Figure 5
From: IER5 generates a novel hypo-phosphorylated active form of HSF1 and contributes to tumorigenesis
IER5 dephosphorylates HSF1 at multiple Ser and Thr residues including sites involved in repression of HSF1 activity.
(A–E) Whole cell extracts and immunoprecipitated samples were analyzed by Western blotting. (A) 293T cells were transfected with HSF1-Flag together with control vector or an IER5 expression vector. Cells were harvested 21 hrs post-transfection. (B) 293T cells were transfected with control vector or HSF1-Flag expression vector, together with control or IER5-targetting siRNAs. Cells were harvested 49 hrs post-transfection. (C) 293T cells were transfected with HSF1-Flag and control vector or IER5. Cells were harvested 27 hrs post-transfection. Cell lysates were immunoprecipitated using anti-Flag antibody and incubated with or without CIAP for 30 min. Total HSF1 and p-Ser320 were detected. (D) 293T cells were transfected with control vector or HA-HSF1 expression vector. Cells were treated with or without heat shock at 42 °C for 3 hrs and harvested 24 hrs post-DNA transfection. (E) 293T cells were transfected with HA-HSF1 and control or IER5-Flag expression vector. Cells were harvested 24 hrs post-DNA transfection. (F) HSF1 modification was analyzed by LC-MS/MS. The experiment was performed nine times and the numbers of analysis that detected each phosphorylation are shown. Significant reductions in phosphorylation at 5 residues (S121, S307, S314, T3232 and T367) were detected. Phosphorylation sites previously reported to be involved in the repression of HSF1 activity (S121 and S307) are shown in blue. (G) 293T cells were transfected with the indicated plasmids and harvested 24 hrs post-transfection. HSPA1A mRNA expression was analyzed by quantitative RT-PCR and other proteins were detected by Western Blotting. (***p < 0.001, #p < 0.0001). (H,I) 293T cells were transfected with control vector or IER5-Flag expression vector. Cells were treated with or without heat shock at 42 °C for 3 hrs and harvested 24 hrs post-DNA transfection. Endogenous HSF1, IER5-Flag protein expression was analyzed by Western blotting (H) and HSPA1A, HSPA6 and HSPA1B mRNA expression was analyzed by quantitative RT-PCR (I). (*p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.0001).
