HSF1 and Protein Kinase A HSF1 antibodies. D) Hsp70.1-Luc activity with or without heat shock in untransfected cells, cells transfected with wtHSF1 or mutant HSF1. HeLa cells were transfected with plasmids encoding HSF1-GFP/HSF1, (S320A)-GFP/HSF1, (S320D)-GFP, b-gal, and pGL3-Hsp70-luciferase. Luciferase assay was performed in triplicate 24 hr following heat treatment and data expressed as mean luciferase level +/2 SD. Comparisons between treatment groups were made using Student’s t test. Experiments were repeated four times with similar results. doi:10.1371/journal.pone.0013830.g004 localization of HSF1 (Fig. 4B). The predominantly nuclear localization of HSF1-GFP (S320D) (Fig. 4 B) resembled that of wt HSF1 after heat shock (Fig. 3 B, F), while the largely cytoplasmic localization of S320A-HSF1 (Fig. 4A, C) resembled localization of wt HSF1 after PKAca knockdown by PKAkd#1 (Fig. 3F). These data support the hypothesis that PKAca-mediated HSF1 phosphorylation at S320 plays a significant role in nuclear localization. To further determine the role of HSF1 phosphory- lation at S320 in co-localization with PKAca, we expressed FLAG-PKAca in HeLa cells and then stained for phospho-S320 HSF1 in control, heat shocked (43uC, 1 hr) and HRG treated cells. We found similar patterns of partial co-localization of phospho-S320-HSF1 and PKAca in perinuclear to nuclear regions as seen previously for total HSF1 in Fig. 3, (Fig. S4). HSF1 may thus dissociate from PKAca but remain phosphorylated on S320. We next examined the transcriptional activity of these HSF1- GFP mutants (Fig. 4D). These experiments were carried out in cells expressing wtHSF1 as we aimed to investigate a potential dominant negative role for the mutant (HSF1-S320A). Exposure to heat shock led to activation of the hsp70.1-luciferase promoter-reporter construct (hsp70.1-Luc) in untransfected cells and such activity was increased in cells transfected with wild-type HSF1 (Fig. 4D). However, after HSF1-S320A transfection, heat shock induced hsp70.1-Luc activity was significantly (p,0.01) decreased below the levels seen in untransfected cells suggesting potential ‘‘dominant negative’’ effects of S320A in quenching activation of the endogenous HSF1 by heat shock. Expression of HSF1-S320D amplified the level of heat shock-induced luciferase activity by a similar magnitude compared to activation by wild- type HSF1 (Fig. 4D). Interestingly, cells expressing HSF1-S320D had significantly elevated mean luciferase levels (p,0.05) even in non-heat conditions suggesting that gain of negative charge at S320 may mediate the activating effects of heat shock (Fig. 4D). (4) Regulatory role for PKAca in stress-mediated localization of HSF1 to the hsp70.1 promoter and in hsp70.1 transcription We next examined whether PKAca is required for HSF1 binding to the hsp70.1 gene in vivo using ChIP analysis (Fig. 5). We measured association of native HSF1 with hsp70.1 chromatin using anti-HSF1 antibodies. Potential HSF1 association with two sequences in hsp70.1 chromatin was investigated, including a region within the promoter containing the most proximal heat shock element (hsp70HSE) and a downstream sequence in the open reading frame (hsp70EXN) (Fig. 5A). Fig. 5B shows ChIP analysis indicating HSF1 binding to hsp70.1 chromatin. In each panel we show PCR analysis of DNA associated with HSF1 in the first lane, a control ChIP analysis with pre-immune IgG in the second lane, as well as an indication of the input in the third lane. Experiments are carried out without and with heat shock in cells treated with the shRNA PKAkd#1 described above (PKAshRNA) or in controls expressing a scrambled RNA sequence (PKAscr). Using this approach we detected HSF1 binding to the HSE region of hsp70.1 after heat shock in PKAscr (Fig. 5B top left panel). HSF1 was not detected in association with the EXN region of hsp70.1 in any of the conditions and did not appear to bind Hsp70HSE in the absence of heat shock or after PKAca knockdown with or without heat shock (Fig. 5B). These experiments were repeated using quanti- tative real time RT-PCR and are shown in Fig. 5C. Heat shock led to an approximate 35-fold enrichment of HSF1 on the HSE region and such binding was inhibited in cells after PKAca knockdown (PKAkd#1) (Fig. 5C). Minimal binding of HSF1 to the HSE was seen in PKAshRNA cells and in the absence of heat shock (Fig. 5C). Minimal association of HSF1 with HSP70 EXN was seen in under each of the conditions (Fig. 5C). We next examined the requirement for PKAca in transcrip- tional activation of HSF1 by heat shock. Modulation of PKAca activity by overexpression or knockdown influenced markedly the activity of the hsp70.1-Luc reporter (Fig. 5). Increasing intracellular PKAca levels by forced expression from a viral promoter (pCMV.FLAG-PKAca) led to a significant (p,0.005) plasmid dose-dependent increase in heat-induced hsp70.1-Luc activity [36] (Fig. 5D (left panel). The increased levels of PKAca expression in these experiments are indicated in the anti-FLAG antibody immunoblot shown in the lower part of the figure and bands are quantitated (in Fig. 5E, bottom). The hsp70.1 promoter was not markedly activated by forced expression of a kinase dead PKAca construct (from transfected pCMV.FLAG-PKAca-K73M), indi- cating a requirement for the catalytic activity of PKA in enhanced HSF1 activation (Fig. 5D, middle panel). We next examined the effect of reducing PKAca levels by RNA interference (using the shRNA construct PKAkd#1) on hsp70.1- Luc activity. Heat shock activated hsp70.1-Luc in control cells expressing scrambled RNA (PKAscr), while PKAca knockdown significantly (p,0.05) reduced both basal and heat-induced hsp70.1-Luc activity (Fig. 5D, right panel). The relative levels of PKAca expressed in cells without and with knockdown by PKAshRNA are indicated in the earlier figure (2 B). Discussion Our experiments therefore suggest a novel mechanism in the activation of hsp70 after stress, involving HSF1 association with PKAca and phosphorylation by this kinase at a novel site (serine 320) (Fig. 1). These conclusions are confluent with earlier studies suggesting a role for PKA in activation of an hsp70 promoter [42]. The mechanisms dictating PKAca binding to HSF1 in stress are not entirely clear but do not appear to involve major roles for adenylate cyclase or cAMP (Fig. 1D). We observed only a minor amount of binding of the regulatory R1 domain of PKA to HSF1 (Fig. 1). It seems likely that activation of phosphorylation by the bound PKAca involves the transition in HSF1 structure that occurs after heat shock, from a compacted form, constrained by intramolecular coiled-coil and molecular chaperone interactions to a more expansive conformation [43,44,45,46,47]. Such a transition may expose domains previously cryptic within the inactive protein. We also observed a basal level of HSF1- PKAca binding and S320 phosphorylation in HeLa and MCF-7 cells (Fig. 1, 2). This may reflect the fact that HSF1 possesses basal transcriptional activity in many human malignant cells lines and is required for survival in these cells [1,2,48]. Previous studies also showed that, when HSF1 is partially activated to a more relaxed, DNA binding form by exposure of cells to salicylate or menadione, it can then be rendered transcriptionally competent by exposure of cells to protein phosphatase inhibitors [14]. In addition our in vitro studies showed a similar two part activation and although ATP is PLoS ONE | www.plosone.org 9 November 2010 | Volume 5 | Issue 11 | e13830