Degradation of the basic helix-loop-helix/Per-ARNT-Sim homology domain dioxin receptor via the ubiquitin/proteasome pathway.
The basic helix-loop-helix/Per-ARNT-Sim homology domain dioxin receptor (DR) translocates to the nucleus upon binding of aromatic hydrocarbon ligands typified by dioxin, whereupon it partners the Ah receptor nuclear translocator and initiates transcription. Concurrently, ligand binding down-regulates receptor levels via an unknown mechanism. In this study we show that receptor levels are dependent upon cellular compartmentalization, with entry into the nucleus leading to the rapid destruction of the DR. Ligand-induced DR translocation was bypassed by adding a heterologous nuclear localization signal to the DR, creating a constitutively nuclear form of the dioxin receptor (DRNLS). The DRNLS protein was shown to be unstable with a half-life of </=1 h whether partnering ARNT or HSP90. Thus, the structural changes induced by ligand binding have no inherent effect on DR stability but are critical in transporting the receptor prior to degradation. The proteolytic pathway that degrades the nuclear receptor is suggested to involve ubiquitination as it was inhibited by the proteasome inhibitor MG132 or co-expression of DRNLS with the ubiquitin mutant UbK48R. Incubation of cells expressing DRNLS with the phosphatase inhibitor calyculin resulted in the rapid phosphorylation and ubiquitination of DRNLS, suggesting that a nuclear kinase is required to trigger receptor proteolysis. Overall, this study demonstrates a novel mechanism of proteolysis whereby the simple relocation of a transcription factor from cytoplasm to nucleus initiates its rapid destruction.
Roberts
B J
BJ
Department of Biochemistry, University of Adelaide, Adelaide, South Australia, Australia 5005. broberts@biochem.adelaide.edu.au
Whitelaw
M L
ML
D000818:Animals; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004147:Dioxins / Q000378:metabolism; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D009097:Multienzyme Complexes / Q000378:metabolism*; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism*; D017384:Sequence Deletion; D015398:Signal Transduction; D014452:Ubiquitins / Q000378:metabolism*
D004147:Dioxins; D009097:Multienzyme Complexes; D018336:Receptors, Aryl Hydrocarbon; D014452:Ubiquitins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex