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

Abstract

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.

ACKNOWLEDGMENTS

This work was supported by NIH Mental Retardation Research Center grant P30-HD18655 (to M.E.G. and J.M.K.), NIH grant CA43855 (to M.E.G.), NIH grant GM55637 (to A.H.), an established investigatorship from the American Heart Association (to A.H.), and an HMS/Affiliated Hospital Collaborative Seed grant (to A.H. and M.E.G.). A.J.S. was supported by an NIH Medical Scientist Training Program fellowship, and S.L.D. was supported by a Charles A. King Trust postdoctoral fellowship.

We thank P. Brindle for providing the GAL4-Myb expression vector and for useful discussions, F. Whipple for providing the 5P2 vector and for helpful advice, R. Goodman and P. Goldman for providing the KID helix αB mutants, and R. Goodman and R. Kwok for providing the HA-CBP expression vector. We also thank A. Bonni, P. Brindle, R. Goodman, and J. Zeig for critical reading of the manuscript. We thank members of the Greenberg and Hochschild laboratories for their help and advice during the course of these studies, and, in particular, we are especially grateful to A. Brunet for her support and generous help in conducting some of the experiments.