Specific ubiquitin-conjugating enzymes promote degradation of specific nuclear receptor coactivators.
Nuclear receptor coactivators (NRCoAs) are nuclear hormone receptor-associated regulatory proteins that interact with members of the nuclear receptor superfamily in the presence of their cognate ligand, enhancing their transcriptional activity. The identification of ubiquitin-proteasome pathway proteins as coactivators provides evidence that ubiquitin-proteasome-mediated protein degradation plays an integral role in eukaryotic gene transcription. It has also been observed that nuclear receptors themselves are ubiquitinated and degraded in a hormone-dependent manner and that ubiquitin-proteasome function is essential for most nuclear receptors to function as transactivators. Here, we show that specific ubiquitin-proteasome pathway enzymes target specific NRCoA proteins in vivo and in vitro. First, using a temperature-sensitive cell line that contains a thermolabile ubiquitin-activating E1 enzyme, we confirmed that NRCoA proteins are targets of the ubiquitin-proteasome pathway. Then using coimmunoprecipitation studies, we also demonstrate that in vivo, NRCoA proteins are ubiquitinated. Finally, we illustrate that in vitro, NRCoA ubiquitination and degradation depend on the ubiquitin-activating enzyme (E1) and on specific ubiquitin-conjugating enzymes (E2) for each of the coactivators.
Yan
Feng
F
Department of Surgery, Medical Microbiology & Immunology and Cancer Center Criss II, Creighton University, Omaha, Nebraska 68178, USA.
Gao
Xiuhua
X
Lonard
David M
DM
Nawaz
Zafar
Z
D000818:Animals; D001665:Binding Sites; D050882:CREB-Binding Protein; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D051548:Histone Acetyltransferases; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009154:Mutation; D009687:Nuclear Proteins / Q000187:drug effects / Q000378:metabolism; D056920:Nuclear Receptor Coactivator 1; D051958:Nuclear Receptor Coactivator 2; D056921:Nuclear Receptor Coactivator 3; D046988:Proteasome Endopeptidase Complex; D011506:Proteins / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D015534:Trans-Activators / Q000187:drug effects / Q000378:metabolism; D014157:Transcription Factors / Q000187:drug effects / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D044764:Ubiquitin-Activating Enzymes / Q000235:genetics / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism*
D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D009097:Multienzyme Complexes; D009687:Nuclear Proteins; D051958:Nuclear Receptor Coactivator 2; D011506:Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D015534:Trans-Activators; D014157:Transcription Factors; D025801:Ubiquitin; D050882:CREB-Binding Protein; C494363:CREBBP protein, human; D051548:Histone Acetyltransferases; C538859:NCOA1 protein, human; D056920:Nuclear Receptor Coactivator 1; D056921:Nuclear Receptor Coactivator 3; D044763:Ubiquitin-Conjugating Enzymes; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D044764:Ubiquitin-Activating Enzymes; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde