HSP90 Interacts with and Regulates the Activity of Heat Shock Factor 1 in Xenopus Oocytes

ABSTRACT Transcriptional activation of heat shock genes is a reversible and multistep process involving conversion of inactive heat shock factor 1 (HSF1) monomers into heat shock element (HSE)-binding homotrimers, hyperphosphorylation, and further modifications that induce full transcriptional competence. HSF1 is controlled by multiple regulatory mechanisms, including suppression by additional cellular factors, physical interactions with HSP70, and integration into different cellular signaling cascades. However, the signaling mechanisms by which cells respond to stress and control the HSF1 activation-deactivation pathway are not known. Here we demonstrate that HSP90, a cellular chaperone known to regulate several signal transduction molecules and transcription factors, functions in the regulation of HSF1. The existence of HSF1-HSP90 heterocomplexes was shown by coimmunoprecipitation of HSP90 with HSF1 from unshocked and heat-shocked nuclear extracts, recognition of HSF1-HSE complexes in vitro by using HSP90 antibodies (Abs), and recognition of HSF1 in vivo by HSP90 Abs microinjected directly into oocyte nuclei. The functional impact of HSP90-HSF1 interactions was analyzed by using two strategies: direct nuclear injection of HSP90 Abs and treatment of cells with geldanamycin (GA), an agent that specifically blocks the chaperoning activity of HSP90. Both HSP90 Abs and GA delayed the disassembly of HSF1 trimers during recovery from heat shock and specifically inhibited heat-induced transcription from a chloramphenicol acetyltransferase reporter construct under control of the hsp70 promoter. HSP90 Abs activated HSE binding in the absence of heat shock, an effect that could be reversed by subsequent injection of purified HSP90. GA did not activate HSE binding under nonshock conditions but increased the quantity of HSE binding induced by heat shock. On the basis of these findings and the known properties of HSP90, we propose a new regulatory model in which HSP90 participates in modulating HSF1 at different points along the activation-deactivation pathway, influencing the interconversion between monomeric and trimeric conformations as well as transcriptional activation. We also put forth the hypothesis that HSP90 links HSF1 to cellular signaling molecules coordinating the stress response.

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Inhibition of the activation of heat shock factor in vivo and in vitro by flavonoids.

Molecular and Cellular Biology ◽

10.1128/mcb.12.8.3490 ◽

1992 ◽

Vol 12 (8) ◽

pp. 3490-3498 ◽

Cited By ~ 151

Author(s):

N Hosokawa ◽

K Hirayoshi ◽

H Kudo ◽

H Takechi ◽

A Aoike ◽

...

Keyword(s):

Heat Shock ◽

Transcriptional Activation ◽

Heat Shock Factor ◽

Mobility Shift ◽

Human Colon Cancer ◽

Heat Shock Element ◽

Mrna Accumulation ◽

Cell Extracts

Transcriptional activation of human heat shock protein (HSP) genes by heat shock or other stresses is regulated by the activation of a heat shock factor (HSF). Activated HSF posttranslationally acquires DNA-binding ability. We previously reported that quercetin and some other flavonoids inhibited the induction of HSPs in HeLa and COLO 320DM cells, derived from a human colon cancer, at the level of mRNA accumulation. In this study, we examined the effects of quercetin on the induction of HSP70 promoter-regulated chloramphenicol acetyltransferase (CAT) activity and on the binding of HSF to the heat shock element (HSE) by a gel mobility shift assay with extracts of COLO 320DM cells. Quercetin inhibited heat-induced CAT activity in COS-7 and COLO 320DM cells which were transfected with plasmids bearing the CAT gene under the control of the promoter region of the human HSP70 gene. Treatment with quercetin inhibited the binding of HSF to the HSE in whole-cell extracts activated in vivo by heat shock and in cytoplasmic extracts activated in vitro by elevated temperature or by urea. The binding of HSF activated in vitro by Nonidet P-40 was not suppressed by the addition of quercetin. The formation of the HSF-HSE complex was not inhibited when quercetin was added only during the binding reaction of HSF to the HSE after in vitro heat activation. Quercetin thus interacts with HSF and inhibits the induction of HSPs after heat shock through inhibition of HSF activation.

