Activation of Heat Shock Factor 1 DNA Binding Precedes Stress-induced Serine Phosphorylation

Abstract

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...

Cell number‐dependent regulation of Hsp70B′ expression: Evidence of an extracellular regulator

“…Hsf-1 is present in the cytosol of unstressed cells in monomeric form and upon stress, trimerizes, translocates to the nucleus, and binds HSEs (Green et al, 1995;Zuo et al, 1995). As binding precedes full activation (Cotto et al, 1996), the next step in the regulation of Hsp gene expression involves hyperphosphorylation of Hsf-1 (specifically, Ser 326 ) (Guettouche et al, 2005) to its transcriptionally active form. Using the PAGen promoter sequence analysis database (http://www.uic.edu/pharmacy/depts/pmpcpd/ pagen/ University of Illinois at Chicago) the 1 kb nucleotide sequence upstream of the HspA6 gene was found to have seven HSE sequences with more than 85% consensus, three of which reside in the proximal promoter region (À110).…”

Inhibition of nuclear factor κB by prostaglandin A1: An effect associated with heat shock transcription factor activation

“…Several HSFs (HSF1-HSF4) have been identified in vertebrate cells (18). In mammalian cells, HSFs exist as an inactive non-DNA-binding form, which is rapidly converted to the DNA-binding form upon exposure to heat shock or other environmental stimuli (18). As described for NF-B, HSF activation is also a multistep process.…”

“…As described for NF-B, HSF activation is also a multistep process. In the case of HSF, activation requires oligomerization, acquisition of DNAbinding activity, localization to the nucleus, and phosphorylation (18).…”

“…Induction is mediated by the activation and translocation to the nucleus of a transregulatory protein, the heat shock transcription factor (HSF), which binds to multiple arrays of heat shock elements (HSEs) located in the promoter of heat shock genes (17). Several HSFs (HSF1-HSF4) have been identified in vertebrate cells (18). In mammalian cells, HSFs exist as an inactive non-DNA-binding form, which is rapidly converted to the DNA-binding form upon exposure to heat shock or other environmental stimuli (18).…”

Proteolytic mapping of heat shock transcription factor domains

“…Trimerization increases the binding affinity of HSF for the HSE by more than 1,000-fold (Kim et al, 1994). It is known that HSF is a phosphoprotein and undergoes hyperphosphorylation after heat stress (Sorger et al, 1987;Larson et al, 1988;Sorger & Pelham, 1988;Sarge et al, 1993), although the functional significance of these modifications is not clear and the positions of phosphorylation have not been mapped completely (Cotto et al, 1996). On the other hand, the heat shock proteins themselves have been proposed to function as suppressors of HSF activity via an autoregulatory loop (for a review, see Morimoto, 1993).…”