Uncoupling Stress-Inducible Phosphorylation of Heat Shock Factor 1 from Its Activation

Abstract

In mammals the stress-inducible expression of genes encoding heat shock proteins is under the control of the heat shock transcription factor 1 (HSF1). Activation of HSF1 is a multistep process, involving trimerization, acquisition of DNA-binding and transcriptional activities, which coincide with several posttranslational modifications. Stress-inducible phosphorylation of HSF1, or hyperphosphorylation, which occurs mainly within the regulatory domain (RD), has been proposed as a requirement for HSF-driven transcription and is widely used for assessing HSF1 activation. Nonetheless, the contribution of hyperphosphorylation to the activity of HSF1 remains unknown. In this study, we generated a phosphorylation-deficient HSF1 mutant (HSF1Δ∼PRD), where the 15 known phosphorylation sites within the RD were disrupted. Our results show that the phosphorylation status of the RD does not affect the subcellular localization and DNA-binding activity of HSF1. Surprisingly, under stress conditions, HSF1Δ∼PRD is a potent transactivator of both endogenous targets and a reporter gene, and HSF1Δ∼PRD has a reduced activation threshold. Our results provide the first direct evidence for uncoupling stress-inducible phosphorylation of HSF1 from its activation, and we propose that the phosphorylation signature alone is not an appropriate marker for HSF1 activity.

ACKNOWLEDGMENTS

We thank Julius Anckar for insightful discussion in the initial stage of the project, Alexandra Elsing for expert help with the statistical analysis, Samu Himanen for help with the ChIP assay, and Emine Lundsten and Cynthia Swan for assistance with writing of the manuscript. All of the members of the Sistonen laboratory are acknowledged for their constructive comments.

This study was financially supported by the Academy of Finland, the Sigrid Jusélius Foundation, the Magnus Ehrnrooth Foundation, Åbo Akademi University Foundation (L.S.), and the Turku Doctoral Program of Biomedical Sciences (M.A.B. and J.J.).