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Inhibition of the activation of heat shock factor in vivo and in vitro by flavonoids

Molecular and Cellular Biology ◽

10.1128/mcb.12.8.3490-3498.1992 ◽

1992 ◽

Vol 12 (8) ◽

pp. 3490-3498

Author(s):

N Hosokawa ◽

K Hirayoshi ◽

H Kudo ◽

H Takechi ◽

A Aoike ◽

...

Keyword(s):

Heat Shock ◽

Transcriptional Activation ◽

Heat Shock Factor ◽

Mobility Shift ◽

Human Colon Cancer ◽

Heat Shock Element ◽

Mrna Accumulation ◽

Cell Extracts

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Phosphorylation of Serine 303 Is a Prerequisite for the Stress-Inducible SUMO Modification of Heat Shock Factor 1

Molecular and Cellular Biology ◽

10.1128/mcb.23.8.2953-2968.2003 ◽

2003 ◽

Vol 23 (8) ◽

pp. 2953-2968 ◽

Cited By ~ 205

Author(s):

Ville Hietakangas ◽

Johanna K. Ahlskog ◽

Annika M. Jakobsson ◽

Maria Hellesuo ◽

Niko M. Sahlberg ◽

...

Keyword(s):

Heat Shock ◽

Phosphorylation Site ◽

Stress Granules ◽

Heat Shock Factor ◽

Transcriptional Level ◽

Heat Shock Factor 1 ◽

Protection Mechanism ◽

Sumo Modification

ABSTRACT The heat shock response, which is accompanied by a rapid and robust upregulation of heat shock proteins (Hsps), is a highly conserved protection mechanism against protein-damaging stress. Hsp induction is mainly regulated at transcriptional level by stress-inducible heat shock factor 1 (HSF1). Upon activation, HSF1 trimerizes, binds to DNA, concentrates in the nuclear stress granules, and undergoes a marked multisite phosphorylation, which correlates with its transcriptional activity. In this study, we show that HSF1 is modified by SUMO-1 and SUMO-2 in a stress-inducible manner. Sumoylation is rapidly and transiently enhanced on lysine 298, located in the regulatory domain of HSF1, adjacent to several critical phosphorylation sites. Sumoylation analyses of HSF1 phosphorylation site mutants reveal that specifically the phosphorylation-deficient S303 mutant remains devoid of SUMO modification in vivo and the mutant mimicking phosphorylation of S303 promotes HSF1 sumoylation in vitro, indicating that S303 phosphorylation is required for K298 sumoylation. This finding is further supported by phosphopeptide mapping and analysis with S303/7 phosphospecific antibodies, which demonstrate that serine 303 is a target for strong heat-inducible phosphorylation, corresponding to the inducible HSF1 sumoylation. A transient phosphorylation-dependent colocalization of HSF1 and SUMO-1 in nuclear stress granules provides evidence for a strictly regulated subnuclear interplay between HSF1 and SUMO.

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Intramolecular Repression of Mouse Heat Shock Factor 1

Molecular and Cellular Biology ◽

10.1128/mcb.18.2.906 ◽

1998 ◽

Vol 18 (2) ◽

pp. 906-918 ◽

Cited By ~ 56

Author(s):

Thomas Farkas ◽

Yulia A. Kutskova ◽

Vincenzo Zimarino

Keyword(s):

