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J. Biol. Chem., Vol. 275, Issue 37, 29147-29152, September 15, 2000

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Glycogen Synthase Kinase 3 Negatively Regulates Both DNA-binding and Transcriptional Activities of Heat Shock Factor 1^*

Ilungo J. Xavier, Phillipe A. Mercier, Christine M. McLoughlin, Adnan Ali^§, James R. Woodgett^§, and Nick Ovsenek^¶

From the  Department of Anatomy and Cell Biology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada and the ^§ Division of Experimental Therapeutics, Ontario Cancer Institute/Princess Margaret Hospital, Toronto, Ontario M5G 2M9, Canada

Received for publication, March 15, 2000, and in revised form, June 6, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Stress activation of heat shock factor (HSF1) involves the conversion of repressed monomers to DNA-binding homotrimers with increased transcriptional capacity and results in transcriptional up-regulation of the heat shock protein (hsp) gene family. Cells tightly control the activity of HSF1 through interactions with hsp90 chaperone complexes and through integration into a number of different signaling cascades. A number of studies have shown that HSF1 transcriptional activity is negatively regulated by constitutive phosphorylation in the regulatory domain by glycogen synthase kinase (GSK3) isoforms /. However, previous studies have not examined the ability of GSK3 to regulate the DNA-binding activity of native HSF1 in vivo under heat shock conditions. Here we show that GSK3 inhibits both DNA-binding and transcriptional activities of HSF1 in heat-shocked cells. Specific inhibition of GSK3 increased the levels of DNA binding and transcription after heat shock and delayed the attenuation of HSF1 during recovery. In contrast, the overexpression of GSK3 resulted in significant reduction in heat-induced HSF1 activities. These results confirm the role of GSK3 as a negative regulator of HSF1 transcription in cells during heat shock and demonstrate for the first time that GSK3 functions to repress DNA binding.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The stress response is a highly conserved cellular reaction leading to rapid expression of cytoprotective heat shock proteins (hsp).¹ Hsps are molecular chaperones that aid in the folding, transport, regulation, and degradation of cellular proteins under normal conditions, and their expression during stress is essential for cell survival (1). In higher organisms the response is controlled at the transcriptional level by transient activation of the heat shock transcription factor (HSF1) (2). Under normal conditions, HSF1 is maintained as latent non-DNA-binding monomers that upon stress assemble into active homotrimers capable of interacting with heat shock elements upstream of hsp genes. Full activation is a multistep process involving additional modifications to the transcriptional activation domain, i.e. transcriptional competence is regulated independently from oligomerization/DNA binding (3). Upon the removal of stress or after the expression of a critical level of hsps, trimers are disassembled back to inactive monomers in a process called attenuation.

The functional domains responsible for oligomeric switching, DNA binding, and transcriptional activation of HSF1 have been determined, and it is known that intrinsic properties of the molecule control its own activity (2). However, the cellular mechanisms governing HSF1 regulation appear to be quite complex and not fully understood. Recent studies have shown that HSF1 is regulated by association with a dynamic series of hsp90-chaperone complexes (4-7). In addition, it is a highly phosphorylated molecule that appears to be integrated into a number of stress-activated signaling cascades. Signaling pathways/kinases that are activated in stressed cells include PKB (8), PKC (9, 10), PKR (for review, see Ref.11), and mitogen-activated protein kinase family members, such as extracellular-regulated kinase (ERK1/2), stress-activated protein kinase/c-Jun N-terminal kinase, and p38/Hog-1 (for review, see Ref. 12). Glycogen synthase kinase (GSK3) isoforms / have also been implicated in the stress response (13-15).

