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J. Biol. Chem., Vol. 282, Issue 27, 19872-19883, July 6, 2007

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

IMPLICATIONS FOR THE MECHANISM OF TRANSCRIPTION FUNCTION^*

From the Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523-1870

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.

Received for publication, February 16, 2007 , and in revised form, May 9, 2007.

^* This study was supported by Grant NCI CA80002 from the National Institutes of Health and the W. M. Keck Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870. Tel.: 970-491-0420; Fax: 970-491-0494; E-mail: Jennifer.Nyborg{at}ColoState.edu.

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