Heat Shock ◽

Transcriptional Activation ◽

Mammalian Cells ◽

Coiled Coil ◽

Heat Shock Factor ◽

Heat Shock Factor 1 ◽

Reticulocyte Lysate ◽

Heptad Repeats ◽

Intramolecular Mechanism

ABSTRACT The pathway leading to transcriptional activation of heat shock genes involves a step of heat shock factor 1 (HSF1) trimerization required for high-affinity binding of this activator protein to heat shock elements (HSEs) in the promoters. Previous studies have shown that in vivo the trimerization is negatively regulated at physiological temperatures by a mechanism that requires multiple hydrophobic heptad repeats (HRs) which may form a coiled coil in the monomer. To investigate the minimal requirements for negative regulation, in this work we have examined mouse HSF1 translated in rabbit reticulocyte lysate or extracted from Escherichia coli after limited expression. We show that under these conditions HSF1 behaves as a monomer which can be induced by increases in temperature to form active HSE-binding trimers and that mutations of either HR region cause activation in both systems. Furthermore, temperature elevations and acidic buffers activate purified HSF1, and mild proteolysis excises fragments which form HSE-binding oligomers. These results suggest that oligomerization can be repressed in the monomer, as previously proposed, and that repression can be relieved in the apparent absence of regulatory proteins. An intramolecular mechanism may be central for the regulation of this transcription factor in mammalian cells, although not necessarily sufficient.

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Cooperative Binding of Heat Shock Factor to the Yeast HSP82 Promoter In Vivo and In Vitro

Molecular and Cellular Biology ◽

10.1128/mcb.19.3.1627 ◽

1999 ◽

Vol 19 (3) ◽

pp. 1627-1639 ◽

Cited By ~ 62

Author(s):

Alexander M. Erkine ◽

Serena F. Magrogan ◽

Edward A. Sekinger ◽

David S. Gross

Keyword(s):

Heat Shock ◽

Single Point ◽

Heat Shock Factor ◽

Single Point Mutation ◽

Heat Shock Element ◽

Cooperative Interactions ◽

Before And After ◽

Order Of Magnitude

ABSTRACT Previous work has shown that heat shock factor (HSF) plays a central role in remodeling the chromatin structure of the yeastHSP82 promoter via constitutive interactions with its high-affinity binding site, heat shock element 1 (HSE1). The HSF-HSE1 interaction is also critical for stimulating both basal (noninduced) and induced transcription. By contrast, the function of the adjacent, inducibly occupied HSE2 and -3 is unknown. In this study, we examined the consequences of mutations in HSE1, HSE2, and HSE3 on HSF binding and transactivation. We provide evidence that in vivo, HSF binds to these three sites cooperatively. This cooperativity is seen both before and after heat shock, is required for full inducibility, and can be recapitulated in vitro on both linear and supercoiled templates. Quantitative in vitro footprinting reveals that occupancy of HSE2 and -3 by Saccharomyces cerevisiae HSF (ScHSF) is enhanced ∼100-fold through cooperative interactions with the HSF-HSE1 complex. HSE1 point mutants, whose basal transcription is virtually abolished, are functionally compensated by cooperative interactions with HSE2 and -3 following heat shock, resulting in robust inducibility. Using a competition binding assay, we show that the affinity of recombinant HSF for the full-length HSP82promoter is reduced nearly an order of magnitude by a single-point mutation within HSE1, paralleling the effect of these mutations on noninduced transcript levels. We propose that the remodeled chromatin phenotype previously shown for HSE1 point mutants (and lost in HSE1 deletion mutants) stems from the retention of productive, cooperative interactions between HSF and its target binding sites.

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Basal-level expression of the yeast HSP82 gene requires a heat shock regulatory element

Molecular and Cellular Biology ◽

10.1128/mcb.9.11.4789-4798.1989 ◽

1989 ◽

Vol 9 (11) ◽

pp. 4789-4798

Author(s):

D McDaniel ◽

A J Caplan ◽

M S Lee ◽

C C Adams ◽

B R Fishel ◽

...

Keyword(s):

Heat Shock ◽

Regulatory Element ◽

Heat Shock Factor ◽

Tata Box ◽

Hypersensitive Site ◽

Heat Shock Element ◽

Hsp82 Promoter ◽

Basal Level Expression

Previous studies have shown that heat shock factor is constitutively bound to heat shock elements in Saccharomyces cerevisiae. We demonstrate that mutation of the heat shock element closest to the TATA box of the yeast HSP82 promoter abolishes basal-level transcription without markedly affecting inducibility. The mutated heat shock element no longer bound putative heat shock factor, either in vitro or in vivo, but still resided within a nuclease-hypersensitive site in the chromatin. Thus, constitutive binding of heat shock factor to heat shock elements in S. cerevisiae appears to functionally direct basal-level transcription.

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