Protein Kinase A Regulates Molecular Chaperone Transcription and Protein Aggregation Yue Zhang . , Ayesha Murshid., Thomas Prince, Stuart K. Calderwood* Department of Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America Abstract Heat shock factor 1 (HSF1) regulates one of the major pathways of protein quality control and is essential for deterrence of protein-folding disorders, particularly in neuronal cells. However, HSF1 activity declines with age, a change that may open the door to progression of neurodegenerative disorders such as Huntington’s disease. We have investigated mechanisms of HSF1 regulation that may become compromised with age. HSF1 binds stably to the catalytic domain of protein kinase A (PKAca) and becomes phosphorylated on at least one regulatory serine residue (S320). We show here that PKA is essential for effective transcription of HSP genes by HSF1. PKA triggers a cascade involving HSF1 binding to the histone acetylase p300 and positive translation elongation factor 1 (p-TEFb) and phosphorylation of the c-terminal domain of RNA polymerase II, a key mechanism in the downstream steps of HSF1-mediated transcription. This cascade appears to play a key role in protein quality control in neuronal cells expressing aggregation-prone proteins with long poly-glutamine (poly-Q) tracts. Such proteins formed inclusion bodies that could be resolved by HSF1 activation during heat shock. Resolution of the inclusions was inhibited by knockdown of HSF1, PKAca, or the pTEFb component CDK9, indicating a key role for the HSF1- PKA cascade in protein quality control. Citation: Zhang Y, Murshid A, Prince T, Calderwood SK (2011) Protein Kinase A Regulates Molecular Chaperone Transcription and Protein Aggregation. PLoS ONE 6(12): e28950. doi:10.1371/journal.pone.0028950 Editor: Michael Sherman, Boston University Medical School, United States of America Received November 15, 2011; Accepted November 17, 2011; Published December 22, 2011 Copyright: 2011 Zhang 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: This work was supported by National Institutes of Health research grants RO-1CA047407, R01CA119045, and RO-1CA094397. 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 . These authors contributed equally to this work. Introduction HSF1 regulates the expression of molecular chaperones and thereby controls one of the most essential arms of the protein quality control network [1,2]. One of the contributing factors to neurological disorders such as Huntington’s disease is the expression of aggregation prone proteins containing long polyglu- tamine (poly-Q) tracts. As age-dependent failure of the protein quality control networks in neuronal cells plays a decisive role in neurodegeneration, understanding the HSF1 transcriptional pathway may thus be significant in identifying potential mecha- nisms of neurodegeneration [3]. Although HSF1-related proteins have been identified in many species, details of its regulation are still incomplete. We have shown in a recent study that that HSF1 binds to the catalytic domain of protein kinase A (PKAca) and becomes phosphorylated on a PKA consensus phosphorylation site required for transcriptional activation of downstream gene expression [4]. In the present investigation, we have examined molecular mechanisms of HSF1 activation by PKA that may be important for regulation of HSP genes on chromatin. As heat shock transcription has been shown to be activated by histone deacetylase inhibitors, we examined the possibility that stress- induced, PKA-dependent HSF1 activation may involve interac- tion with histone acetylases [5,6]. In addition, activation of the hsp70 gene by stress in Drosophila requires HSF association with positive translation elongation factor 1 p-P-TEFb and downstream hyperphosphorylation of the C-terminal domain (CTD) of RNA polymerase II [7]. The p-TEFb is activated by release from a complex containing the regulatory 7SK snRNA [8] and controls a major transcriptional checkpoint permitting elongation, processing and transport of mRNA as well as synthesis [9,10,11,12]. Here we demonstrate that PKAca is required for coordinate p300 binding and p-TEFb association with HSF1 on the chromatin of heat shock-induced genes and for stress-induced transcription. This cascade was required for phosphorylation at the 2 position in the repeat sequences of the C-terminal tail of RNA poll II on heat-inducible promoters. In addition, we found that activation of HSF1 by protein stress depletes the levels of intracellular protein inclusion bodies containing aggregation– prone, poly-Q containing proteins in neuronal cells an effect abrogated by targeting HSF1, PKAca or the component CDK9 of p-TEFb. Materials and Methods Reagents and Antibodies Rat monoclonal anti-HSF1 antibodies were from Assay designs (Enzo Life Sciences). In addition we used rabbit polycolonal anti- PKAca antibodies (Cell Signaling), anti-HSF1 (Stressgen), anti-p300 (Millipore), anti-RNA Pol II (Millipore), anti-pCDK9 (Abcam), anti- phosphoserine-2-RNA Pol II (Abcam), anti-phosphoserine -5-RNA Pol II (Abcam), anti-acetylated histone 4 and anti-total histone 4 (Millipore) antibodies. PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e28950