Abstract
BackgroundHeat shock factor (HSF/HSF1) not only is the transcription factor primarily responsible for the transcriptional response of cells to physical and chemical stress but also coregulates other important signaling pathways. The factor mediates the stress-induced expression of heat shock or stress proteins (HSPs). HSF/HSF1 is inactive in unstressed cells and is activated during stress. Activation is accompanied by hyperphosphorylation of the factor. The regulatory importance of this phosphorylation has remained incompletely understood. Several previous studies on human HSF1 were concerned with phosphorylation on Ser303, Ser307 and Ser363, which phosphorylation appears to be related to factor deactivation subsequent to stress, and one study reported stress-induced phosphorylation of Ser230 contributing to factor activation. However, no previous study attempted to fully describe the phosphorylation status of an HSF/HSF1 in stressed cells and to systematically identify phosphoresidues involved in factor activation. The present study reports such an analysis for human HSF1 in heat-stressed cells.ResultsAn alanine scan of all Ser, Thr and Tyr residues of human HSF1 was carried out using a validated transactivation assay, and residues phosphorylated in HSF1 were identified by mass spectrometry and sequencing. HSF1 activated by heat treatment was phosphorylated on Ser121, Ser230, Ser292, Ser303, Ser307, Ser314, Ser319, Ser326, Ser344, Ser363, Ser419, and Ser444. Phosphorylation of Ser326 but none of the other Ser residues was found to contribute significantly to activation of the factor by heat stress. Phosphorylation on Ser326 increased rapidly during heat stress as shown by experiments using a pSer326 phosphopeptide antibody. Heat stress-induced DNA binding and nuclear translocation of a S326A substitution mutant was not impaired in HSF1-negative cells, but the mutant stimulated HSP70 expression several times less well than wild type factor.ConclusionTwelve Ser residues but no Thr or Tyr residues were identified that were phosphorylated in heat-activated HSF1. Mutagenesis experiments and functional studies suggested that phosphorylation of HSF1 residue Ser326 plays a critical role in the induction of the factor's transcriptional competence by heat stress. PhosphoSer326 also contributes to activation of HSF1 by chemical stress. To date, no functional role could be ascribed to any of the other newly identified phosphoSer residues.
Highlights
Heat shock factor (HSF/heat shock factor 1 (HSF1)) is the transcription factor primarily responsible for the transcriptional response of cells to physical and chemical stress and coregulates other important signaling pathways
Transactivation by LEXA-HSF1 was assessed by dual luciferase assay of cells co-transfected with a firefly luciferase gene responsive to LEXA-HSF1 (LEXA-fLUC) and a constitutively expressed Renilla luciferase gene
The present article is concerned with regulation of human HSF1, which is a key factor mediating the transcriptional response of human cells to physical and chemical stresses and a coregulator of other important signaling pathways (e.g.. [35,36,37,38])
Summary
Heat shock factor (HSF/HSF1) is the transcription factor primarily responsible for the transcriptional response of cells to physical and chemical stress and coregulates other important signaling pathways. During periods of physical or chemical stress, transcription of genes encoding cytoprotective heat shock or stress proteins (HSPs) is increased This enhanced expression is primarily mediated by heat shock factor 1 (HSF1) in vertebrate cells or by a homologous factor (HSF) in non-vertebrate cells. A first step results in formation of factor homotrimers that are capable of binding so-called heat shock element (HSE) sequences present in hsp genes but essentially lack transcriptional activity. In cells exposed to heat, acquisition of HSE DNA-binding activity was observed to precede hyperphosphorylation of HSF1 [9]. This result suggested that hyperphosphorylation could play a regulatory role in the second activation step that renders the factor transactivation-competent. For the inhibitors investigated it was found that they did not affect HSF1 DNA-binding activity [11] (see [18])
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