modifications are converted into intracellular functions are not clear [16,18,24]. The inhibitory modifications at serines 303, 307 and 363 have each been attributed to accelerated nuclear export [24,26]. This effect has, in the case of serines 303 and 307 been attributed to recruitment of 14-3-3 to Phospho-S303, S307-HSF1 and stimulation of nuclear export through a pathway involving nuclear export protein CRM1/exportin1 [24]. In addition, S303 phosphorylation has been shown to lead to a secondary posttranslational modification, HSF1 sumoylation at lysine 298 [27]. Another curious aspect of HSF1 regulation during stress is that, while HSF1 phosphorylation at S303 and S307 and sumolylation at K298 are inhibitory to HSF1 function when assayed at 37uC, during heat shock these inhibitory signals are evidently inoperative and HSP transcription proceeds [28]. It seems likely that an override mechanism exists to promote rapid activation of the stress response. Persistence of inhibitory signaling may permit rapid turn off of transcription in recovery from stress after the override mechanisms of stress subside. Much therefore remains to be learned regarding the activating roles of posttranslational modifications in HSF1 regulation, their role in stress mediated transcription and the mechanisms by which such modifications are recognized by regulatory pathways in cells and converted into altered function. We therefore began a screen of intracellular proteins that can interact with HSF1 and modify function and observed binding to a number of proteins including the catalytic subunit of 39-59-cyclic adenosine monophosphate (cAMP)- dependent protein kinase (PKAc). Protein kinase A is a versatile regulator of cell metabolism and gene transcription and consists of two main subunits, PKAc and PKAR (PKAR is the regulatory subunit of PKA) [29]. Before cellular activation, PKAc and PKAR bind in an inactive complex that can be induced by the low molecular weight signaling molecule cAMP [30]. Cyclic AMP is a second messenger molecule generated both at the cell surface and in the cytoplasm by numerous isoforms of the enzyme adenylate cyclase [31]. Binding of cAMP to the PKA complex liberates PKAc to interact with a wide range of protein substrates, phosphorylating them on serine or threonine residues, usually within the conserved motif (RXXS/T), (where X can be any amino acid, R is arginine, S/T serine or threonine) [32]. However, a significant exception to this mechanism is found in the regulation of NFkB. PKAc binds directly to the substrate (NFkB/p65 transcription factor), phosphorylates p65 when NFkB is induced by cytokines and activates kB gene transcription independently of cAMP [20]. However, cAMP–dependent phosphorylation of p65 has also been observed [33]. We have investigated binding of the a form of PKAC (PKAca) to HSF1 and its role in hsp70.1 transcription. PKAca bound to HSF1 and led to phosphorylation on a novel site (serine 320). Reduction of intracellular PKAca levels by RNA interference inhibited HSF1-S320 phosphorylation, prevented accumulation of HSF1 in the nucleus and binding to the hsp70.1 promoter and decreased heat shock transcription. Thus PKAca may play a novel role in activation of HSF1 and stress-induced transcription. Methods Antibodies and Reagents Rat and Rabbit polyclonal anti-HSF1 clone 4B4/10H8 were from the Abcam and Assay Designs (Enzo Life Sciences, Inc.). Hsp70 antibody was purchased from Assay Designs (Enzo Life Sciences, Inc.). Rabbit monoclonal anti-HSF1 phospho-S320 and HSF1 phospho-S326 (Abcam), mouse monoclonal anti-HA (Covance), mouse monoclonal anti-FLAG (M2, Sigma), Rabbit polyclonal anti-PKAca (Cell Signaling) and rabbit anti-PKA HSF1 and Protein Kinase A phospho-substrate (Cell Signaling) antibodies were also employed. The rabbit polyclonal PKARI antibody was from Cell Signaling and Rabbit polyclonal anti-PKRII antibody was from Santa Cruz Antibodies. The secondary antibodies were goat anti-mouse Alexa 488 (Invitrogen, USA), goat anti-rabbit Cy3 (Jackson Immur- esearch Laboratories), goat anti-rabbit Cy5 (Jackson Immure- search Laboratories), goat anti-rat Cy3 (Jackson Immuresearch Laboratories) and HRP-goat anti-mouse IgG, HRP-goat anti-rat IgG, HRP-goat anti-rabbit IgG, goat anti-Rabbit IR Dye 680 (Santa Cruz and Licor) for use in Western blotting. Heregulin, IGF-1, 17AAG, MG132, leptomycin B and t-busate were purchased from Sigma-Aldrich. Cells, Culture conditions and Transfection HeLa, MCF-7, and Du145 cells were obtained from the American Type Tissue Culture Collection while HEK293FT were purchased from the Invitrogen Corporation. Cells were main- tained in DMEM supplemented with 10% heat inactivated FBS, 1000 U of penicillin/streptomycin, 2 mM L-glutamine. Cells were grown in petri dishes at 37uC in a 5% CO2 humidified incubator. Cells were grown to 50–60% confluence on glass coverslips in six- well plates for immunofluorescence microscopy, and transfected using the Fugene TM6 reagent (Roche) according to manufac- turer’s instructions. All plasmids were purified using Qiagen plasmid purification kits. To generate PKAca knockdown cells, envelope plasmid, packaging plasmid (Open Biosystems) and shRNA expressing plasmid were co-transfected into HEK293FT cells. Virus-containing medium was collected 48 and 72 hr after transfection. HeLa cells were infected by incubation with the lentivirus-containing medium and cells were treated with puro- mycin for selection of knockdown cells. DNA constructs and mutagenesis FLAG-HSF1, HSF1-EGFP-N3, pGL3-Hsp70-LUC, pCMV-b- galactosidase constructs were prepared in-house and their construction is described in earlier publications [24,28]. FLAG- PKAca was made by PCR Topo-cloning (Invitrogen) the open reading frame of PKAca (NM_002730) into pcDNA3.1 (Invitro- gen). HSF1-EGFP (S320A, S320D), and FLAG-PKA (K73M) mutants were constructed using the Quickchange site-directed mutagenesis approach (Stratagene). Hairpin pLKO.1 control vector and shRNA expressing plasmids for knocking down PKAca expression plasmids (PKAca kd #1 and #2) were obtained from Open Biosystems. Envelope plasmid (VSV-G/Pmd2.G) and packaging plasmid (pCMV-R8.74 psPAX2) were used to generate shRNA-expressing lentivirus. Immunoprecipitation Cells were solubilized in ice-cold Lysis Buffer (150 mM NaCl, 1% Triton X-100, PMSF (phenyl methyl sulfonyl fluoride) protease inhibitor cocktail mix (Roche) and sodium orthovana- date). Cell lysates were probed by antibody-protein-A-sepharose cascade (GE-Healthcare), and primary and secondary precipitated proteins assayed by SDS-PAGE/immunoblot as described [34]. Immunofluorescence and Microscopy Cells fixed with 4% para-formaldehyde in PBS, were washed twice with 1X PBS, permeabilized using 0.1% Triton X-100 and placed in 3% normal goat serum (NGS) for 60 minutes. Cells were analyzed using a Zeiss 510 LSM confocal microscope (Carl Zeiss GmbH, Jena, Germany) using 1.4 63X oil immersion objective set with the pinhole set at 0.7–0.9 Airy units. GFP/ Alexa 488 was visualized using 488 nm excitation and BP 505– PLoS ONE | www.plosone.org 2 November 2010 | Volume 5 | Issue 11 | e13830