Roles of phosphorylation and helix propensity in the binding of the KIX domain of CREB-binding protein by constitutive (c-Myb) and inducible (CREB) activators.
cAMP-response element-binding protein (CREB)-binding protein (CBP) is a general transcriptional co-activator that mediates interactions between transcription factors and the basal transcription machinery. To obtain insights into the mechanism by which the KIX domain of CBP can recognize the transactivation domains of many different transcription factors, we have used NMR and biochemical analyses to study the interactions of KIX with the transactivation domain from the constitutive activator c-Myb and with the kinase-inducible transactivation domain (KID) from CREB. NMR chemical shift mapping shows that both activation domains bind to the same surface of KIX. In the unbound state, both the phosphorylated KID and c-Myb activation domains are only partly structured, and binding to KIX is coupled with folding to form an amphipathic helix. Helix-destabilizing mutations significantly impair binding, whereas mutations that increase the intrinsic secondary structure content of the free phosphorylated KID peptide have only a small influence on binding affinity. Low affinity but specific binding of unphosphorylated KID to KIX was measured by ITC and was also observed in Western blot assays and by a fluorescence resonance energy transfer experiment in living cells. The large increase in the affinity for phosphorylated KID is due to favorable intermolecular interactions involving the phosphate moiety. After induction by phosphorylation, CREB is able to compete effectively with other transcriptional activators for binding to CBP.
Department of Molecular Biology and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Tsaffrir
T
Zor
Bernhard M
BM
Mayr
H Jane
HJ
Dyson
Marc R
MR
Montminy
Peter E
PE
Wright
D000595:Amino Acid Sequence; D050882:CREB-Binding Protein; D002942:Circular Dichroism; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D017433:Protein Structure, Secondary; D020598:Proto-Oncogene Proteins c-myb / Q000378:metabolism*; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism*; D015533:Transcriptional Activation
D017362:Cyclic AMP Response Element-Binding Protein; D009687:Nuclear Proteins; D020598:Proto-Oncogene Proteins c-myb; D015534:Trans-Activators; D050882:CREB-Binding Protein