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Analysis of an activator:coactivator complex reveals an essential role for secondary structure in transcriptional
D Parker1, U S Jhala, I Radhakrishnan
1Joslin Diabetes Center, Research Division, Boston, Massachusetts, USA.
Abstract:
Ser-133 phosphorylation of CREB within the kinase-inducible domain (KID) promotes target gene activation via complex formation with the KIX domain of the coactivator CBP. Concurrent phosphorylation of CREB at Ser-142 inhibits transcriptional induction via an unknown mechanism. Unstructured in the free state, KID folds into a helical structure upon binding to KIX. Using site-directed mutagenesis based on the NMR structure of the KID:KIX complex, we have examined the mechanisms by which Ser-133 and Ser-142 phosphorylation regulate CREB activity. Our results indicate that phospho-Ser-133 stablizes whereas phospho-Ser-142 disrupts secondary structure-mediated interactions between CREB and CBP. Thus, differential phosphorylation of CREB may form the basis by which upstream signals regulate the specificity of target gene activation.
Insights
Phosphorylation of CREB at Ser-133 promotes gene activation, while Ser-142 phosphorylation inhibits it. Differential phosphorylation regulates gene activation specificity by altering CREB-CBP interactions.
Area of Science:
- Molecular Biology
- Gene Regulation
- Protein Phosphorylation
Background:
- CREB (cAMP response element-binding protein) is a transcription factor crucial for gene activation.
- Phosphorylation at Ser-133 by protein kinase A promotes CREB transcriptional activity.
- Phosphorylation at Ser-142 inhibits CREB activity, but the mechanism remains unclear.
Purpose of the Study:
- To investigate the distinct roles of Ser-133 and Ser-142 phosphorylation in regulating CREB activity.
- To elucidate the structural mechanisms underlying CREB-CBP complex formation and regulation.
Main Methods:
- Site-directed mutagenesis based on Nuclear Magnetic Resonance (NMR) structure of the CREB kinase-inducible domain (KID):KIX complex.
- Analysis of CREB-CBP interactions and secondary structure changes.
Main Results:
- Phosphorylation at Ser-133 stabilizes the helical structure of KID, enhancing CREB-CBP complex formation.
- Phosphorylation at Ser-142 disrupts secondary structure-mediated interactions between CREB and CBP.
- Differential phosphorylation at Ser-133 and Ser-142 dictates CREB transcriptional activity.
Conclusions:
- The study reveals that differential phosphorylation of CREB at Ser-133 and Ser-142 differentially regulates its interaction with CBP.
- These phosphorylation events modulate CREB's secondary structure, impacting its ability to form complexes and activate transcription.
- This provides a mechanistic basis for how upstream signals control the specificity of CREB-mediated gene activation.