HSF1 is constitutively phosphorylated on several serine residues, and the molecule is hyperphosphorylated after heat shock (13, 16-18). Upon stress, some residues remain constitutively phosphorylated, some become dephosphorylated, and several others are phosphorylated. The magnitude and complex dynamics of HSF1 phosphorylation throughout the activation/deactivation process has made it difficult to ascertain the functional role of these modifications, and the links to the various kinases still remain unclear. There is a great deal of contradictory evidence in the literature regarding the role of hyperphosphorylation. For example, hyperphosphorylation has been shown in some studies to accompany transcriptional activation, thus suggesting its importance for this process (14, 16, 19, 20). In support of this, HSF1 is not hyperphosphorylated by salicylate, which activates DNA binding but not transcription (21). Hsp expression is either reduced by serine/threonine kinase inhibitors (H7, staurosporine, 2-aminopurine, calphostin) or increased by serine/threonine phosphatase inhibitors (calyculin A, okadaic acid, sodium fluoride) (17, 22-26). In contrast, other studies suggest that hyperphosphorylation functions to deactivate transcription after heat shock (27) or even that it plays no role in regulating HSF1 activity (28, 29).

A number of studies using general kinase inhibitors to examine the potential regulatory role of phosphorylation on the DNA-binding activity of HSF1 have also yielded contradictory information. For example the serine/threonine kinase inhibitor H7 was reported to either inhibit DNA binding (26) or have no effect (17, 28). Other general kinase inhibitors, such as GF-X, staurosporine, K252b, and KT5720, have no effect on DNA binding (17, 23, 28). General phosphatase inhibitors have been shown to have inconsistent effects. They increase activation (25), delay activation (20), or delay attenuation of DNA binding (17, 28).

Previous studies have clearly demonstrated that amino acids 303, 307, and 363 in the regulatory domain of human HSF1 are constitutively phosphorylated by GSK3, ERK1/2, and PKC, respectively, and in vivo analyses of specific mutants have demonstrated that these phosphorylations function to repress the transcriptional activity of HSF1 (13, 14, 16, 30, 31). Repression can be overcome by heat shock, suggesting that phosphorylations in the regulatory domain, including those by GSK3, act in concert with other negative regulatory mechanisms to tightly control HSF1 activity in unshocked cells. However, the potential role for regulatory domain phosphorylations in controlling trimerization and DNA binding has never been directly examined. This is because past work has involved phosphorylation mutants of either GAL4- or LexA-HSF1 chimeras that did not contain the DNA-binding and oligomerization domains of HSF1 (16, 30, 31) or were overexpressed but constitutively active (under non-shock conditions) full-length HSF1 mutants (13, 14).

Here, we directly examined the in vivo role of GSK3 on the regulation of endogenous HSF1 under physiological stress conditions. In microinjection experiments with Xenopus oocytes, overexpression of GSK3 repressed both the DNA-binding and transcriptional activity of endogenous HSF1. In contrast, inhibition of GSK3 with LiCl or by overexpression of a GSK3-binding protein (GBP) increased DNA binding and transcription and significantly delayed the attenuation of DNA binding during recovery. These results confirmed that GSK3 acts as a negative regulator of HSF1 transcription in vivo during heat shock and demonstrated that GSK3 represses the DNA-binding activity of HSF1.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Oocyte Manipulations-- Xenopus laevis frogs were purchased from Xenopus I (Ann Arbor, MI). Ovaries were surgically removed from adult female frogs, and follicular cells were removed from oocytes by treatment in a calcium-free OR2 buffer (82.5 mM NaCl, 2.5 mM KCl, 1 mM MgCl_2, 1 mM NaH₂PO₄, 5 mM Hepes, pH 7.8, 10 mg/liter streptomycin sulfate, 10 mg/liter benzyl penicillin) that contained 2 mg/ml collagenase (type II, Sigma) for 3 h at 18 °C. Oocytes were washed extensively and were allowed to recover for 4 h in the OR2 buffer (as noted above with 1 mM CaCl₂ added) at 18 °C. These oocytes were maintained in the OR2 buffer during experimental treatments. Only stage VI oocytes were selected for experiments. Nuclei and cytoplasms were obtained by scoring animal hemispheres with a needle and by gently squeezing the equatorial region with watchmaker's forceps.

