CUEDC2 interacts with PRB in vitro and in vivo. (A) GST-CUEDC2 or GST was incubated with total cell lysates expressing Myc-PRB with or without 100 nM progesterone for 2 h (hours), and then detected using an anti-Myc. (B) 293T cells were transfected as indicated, and treated with progesterone as in (A). Cell lysates were immunoprecipitated (IP) using anti-HA, and the immunoprecipitates were probed with anti-Myc. (C) T47D cells were treated with progesterone as in (A). Lysates were subjected to IP with anti-PR or a control mouse IgG. The immunoprecipitates were analyzed by immunoblotting (IB) with anti-CUEDC2 and anti-PR. (D) 293T cells were transfected as indicated, and treated as in (B). The localization of either PRB or CUEDC2 was analyzed by confocal microscopy. Scale bar: 20 μm. (E) 293T cells were treated as in (D), except that cells were cotransfected with RFP-PRB and GFP-CUEDC2. Scale bar: 20 μm. (F) The yeast strain AH109 was cotransformed with Gal4-AD-PRB or its mutants plus the Gal4-DBD-CUEDC2. A cotransformant from each plate was streaked on SD/−Trp/−Leu/−His/−Ade medium with β-galactosidase (+, positive interaction; −, no interaction). At the top of the graph is a schematic diagram of the PRA/B protein, illustrating the locations of various domains. (G) 293T cell lysates expressing Myc-PRB or its mutants were incubated with GST or GST-CUEDC2, and interacting proteins were detected with an anti-Myc. (H) 293T cell lysates expressing Myc-PRB were incubated with GST, GST-CUEDC2 or its mutants as indicated, and interacting proteins were detected with anti-Myc. At the top of the graph is a schematic diagram of the CUEDC2 protein, illustrating the regions of various mutants.

CUEDC2 promotes PRB degradation by ubiquitin–proteasome. (A, B) 293T cells were cotransfected with Myc-PRB (0.5 μg) and HA-CUEDC2 (2.0 μg). At 12 h after transfection, the cells were treated with the proteasome inhibitor ALLN (30 μM) or lactacystin (10 μM) for 30 min before treatment with progesterone (10 nM). Cells were cultured for additional 12 h and Western blot was performed with anti-Myc and anti-HA. (C) 293T cells were transfected as indicated and treated as in (A). Lysates were immunoprecipitated and detected with anti-Myc. (D) 293T cells were transfected with either CUEDC2 siRNA or control siRNA. At 48 h post-transfection, cells were cotransfected with Myc-PRB and His-ubiquitin plasmid and then treated as in (A).

CUEDC2 downregulates expression of PR target genes. (A, B) T47D cells were transiently transfected as indicated and treated with 10 nM progesterone for 6 h (cyclin D1) or 24 h (p21). The cells were harvested and total RNA was extracted. Real-time RT–PCR was performed as described. Cyclin D1 and p21 mRNA levels were assessed and normalized to GAPDH mRNA. (C, D) T47D cells were transfected and treated as described in (A) and (B); cyclin D1, p21, and Bax expression was detected using specific antibodies. (E) T47D cells were transiently transfected as indicated. At 18 h post-transfection, cells were treated with 10 nM progesterone for 6 h, and the proteasome inhibitor ALLN (30 μM) was added 30 min before progesterone treatment. Cells were cultured and Western blot was performed with anti-cyclin D1 and anti-HA. (F, G) p21-Luc (0.2 μg) and HA-CUEDC2 (2.0 μg) were cotransfected into T47D and HeLa cells as indicated. The cells were treated as in (A) and (B). Cell lysates were prepared and measured for luciferase activity as described above.

CUEDC2 inhibits progesterone-induced cell proliferation. (A) T47D cells were transfected. After 6 h, the cultures were treated with 10 nM progesterone for 24 h and percentage of cells in S phase was determined by PI staining and FACS. (B) T47D cells were transfected as indicated. At 48 h after transfection, cells were treated and analyzed as in (A). (C) T47D cells were transfected with HA-CUEDC2 and treated with progesterone as indicated. Cells were harvested and detected by antibodies as indicated. (D, E) Expression of CUEDC2 in human breast cancer and other tumor cells. RT–PCR from the selected cell lines was performed as described. The tumor cells were also prepared and analyzed by anti-CUEDC2.

