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Molecular and Cellular Biology, December 2000, p. 9409-9422, Vol. 20, No. 24
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Magnitude of the CREB-Dependent Transcriptional Response Is Determined by the Strength of the Interaction between the Kinase-Inducible Domain of CREB and the KIX Domain of CREB-Binding Protein

Adam J. Shaywitz,1,2 Simon L. Dove,3 Jon M. Kornhauser,2,4 Ann Hochschild,3,* and Michael E. Greenberg2,4,*

Program in Biological and Biomedical Sciences1 and the Departments of Microbiology and Molecular Genetics3 and Neurobiology,4 Harvard Medical School, and Division of Neuroscience, Children's Hospital,2 Boston, Massachusetts 02115

Received 3 July 2000/Returned for modification 21 August 2000/Accepted 26 September 2000

The activity of the transcription factor CREB is regulated by extracellular stimuli that result in its phosphorylation at a critical serine residue, Ser133. Phosphorylation of Ser133 is believed to promote CREB-dependent transcription by allowing CREB to interact with the transcriptional coactivator CREB-binding protein (CBP). Previous studies have established that the domain encompassing Ser133 on CREB, known as the kinase-inducible domain (KID), interacts specifically with a short domain in CBP termed the KIX domain and that this interaction depends on the phosphorylation of Ser133. In this study, we adapted a recently described Escherichia coli-based two-hybrid system for the examination of phosphorylation-dependent protein-protein interactions, and we used this system to study the kinase-induced interaction between the KID and the KIX domain. We identified residues of the KID and the KIX domain that are critical for their interaction as well as two pairs of oppositely charged residues that apparently interact at the KID-KIX interface. We then isolated a mutant form of the KIX domain that interacts more tightly with wild-type and mutant forms of the KID than does the wild-type KIX domain. We show that in the context of full-length CBP, the corresponding amino acid substitution resulted in an enhanced ability of CBP to stimulate CREB-dependent transcription in mammalian cells. Conversely, an amino acid substitution in the KIX domain that weakens its interaction with the KID resulted in a decreased ability of full-length CBP to stimulate CREB-dependent transcription. These findings demonstrate that the magnitude of CREB-dependent transcription in mammalian cells depends on the strength of the KID-KIX interaction and suggest that the level of transcription induced by coactivator-dependent transcriptional activators can be specified by the strength of the activator-coactivator interaction.

* Corresponding author. Mailing address for M. E. Greenberg: Division of Neuroscience, Children's Hospital, 250 Enders Research Building, 300 Longwood Ave., Boston, MA 02115. Phone: (617) 355-8344. Fax: (617) 738-1542. E-mail: greenberg{at}a1.tch.harvard.edu. Mailing address for Ann Hochschild: Department of Microbiology and Molecular Genetics, Harvard Medical School, Building D1, 200 Longwood Ave., Boston, MA 02115. Phone: (617) 432-1986. Fax: (617) 738-7664. E-mail: ahochschild{at}hms.harvard.edu.

Molecular and Cellular Biology, December 2000, p. 9409-9422, Vol. 20, No. 24
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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