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. 2008 Jan 17;22(4):823–837. doi: 10.1210/me.2007-0437

Figure 3.

Figure 3

Functional Analysis of S345A Phospho-Mutant PR-B and MAPK Dependence of Nonclassical PR Gene Targets

A, T47D PR-null cells or T47D cells stably expressing either wt or S345A PR-B were treated with R5020 for the indicated times. Phospho-Ser345 and total PR were visualized using phospho-PR and total PR Ab. Phospho-Ser345 blotting demonstrates the specificity of phosphospecific Ab. Total Erk1/2 blots were included as protein loading controls. B, T47D cells expressing wt or mutant S345A PR-B were treated for 10 min with R5020. Whole-cell lysates were subjected to phospho-Erk1/2 and total Erk1/2 Western blotting. C, PR-null HeLa cells were transiently transfected with either wt or S345A PR-B and PRE-luciferase and renilla-luciferase (control) reporter constructs. Luciferase activity was measured in whole-cell lysates after EtOH or R5020 treatment (24 h) and represented as firefly/renilla. Similar results were obtained with T47D cell lines stably expressing wt or S345A PR-B. Error bars represent sd (n = 3). D, T47D PR-null cells or T47D cells stably expressing S345A or wt PR-B were treated for 8 h with R5020. Cells expressing wt PR-B were pretreated with DMSO, PP2, or AG1478. Cell lysates were subjected to Western blotting to detect EGFR, p21, and cyclin D1. Total PR blotting indicates PR expression levels and ligand-dependent PR down-regulation. Total Erk1/2 was included as a loading control. E and F, T47D-YB cells were treated with or without R5020 (18 h) in the presence of vehicle control (DMSO) or the MEK inhibitor U0126. Real-time quantitative PCR was then used to measure p21 (E) or EGFR (F) mRNA. Values were normalized to β-actin. Error bars represent sd of triplicate cultures. G and H, T47D-YB cells were treated with or without R5020 (18 h) in the presence of vehicle control (DMSO), antiestrogen ICI 182,780 (ICI; 1.0 μm), or antiprogestin RU486 (RU; 10 μm). Real-time quantitative PCR was then used to measure EGFR (G) or Sgk (H) mRNA. Values were normalized to β-actin. Error bars represent sd of triplicate cultures. G, Inset, Western blot showing progestin-induced PR Ser345 phosphorylation in the presence or absence of ICI 182,780 (1.0 μm). All experiments were repeated at least three times with similar results.