor NF-1, could be inhibited by dephosphorylation with a specific phosphotyrosine phosphatase. We found that both polyclonal and monoclonal phosphotyrosine-specific antibodies blocked binding of the repressor protein to the BEF-1 site. Moreover, BEF-1 activity could be adsorbed on an anti-phosphotyrosine antibody column and specially eluted with phosphotyrosine. In transfection studies in HeLa cells, which contain high levels of BEF-1, we show that E1a-induced repression mediated by BEF-1 was relieved with the tyrosine kinase inhibitors genistein and tyrphostin. Together, these results demonstrate that a phosphotyrosine on the BEF-1 repressor protein regulates DNA binding activity and thus regulates repression of the BKV-P2 enhancer. This report represents the first demonstration that the phosphorylated state of a tyrosine can control gene expression by altering the DNA binding activity of a transcription factor.
Reifel-Miller
A E
AE
Department of Diabetes, Lilly Research Laboratories, Indianapolis, Indiana 46285.
Calnek
D S
DS
Grinnell
B W
BW
D001739:BK Virus; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D051839:NFI Transcription Factors; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism*; D014157:Transcription Factors; D014158:Transcription, Genetic; D014443:Tyrosine / Q000276:immunology / Q000378:metabolism*; D051840:Y-Box-Binding Protein 1
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human; D014443:Tyrosine