DNA binding and phosphorylation induce conformational alterations in the kinase-inducible domain of CREB. Implications for the mechanism of transcription function.
CREB-mediated activation of target gene transcription is stimulated by protein kinase A (PKA) phosphorylation at serine 133. This is followed by recruitment of the coactivators CREB-binding protein (CBP) or p300. Conversely, the decline in expression during the attenuation phase is linked to CREB dephosphorylation by nuclear phosphatases. The CREB bZIP domain, which promotes dimerization and promoter binding, as well as the kinase-inducible domain (KID), which interacts with the KIX domain of CBP/p300, are both largely unstructured in solution and become more structured once bound to their respective ligands. In this study, we biochemically characterize DNA- and phosphorylation-induced conformational alterations in CREB that may play a role in its transcriptionally poised, activated state. We find that sequence-specific DNA binding of pCREB renders the protein resistant to serine 133 dephosphorylation by protein phosphatase 1. Paradoxically, CREB bound to DNA and chromatin is efficiently phosphorylated by PKA, indicating that the KID region exists in a different conformation depending on its phosphorylation state. Consistent with this observation, we find that phosphorylation of DNA-bound CREB promotes an alternate conformation characterized by an apparent increase in the size or asymmetry of the complex and a qualitative change in proteolytic sensitivity. Together, our data indicate that DNA binding promotes a global conformational change in CREB that alters the structure of KID. PKA phosphorylation of KID in the DNA-bound state induces a phosphatase-resistant conformation that may prolong transcriptional activity.
Sharma
Neelam
N
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523-1870, USA.
Lopez
Dinaida I
DI
Nyborg
Jennifer K
JK
D000818:Animals; D050882:CREB-Binding Protein / Q000737:chemistry / Q000378:metabolism; D002474:Cell-Free System; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000737:chemistry / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004330:Drosophila; D019476:Insect Proteins / Q000737:chemistry / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D054645:Protein Phosphatase 1; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D017434:Protein Structure, Tertiary; D014158:Transcription, Genetic / Q000502:physiology*
D002843:Chromatin; D017362:Cyclic AMP Response Element-Binding Protein; D019476:Insect Proteins; D004247:DNA; D050882:CREB-Binding Protein; D017868:Cyclic AMP-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1