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.
Wagner
B L
BL
Peptide Biology Laboratories, Salk Institute for Biological Studies, La Jolla, California 92037, USA.
Bauer
A
A
Schütz
G
G
Montminy
M
M
D000595:Amino Acid Sequence; D000918:Antibody Specificity; D050882:CREB-Binding Protein; D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism*; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D015290:Second Messenger Systems; D015534:Trans-Activators / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D017362:Cyclic AMP Response Element-Binding Protein; D009687:Nuclear Proteins; D010446:Peptide Fragments; D015534:Trans-Activators; D000242:Cyclic AMP; D050882:CREB-Binding Protein