| pmid | 10196247 | |||||
|---|---|---|---|---|---|---|
| title | Characterization of an E1A-CBP interaction defines a novel transcriptional adapter motif (TRAM) in CBP/p300. | |||||
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| journal | J Virol | |||||
| year | 1999 | |||||
| full_text_available | true | |||||
| full_text_extraction_method | html | |||||
| pmcid | PMC104130 | |||||
| doi | 10.1128/JVI.73.5.3574-3581.1999 |
Characterization of an E1A-CBP interaction defines a novel transcriptional adapter motif (TRAM) in CBP/p300.
Authors: O'Connor MJ, Zimmermann H, Nielsen S, Bernard HU, Kouzarides T Journal: J Virol (1999) DOI: 10.1128/JVI.73.5.3574-3581.1999 PMC: PMC104130
- J Virol. 1999 May;73(5):3574-81. doi: 10.1128/JVI.73.5.3574-3581.1999.
Characterization of an E1A-CBP interaction defines a novel transcriptional adapter motif (TRAM) in CBP/p300.
O'Connor MJ(1), Zimmermann H, Nielsen S, Bernard HU, Kouzarides T.
Author information: (1)Institute of Molecular and Cell Biology, Singapore 117 609, Singapore. mcbomark@imcb.nus.edu.sg
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.
DOI: 10.1128/JVI.73.5.3574-3581.1999 PMCID: PMC104130 PMID: 10196247 [Indexed for MEDLINE]
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.
DISCUSSION
The identification of a small TRAM within the C/H3 region of CBP explains to a large extent why this region represents a hot spot for transcription factor binding. In addition to interacting with E1A, p53, E2F, and TFIIB, the CBP TRAM also binds to the cellular transcription factors MyoD, YY1, c- fos , c- jun , and P/CAF ( 40 ). In principle, E1A could inhibit the binding of these and other factors to the CBP TRAM, whether or not they interact with residues similar to the FXE/DXXXL sequence. At least for MyoD ( 47 ) and YY1 ( 40 ), sequences similar to those in E1A, p53, and E2F have been shown to be necessary for the binding of CBP. In MyoD, the sequence has been referred to as the FYD motif, which is present in the N-terminal region of a number of myogenic transcription factors ( 36 ).
The modification and/or concentration of any given protein interacting with the CBP TRAM is likely to be important because the increased binding of one transcription factor may alter cellular responses by competing for a limiting TRAM-containing regulator, as has been suggested by recent studies ( 17 , 18 , 23 ). This strategy would appear to have been adopted by adenoviruses, which express high levels of the FXE/DXXXL-containing E1A protein; this protein competitively inhibits the binding of CBP to cellular proteins, resulting in both the inhibition of cellular differentiation and the activation of cell proliferation. Our characterization of the E1A-CBP interaction now provides a molecular basis for these observations.
One cellular regulator targeted by E1A in this way is p53. Previous studies have demonstrated that p53-dependent transcription is activated by CBP/p300 and that this activation is abrogated by E1A. Stimulation of p53 activity by CBP/p300 has been attributed to the properties of these transcriptional adapters, namely, the ability to provide AT activity ( 12 ) and/or to bridge the gap between p53 and components of the basal transcription machinery. In this study, we show for the first time that binding per se is an important part of the mechanism by which CBP/p300 activates p53-dependent transcription. Consequently, E1A binding to the CBP TRAM and the concomitant prevention of a p53-CBP interaction may be sufficient to explain the abrogation of CBP-mediated p53 transcriptional activity.
The mechanism by which p53-dependent transcription is activated by a relatively small region of CBP (residues 1808 to 1852) does not appear to involve either the acetylation of p53 (since CBP sequences responsible for AT activity are not present) or bridging to components of the basal transcription machinery. Rather, activation results from the competitive inhibition of MDM2 binding to p53 and the resulting stabilization of cellular p53 protein levels (as demonstrated in Fig. 6 ). Similar observations were made in vivo when the MDM2-p53 interaction was blocked by an alternative mechanism: that is, through the expression of a small molecule (thioredoxin) that contains the MDM2-binding domain of p53 in its active-site loop ( 4 ). From these observations, we could predict that any protein or protein fragment that inhibits the binding of full-length MDM2 to p53 should be able to activate p53-dependent transcription. We tested this hypothesis by expressing the N-terminal p53-binding domain of MDM2 that lacks the sequences required to target p53 for degradation ( 24 ). As predicted, the expression of the MDM2 N-terminal region from residues 1 to 125 in U-2 OS cells resulted in an almost identical level of activation of p53-dependent transcription as the expression of the CBP TRAM fragment (residues 1808 to 1852) ( 40 ).
Interestingly, E1A is not the only viral oncoprotein to bind the CBP TRAM. We have recently shown that human papillomavirus E6 proteins also target CBP/p300 and can interact directly with CBP amino acids 1808 to 1826 ( 58 ). This interaction results in the down-regulation of p53 transcriptional activity to a level comparable to E1A-mediated repression and is limited to E6 proteins from human papillomaviruses associated with cervical cancer ( 58 ). Moreover, given that the simian virus 40 large T antigen also binds the C/H3 region of CBP ( 9a ) and down-regulates p53-dependent transcription ( 32a ), it is likely that all three of these small DNA tumor virus oncoproteins target the CBP TRAM, strongly suggesting an important role for this motif in cell cycle regulation.
Other inhibitors of the cell cycle that bind the CBP/p300 TRAM and have been shown to be targeted by adenovirus E1A include MyoD and P/CAF. The interaction of MyoD with CBP/p300 has been shown to be essential for cell cycle arrest and muscle-specific gene expression ( 45 ). E1A, by binding to CBP/p300, inhibits these processes ( 5 , 35 ). Our results suggest this activity may involve an E1A FPESLIL-TRAM interaction. Similarly, our unpublished finding that P/CAF binds CBP amino acids 1808 to 1826 suggests another TRAM interaction targeted by E1A. Consistent with this idea is the previously described ability of E1A to displace P/CAF from the C/H3 region of CBP ( 55 ). Like MyoD and p53, P/CAF possesses cell cycle inhibition and cellular differentiation properties ( 46 , 55 ). Thus, by targeting the CBP/p300 TRAM, E1A may affect multiple cellular factors whose role it is to inhibit cell cycle progression.
Future studies that make use of TRAM mutants in the context of full-length CBP/p300 should prove useful in the analysis of CBP/p300-mediated integration of different signalling pathways. Such an approach may be facilitated by the interesting finding that not all CBP TRAM-interacting transcription factors are affected to the same extent by the same point mutations within the CBP TRAM sequence ( 40 ). Thus, by use of variants of the TRAM sequence, it might be possible to selectively block the binding of certain transcription factors to CBP/p300.
In summary, we initiated this study in order to gain greater insight into the molecular mechanism of E1A action and how this protein functions to subvert cellular pathways. Through detailed mapping of the E1A-CBP C/H3 interaction, we have identified a TRAM that is conserved in all CBP/p300 proteins. This motif, targeted by E1A, is used by various cellular factors and may prove to play an important role in the integration of multiple signal transduction pathways.