Identification and purification of a novel 120-kDa protein that recognizes the cAMP-responsive element.
The TGACGTCA (CRE) motif required for function by a number of cellular (somatostatin, enkephalin, alpha-human chorionic gonadotropin) and viral (Ad5 E1A-inducible, HTLV-1 TAX-inducible) genes is the site of interaction of multiple sequence-specific complexes. A protocol has been developed for the fractionation and purification of these activities. We report here the purification from HeLa nuclear extracts of a novel 120-kDa polypeptide which by Southwestern blots, gel retardation, and UV cross-linking assays displays CRE-specific binding. The CRE-affinity purified 120-kDa protein displays properties distinct from those of the 43-kDa CREB/ATF polypeptide. The 120-kDa protein is readily phosphorylated in vitro by protein kinase C but not by protein kinase A, suggesting that this molecule may mediate cellular signals distinct from the cAMP-responsive pathway. In vitro transcription-complementation assays utilizing the purified 120-kDa protein failed to transactivate the cAMP-responsive somatostatin promoter suggesting that the mode of action of this 120-kDa polypeptide may require an activation step distinct from the cAMP-signaling pathway.
Andrisani
O
O
Department of Biochemistry, Purdue University, West Lafayette, Indiana.
Dixon
J E
JE
D001483:Base Sequence; D001973:Bromodeoxyuridine; D002467:Cell Nucleus / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005816:Genetic Complementation Test; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015345:Oligonucleotide Probes; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000378:metabolism; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D015345:Oligonucleotide Probes; D011494:Protein Kinases; D001973:Bromodeoxyuridine