Characterization of motifs which are critical for activity of the cyclic AMP-responsive transcription factor CREB.
Cyclic AMP mediates the hormonal stimulation of a number of eukaryotic genes by directing the protein kinase A (PK-A)-dependent phosphorylation of transcription factor CREB. We have previously determined that although phosphorylation at Ser-133 is critical for induction, this site does not appear to participate directly in transactivation. To test the hypothesis that CREB ultimately activates transcription through domains that are distinct from the PK-A site, we constructed a series of CREB mutants and evaluated them by transient assays in F9 teratocarcinoma cells. Remarkably, a glutamine-rich region near the N terminus appeared to be important for PK-A-mediated induction of CREB since removal of this domain caused a marked reduction in CREB activity. A second region consisting of a short acidic motif (DLSSD) C terminal to the PK-A site also appeared to synergize with the phosphorylation motif to permit transcriptional activation. Biochemical experiments with purified recombinant CREB protein further demonstrate that the transactivation domain is more sensitive to trypsin digestion than are the DNA-binding and dimerization domains, suggesting that the activator region may be structured to permit interactions with other proteins in the RNA polymerase II complex.
Gonzalez
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GA
Clayton Foundation Laboratories for Peptide Biology, Salk Institute, La Jolla, California 92037.
Menzel
P
P
Leonard
J
J
Fischer
W H
WH
Montminy
M R
MR
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000502:physiology; D017362:Cyclic AMP Response Element-Binding Protein; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000502:physiology*; D005786:Gene Expression Regulation; D007158:Immunologic Techniques; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000502:physiology; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000502:physiology; D051381:Rats; D011994:Recombinant Proteins; D012045:Regulatory Sequences, Nucleic Acid; D013004:Somatostatin / Q000235:genetics; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic
D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D014157:Transcription Factors; D013004:Somatostatin; D000242:Cyclic AMP; D011494:Protein Kinases