Nuclear receptor coactivator thyroid hormone receptor-binding protein (TRBP) interacts with and stimulates its associated DNA-dependent protein kinase.
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.
Ko
Lan
L
Department of Gene Regulation, Bone and Inflammation Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, USA. kol@lilly.com
Chin
William W
WW
D002352:Carrier Proteins / Q000378:metabolism*; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D009687:Nuclear Proteins; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011995:Recombination, Genetic; D013379:Substrate Specificity; D013963:Thyroid Hormones / Q000378:metabolism*; D014158:Transcription, Genetic; D000097810:Thyroid Hormone-Binding Proteins
D002352:Carrier Proteins; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D009687:Nuclear Proteins; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D013963:Thyroid Hormones; D000097810:Thyroid Hormone-Binding Proteins; C497263:PRKDC protein, human