Stimulus-specific interaction between activator-coactivator cognates revealed with a novel complex-specific antiserum

A number of second messenger pathways propagate inductive signals via protein-protein interactions that are phosphorylation-dependent. The second messenger, cAMP, for example, promotes cellular gene expression via the protein kinase A-mediated phosphorylation of cAMP-response element-binding protein (CREB) at Ser(133), and this modification in turn stimulates the association of CREB with the co-activator, CREB-binding protein (CBP). The solution structure of the CREB.CBP complex, using relevant interaction domains, kinase inducible domain and kinase-induced domain interacting domain, referred to as KID and KIX, respectively, shows that KID undergoes a coil to helix transition, upon binding to KIX, that stabilizes complex formation. Whether such changes occur in the context of the full-length CREB and CBP proteins, however, is unclear. Here we characterize a novel antiserum that specifically binds to the CREB. CBP complex but to neither protein individually. Epitope mapping experiments demonstrate that the CREB.CBP antiserum detects residues in KID that undergo a conformational change upon binding to KIX. The ability of this antiserum to recognize full-length CREB.CBP complexes in a phospho-(Ser(133))-dependent manner demonstrates that the structural transition observed with the isolated KID domain also occurs in the context of the full-length CREB protein. To our knowledge, this is the first report documenting formation of endogenous cellular protein-protein complexes in situ.     

Mutational analysis of the KIX domain of CBP reveals residues critical for SREBP binding

Structure-based mutagenesis was used to probe the binding surface for the activation domain of sterol-responsive element binding protein (SREBP) in the KIX domain of CREB binding protein. A set of conserved residues scattering in the alpha2 helix and the extended C-terminal region of alpha 3 helix in the KIX domain including two arginines previously characterized as a hot spot for cofactor-mediated methylation was shown to be crucial for SREBP-KIX interaction, and was not essential for phosphorylated KID recognition. Therefore, our results suggest the existence of a SREBP binding site formed by positively charged residues in the C-terminal part of the extended alpha 3 helix of the KIX domain distinct from the previously identified phosphorylated KID binding site.