ANALYSIS OF THE STRUCTURAL-PROPERTIES OF CAMP-RESPONSIVE ELEMENT-BINDING PROTEIN (CREB) AND PHOSPHORYLATED CREB

The transcription factor CREB (cAMP responsive element binding protein ) is activated by protein kinase A (PKA) phosphorylation of a single s erine residue. To investigate possible mechanisms of CREB regulation b y phosphorylation, we initiated a structural and biophysical character ization of the full-length, wild-type CREB protein, an altered CREB pr otein (CREB/SER) in which the three cysteine residues in the DNA-bindi ng domain were replaced with serine residues and a truncated protein ( ACT265) which encompasses the entire activation domain of CREB. Circul ar dichroism (CD) reveals that CREB and CREB/SER have identical second ary structures and contain approximately 20% alpha-helix, 9% beta-stra nd, 34% beta-turn, and 37% random coil structures. PKA phosphorylation does not alter the CD spectra, and therefore the secondary structure, of CREB or of CREB bound to DNA. Protease cleavage patterns indicate that PKA phosphorylation does not induce a global conformational chang e in CREB. Furthermore, PKA phosphorylation does not change the DNA bi nding affinity of CREB for either canonical or non-canonical CRE seque nces as measured by a fluorescence anisotropy DNA binding assay. Since PKA phosphorylation of CREB results in its specific binding to the tr anscriptional coactivators CREB-binding protein and p300, we suggest t hat the PKA activation of CREB occurs by the production of specific, c omplementary interactions with these proteins, rather than through the previously proposed mechanisms of a phosphorylation-dependent conform ational change or increased DNA binding affinity.