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TGF-beta-induced phosphorylation of Smad3 regulates its interaction with coactivator p300/CREB-binding protein
1Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Abstract:
Smads are intermediate effector proteins that transduce the TGF-beta signal from the plasma membrane to the nucleus, where they participate in transactivation of downstream target genes. We have shown previously that coactivators p300/CREB-binding protein are involved in TGF-beta-mediated transactivation of two Cdk inhibitor genes, p21 and p15. Here we examined the possibility that Smads function to regulate transcription by directly interacting with p300/CREB-binding protein. We show that Smad3 can interact with a C-terminal fragment of p300 in a temporal and phosphorylation-dependent manner. TGF-beta-mediated phosphorylation of Smad3 potentiates the association between Smad3 and p300, likely because of an induced conformational change that removes the autoinhibitory interaction between the N- and C-terminal domains of Smad3. Consistent with a role for p300 in the transcription regulation of multiple genes, overexpression of a Smad3 C-terminal fragment causes a general squelching effect on multiple TGF-beta-responsive reporter constructs. The adenoviral oncoprotein E1A can partially block Smad-dependent transcriptional activation by directly competing for binding to p300. Taken together, these findings define a new role for phosphorylation of Smad3: in addition to facilitating complex formation with Smad4 and promoting nuclear translocation, the phosphorylation-induced conformational change of Smad3 modulates its interaction with coactivators, leading to transcriptional regulation.
Insights
Transforming growth factor-beta (TGF-β) signaling involves Smad proteins interacting with p300 coactivators. This interaction, regulated by Smad3 phosphorylation, is crucial for TGF-β-mediated gene transcription.
Area of Science:
- Molecular Biology
- Cell Signaling
- Gene Regulation
Background:
- Smads are key mediators of Transforming Growth Factor-beta (TGF-β) signaling.
- p300/CREB-binding protein (CBP) coactivators are known to be involved in TGF-β-induced gene expression.
- The precise mechanism of Smad interaction with coactivators in TGF-β signaling requires further elucidation.
Purpose of the Study:
- To investigate the direct interaction between Smad proteins and the p300 coactivator.
- To determine the role of Smad3 phosphorylation in modulating this interaction.
- To understand how this interaction contributes to TGF-β-mediated transcriptional regulation.
Main Methods:
- Co-immunoprecipitation assays to detect protein-protein interactions.
- Analysis of Smad3 phosphorylation status.
- Reporter gene assays to assess transcriptional activity.
- Expression of Smad3 fragments and adenoviral E1A protein.
Main Results:
- Smad3 directly interacts with a C-terminal fragment of p300.
- TGF-β-mediated phosphorylation of Smad3 enhances its association with p300.
- This phosphorylation event induces a conformational change in Smad3, relieving autoinhibition.
- Overexpression of a Smad3 fragment causes a general squelching effect on TGF-β reporters.
- Adenoviral E1A protein competes with Smads for p300 binding, inhibiting transcription.
Conclusions:
- Smad3 phosphorylation is critical for modulating its interaction with p300 coactivators.
- This phosphorylation-induced conformational change is essential for efficient transcriptional regulation by TGF-β.
- Smad-p300 interaction is a key step in the TGF-β signaling pathway, targeted by viral oncoproteins.