Figure 2: BIG3-PKA-PP1Cα regulates the suppressive ability of PHB2. | Nature Communications

Figure 2: BIG3-PKA-PP1Cα regulates the suppressive ability of PHB2.

From: A-kinase anchoring protein BIG3 coordinates oestrogen signalling in breast cancer cells

Figure 2

(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).

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