Oocyte Microinjection and Stress Treatments-- Capped mRNAs encoding active or inactive (kinase-dead) forms of GSK3 and GBP were synthesized in vitro from pXG73/CS2, pXG114/CS2, and pBP20 (32), respectively, by using SP6 RNA polymerase (Amersham Pharmacia Biotech). pXG73/CS2-encoding active GSK3 was generated by polymerase chain reaction (PCR) cloning from XG73 (kindly provided by D. Kimelman) by using PCR primers, 5'-cgcggatccatgtcgggaaggccgagaac-3' and 5'-cgcggatcctcaggaggagttggaggcag-3' (33). The PCR product was ligated into the PCR TA3.1 cloning vector (Invitrogen, San Diego, CA), and the BamHI insert was further isolated and cloned into the pCS2 expression vector. pXG114/CS2 encoding the kinase dead mutant that contained a lysine to arginine substitution at position 114 was constructed as above by PCR cloning from XG114 (provided by D. Kimelman). For expression in oocytes, 20 nl of respective mRNA solutions (2 mg/ml) were injected directly into the cytoplasm. For immediate elevation of nuclear levels of GSK3, purified recombinant rabbit skeletal muscle GSK3 (New England Biolabs, Inc., Beverly, MA) was injected directly into the oocyte nuclei. Cells injected with mRNA were incubated for 12 h at 18 °C to allow for the translation of expressed protein before heat shock. Cells injected with purified enzyme were incubated for 1 h at 18 °C before heat shock. For stress treatments with LiCl, oocytes were incubated in the OR2 buffer that contained 10, 25, and 50 mM LiCl for 1 h before heat shock in LiCl-free OR2. Heat shock was at 33 °C for 1 h (unless otherwise indicated). For recovery, oocytes were transferred to the OR2 buffer at 18 °C for the indicated times. In all experiments, a minimum of 25 oocytes was used for each sample.

Protein Extracts and Gel Mobility Shift Assays-- Protein extracts were prepared by homogenizing oocytes in Buffer C (50 mM Tris-Cl, pH 7.9, 20% glycerol, 50 mM KCl, 0.1 mM EDTA, 2 mM dithiothreitol, 10 µg/ml aprotinin, and 10 µg/ml leupeptin) in a volume of 10 µl of buffer per oocyte. Homogenates were transferred to Eppendorf tubes and centrifuged for 5 min at 15,000 × g (4 °C). The resulting supernatants were immediately frozen in liquid nitrogen and stored at 80 °C.

DNA mobility shift assays were performed by using radiolabeled oligonucleotide probes as described previously (34, 35). DNA-binding reactions contained 10 µl of extract (one oocyte equivalent by volume or 20 µg of soluble proteins). Binding reactions were performed with 1 µg of poly(dI·dC), 10 mM Tris, pH 7.8, 50 mM NaCl, 1 mM EDTA, 0.5 mM dithiothreitol, and 5% glycerol in a final volume of 20 µl. Reactions were incubated on ice for 20 min and immediately loaded onto 5% non-denaturing polyacrylamide gels that contained 6.7 mM Tris-Cl, pH 7.5, 1 mM EDTA, 3.3 mM sodium acetate. Gels were electrophoresed for 2.5 h at 150 V, dried, and exposed to x-ray film. Quantitation of DNA-binding activity was performed by using an NIH Image (Version 1.6.1) and was expressed in arbitrary densitometry units.

Immunoblotting-- Protein extracts were fractionated by SDS-polyacrylamide gel electrophoresis (10% acrylamide), then electroblotted onto polyvinylidene difluoride membranes. Blots were blocked for 2 h in TBST (20 mM Tris-Cl, pH 7.6, 137 mM NaCl, 0.1% (v/v) Tween 20) containing 5% milk powder. Blots were incubated in primary antibody (1:5000) in TBST with 2.5% milk and for 2 h, were washed in TBST and incubated with a secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG, Bio-Rad) diluted 1:10,000 in TBST and 2.5% milk. Blots were washed and proteins were visualized by chemiluminescence (Renaissance system, NEN Life Science Products). Antibodies against GSK3, IB, FLAG tag, and PCNA were obtained from Santa Cruz Biotechnology.

