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Stimulus-specific interaction between activator-coactivator cognates revealed with a novel complex-specific
B L Wagner1, A Bauer, G Schütz
1Peptide Biology Laboratories, Salk Institute for Biological Studies, La Jolla, California 92037, USA.
The Journal of Biological Chemistry
|March 18, 2000
Summary
Researchers developed a novel antiserum to detect protein-protein interactions, specifically the CREB-CBP complex. This tool confirms that a key structural change in CREB occurs in full-length proteins, advancing our understanding of cellular signaling pathways.
Area of Science:
- Molecular Biology
- Cellular Signaling
- Protein Interactions
Background:
- Second messenger pathways, like cAMP, regulate gene expression through phosphorylation-dependent protein-protein interactions.
- Cyclic AMP (cAMP) promotes gene expression by activating protein kinase A, which phosphorylates cAMP-response element-binding protein (CREB) at Serine 133.
- Phosphorylated CREB (pCREB) binds to the co-activator CREB-binding protein (CBP), a crucial step in signal transduction.
Purpose of the Study:
- To investigate whether the conformational changes observed in isolated CREB-binding protein (CBP) interaction domains (KID and KIX) occur in full-length CREB and CBP proteins.
- To develop and characterize a novel antiserum capable of specifically detecting the CREB-CBP complex in situ.
- To confirm the phosphorylation-dependent nature of the CREB-CBP complex formation and associated structural transitions.
Main Methods:
- Characterization of a novel antiserum raised against the CREB-CBP complex.
- Epitope mapping to identify the specific region of CREB recognized by the antiserum.
- Assessing the antiserum's ability to detect full-length CREB-CBP complexes in a phosphorylation-dependent manner.
Main Results:
- A novel antiserum was generated that specifically binds to the CREB-CBP complex, not to individual CREB or CBP proteins.
- Epitope mapping revealed the antiserum recognizes a conformational change in the CREB kinase-inducible domain (KID) upon binding to CBP's KIX domain.
- The antiserum successfully detected full-length CREB-CBP complexes in a phospho-Serine 133-dependent manner, confirming the conformational transition occurs in the context of the intact proteins.
Conclusions:
- The structural transition of the CREB KID domain upon binding to CBP's KIX domain is conserved in full-length proteins.
- The developed antiserum provides a specific tool for detecting endogenous CREB-CBP complexes in situ.
- This study provides the first evidence of detecting endogenous cellular protein-protein complexes in situ, validating a key step in cAMP-mediated gene regulation.