HSF1 and Protein Kinase A chain to indicate the equal loading of immunoprecipitates. In the lower panel equal amounts of protein lysates were loaded for measurement of HSF1, PKAca, b-actin and Hsp70 in cells prior to immunoprecipitation (B, lower panel). The whole-cell lysates were analyzed by 4–15% gradient gels to demonstrate the presence of hyperphosphorylated HSF1 in the preparations more effectively. In addition we analyzed the expression of Hsp70 in cells 6 and 24 hr after the treatments to determine their effectiveness in inducing Hsp70 expression. Experiments were carried out in duplicate reproducibly. C) MCF-7 cells were incubated with or without heat shock for 1 hour at 43uC. Additional cell cultures were also treated with growth factors (30 mM HRG for 24 hrs, 100 ng/ml IGF-1 for 24 hrs) and inhibitors (2 mM 17AAG for 24 hrs, 5 mM MG132 for 6 hrs) at 37uC, as indicated in panels 2–5. Cells were then lysed and extracts collected and equalized for protein concentration. Immunoprecipitation was performed with anti- PKAca antibodies prior to anti-HSF1 immunoblot. Equal amounts of protein were loaded to measure quantitatively the level of HSF1, and PKAca. Levels of HSF1, PKAca and b-actin in cell lysates prior to immunoprecipitation are shown in the lower panels. The experiments were carried out three times with similar findings. D) HSF1, adenylate cyclase and PKAca binding. Control cells or cells given 1 hr at 43uC were treated with or without t- busate (lane 2) and then lysed on ice and immunoprecipitated with anti- PKAca antibody. Immunoprecipitates were then fractionated by 10% SDS- PAGE and analyzed by immunoblot with anti-HSF1 antibodies. In addition, we assayed the cAMP levels in these cell lysates by EIA and these are plotted as means +/2 standard deviation (right panel). Amount of cAMP was normalized to 100% of the cAMP concentration in control HeLa cells (no heat). We also measured intracellular cAMP levels (mean +/2 SD) after heat shock, HRG, IGF1, MG-132 and 17-AAG treatment, using the conditions described in Fig. 1B. The cAMP levels after each of the treatments were not significantly increased compared to control. Each experiment was carried out in duplicate with similar results. doi:10.1371/journal.pone.0013830.g001 intracellular cAMP levels after heat shock at 43uC or with the other treatments (Fig. 1D, lower panel). However some studies have shown transient activation of adenylate cyclase, within seconds or minutes after the stress, that decay over time [38,39]. Elevating cAMP levels with the drug forskolin in the absence of stress failed to activate HSF1-PKAca binding (A. Murshid & SK Calderwood, unpublished data). Thus although basal cAMP may be required for HSF1-PKAca binding, alterations in cAMP after HSF1 activation do not seem to be involved. (2) HSF1 is phosphorylated on serine 320 by PKAca We next investigated whether PKAca could phosphorylate HSF1 when the two proteins were incubated together in vitro. Purified GST-HSF1 was mixed with purified PKAca and ATP for a range of incubation times to maximize the extent of HSF1 phosphorylation. The reaction mixtures were then fractionated by SDS-PAGE and analyzed by immunoblot for HSF1 and for HSF1 phosphorylation on PKA consensus phosphorylation motif RXXpS, using anti-RXXpS antibodies (Fig. 2A). Abundantly Figure 2. HSF1 is phosphorylated on serine 320 by PKAca. A) Purified GST-tagged HSF1 was phosphorylated in vitro by PKAca. Samples were then fractionated by 10% SDS-PAGE and initially immunoblotted for PKAca phospho-substrate. The membranes were then stripped and re-blotted with anti-HSF1 antibodies. The identities of the PKAca - modified phospho-peptides were determined by isolating the PKA-phosphorylated GST-HSF1 with 10% SDS-PAGE, trypsin digestion of the GST-HSF1 band and identification of peptides by LTQ-Orbitrap MS and database analysis. B) PKAca expression in control (PKA scr) and PKA knockdown (PKA kd,#1 and 2) HeLa cells with or without heat shock. PKAca levels with or without shRNA knockdown were measured by anti-PKAca antibody immunoblot. C) Phosphorylation of HSF1 S320 and S326 in response to heat. Control (PKA scr) and PKAca knockdown (PKA kd#1 and 2) HeLa cells were treated with or without heat shock as indicated. Phospho-HSF1 expression levels were then evaluated by western blot using anti-phospho-HSF1 antibodies (anti-pS320 and anti-pS326). b-actin expression was also measured as loading control and for quantitative normalization. Experiments (A–C) were carried out in duplicate with similar results. doi:10.1371/journal.pone.0013830.g002 PLoS ONE | www.plosone.org 5 November 2010 | Volume 5 | Issue 11 | e13830