Transcription and Kinase Assays-- CAT assays were performed by using one oocyte equivalent to a whole cell extract as described previously (35). CAT expression vectors (36) and CAT assay protocols were as previously used in Ref. 5. Heat shock treatments were performed 4 h after CAT plasmid injections, then oocytes were incubated at 18 °C for 12 h to allow for CAT expression. GSK3 activity was assayed as described previously (37). Activity was measured by scintillation counting of label transfer from [-³²P]ATP to the cAMP-response element-binding protein phosphopeptide substrate (New England Biolabs, Inc.) and was expressed as a percentage of activity in uninjected, unshocked cells.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

GSK3 Represses HSF1-mediated DNA Binding-- To examine whether GSK3 modulates HSF1 in vivo under relevant stress conditions, we overexpressed GSK3 and observed the effects on HSF1 activity in Xenopus oocytes. Overexpression was achieved either by microinjection of mRNA into the cytoplasm or by direct injection of purified GSK3 into the nucleus. Increased levels and activity of GSK3 in cells were confirmed by immunoblotting and by specific kinase assays (Fig. 1A). We typically attained a 4- to 5-fold increase in GSK3 protein and activity levels over uninjected control cells (Fig. 1A). By comparison, heat shock for 1 h resulted in a 2-fold increase in endogenous GSK3 activity. In repeated experiments, overexpression of GSK3 consistently resulted in a 5-fold decrease in the amount of HSF1-mediated DNA-binding activity after 1 h of heat shock (Fig. 1B). Microinjection of purified GSK3 enzyme also resulted in decreased DNA binding after heat shock (Fig. 1B). Heat-inducible DNA-binding activity was almost completely inhibited after injection of 0.5 unit of GSK3. To control for potential nonspecific effects on HSF1 because of the microinjection procedure and to confirm that the effects observed were attributable specifically to alterations in GSK3 activity, we performed similar experiments with an inactive GSK3 mutant lacking kinase activity (kinase-dead GSK3) (38). Expression of kinase-dead enzyme did not change the total GSK3 kinase activity (Fig. 1A) and had no measurable effect on the amount of DNA binding by HSF1 after heat shock (Fig. 1B). Comparisons between HSF1 and GSK3 activation during the time course of heat shock showed rapid induction of heat shock element binding (at 5 min) well before the increase in GSK3 activity, which was initially observed after 30 min (Fig. 1C). Control experiments showed that endogenous HSF1 was not activated by the injection procedure itself and that the manipulation of GSK3 had no apparent effect on other DNA-binding activities. (Fig. 1D). Therefore, the results of these experiments appear to be specific effects of GSK3 on the DNA-binding activity of HSF1.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Overexpression of GSK3 results in decreased DNA binding. A, expression and activity of kinases from in vitro synthesized mRNA. Immunoblot of whole cell extracts of control and heat-shocked (33 °C, 1 h) oocytes injected with mRNA coding for active and inactive (kinase-dead) GSK3 and purified enzyme (0.5 unit). The activity of overexpressed GSK3 was confirmed by specific kinase assay. Kinase activity is expressed as a percentage of uninjected control (100%) and are means of values obtained from five experiments. B, gel mobility shift assay of non-shocked (NS) and heat-shocked (HS, 33 °C) stage VI oocytes. Oocytes were microinjected (cytoplasmic) with in vitro synthesized mRNA coding for active or inactive GSK3 or were microinjected (nuclear) with different concentrations of purified GSK3 30 min before 60 min of heat shock. Results shown are representative of at least five separate experiments performed with different batches of oocytes. Quantitation of DNA binding is shown below each panel. C, a comparison between HSF1 activation and GSK3 activity during time course of heat shock induction. Top panel, gel mobility shift assay; bottom panel, GSK3 activity. D, gel mobility shift assays showing a comparison of heat shock element binding in uninjected (control) and H2O-injected oocytes (upper) and the effects of GSK manipulation on CCAAT-(center) and Sp1-binding activities (lower).

