Partial agonist activity of the progesterone receptor antagonist RU486 mediated by an amino-terminal domain coactivator and phosphorylation of serine400

Abstract

Jun dimerization protein-2 (JDP-2) is a progesterone receptor (PR) coregulatory protein that acts by inducing structure and transcriptional activity in the disordered amino-terminal domain (NTD) of PR. JDP-2 can also potentiate the partial agonist activity of the PR antagonist RU486 by mechanisms that have not been defined. Functional mutagenesis experiments revealed that a subregion of the NTD (amino acids 323-427) was required for the partial agonist activity of RU486 induced by PR interaction with JDP-2. However, this subregion was not required for JDP-2 enhancement of the activity of progestin agonists. Mutation of phosphorylation sites within this region of the NTD showed that phosphorylation of serine 400 was required for the partial agonist activity of RU486 stimulated by JDP-2, but was not required for activity of hormone agonist, either in the presence or absence of JDP-2. Cyclin-dependent kinase 2 (Cdk2)/cyclin A is a novel PR coregulator that binds the NTD and acts by phosphorylating steroid receptor coactivator-1 and modulating steroid receptor coactivator-1 interaction with PR. Cdk2/cyclin A also potentiated the partial agonist activity of RU486; however, phosphorylation of serine 400 was not required, indicating that JDP-2 and Cdk2/cyclin A act by distinct mechanisms. We conclude that PR bound to RU486 and associated with JDP-2 adopts an active conformation in a subregion of the NTD requiring phosphorylation of serine 400 that is distinct from that promoted by progestin agonists. These data underscore the structural flexibility of the NTD of PR, and the ability of steroid ligands together with interacting proteins to affect the conformation and activity of the NTD.

Figure 1

JDP-2 potentiation of the partial agonist activity of RU486 is dependent on the NTD of PR. A, COS-1 cells were transfected with PRE₂-TATA-Luc (200 ng) and phPR-B (1.5 ng) or phPR-A (15 ng) together with pCR3.1-JDP-2 (100 ng) or an empty vector. B, COS-1 cells were transfected with PRE₂-TATA-Luc (200 ng) and DBD-hLBD (10 ng) or phPR-B (1.5 ng) together with pCR3.1-SRC-1 (200 ng) or pCR3.1-JDP-2 (137 ng). C, COS-1 cells were transfected with PRE₂-TATA-Luc (200 ng) and phPR-B (1.5 ng) together with pCR3.1-SRC-1 (150 ng) or pCR3.1-JDP-2 (100 ng). Cells were treated for 24 h with vehicle, 10 nm R5020, 10 nm RU486, or 100 nm ZK98299 as indicated in panels A–C. Fold luciferase induction was calculated as a ratio of relative luciferase activation of ligand-treated cells divided by the corresponding vehicle-treated samples. Fold enhancement was calculated by setting luciferase induction of each receptor construct in each treatment group to 1.0, and corresponding values in the presence of JDP-2 or SRC-1 were calculated as folds relative to 1.0. This is a single representation of triplicate experiments.

Figure 2

NTD (N) sequence between AF1 and ID is required for JDP-2 potentiation of the partial agonist activity of RU486. A, Schematic illustration of PR-B internal deletion constructs. B, COS-1 cells were transfected with PRE₂-TATA-Luc (200 ng) together with varying doses of wild-type or deletion PR-B constructs expressed from pCDNA1 (1–12.5 ng). Cells were treated with vehicle or R5020 (10 nm) for the final 24 h of transfection. C, COS-1 cells were transfected with PRE₂-TATA-Luc (200 ng) together with a single dose of phPR-B expressing wild-type PR-B, Δ323 PR-B, or Δ475 PR-B (1.5 ng each) in the presence or absence of pCR3.1-JDP-2 (100 ng). Cells were treated for the final 24 h of transfection with vehicle, or 10 nm R5020 or RU486, as indicated. Fold luciferase induction by ligands and fold enhancement by JDP-2 were calculated as in Fig. 1. Values are averages ± sem of at least three independent experiments. WT, Wild type.

