Identification of the functional domains of the transcriptional regulator CRE-BP1.
We present evidence that CRE-BP1 binding to the cyclic AMP (cAMP) response element (CRE) is a transcriptional activator. Transcriptional activation was assayed by cotransfection into CV-1 cells of a CRE-BP1 expression plasmid together with a reporter plasmid in which the thymidine kinase promoter and four tandem repeats of CRE were linked to the chloramphenicol acetyltransferase (CAT) gene. Cotransfection with the CRE-BP1 expression plasmid caused an 8-fold stimulation of CAT activity, while cotransfection with the plasmids to express CRE-BP1 and c-Jun induced a 32-fold stimulation of CAT activity, suggesting that a heterodimer of CRE-BP1 with c-Jun is a stronger trans-activator than a homodimer of CRE-BP1. By using a series of deletion and point mutants of CRE-BP1 in this cotransfection assay, two functional domains of CRE-BP1 were identified: the putative metal finger structure in the amino-terminal region and the leucine zipper motif linked to a cluster of basic amino acids in the carboxyl-terminal region. The former was a transcriptional activation domain in the absence of c-Jun. The latter was a DNA-binding domain, and was essential in both the presence and absence of c-Jun.
Matsuda
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S
Laboratory of Molecular Genetics, Tsukuba Life Science Center, Institute of Physical and Chemical Research (RIKEN), Ibaraki, Japan.
Maekawa
T
T
Ishii
S
S
D000595:Amino Acid Sequence; D015153:Blotting, Western; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010957:Plasmids; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D016755:Proto-Oncogene Proteins c-jun; D015500:Chloramphenicol O-Acetyltransferase