TY - JOUR
T1 - Uncoupling Stress-Inducible Phosphorylation of Heat Shock Factor 1 from Its Activation
AU - Budzyński, Marek A
AU - Puustinen, Mikael C
AU - Joutsen, Jenny
AU - Sistonen, Lea
N1 - Copyright © 2015, American Society for Microbiology. All Rights Reserved.
PY - 2015/7
Y1 - 2015/7
N2 - 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.
AB - 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.
KW - Animals
KW - Binding Sites/genetics
KW - Blotting, Western
KW - Cell Movement/genetics
KW - Cells, Cultured
KW - DNA/genetics
KW - DNA-Binding Proteins/genetics
KW - Embryo, Mammalian/cytology
KW - Fibroblasts/cytology
KW - HeLa Cells
KW - Heat Shock Transcription Factors
KW - Heat-Shock Proteins/genetics
KW - Hot Temperature
KW - Humans
KW - Mice, Knockout
KW - Microscopy, Confocal
KW - Mutant Proteins/genetics
KW - Phosphorylation
KW - Protein Binding/genetics
KW - Regulatory Sequences, Nucleic Acid/genetics
KW - Reverse Transcriptase Polymerase Chain Reaction
KW - Stress, Physiological
KW - Transcription Factors/genetics
KW - Transcriptional Activation
U2 - 10.1128/MCB.00816-14
DO - 10.1128/MCB.00816-14
M3 - Article
C2 - 25963659
SN - 0270-7306
VL - 35
SP - 2530
EP - 2540
JO - Molecular and Cellular Biology
JF - Molecular and Cellular Biology
IS - 14
ER -