Heat shock transcription factor 1 (HSF1) is constitutively expressed in mammalian cells and negatively regulated for DNA binding and transcriptional activity. Upon exposure to heat shock and other forms of chemical and physiological stress, these activities of HSF1 are rapidly induced. In this report, we demonstrate that constitutive phosphorylation of HSF1 at serine residues distal to the transcriptional activation domain functions to repress transactivation. Tryptic phosphopeptide analysis of a collection of chimeric GAL4-HSF1 deletion and point mutants identified a region of constitutive phosphorylation encompassing serine residues 303 and 307. The significance of phosphorylation at serines 303 and 307 in the regulation of HSF1 transcriptional activity was demonstrated by transient transfection and assay of a chloramphenicol acetyltransferase reporter construct. Whereas the transfected wild-type GAL4-HSF1 chimera is repressed for transcriptional activity and derepressed by heat shock, mutation of serines 303 and 307 to alanine results in derepression to a high level of constitutive activity. Similar results were obtained with mutation of these serine residues in the context of full-length HSF1. These data reveal that constitutive phosphorylation of serines 303 and 307 has an important role in the negative regulation of HSF1 transcriptional activity at control temperatures.A hallmark of the heat shock response is the rapid, inducible transcription of genes that encode heat shock proteins and molecular chaperones. These events are mediated through heat shock transcription factor 1 (HSF1), a ubiquitous and constitutively expressed protein which is activated upon exposure to heat shock and other chemical, environmental, and physiological stresses (29,31,32,46). Under normal growth conditions, HSF1 exists in a repressed state for both DNA binding and transcriptional activities. Activation is associated with transitions in the oligomeric state of HSF1 from a repressed monomer to a nucleus-localized trimer which possesses DNA binding activity (4,34,38,39,42,44,47). Treatment of cells with salicylate and other nonsteroidal anti-inflammatory drugs induces formation of the HSF1 DNA binding trimer in the absence of transcriptional activity (10,(18)(19)(20), demonstrating that the DNA binding and transcriptional activities of HSF1 can be uncoupled in mammalian cells and that the two activities may be silenced via distinct mechanisms.The mechanism by which HSF1 is negatively regulated is complex. For example, the analysis of chimeric HSF1 proteins, in which a heterologous DNA binding domain of either LexA or GAL4 is fused to HSF1, reveals that the transcriptional activation domain remains repressed despite the ability of chimeric HSF1 to bind DNA constitutively (13,35,40,48). In these chimeric HSF1 constructs, the transactivation domain is derepressed either by exposure to heat shock or upon deletion of the negative regulatory domain (40). These results reveal that chimeric HSF1 retains the ability to regulate the transac...

Exposure of mammalian cells in culture to the antiinflammatory drugs sodium salicylate or indomethacin results in activation of heat shock factor 1 (HSF1) DNA binding activity. We have previously shown that the drug-induced HSF1 becomes associated with the heat shock elements of the hsp70 promoter, yet transcription of the hsp70 gene is not induced (Jurivich, D. A., Sistonen, L., Kroes, R. A., and Morimoto, R. I. (1992) Science 255, 1243-1245). In this study, we have examined the basis for uncoupling the heat shock transcriptional response. Comparison of heat shock and drug-induced forms of HSF1 has revealed that the transcriptionally inert drug-induced HSF1 is constitutively but not inducibly serine-phosphorylated, whereas heat shock-induced HSF1 is both constitutively and inducibly serinephosphorylated. The transcriptionally inert intermediate represented by drug-induced HSF1 can be converted to the transcriptionally active state by a subsequent exposure to heat shock. The only detectable change in HSF1 is the acquisition of inducible serine phosphorylation. These data reveal that acquisition of the trimeric DNA binding state of HSF1 is independent of and precedes inducible phosphorylation and furthermore that inducible phosphorylation correlates with transcriptional activation.The genes that encode heat shock proteins are coordinately regulated in response to acute exposure of cells to a range of physiological and environmental trauma, including heat shock, amino acid analogues, heavy metals, oxidative stress, antiinflammatory drugs, and arachidonic acid (2-5). The transcriptional induction of heat shock genes in eukaryotic cells is mediated by pre-existing heat shock transcription factors (HSF) 1 which, upon activation, bind as trimers to multiple arrays of the heat shock element (HSE) which are located in the promoter region of genes encoding heat shock proteins and molecular chaperones (6 -18). In vertebrate cells, the HSF gene family is comprised of four distinct HSF genes (HSF1-HSF4) which have been cloned in chicken (HSF1-HSF3), mouse (HSF1 and -2), and humans (HSF1, -2, and -4) (19 -22).2 The co-expression of multiple HSFs within the same cell type has led to the suggestion that these multiple factors could mediate the response to different forms of stress. Indeed, it has been shown that HSF1 corresponds to the stress-responsive factor, whereas HSF2 is not stress-responsive and is induced during early embryogenesis, spermatogenesis, and erythroid differentiation (23-28).Overall, the HSFs exhibit a similar structure with a conserved amino-terminal localized DNA binding domain, multiple arrays of hydrophobic heptad repeats, and a carboxyl-terminal transcription activation domain (5, 13, 19 -22, 29 -33). HSF1 is maintained in the latent control state as a non-DNA binding monomer that is constitutively phosphorylated (16,24,34). Intramolecular negative regulation appears to be specified through interactions between leucine zippers 1-3 and 4, whereas zippers 1-3 have an additional function as an extended co...

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