Specificity in the actions of the UBR1 ubiquitin ligase in the degradation of nuclear receptors

Abstract

The UBR1 ubiquitin ligase promotes degradation of proteins via the N-end rule and by another mechanism that detects a misfolded conformation. Although UBR1 was shown recently to act on protein kinases whose misfolding was promoted by inhibition of Hsp90, it was unknown whether this ubiquitin ligase targeted other client types of the chaperone. We analyzed the role of UBR1 in the degradation of nuclear receptors that are classical clients of Hsp90. Our results showed that UBR1 deletion results in impaired degradation of the glucocorticoid receptor and the androgen receptor but not the estrogen receptor α. These findings demonstrate specificity in the actions of the UBR1 ubiquitin ligase in the degradation of Hsp90 clients in the presence of small molecule inhibitors that promote client misfolding.

Keywords: AR, androgen receptor; CHIP, C-terminal Hsp interacting protein; DMSO, dimethylsulfoxide; Degradation; ER, estrogen receptor; GA, geldanamycin; GR, glucocorticoid receptor; HA, hemagglutinin; Hsp90; MEF, mouse embryonic fibroblasts; Molecular chaperone; Quality control; UBR1; UPS, ubiquitin proteasome system; Ubiquitin ligase.

Graphical abstract

Fig. 1

Geldanamycin dependent degradation of GR in WT, Ubr1^−/− and Chip^−/− MEF cells. (A) WT, Ubr1^−/− and CHIP^−/− MEF cells were treated with 100 nM of GA for indicated times. 40 μg of total protein from each cell line were analyzed by SDS–PAGE and probed with anti-GR; PI3K was used as a loading control. (B) WT, Ubr1^−/− and CHIP^−/− MEF cells were treated with different concentration of GA for 6 hours. 40μg of total protein from each cell line were fractionated by SDS–PAGE and probed with anti-GR. PI3K levels were analyzed as a loading control.

Fig. 2

Effect of UBR1 overexpression on GR levels. (A) Ubr1^−/− cells were transfected with human HA-tagged GR (HA-hGR) and pCMV empty vector or rat UBR1. After 24 h of transfection the cells were treated with 50 nM of GA or DMSO for the indicated time points and harvested. 40 μg of cell lysates were analyzed in SDS–PAGE and probed with anti-HA (α-HA) and UBR1 antisera. PI3K was used as loading control. * in the PI3K blot indicates a non- specific band. (B) Ubr1^−/− cells were transfected with HA-hGR and empty vector or rat UBR1. After transfection cells were treated with DMSO or MG-132 (50 μM) for 18 h and GA (100 nM) for 6 h. Cells were harvested 24 h after transfection. 40 μg of cell lysates were analyzed in SDS–PAGE and probed with anti-HA (α-HA), UBR1 and PI3K. (C) Ubr1^−/− cells were transfected with HA-hGR and different amounts of rat E3 ligase UBR1 plasmid DNA (0, 0.5, 2 and 4 μg). Cell were harvested 24 h after transfection. 40 μg of total cell lysates were fractionated by SDS–PAGE and probed with anti-HA (α-HA) and UBR1 antisera. PI3K was used as a loading control. NT represents non-transfected Ubr1^−/− MEF cells in A–C.

Fig. 3

Effects of UBR1 overexpression on AR and ER-α. (A) Ubr1^−/− cells were transfected with the AR expressing plasmid and pCMV empty vector or rat UBR1. After 24 h of transfection the cells were treated with different concentrations of GA or vehicle for 24 h. 40 μg of cell lysates were analyzed in SDS–PAGE and probed with AR and UBR1 antisera. PI3K was used as loading control. (B) Ubr1^−/− cells were transfected with AR and pCMV empty vector or rat UBR1. After transfection the cells were treated with DMSO, MG-132 (50 μM) for 18 h and GA (100 nM) for 6 h. The cells were harvested 24 h after transfection and 40 μg of cell lysates were analyzed by SDS–PAGE and probed with anti AR, UBR1 and PI3K. (C) Ubr1^−/− cells were transfected a plasmid expressing ER-α or pCMV empty vector or rat UBR1. After 24 h of transfection, the cells were treated with different concentrations of GA or DMSO for 24 h. 40 μg of cell lysates were analyzed by SDS–PAGE and probed with AR and UBR1 antisera. PI3K was used as loading control. NT represents non-transfected Ubr1^−/− MEF cells in A–C.