In converse experiments, we inhibited endogenous GSK3 by pretreating cells with LiCl (39) or by overexpression of GBP, a Xenopus protein, which specifically binds to and inactivates GSK3 (32). LiCl and GBP inactivate both GSK3 and - subtypes. Endogenous oocyte GSK3 activity was reduced to less than 10% of the controls after treatment with LiCl (25 mM) or after overexpression of GBP (Fig. 2A). Under non-shock conditions, the DNA-binding activity of HSF1 was unchanged by GBP expression but was significantly increased at different time points during heat shock induction (Fig. 2B). Inhibition by LiCl also resulted in a dose-dependent increase in DNA binding. DNA binding was apparently activated in the absence of heat shock at 50 mM LiCl, suggesting that GSK3 may function to repress HSF1 under non-shock conditions.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Inhibition of GSK3 elevates heat shock-induced DNA binding of HSF1. A, expression of GBP from in vitro synthesized mRNA. Immunoblot of whole cell extracts from oocytes injected with mRNA coding for FLAG-GBP. The inhibition of GSK3 by LiCl and GBP was confirmed using kinase assays as described in Fig. 1. B, gel mobility shift assay of heat-shocked oocytes pretreated with various concentrations of LiCl in OR2 buffer for 1 h before heat shock (upper panel). The pretreated oocytes were heat-shocked at 33 °C for 1 h. Separate sets of oocytes were microinjected with mRNA coding for GBP (lower panel). Quantitation of DNA binding is shown below each panel. NS, non-shocked; HS, heat-shocked.

GSK3 has been hypothesized to facilitate the inactivation of HSF1 transcription and the dispersal of stress granules following heat stress (15). Therefore, we next determined the potential role of GSK3 on the DNA-binding activity of HSF1 during recovery. Inhibition of GSK3 by LiCl or GBP delayed attenuation of DNA-binding activity relative to untreated cells (Fig. 3A). Overexpression of GSK3 appeared to accelerate recovery (Fig. 3A), an opposite effect to what was seen with GSK inhibition. Kinase assays confirmed the magnitude of changes in the levels of GSK3 activity in these experiments (Fig. 3B). The prolonged retention of HSF1 activity in LiCl and GBP inhibition experiments and the accelerated recovery after overexpression suggest that GSK3 functions normally in efficient disassembly of trimers during attenuation. These results, combined with previously reported evidence of elevated GSK3 activity in cells recovering from heat shock (15), suggest that one of the physiological roles of HSF1 phosphorylation by GSK3 might be to inactivate DNA binding and transcription at the later phases of the activation process. Overall, the results of experiments shown in Figs. 1-3 clearly demonstrate that GSK3 represses the DNA-binding activity of HSF1 during and after heat shock.

View larger version (40K): [in this window] [in a new window]   Fig. 3.   Inhibition of GSK3 delays attenuation of HSF1 during recovery from heat shock. A, oocytes pretreated with LiCl (25 mM, 1 h), overexpressing GBP, or overexpressing active GSK3 were heat-shocked at 33 °C for 1 h and then allowed to recover at 18 °C for the times indicated, and the DNA-binding activity of HSF1 was analyzed by gel mobility shift assay. Quantitation of DNA binding is shown below each panel. B, relative GSK3 activity in control and treatment groups (as indicated) during recovery (time of recovery below panel). Kinase activity is expressed as a percentage of uninjected control (100%) and are means of values obtained from five experiments.

