Three distinct regions within the constitutive activation domain of cAMP regulatory element-binding protein (CREB) are required for transcription activation.

The transcription factor cAMP regulatory element-binding protein (CREB) mediates both constitutive and cAMP-induced gene expression through distinct, independently acting domains. The constitutive activation domain (CAD) (amino acids (aa) 165-252) encompasses and overlaps exon 9 of the CREB gene (E9, aa 180-243). In the present study, deletion of either the CAD or exon 9 from CREB-GAL4 (CRG) reduced constitutive activity to less than 2-fold, without affecting kinase inducible activity. However, fusion of the CAD to the GAL4 DNA binding domain (CAD-G4) stimulated transcription, whereas fusion of exon 9 sequences did not. Deletion of the amino-terminal flanking region of exon 9 (aa 165-180), but not COOH-terminal flanking sequences (aa 243-252), decreased constitutive activation in either the CAD-G4 or CRG background. Deletion of the previously characterized glutamine-rich region (Q3, aa 218-252) or of a region containing a hydrophobic cluster of amino acids (HC, aa 180-218) also reduced constitutive activation by either CAD-G4 or CRG. No single mutation of hydrophobic residues within HC impaired activity of the CAD, but double and triple mutations did, suggesting that multiple weak interactions are involved in function of the HC region. Thus, exon 9 of the CREB gene is necessary but not sufficient for constitutive activation. The CAD requires three distinct regions for function, suggesting that CREB may interact with multiple targets in the RNA polymerase II complex.