Protein Kinase A Binds and Activates Heat Shock Factor 1 Ayesha Murshid1, Shiuh-Dih Chou1, Thomas Prince1, Yue Zhang 1 , Ajit Bharti2, Stuart K. Calderwood 1 * 1 Molecular and Cellular Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America, 2 Stress Response Center, Boston University Medical Center, Boston, Massachusetts, United States of America Abstract Background: Many inducible transcription factors are regulated through batteries of posttranslational modifications that couple their activity to inducing stimuli. We have studied such regulation of Heat Shock Factor 1 (HSF1), a key protein in control of the heat shock response, and a participant in carcinogenisis, neurological health and aging. As the mechanisms involved in the intracellular regulation of HSF1 in good health and its dysregulation in disease are still incomplete we are investigating the role of posttranslational modifications in such regulation. Methodology/Principal Findings: In a proteomic study of HSF1 binding partners, we have discovered its association with the pleiotropic protein kinase A (PKA). HSF1 binds avidly to the catalytic subunit of PKA, (PKAca) and becomes phosphorylated on a novel serine phosphorylation site within its central regulatory domain (serine 320 or S320), both in vitro and in vivo. Intracellular PKAca levels and phosphorylation of HSF1 at S320 were both required for HSF1 to be localized to the nucleus, bind to response elements in the promoter of an HSF1 target gene (hsp70.1) and activate hsp70.1 after stress. Reduction in PKAca levels by small hairpin RNA led to HSF1 exclusion from the nucleus, its exodus from the hsp70.1 promoter and decreased hsp70.1 transcription. Likewise, mutation of HSF1 at S320 by alanine substitution for serine led to an HSF1 species excluded from the nucleus and deficient in hsp70.1 activation. Conclusions: These findings of PKA regulation of HSF1 through S320 phosphorylation add to our knowledge of the signaling networks converging on this factor and may contribute to elucidating its complex roles in the stress response and understanding HSF1 dysregulation in disease. Citation: Murshid A, Chou S-D, Prince T, Zhang Y, Bharti A, et al. (2010) Protein Kinase A Binds and Activates Heat Shock Factor 1. PLoS ONE 5(11): e13830. doi:10.1371/journal.pone.0013830 Editor: Mikhail V. Blagosklonny, Roswell Park Cancer Institute, United States of America Received June 2, 2010; Accepted October 18, 2010; Published November 9, 2010 Copyright: 2010 Murshid et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Studies were supported by NIH research grants 2RO1CA047407 and RO1CA077465. These are peer-reviewed National Institutes of Health RO-1 grants intended to investigate the role of cell signaling in the heat shock/stress response. The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: scalderw@bidmc.harvard.edu Introduction HSF1 is a primary regulator of the heat shock response and a factor in a number of human pathologies including cancer and neurodegenerative diseases [1,2,3,4,5]. Curiously, although both diseases are associated with advancing age, HSF1 loses activity in the progression of neurodegenerative diseases while being activated in cancer [1,2,6]. It would seem apparent therefore that understanding the molecular basis of HSF1 up- and down- regulation in disease would provide valuable insights. HSF1 belongs to the multi-gene HSF family present in all eukaryotes [7]. Initial studies were carried out on the single HSF gene of the yeast S. cereviseae [8]. These studies indicated that, unique among transcription factors HSF undergoes trimer formation on activation and that such oligomerization governs binding to the heat shock elements (HSE) on the promoters of heat shock protein (HSP) genes [9,10]. The findings in yeast were confirmed in mammalian cells in which trimerization was shown to be a requirement for binding to HSP promoters [7]. Another unusual feature associated with HSF is that trimerization and binding to HSE can be dissociated from trans-activation in studies carried out both in vitro and in vivo; DNA binding alone is evidently insufficient to drive transcription and other, binding independent processes are involved [11,12,13,14]. Early studies suggested that these may include posttranslational modification of HSF1 [10,13,15]. Indeed yeast HSF and mammalian HSF1 appear to undergo heavy phosphorylation on serine and threonine residues when activated [10,15,16]. In addition intracellular HSF1 under- goes other modifications such as sumoylation and acetylation after stress [17]. Alterations in HSF1 phosphorylation appear to be important in the ‘‘second step’’ of HSF1 activation and stress and trans-activation of HSP genes can be inhibited by kinase inhibitors, while inactive HSF1 trimers can be rendered active in vivo by exposure to phosphatase inhibitors [14]. The sites of HSF1 phosphorylation have been studied by phosphopeptide mapping and a partial list of such sites exists. HSF1 is known to be phosphorylated on serines residues at 121, 230, 303, 307, 326, 363 [16,18,19,20,21,22,23]. The role of these sites in HSP transcrip- tion have been attributed mainly by point mutation studies and these experiments suggest that phosphorylation of serine 121, 303, 307, or 363 can inhibit HSP transcription [16,18,24,25]. S230 and S326 are the only currently known phosphorylation sites associated with stimulation of transcription by HSF1. In addition, the regulatory mechanisms through which these posttranslational PLoS ONE | www.plosone.org 1 November 2010 | Volume 5 | Issue 11 | e13830