GSK 3 Represses HSF1-mediated Transcription during Heat Shock in Vivo-- The role of GSK3 in regulating HSF1-dependent transcription was determined in oocytes by measuring CAT expression from a microinjected reporter construct, hsp70-CAT. This construct was under control of the hsp70 promoter, which is known to be switched on by heat shock and by various other stresses in oocytes (36, 40, 41). The heat shock-induced transcriptional activity of HSF1 was significantly reduced in cells with elevated levels of GSK3 activity that were achieved by microinjection of either mRNA or active kinase (Fig. 4). Kinase-dead GSK3 had no apparent effect on HSF1-mediated transcription. Conversely, inhibition of GSK3 activity by LiCl or microinjection of GBP elevated the levels of HSF1-mediated transcription in heat-shocked cells. The possibility that GSK3 manipulation caused a general inhibition of transcription was ruled out by the controls in which equal expression from the cytomegalovirus promoter was observed after each treatment (Fig. 4). It is possible that the decreased transcription observed in oocytes with elevated GSK3 activity was because of decreased DNA binding; however, in the experiment in which GSK3 was expressed from microinjected mRNA, DNA binding was not completely inhibited (Fig. 1B), whereas the transcription was nearly abolished in similarly treated oocytes (Fig. 4). This suggests that GSK3 has dual effects on DNA binding and transcription. Therefore, these results confirm that GSK3 serves to repress HSF1-mediated transcription under stress conditions in vivo.

View larger version (66K): [in this window] [in a new window]   Fig. 4.   GSK3 modulates HSF1-mediated transcription in heat-shocked cells. CAT assays of oocytes microinjected with hsp70-CAT or cytomegalovirus (CMV)-CAT. Cells were injected with purified GSK3 (0.5 unit), with mRNA coding for active and inactive GSK3, or with mRNA encoding GBP, or oocytes were pretreated with 25 mM LiCl for 1 h before heat shock (HS). In each experiment, the same batch of oocytes was used to minimize variation. Each experiment was repeated at least five times with different batches of oocytes, and representative assays are shown. The lower panel expresses relative heat shock-inducible CAT activity as a ratio between control (untreated) and treated groups. NS, non-shocked.

Intracellular Localization of Endogenous GSK3 during Heat Shock-- We have previously shown that HSF1 is a nuclear protein in oocytes before and after heat shock (42). Therefore, direct phosphorylation of HSF1 by GSK3 would require colocalization of these proteins during activation or deactivation. To assess the intracellular distribution of GSK3 before and after heat shock, nuclear and cytoplasmic extracts from manually enucleated cells were analyzed by immunoblotting and kinase assays. GSK3 protein and GSK3 activity were mostly cytoplasmic under non-shock and heat shock conditions, and they were barely detectable in the nuclei of unshocked cells (Fig. 5). In intact cells, GSK3 activity was elevated by heat shock. The nuclear levels of GSK3 protein were elevated after heat shock, and nuclear kinase activity rose accordingly (Fig. 5). No significant change in GSK3 activity was seen after the inhibition of de novo protein synthesis with cycloheximide in both non-shock and heat shock conditions, which suggests that heat shock up-regulates endogenous kinase activity. However, the increase in nuclear GSK3 activity could have been caused by a combination of nuclear translocation and activation of the enzyme. To demonstrate that the nuclear preparations used in these assays were free of contaminating cytoplasms, we assayed for the presence of IB and PCNA, which are strictly cytoplasmic and nuclear proteins, respectively. The cytoplasmic marker IB was not detected in the nuclear extracts, and PCNA was not detected in cytoplasmic extracts (Fig. 5).

