HSF1 and Protein Kinase A to heat shock. HeLa cells were transfected with plasmids expressing either wild type PKAca (FLAG-PKA) or kinase dead PKAca (FLAG-PKA K73M). The amounts of plasmid transfected under each condition are indicated on the abscissa. We also measured hsp70.1-Luc activity in cells without or with heat shock both under control conditions in cells expressing scrambled RNA (PKA scr) or after PKAca knockdown (PKA kd #1). Luciferase assays were then performed in triplicate following heat treatment and activity plotted as mean +/2 standard deviation. Statistical significance of differences in hsp70.1-Luc activity under the various conditions was compared by Student’s t test. Experiments were performed three times with similar findings. (E) Anti-FLAG immunoblot analysis was carried out to demonstrate the relative levels of intracellular PKAca expression achieved under these transfection conditions and are shown beneath the luciferase panels. b-actin expression under these conditions is shown as loading control and was used for quantitative normalization. Experiments were carried out 3–5 times with consistent findings. doi:10.1371/journal.pone.0013830.g005 not required for the formation of HSF1 trimers and for DNA binding it is essential for full transcriptional activation. Serine 320 is within the central region of HSF1 that is regulated by heat shock independently of trimerization or DNA binding [49,50]. The regulatory domain of HSF1 is repressive to transcription under normal temperatures while stress not only overturns repression but leads to trans-activation considerably greater in magnitude than might be accounted for by reversal of repression [49,50]. The original studies suggested that regulatory function is bounded by amino acids 221–310 although the discovery of activating phosphorylation at serines 320 and 326 suggests extending the domain to at least serine 326 (Fig. 2) [21,49,50]. The regulatory domain of HSF1 could function as a signaling platform in response to protein kinases as it contains multiple serine and threonine residues [16,51]. This region contains serines 303 and 307 that are repressive when phosphorylated and could exert some of the inhibitory functions of the regulatory domain seen at normal temperatures. Inhibition of trans activation exerted through this region appears to involve a pathway in which dual phosphoryla- tion of HSF1 at serines 307 and 303 by ERK1 and GSK3, respectively leads to nuclear export of HSF1 through a mechanism involving recruitment of 14-3-3 family members, thus removing HSF1 from HSP promoters [20,24,26,52]. In addition an adjacent residue at lysine 298 can be sumoylated in a mechanism also dependent on serine 303 phosphorylation [27,53]. These inhib- itory posttranslational modifications can be overridden in heat shocked cells even though serine 303 remains abundantly phosphorylated, 14-3-3 remains bound and K298 is sumoylated [28]. The central regulatory region also contains positively acting phosphorylation sites at serines 230, 326 and, as shown here at S320 (Fig. 2) [19,21]. It is not clear how posttranslational modifications in this domain during stress might override the inhibitory influence of S303 phosphorylation and stimulate HSF1 trans activation. However, the current studies suggest a role for PKAca and S320 in permitting HSF1 to accumulate in the nucleus and in nuclear stress granules, bind to the hsp70.1 promoter and activate hsp70.1 transcription (Figs. 3, 4, 5). PKAca phosphorylation of S320 may be involved in the accumulation in nuclei by reversing of nuclear export [28]. The exact mechanism involved is, however not clear. Nuclear localization depends on relative rates of nuclear import and nuclear export. Human HSF1 contains bipartite nuclear localization signals (NLS) adjacent to leucine zipper domains, N-terminal to the regulatory domain [54]. Earlier studies showed that nuclear import involving one or more of these NLS sequences occurs continuously with or without stress and that nuclear accumulation is most likely due to blockade of nuclear export [55]. A role for reversible binding of HSF1 to the major nuclear export protein CRM1/exportin1 in stress seems unlikely as this protein binds avidly to HSF1 with or without heat shock (A. Murshid & S.K. Calderwood, unpublished data). It is also notable that depletion of PKAca did not completely inhibit HSF1 (Fig. 5 D). This may reflect the incomplete reduction in PKAca levels after expression of shRNA in the cells (Fig. 2 C). It seems however likely that other posttranslational modifications in the regulatory domain also contribute to HSF1 activation, notably phosphorylation at serines 230 and 326 [21,27]. One suggested mechanism for differential regulation of HSF1 in stressed and unstressed conditions is that an altered phosphorylation landscape in the central regulatory domain may permit a switch between activator and inhibitor activity. This region of HSF1, rich in proline and serine residues, would be predicted to lack secondary structure motifs and phosphorylation could impose alternative structures due to changes in charge distribution. In addition, such phosphorylation patterns might also form binding sites for (as yet undetected) regulatory proteins. We did not examine here the potential effects of HSF1 binding on the kinase activity of PKA. However, one recent study indicates that HSF1 may be involved in upstream activation of PKA during cell transformation [1]. Although we did not study HSF1 mediation of intracellular PKA activity, our studies do indicate sturdy interaction of HSF1 and PKAca after treatment with transforming growth factor HRG (Figs. 1, 2). Previously we showed that, HRG directly activates HSF1 and that HSF1 mediates some of its tumorigenic effects [56]. In conclusion therefore, we have demonstrated physical and functional interaction between protein kinase A and HSF1. The PKA catalytic subunit PKAca binds and phosphorylates HSF1 on serine 320, a modification essential for nuclear localization, association with the hsp70.1 promoter and transcription. A role for PKAca in the network of signaling events leading to the activation of HSF1 during the stress response is suggested. Supporting Information Figure S1 HSF1 interacts with PKAca. HeLa proteins adsorbed to GST (control) or GST-HSF1 were eluted from GSH-4B beads by reduced glutathione, and proteins analyzed by SDS-PAGE and silver staining. Silver-stained bands were digested in-gel with trypsin and eluted peptides analyzed by matrix-assisted laser desorption/ionization-time of flight-mass spectroscopy using a Voyager DE-PRO (Applied Bio-systems) and proteins identified by mass fingerprinting using database (SwissProt.8.17.2002). Peptides (AKEDFLK, VMLVKHK, QIEHTLNRK) with an exact match to murine (8.66), rat (8.64), human (8.65) PKAca and bovine PKAcb (8.65) were found. (MOWSE scores are shown in parenthesis.) Mass spectrometry data were confirmed by immu- noblot assay shown here carried out here on eluates from either unmodified GST or GST-HSF1, using anti-PKAc antibodies. Experiments were performed in duplicate with similar findings. Found at: doi:10.1371/journal.pone.0013830.s001 (0.04 MB TIF) Figure S2 Effects of LMB on localization of HSF1 with or without PKAca knockdown. A–D) HeLa cells were treated without or with LMB for 16 hours. Cells were later probed for PKAca and HSF1 with anti-PKAca antibodies and anti-HSF1 antibodies respectively. Cells were later stained with second antibodies goat anti-rabbit Alexa 488 (for PKAca, green) and goat anti-rat Cy3 (for HSF1, red). Nuclei were visualized using DAPI (blue) staining. C, D, E) HeLa (PKAscr) and HeLa (PKAkd#1) cells were treated with LMB for 16 hours before heat shock (43oC, PLoS ONE | www.plosone.org 11 November 2010 | Volume 5 | Issue 11 | e13830