Related Experiment Videos
Characterization of an E1A-CBP interaction defines a novel transcriptional adapter motif (TRAM) in CBP/p300
M J O'Connor1, H Zimmermann, S Nielsen
1Institute of Molecular and Cell Biology, Singapore 117 609, Singapore. mcbomark@imcb.nus.edu.sg
Abstract:
The adenovirus E1A protein subverts cellular processes to induce mitotic activity in quiescent cells. Important targets of E1A include members of the transcriptional adapter family containing CBP/p300. Competition for CBP/p300 binding by various cellular transcription factors has been suggested as a means of integrating different signalling pathways and may also represent a potential mechanism by which E1A manipulates cell fate. Here we describe the characterization of the interaction between E1A and the C/H3 region of CBP. We define a novel conserved 12-residue transcriptional adapter motif (TRAM) within CBP/p300 that represents the binding site for both E1A and numerous cellular transcription factors. We also identify a sequence (FPESLIL) within adenovirus E1A that is required to bind the CBP TRAM. Furthermore, an E1A peptide containing the FPESLIL sequence is capable of preventing the interaction between CBP and TRAM-binding transcription factors, such as p53, E2F, and TFIIB, thus providing a molecular model for E1A action. As an in vivo demonstration of this model, we used a small region of CBP containing a functional TRAM that can bind to the p53 protein. The CBP TRAM binds p53 sequences targeted by the cellular regulator MDM2, and we demonstrate that an MDM2-p53 interaction can be disrupted by the CBP TRAM, leading to stabilization of cellular p53 levels and the activation of p53-dependent transcription. Transcriptional activation of p53 by the CBP TRAM is abolished by wild-type E1A but not by a CBP-binding-deficient E1A mutant.
Insights
Adenovirus E1A protein disrupts cell cycles by binding CBP/p300. A novel TRAM motif in CBP/p300 is key for E1A and transcription factor binding, offering a model for E1A
Area of Science:
- Molecular biology
- Virology
- Cellular biology
Background:
- Adenovirus E1A protein induces cell proliferation in quiescent cells.
- CBP/p300 proteins are key transcriptional adapters and targets of E1A.
- E1A may manipulate cell fate by competing for CBP/p300 binding sites.
Purpose of the Study:
- To characterize the interaction between adenovirus E1A and CBP/p300.
- To identify the binding sites and molecular mechanisms involved in E1A-mediated cellular manipulation.
Main Methods:
- Protein interaction studies focusing on the C/H3 region of CBP.
- Identification and characterization of a novel 12-residue transcriptional adapter motif (TRAM).
- Peptide-based inhibition assays and in vivo studies using CBP TRAM fragments and p53.
Main Results:
- A novel 12-residue TRAM within CBP/p300 serves as a binding site for E1A and transcription factors like p53, E2F, and TFIIB.
- A specific sequence (FPESLIL) in E1A is essential for binding the CBP TRAM.
- E1A peptides containing FPESLIL disrupt E1A-CBP interactions, inhibiting p53, E2F, and TFIIB binding.
- In vivo, the CBP TRAM binds p53, disrupts MDM2-p53 interaction, stabilizing p53 and activating transcription.
- Wild-type E1A, but not a binding-deficient mutant, inhibits E1A TRAM-mediated p53 transcriptional activation.
Conclusions:
- The study defines a TRAM motif in CBP/p300 as a critical interaction hub for E1A and cellular transcription factors.
- A molecular model for E1A's manipulation of cell fate via CBP/p300 interaction is proposed.
- E1A antagonizes p53-mediated transcription by interfering with the CBP TRAM-p53 interaction.