View larger version (41K): [in this window] [in a new window]   Fig. 5.   Intracellular localization of GSK3. Western immunoblot of whole cell, cytoplasmic, and nuclear fractions and detection with anti-GSK3 antibody. Control blots with nuclear (PCNA) and cytoplasmic (IB) marker proteins are also shown. One oocyte equivalent of protein extract was used for whole cell and cytoplasmic samples. One nuclear equivalent was loaded for detection of GSK3 and PCNA, but 10 nuclear equivalents of protein were used for detection of IB. Cycloheximide treatment was at 150 µg/ml in OR2 for 1 h. GSK3 activities are as described in Fig. 1. NS, non-shocked; HS, heat-shocked.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

GSK3 is a proline-directed serine/threonine kinase that was originally discovered as the major kinase-phosphorylating glycogen synthase (43). Various other GSK3 substrates have since been identified, including protein phosphatases, kinases, adhesion molecules, myelin basic proteins, and several transcription factors including c-Jun/AP-1, JunD, c-Myb, c-Myc, L-Myc (44). The results shown here clearly demonstrate for the first time that GSK3 negatively regulates the DNA-binding activity of HSF1 in vivo and confirms that it functions to repress transcription in cells that are exposed to heat shock. It is interesting that GSK3 phosphorylates c-Jun in the DNA-binding domain and down-regulates its DNA-binding activity (45), and that it negatively regulates Myc and Myb family members (46). Therefore, it appears that the inhibitory action of GSK3 may be common to a variety of transcription factors including HSF1.

The role of GSK3 as a negative modulator of HSF1 was supported here by observations that heat-induced DNA binding was inhibited in cells overexpressing GSK3 and conversely that DNA binding was stimulated after the inhibition of GSK3. In addition to the effects on DNA binding, our results confirmed the role of GSK3 in modulating transcription. HSF1-mediated transcription was either decreased after increasing GSK3 or increased after the inhibition of GSK3. In each experiment in which GSK3 was either increased or decreased, we observed consistent and reciprocal effects on HSF1 activities. The degree to which GSK3 is important for HSF1 regulation in vivo is illustrated by the observation that nuclear enzyme injection almost completely inhibited DNA binding in response to heat shock. Transcriptional effects on HSF1 by manipulating GSK3 were likely not due to changes in the ability of HSF1 to form trimers and bind DNA. This was because we observed a complete transcriptional repression but an incomplete repression of DNA binding with 4-fold increases in GSK3 activity. These repressions lead us to suggest that GSK3 modulates both DNA-binding and transcriptional activities and that this could be through targeting at single or multiple sites on the molecule. The conclusion that GSK3 regulates both activities separately is consistent with the uncoupling of DNA binding and transcription as seen when HSF1 is activated by anti-inflammatory agents, such as salicylate and indomethacin (21).

When during the activation-deactivation process does GSK3 exert its influence on HSF1? Previous work suggests that GSK3 might act constitutively to repress HSF1 (13, 14, 16, 30). Consistent with this theory, we found that HSF1 was inhibited in GSK3-overexpressing cells during the early phases of the activation process. We also observed the activation of HSF1-DNA binding under non-shock conditions after inhibiting GSK3 with LiCl, although it is possible that this effect was caused by toxic effects of LiCl. Therefore, it appears that GSK3 represses the DNA-binding and transcriptional activity of HSF1 under normal conditions, and so it is important for the maintenance of the transcriptionally inactive monomeric conformation. However, several observations lead us to suggest that its primary role is in the later stages of heat shock and attenuation. Our experiments showed that the total level of GSK3 activity had increased in response to heat shock well after HSF1 was activated, the nuclear levels of GSK had increased after 1 h of heat shock, and the inhibition of GSK3 delayed attenuation during recovery. Our observations are consistent with the findings of He et al. (15) that show the localization of GSK3 to stress granules in vivo and the disappearance of transcriptionally active HSF1 granules. The current data suggest that GSK3 is required for the efficient recovery of HSF1 following resumption of normal conditions.

