phosphorylated samples of HSF1, detected by this antibody were then digested in-gel with trypsin and analyzed by mass spectrometry (Fig. 2A). Database analysis of the trypsin fragments indicated the generation of two principal phosphopeptide species, V310-R336 containing a potential site for PKAca at S320 within a consensus PKA motif and K118-K126 containing S121 (Fig. 2A). We have shown that the latter site (S121) is, when phosphorylated, inhibitory for HSF1 under resting conditions. However, the influence of phospho-S121-HSF1 is overridden during heat shock under which conditions it does not influence transcription and becomes dephosphorylated [18]. We therefore concentrated on the novel S320 phosphorylation site. We examined the role of heat shock and PKAca expression on HSF1-S320 phosphorylation using commercial antibodies specific for this phosphorylated form of HSF1 (Fig. 2B, C). Cells apparently contain significant background levels of phospho-S320-HSF1 and phosphorylation increased immediately after heat shock, before decaying back to basal levels by 2 hr recovery at 37uC, correlating well with the period of increased trans-activation by heat shock (Fig. 2C). We then carried out experiments in which intracellular PKAca levels were reduced by stable knockdown with small hairpin RNA packaged in lentivirus (Fig. 2B). We employed two such shRNA species, PKAkd#1 and PKAkd#2, one of which (PKAkd#1) efficiently reduced PKAca levels in infected cells while the other (PKAkd#2) was relatively less effective (Fig. 2B). Phospho-S320- HSF1 levels were significantly reduced in cells expressing PKAkd#1 compared to cells expressing PKAkd#2 or control cells infected with lentivirus enclosing a scrambled RNA species (PKA scr) (Fig. 2C). As a further control, we investigated HSF1 phosphorylation at the adjacent serine 326 using antibodies specific to this phosphorylated motif. HSF1 phosphorylation at this site was also increased by heat shock, although knockdown of PKAca had minimal effects on S326 phosphorylation (Fig. 2C). Phosphorylation of HSF1 at these two adjacent serine residues, although induced with similar kinetics by heat shock, is evidently regulated independently. It is notable that HSF1 hyperpho- sphorylation was not markedly ablated by PKAca knockdown conditions that led to reduced phospho-S320-HSF1 suggesting that other posttranslational modifications may contribute to the electrophoretic mobility shift seen in HSF1 after heat shock (Fig. 2 C). (3) Intracellular localization of HSF1 and PKAca without and with stress We next examined the intracellular localization of HSF and PKAca before and after stress using indirect immunofluorescence and confocal microscopy. In the absence of stress, PKAca was found in punctuate foci in the perinuclear and nuclear regions of cells and HSF1 largely in the nucleus as described previously [24,28] (Fig. 3A). After heat shock, HSF1 was also localized to nuclei and was concentrated in a number of punctuate foci (stress granules) also as observed previously (Fig. 3B). Co-localization or partial co-localization of the two proteins was observed in the nuclei in many cells after heat shock, although a large proportion of PKAca remained in the perinuclear as well as in the cytosol in some cells (Fig. 3B). Quantitation by cell counting indicated that in approximately 30% of cells a proportion of the HSF1 was associated with PKAca before heat shock, and this proportion increased to approximately 70% after heat shock (Fig. 3A, 3B, 3E). Heat shock did not however cause quantitative translocation of the PKAca to the nucleus, suggesting that stable interaction after heat shock involves only a fraction of the cytosolic PKAca or that perinuclear PKAca may be capable of phosphorylating HSF1 in that region and help in translocation of HSF1 to the nucleus. HSF1 and Protein Kinase A Some cells showed nuclear localization of PKAca and partial co- localization with HSF1 suggesting direct association between them as indicated in the earlier immunoprecipitation experiments. Not all PKAca-containing punctate structures were associated with HSF1 in the nucleus, a finding which is not unexpected as nuclear PKAca can associate with and phosphorylate other key compo- nents such as CREB (cyclic AMP responsive factor) after heat shock (A. Murshid & SK Calderwood, in preparation). Although HRG and IGF-1 activate transcription through HSF1, we did not observe the accumulation of the factor in stress granules after these treatments in most cells. However HSF1 association with PKAca in nuclei was increased from 30% to approximately 60% after treatment with both HRG (Fig. 3C, 3E) and IGF-1 (Fig. 3D, 3E). (Stress granules appear to be involved in regulation of Satellite RNA during heat shock rather than HSP-encoding genes) [40]. We next examined a potential role for PKA in the nuclear localization of HSF1 after heat shock. In controls with unper- turbed levels of PKAca, HSF1 was mostly nuclear and after heat shock localized to stress granules in the nucleus (Fig. 3A, B) as described above [24,41]. After PKAca knockdown as in Fig. 2C, (PKA kd #1), the majority of the HSF1 was banished to punctate aggregates in the cytoplasm of both control cells and heat shocked cells (Fig. 3F, G). PKAca levels were markedly reduced in these cells expressing the shRNA, as would be predicted (Fig. 3G). In control cells expressing the scrambled RNA sequence (PKA scr) HSF1 distributions in unheated and heat shocked cells were similar to those in wt cells indicating that the effects of PKA kd #1 are due to PKAca knockdown rather than an artifact of the treatment conditions (Fig. S2, S3). Intracellular localizations of HSF1 in control and knockdown (PKA kd #1) cells were quantitated by cell counting and also shown in Fig. S3. As previous studies suggest that HSF1 is regulated by nucleocytoplasmic shuttling and that nuclear export is an important component in this process, we next examined the potential role of the principal exportin, CRM1/exportin-1 in PKAca-regulated localization of HSF1 under control and stress conditions. A role for CRM1 was examined using the inhibitor leptomycin-B (LMB). As described above, HSF1 was localized by immunofluorescence mainly in the nuclear compartments of cells with or without heat shock (Fig. 3A, B, Fig. S2, S3). We next examined whether LMB could reverse the inhibitory effects on HSF1 nuclear localization of PKAca knockdown. Our hypothesis was that HSF1 phosphorylation by PKAca might be required for nuclear accumulation of HSF1 by decreasing CRM1-mediated nuclear export. However, addition of LMB failed to reverse the effects of PKAca knockdown on redistributing HSF1 to the cytoplasm in heat shocked cells (Fig. S2 D, E). Curiously, LMB was effective in permitting nuclear localization of HSF1 in unstressed cells even under conditions of PKAca knockdown (Fig. S3). However, nuclear localization of HSF1 in stress conditions appears to be regulated in a PKAca-dependent manner by an unconventional pathway that does not involve a major role for CRM1/exportin1. Indeed we were able to detect by co- immunoprecipitation analysis significant binding of CRM1 to HSF1 that was not modulated by heat shock (A Murshid & SK Calderwood, unpublished data). We then investigated the potential role of HSF1 phosphoryla- tion at serine residue 320 in intracellular localization (Fig. 4). Mutant versions of GFP-tagged HSF1 were prepared in which S320 was changed either to aspartate (S320D) or alanine (S320A) and these constructs were then expressed in cells from a CMV- based promoter. S320A, being refractory to phosphorylation, would function as a mutation while in S320D the negative charge of the carboxyl side chain of aspartate might mimic that of PLoS ONE | www.plosone.org 6 November 2010 | Volume 5 | Issue 11 | e13830