CUEDC2 negatively regulates PRB expression. (A) HA-CUEDC2 (1.0 or 2.0 μg) was transfected into T47D cells and treated with 10 nM progesterone for 24 h. Protein levels were assessed by Western blot as indicated. (B) 293T cells were cotransfected with 0.5 μg of Myc-PRB and HA-CUEDC2 (1.0 or 2.0 μg) and treated as in (A), and protein levels were assessed as indicated. (C) T47D cells were transiently transfected with 2.0 μg of HA-CUEDC2 and treated with or without 10 nM estradiol for 24 h and then treated with 10 nM progesterone for another 24 h. The cell lysates were analyzed by immunoblot using anti-PR and anti-HA. (D) T47D cells were transfected with CUEDC2 siRNA or control siRNA. At 48 h after transfection, cells were treated as in (A), and protein levels were measured by Western blot as indicated. (E) T47D cells were transfected with HA-CUEDC2 (2.0 μg) and treated with 10 nM progesterone for 24 h. Cells were harvested and PR mRNA levels were assessed by real-time RT–PCR. (F) The PRB protein levels in 293T cells cotransfected with Myc-PRB (0.5 μg) and HA-CUEDC2 (2.0 μg) were detected by immunoblotting at different times after exposure to the protein synthesis inhibitor cycloheximide (CHX, 20 μg/ml) as indicated. The graph shows the quantification of the immunoblotting data (lower panel) (–⧫−P, –▪–+P, –▴–C+P mean without progesterone, with progesterone, or with HA-CUEDC2 and progesterone, respectively).

CUEDC2 strongly inhibits the transactivation of PR. (A) Inhibition of PRB transcriptional activity by CUEDC2 in HeLa cells. All treatments were as follows, except when noted: cells were transiently transfected with pMMTV luciferase reporter (0.2 μg) together with Myc-PRA or PRB (0.05 μg), or various amounts of HA-CUEDC2 vectors, alone or in combination as indicated. After transfection, cultures were treated with or without 10 nM progesterone for 24 h, and luciferase activity was measured. (B) Inhibition of PR transactivation by CUEDC2 in T47D cells. (C) T47D cells were transfected with either CUEDC2 siRNA or control siRNA, and were cotransfected with pMMTV-luc reporter plasmid 48 h later and then treated with 10 nM progesterone or ethanol for 24 h, and luciferase reporter assays were performed. Western blots were analyzed with antibodies to CUEDC2 and actin. (D) Inhibition of PRB transactivation by CUEDC2 or its mutants in 293T cells. The results are means±s.d. of three independent determinations.

Mutated PRB resists the inhibitory effect of CUEDC2. (A) 293T cells were transfected with 0.2 μg of pMMTV-Luc reporter, 1.0 μg of HA-CUEDC2, and 0.05 μg of Myc-PRB or its mutant, and treated with or without 10 nM progesterone for 24 h. Cells were harvested and luciferase activity was measured, and cell lysates were probed with anti-HA and anti-Myc (lower panel). (B) HEK-293 cells stably expessing wild-type and mutant PRB K388R were treated as in (A). (C) Total cell lysates expressing Myc-PRB or its mutant were incubated with GST-CUEDC2 or GST. Bound proteins were detected by anti-Myc. (D) 293T cells were transfected as indicated and treated with 10 nM progesterone for 12 h, and the proteasome inhibitor ALLN (30 μM) was added 30 min before progesterone treatment. Cells were cultured for an additional 12 h and Western blot was performed with antibodies as indicated. (E) 293T cells were transfected with 1.0 μg Myc-PRB, 1.0 μg HA-SUMO, and 1.0 μg His-ubiquitin as indicated. Cells were treated as in (D) and cultured for an additional 12 h. Western blot was performed with antibodies as indicated. (F) 293T cells were transfected with 1.0 μg Myc-PRB, 1.0 μg HA-SUMO, 1.0 μg His-ubiquitin, and 0.5 μg HA-CUEDC2 as indicated. Cells were treated as in (D) and cultured for an additional 12 h. Western blot was performed with antibodies as indicated and ubiquitination of PRB was detected as in (D). P+C means HA-CUEDC2 and progesterone.