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Nuclear receptor coactivator thyroid hormone receptor-binding protein (TRBP) interacts with and stimulates its
1Department of Gene Regulation, Bone and Inflammation Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, USA. kol@lilly.com
Abstract:
Nuclear receptors mediate gene activation through ligand-dependent interaction with coactivators. We previously cloned and characterized thyroid hormone receptor-binding protein, TRBP (NcoA6: AIB3/ASC-2/RAP250/PRIP/TRBP/NRC), as an LXXLL-containing coactivator that associates with coactivator complexes through its C terminus. To search for protein factors involved in TRBP action, we identified a distinct set of proteins from HeLa nuclear extract that interacts with the C terminus of TRBP. Analysis by mass spectrometric protein sequencing revealed a DNA-dependent protein kinase (DNA-PK) complex including its catalytic subunit and regulatory subunits, Ku70 and Ku86. DNA-PK is a heterotrimeric nuclear phosphatidylinositol 3-kinase that functions in DNA repair, recombination, and transcriptional regulation. DNA-PK phosphorylates TRBP at its C-terminal region, which directly interacts with Ku70 but not Ku86 in vitro. In addition, in the absence of DNA, TRBP itself activates DNA-PK, and the TRBP-stimulated DNA-PK activity has an altered phosphorylation pattern from DNA-stimulated activity. An anti-TRBP antibody inhibits TRBP-induced kinase activity, suggesting that protein content of TRBP is responsible for the stimulation of DNA-independent kinase activity. Furthermore, in DNA-PK-deficient scid cells, TRBP-mediated transactivation is significantly impaired, and nuclear localization of TRBP is altered. The activation of DNA-PK in the absence of DNA ends by the coactivator TRBP suggests a novel mechanism of coactivator-stimulated DNA-PK phosphorylation in transcriptional regulation.
Insights
Thyroid hormone receptor-binding protein (TRBP), a coactivator, activates DNA-dependent protein kinase (DNA-PK) independently of DNA. This novel interaction impacts TRBP
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Nuclear receptors regulate gene expression via ligand-dependent coactivator interactions.
- Thyroid hormone receptor-binding protein (TRBP) is an LXXLL-containing coactivator crucial for transcriptional regulation.
- DNA-dependent protein kinase (DNA-PK) is a key enzyme in DNA repair and transcriptional regulation.
Purpose of the Study:
- To identify protein factors interacting with the C terminus of TRBP.
- To investigate the functional relationship between TRBP and DNA-PK.
- To elucidate a novel mechanism of coactivator-stimulated kinase activity in gene regulation.
Main Methods:
- Mass spectrometric protein sequencing to identify interacting proteins.
- In vitro kinase assays to assess DNA-PK activity and phosphorylation.
- Studies in DNA-PK-deficient scid cells to evaluate TRBP function.
Main Results:
- DNA-dependent protein kinase (DNA-PK) complex, including Ku70 and Ku86, was identified as a TRBP-interacting protein.
- TRBP directly interacts with Ku70 and is phosphorylated by DNA-PK in its C-terminal region.
- TRBP activates DNA-PK in a DNA-independent manner, altering phosphorylation patterns.
- TRBP-mediated transactivation and nuclear localization are impaired in DNA-PK-deficient cells.
Conclusions:
- TRBP activates DNA-PK independently of DNA, suggesting a novel regulatory mechanism.
- Coactivator-stimulated DNA-PK phosphorylation plays a role in transcriptional regulation.
- This interaction highlights a new pathway linking coactivators and DNA repair/transcription machinery.