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The putative cofactor TIF1alpha is a protein kinase that is hyperphosphorylated upon interaction with liganded
R A Fraser1, D J Heard, S Adam
1Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/Université Louis Pasteur/Collège de France, B. P. 163, 67404 Illkirch Cedex, Strasbourg, France.
Abstract:
Ligand-induced gene activation by nuclear receptors (NRs) is a complex process requiring dissociation of corepressors and recruitment of coactivators. The putative transcriptional intermediary factor TIF1alpha has been previously characterized as a nuclear protein that interacts directly with the AF-2 ligand-dependent activating domain present in the ligand-binding domain of numerous steroid and nonsteroid receptors, including the estrogen (ERalpha) and retinoid X (RXRalpha) receptors. We report here that TIF1alpha is both a phosphoprotein and a protein kinase. TIF1alpha coexpressed in COS-1 cells with RXRalpha or ERalpha is phosphorylated and becomes hyperphosphorylated upon ligand treatment. This hyperphosphorylation requires the binding of TIF1alpha to transcriptionally active NRs since it is prevented by mutations either in the core (alpha-helix 12 of the ligand-binding domain) of the AF-2 activating domains of RXRalpha and ERalpha or in the NR box of TIF1alpha that are known to prevent TIF1alpha-NR interactions. Thus, TIF1alpha is a phosphoprotein that undergoes ligand-dependent hyperphosphorylation as a consequence of nuclear receptor binding. We further show that purified recombinant TIF1alpha possesses intrinsic kinase activity and that, in addition to autophosphorylation, TIF1alpha selectively phosphorylates the transcription factors TFIIEalpha, TAFII28, and TAFII55 in vitro. These latter results raise the possibility that TIF1alpha may act, at least in part, by phosphorylating and modifying the activity of components of the transcriptional machinery.
Insights
TIF1alpha is a nuclear protein that acts as a protein kinase. Upon binding to nuclear receptors (NRs), it undergoes ligand-dependent hyperphosphorylation and phosphorylates other transcription factors, potentially regulating gene activation.
Area of Science:
- Molecular Biology
- Gene Regulation
- Protein Kinase Activity
Background:
- Nuclear receptors (NRs) mediate ligand-induced gene activation through corepressor dissociation and coactivator recruitment.
- TIF1alpha is a nuclear protein that interacts with the AF-2 domain of NRs like ERalpha and RXRalpha.
Purpose of the Study:
- To investigate the role of TIF1alpha in nuclear receptor-mediated gene activation.
- To determine if TIF1alpha possesses kinase activity and how it is regulated by NRs.
Main Methods:
- Co-expression of TIF1alpha with ERalpha or RXRalpha in COS-1 cells.
- Analysis of TIF1alpha phosphorylation status upon ligand treatment.
- In vitro kinase assays using purified recombinant TIF1alpha.
Main Results:
- TIF1alpha is a phosphoprotein that undergoes ligand-dependent hyperphosphorylation when bound to NRs.
- Mutations preventing TIF1alpha-NR interaction abolish hyperphosphorylation.
- Purified TIF1alpha exhibits kinase activity, phosphorylating TFIIEalpha, TAFII28, and TAFII55 in vitro.
Conclusions:
- TIF1alpha is a nuclear receptor-binding phosphoprotein and protein kinase.
- Ligand binding to NRs induces TIF1alpha hyperphosphorylation.
- TIF1alpha may regulate transcriptional machinery by phosphorylating components like TFIIEalpha, TAFII28, and TAFII55.