PPAR-gamma AF-2 domain functions as a component of a ubiquitin-dependent degradation signal.
The nuclear hormone receptor peroxisome proliferator-activated receptor-gamma (PPAR-gamma) functions as the "master switch" in adipocyte development and is important in regulating glucose metabolism. PPAR-gamma is rapidly degraded in adipocytes by the ubiquitin proteasome pathway under basal and ligand-activated conditions. Proteasome inhibition increases PPAR-gamma activity, indicating disposal of PPAR-gamma by the ubiquitin proteasome system regulates PPAR-gamma activity. However, the signals and factors required for recognition of PPAR-gamma by the ubiquitin proteasome pathway are unknown. To begin understanding how the ubiquitin-proteasome pathway interacts with PPAR-gamma, we designed a series of constructs containing each PPAR-gamma domain expressed as a fusion protein with the GAL4 DNA-binding domain. The ability of each PPAR-gamma domain to alter the stability of the GAL4 DNA-binding domain and to undergo ubiquitylation was assessed via western blot analysis. In addition, luciferase reporter assays were used to assay PPAR-gamma transcriptional activity. Using this approach, we determined that the AF-1 and ligand-binding domains (LBDs) of PPAR-gamma are targeted to the proteasome for degradation. However, only the LBD is conjugated to ubiquitin. The AF-2 helix of the LBD is required for maximum ubiquitylation, but is not essential for ligand-dependent ubiquitin conjugation. Finally, luciferase reporter assays show a fully functional ubiquitin system is required for PPAR-gamma activation. These results indicate that the ubiquitin-proteasome pathway is an integral determinant of PPAR-gamma activity, targeting PPAR-gamma for proteasomal degradation via ubiquitin independent and ubiquitin dependent mechanisms.
Kilroy
Gail E
GE
Ubiquitin Biology Laboratory, Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, Louisiana, USA.
Zhang
Xiaoying
X
Floyd
Z Elizabeth
ZE
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000166:cytology / Q000502:physiology*; D000818:Animals; D005947:Glucose / Q000378:metabolism; D008024:Ligands; D051379:Mice; D023421:Models, Animal; D041681:NIH 3T3 Cells; D047495:PPAR gamma / Q000502:physiology*; D046988:Proteasome Endopeptidase Complex / Q000502:physiology*; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*; D015533:Transcriptional Activation / Q000502:physiology; D025801:Ubiquitin / Q000502:physiology*
D008024:Ligands; D047495:PPAR gamma; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex; D005947:Glucose