A eukaryotic nuclear protein of 130 kDa binds to a bacterial cAMP responsive element.
It has been known that one of the signal transduction mechanisms in Escherichia coli is mediated by cAMP which binds to the receptor protein (CAP), and that CAP complexed with cAMP facilitates gene expression by binding to the specific sequences. To identify a molecular mechanism in eukaryotes similar to a cAMP-mediated pathway in E. coli, the function of the CAP binding site of lac gene in E. coli and the protein(s) interacting with it were examined in a mammalian system. From transient expression studies of the fusion gene between the chloramphenicol acetyltransferase and lac genes, it was found that the lacCAP binding site could act as an enhancer activity on the SV40 promoter, and also as an additive enhancer activity to the SV40 enhancer in HeLa cells. However, the activity was not stimulated by cpt-cAMP (a highly stable analogue of cAMP) in HeLa cells, although it was induced in PC12 cells. These results suggest that a bacterial cAMP responsive element may function also in eukaryotes as a cis-acting element in a cell type dependent manner. Results from gel mobility shift assays showed that a protein(s) exists that specifically binds to the lacCAP binding site in eukaryotic nuclear extracts. As one of the proteins binding to the above site, we have identified a 130 kDa protein by using the Southwestern method. Although a function of the 130 kDa protein has not yet been understood, there is a possibility that the 130 kDa protein may play a role in the regulation of cAMP-dependent gene expression.
Kihara
F
F
Institute of Medical Science, University of Tokyo, Japan.
Ariga
H
H
D001483:Base Sequence; D001665:Binding Sites; D015139:Blotting, Southern; D015153:Blotting, Western; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D000242:Cyclic AMP / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005798:Genes, Bacterial; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D010957:Plasmids; D015398:Signal Transduction; D014158:Transcription, Genetic
D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D000242:Cyclic AMP; D015500:Chloramphenicol O-Acetyltransferase