Figure 2: BIG3-PKA-PP1Cα regulates the suppressive ability of PHB2.
From: A-kinase anchoring protein BIG3 coordinates oestrogen signalling in breast cancer cells

(a) The schematic representation of human PHB2. The black inverted triangle indicates S39 of PHB2. (b) The subcellular localization of phospho-PHB2 S39 after siBIG3 treatment in the presence of E2. α/β-Tubulin (tublin) and laminin B (lamin) were used as loading controls for the cytoplasmic and nuclear fractions, respectively. (c) Upper, Luciferase assays showing the inhibitory activity of phospho-PHB2 S39. The indicated PHB2 (WT, S39A), ERα, ERE-luciferase, and pRL-TK construct-transfected HEK293T cells were exposed to E2 for 24 h and analysed for luciferase and Renilla-luciferase activities. Data are normalized to Renilla-luciferase and represent the means±s.e.m. of three independent experiments. Lower, PHB2 S39-phosphorylation. HA-PHB2 and FLAG-ERα-transfected HEK293T cells were stimulated by E2 for 24 h. (d) The inhibitory effects of PHB2-S39 on IGF-1Rβ phosphorylation. The indicated PHB2 (WT, S39A) or ERα-transfected HEK293T cells were exposed to E2 for 24 h, followed by immunoprecipitation with IGF-1Rβ antibody. (e) Phosphatase activities of BIG3 against phospho-PHB2-peptide. The indicated FLAG-BIG3 (WT, S305A and S1208A) and HA-ERα-transfected HEK293T cells were treated with E2 for 24 h, followed by immunoprecipitation using an anti-FLAG antibody. Phosphatase activity of the immunoprecipitates was measured as described in Methods section. These data represent the means±s.e.m. of three independent experiments. (f) The effects of siPKA and siPP1Cα on BIG3 S305 and S1208 phosphorylation, and PHB2 S39-phosphorylation in MCF-7 cells after E2 stimulation for 24 h. ***P<0.001 (two-sided Student’s t-test).