Abstract
BackgroundMany 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 FindingsIn 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, (PKAcα) 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 PKAcα 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 PKAcα 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, null 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.ConclusionsThese 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.
Highlights
Heat Shock Factor 1 (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]
Because HSF1 can be activated by a number of other agents including the Hsp90 inhibitor 17AAG, the proteasome inhibitor MG-132 and growth factors such as heregulin (HRG) and insulin-like growth factor (IGF-1), we examined the effect of these molecules on HSF1-PKAca association
As protein kinase A (PKA) can be induced through the activation of adenylate cyclase, we examined a scenario in which HSF1-binding to PKAca is regulated through the product of adenylate cyclase activity, cyclic adenosine monophosphate (cAMP) (Fig. 1D)
Summary
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]. 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 inoperative and HSP transcription proceeds [28]. 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
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