DNA Binding and Phosphorylation Induce Conformational Alterations in the Kinase-inducible Domain of CREB

Abstract

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 sequencespecific 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.The cyclic AMP response element-binding protein (CREB) 2 induces expression of many cellular genes in response to phosphorylation by several kinases including the cAMP-dependent kinase (PKA) (1). CREB binds via its basic leucine zipper (bZIP) domain as a dimer to cAMP response elements (CREs) containing the 5Ј-TGACGTCA-3Ј consensus motif. CREB has been shown to be constitutively bound to promoters carrying CREs in vivo (2-4). Activation of transcription by CREB occurs cooperatively via the kinase-inducible domain (KID) and the constitutive glutamine-rich domain (Q2) (5-7). Following PKA phosphorylation at serine 133 (Ser 133 ) of KID, CREB recruits CREB-binding protein (CBP) or its paralogue, p300 (8, 9). CREB-mediated activation of transcription follows "burstattenuation" kinetics and maximal transcription coincides with maximal levels of phosphorylation (10, 11). Two major serine/ threonine protein phosphatases, PP1 and PP2A, have been implicated as the enzymes responsible for CREB dephosphorylation and transcriptional attenuation (10, 12, 13).Many transcription factors are thought to undergo induced conformational changes in a ligand-dependent fashion. DNAinduced conformational changes have been described for several bZIP family proteins, including CREB, GCN5, C/EBP, c-Jun, and c-Fos (14 -16). In CREB, the DNA-binding bZIP domain displays a 20% increase in helical structure following sequence-specifi...

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