What is the mechanism by which GSK3-mediated phosphorylation represses HSF1? GSK3 has been shown to phosphorylate human HSF1 at serine 303 (13, 14), and so it is likely that it acts directly on HSF1 in oocytes. As with several other GSK3 substrates (47, 48), phosphorylation at this site on HSF1 depends on the hierarchical phosphorylation at an upstream site, serine 307, by ERK1/2 (13, 14). Sequence alignment of human and Xenopus HSF1 (Fig. 6) reveals that the (Thr-286) of frog aligns with serine 303 and matches the consensus phosphorylation site for GSK3 ((S/T)*XXXpS) (47, 48). The effects of GSK3 manipulation observed in these experiments could have been through phosphorylation at this site or other site(s) in the Xenopus HSF1 molecule. It will be interesting to address whether the dual regulation of HSF1 by GSK3 involves targeting of single or multiple sites and to determine the dynamics of phosphorylations throughout the activation profile.

View larger version (32K): [in this window] [in a new window]   Fig. 6.   Sequence alignment of human and Xenopus HSF1. Human serine 303 aligns with threonine 286 as indicated by the closed triangle.

We hypothesize that GSK3 acts as the terminal kinase of several stress signal transduction pathways that regulate HSF1 activity. GSK3 is known to be the terminal effector kinase of a number of signal transduction pathways. The upstream activators of GSK3 have not been identified, but it has been shown that the phosphatideylinositol 3-kinase-PKB pathway, p70^S6K, p90^rsk, and certain isotypes of PKC phosphorylate and down-regulate GSK3 (49-51). Of them, PKC and PKB are known to be activated by stress, but only PKC has been implicated as a negative regulator of HSF1. Treatment of human erythroleukemia K562 cells with 12-O-tetradecanoylphorbol 13-acetate), a specific activator of PKC, has been shown to enhance heat-induced activity and accelerate attenuation (52), and this effect was abolished by the concurrent treatment of cells with a specific PKC inhibitor (53). In addition, PKC has been shown to directly phosphorylate human HSF1 near the transcriptional activation domain and inhibit its transcription (14). It is difficult with the current evidence to determine the relationship between HSF1, PKC, and GSK3 at different phases of the stress response. It is interesting that PKC and GSK3 are both activated by stress and are negative regulators of HSF1. Therefore, initial events in the activation of HSF1 probably involve integration of multiple signals and a complex series of hsp90 chaperone interactions (5-7).

Complete understanding of the role of HSF1 phosphorylation will require incorporation of the effects of other kinases known to phosphorylate HSF1, such as ERK1/2 and PKC. What remains to be answered is whether the constitutively phosphorylated sites in the regulatory domain are actually modified during stress or if GSK3 acts through other sites on the HSF1 molecule. This is part of the larger question concerning the role of multiple signaling pathways and phosphorylation events in the activation and attenuation of HSF1.

	ACKNOWLEDGEMENTS

We thank D. Kimelman for GBP expression vector and A. Wolffe for CAT constructs.

	FOOTNOTES

^* This work was supported by Medical Research Council (MRC) Grants MT-13110 (to N. O.) and MT-12043 (to J. R. W.), postdoctoral fellowship scholarships from Health Services Utilization and Research Commission (to I. J. X.) and Natural Sciences and Engineering Research Council (NSERC) (to A. A.), a MRC graduate scholarship (to P. M.), and a NSERC summer studentship (to C. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Anatomy and Cell Biology, College of Medicine, University of Saskatchewan, 107 Wiggins Rd., Saskatoon, Saskatchewan S7N 5E5, Canada. Tel.: 306-966-4069; Fax: 306-966-4298; E-mail: ovsenekn@duke.usask.ca.

Published, JBC Papers in Press, June 15, 2000, DOI 10.1074/jbc.M002169200

	ABBREVIATIONS

The abbreviations used are: hsp, heat shock protein; HSF1, heat shock factor; GSK3, glycogen synthase kinase; ERK1/2, extracellular signal-regulated kinase; GBP, GSK3-binding protein; CAT, chloramphenicol acetyltransferase; PCR, polymerase chain reaction; PK, protein kinase; PCNA, proliferating cell nuclear antigen.

	REFERENCES

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