Figure 3

The sequence region of the PR NTD (N) between aa 323 and aa 475 is necessary, but not sufficient, for the partial agonist activity of RU486. A, Schematic illustration of PR-B constructs including WT-PR-B, truncation 350-DhLBD, and 323-427-DhLBD constructs. B, COS-1 cells were transfected with PRE₂-TATA-Luc (200 ng) together with single concentrations of WT-PR-B and PR constructs in the presence or absence of pCR3.1-JDP-2 (100 ng). Cells were treated for the final 24 h with vehicle or 10 nm RU486, as indicated. Fold enhancement by JDP-2 was calculated as in Fig. 1. Values are averages ± sem of at least three independent experiments. WT, Wild type; DhLBD, DBD-hLBD.

Figure 4

NTD (N) between AF1 and ID acts as a spacer for hormone agonist activity. A, Schematic illustration of PR-B constructs including WT-PR-B, internal deletions (Δ323-475) (Δ475-534), and AF1 duplication. B, COS-1 cells were transfected with PRE₂-TATA-Luc (200 ng) together with phPR-B expressing wild-type or PR-B constructs depicted in panel A (0.5–25 ng). Cells were treated with vehicle or 10 nm R5020 for the final 24 h of transfection. C, COS-1 cells were transfected with PRE₂-TATA-Luc (200 ng) together with single concentrations (1.5) of WT-PR-B and PR constructs in the presence or absence of pCR3.1-JDP-2 (100 ng). Cells were treated for the final 24 h with vehicle, 10 nm R5020, or RU486, as indicated. Fold luciferase induction by ligand and fold enhancement by JDP-2 was calculated as in Fig. 1. Values are averages ± sem of at least three independent experiments. WT, Wild type.

Figure 5

Phosphorylation of serine 400 in NTD (N) of PR influences JDP-2 potentiation of partial agonist activity of RU486. A, Schematic illustration of characterized serine phosphorylation sites in PR. Hormone-induced sites include serines 102, 294, and 345; other sites are basally phosphorylated and increased by ligand treatment of cells. B, COS-1 cells were transfected with PRE₂-TATA-Luc (200 ng) together with phPR-B expressing wild-type or serine 345 or serine 400 phosphorylation mutants (1.5–25 ng). Cells were treated with vehicle or 10 nm R5020 for the final 24 h of transfection. C, COS-1 cells were transfected with PRE₂-TATA-Luc (200 ng) together with 1.5 ng of phPR-B expressing wild-type PR-B or phosphorylation mutants, in the presence or absence of pCR3.1-JDP-2 (100 ng). Cells were treated for the final 24 h of transfection with vehicle or 10 nm R5020 or RU486, as indicated. Fold luciferase induction by ligand and fold enhancement by JDP-2 were calculated as in Fig. 1. Values are averages ± sem of at least three independent experiments. h, Hinge; WT, wild type.

Figure 6

Cyclin A/Cdk2 potentiation of partial agonist activity of RU486. HeLa cells were transfected with PR-B (panels A and B) or PR-B/S400A (panel B) along with 1 μg empty vector or 0.5 μg cyclin A, 0.5 μg Cdk2 or cyclin A + Cdk2 together, and 0.25 μg of E1B-TATA-luciferase reporter. The cells were treated 24 h after transfection with 10 nm RU486 for an additional 24 h. The cells were harvested, and luciferase activity was measured and normalized to β-Gal activity. The results are expressed as fold change from vector-transfected vehicle-treated samples. Values are averages ± sem of three independent experiments.

Figure 7

Model of JDP-2 potentiation of RU486 activity. JDP-2 interaction with the DBD, through interdomain allosteric coupling, induces distinct structures in the NTD of PR bound to progesterone or RU486.