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(The FASEB Journal. 1998;12:451-459.)
© 1998 FASEB

RESEARCH COMMUNICATION

Overexpression of HSP-70 inhibits the phosphorylation of HSF1 by activating protein phosphatase and inhibiting protein kinase C activity

Xuan Z. Dinga, George C. Tsokosa, and Juliann G. Kianga,1

a Department of Clinical Physiology, Division of Medicine, Walter Reed Army Institute of Research, Washington, DC 20307–5100, USA


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This laboratory reported previously that overexpressed heat shock protein 70 kDa (HSP-70) inhibited the activation of its transcriptional factor, HSF1. We had conducted experiments to understand the mechanisms whereby HSP-70 down-regulated the activation of HSF1. Genetically overexpressed HSP-70 had no effects on the HSF1 level in cytosol, but significantly inhibited phosphorylation of HSF1 in the nucleus. Transfection of cells with HSF1 cDNA resulted in increases in the unphosphorylated, but not phosphorylated, HSF1 levels in both the cytosol and nucleus. Because serine phosphorylation of various proteins was reduced in HSP-70 cDNA-transfected cells, we measured the activity of enzymes involved in serine phosphorylation. Overexpressed HSP-70 significantly inhibited the enzymatic activities of protein kinase A (PKA by 73 and 62% in the cytosol and membrane-bound fraction, respectively) and protein kinase C (PKC by 61% in membrane-bound fraction), whereas it activated that of protein phosphatase (PP by 33 and 86% in the cytosol and the membrane-bound fraction, respectively). Forskolin (a PKA stimulator), PMA (a PKC stimulator), and okadaic acid (an inhibitor of PP) were used to investigate whether HSP-70-induced changes in PKA, PKC, and PP were responsible for the HSF1 dephosphorylation. Forskolin did not change nuclear HSF1 phosphorylation, suggesting that decreases in PKA activity in HSP-70 overexpressing cells is not associated with HSF1 phosphorylation. PMA and okadaic acid induced an increase in HSF1 phosphorylation in both vector- and HSP-70 cDNA-transfected cells, although levels of phosphorylated HSF1 in HSP-70 cDNA-transfected cells were lower than those in vector-transfected cells. The PMA-induced increase in HSF1 phosphorylation in HSP-70 cDNA-transfected cells was blocked by pretreatment with staurosporine, a PKC inhibitor. These results suggest that overexpression of HSP-70 inhibits phosphorylation of HSF1 at serine residues by activating PP and inhibiting PKC activity.—Ding, X. Z., Tsokos, G. C., Kiang, J. G. Overexpression of HSP-70 inhibits the phosphorylation of HSF1 by activating pro~tein phosphatase and inhibiting protein kinase C activity FASEB J. 12, 451–459 (1998)


Key Words: gene transfection • heat shock protein • gene transcription • PP activity • PKC activity


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HEAT SHOCK PROTEIN 70 kDa (HSP-70)2 has been shown to be widely present in prokaryotic and eukaryotic cells and functions as an intracellular molecular chaperone (1, 2). Overexpression of HSP-70 protects cells, tissues, and organs from harmful assaults such as lethal temperature (3). It has been reported that no additional HSP-70 synthesis occurs if the cells are reexposed to the same or a different type of stimulation (4). One possible mechanism for this retardation of HSP-70 synthesis is that overexpression of HSP-70 down-regulates its gene transcription and expression. HSP-70 gene transcription is initiated by a group of transcriptional factors named heat shock factors (HSFs) (5). Among these HSFs, HSF1 is known to have a binding domain for the promoter region of the HSP-70 gene and to be responsible for the heat shock-induced increase in HSP-70 gene expression (6–8). Like other transcription factors, HSF1 needs to be activated before promoting HSP-70 gene expression (5, 6). The activation of HSF1 involves a series of processes including phosphorylation, translocation from the cytosol to the nucleus, formation of a trimer, binding to heat shock elements (HSE), and initiating HSP-70 gene expression (5, 9–13). Recently, our laboratory and others found that overexpression of HSP-70 elicits an inhibitory effect on HSF1 activation (14–18). This down-regulatory effect of HSP-70 on HSF1 probably explains why no additional induction of HSP-70 is observed in HSP-70 overexpressing cells after stimulation. However, the mechanism whereby HSP-70 down-regulates HSF1 activation is not clear.

Phosphorylation–dephosphorylation is essential for cellular homeostasis. For example, an abnormal ratio of phosphorylation–dephosphorylation may lead to apoptosis (19–21). Protein phosphorylation that occurs at serine and threonine residues is mediated by two major protein kinases, cAMP-dependent protein kinase A (PKA) and the calcium- and phospholipid-dependent protein kinase C (PKC; 22–24). The enzyme that catalyzes dephosphorylation is protein phosphatase (PP) (25, 26). PKs and PP have both been found to be involved in the phosphorylation–dephosphorylation of HSPs and HSFs (27–40). Furthermore, PKC and intracellular Ca2+ have been known to be involved in the regulation of HSF1 phosphorylation and activation (41, 42). Therefore, we hypothesized that inhibition of HSP-70 synthesis in HSP-70 overexpressing cells is a result of decreased HSF1 phosphorylation due to altered activities of PKs and PP.

In this study, we transfected HSP-70 cDNA into human epidermoid A-431 cells and investigated the ability of overexpressed HSP-70 to regulate HSF1 phosphorylation. We found that the increased HSP-70 attenuated protein phosphorylation, including HSF1 phosphorylation, at serine residues by inhibiting the enzymatic activity of PKC and increasing that of PP.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
Human epidermoid A-431 cells (American Type Culture Collection, Rockville, Md.) were grown in 150 cm2 flasks incubated at 37°C in an atmosphere of 5% CO2 in air. The tissue culture medium was Dulbecco's modified Eagle medium containing 0.03% glutamine, 4.5 g/l glucose, 25 mM HEPES, 10% fetal bovine serum, 50 µg/ml penicillin, and 50 U/ml streptomycin (Gibco BRL, Gaithersburg, Md.). Cells were fed every 3–4 days; cells from passages 28–45 were used for experiments. All experiments were performed at least three times.

Gene transfection
The full length of HSP-70 cDNA (a gift from Dr. Richard I. Morimoto) was excised with enzymes Bam HI and Eco.RI from pH 2.3 plasmid (43). The HSF1 cDNA in pCMVHuHSFB plasmid (a gift from Dr. Carl Wu) was excised with enzymes Eco.RI and Bgl II (44). The vector used to carry these cDNAs was pcDNA3 (Invitrogen Co., San Diego, Calif.). This 5.4 kb of vector has enhancer-promoter sequences from the human cytomegalovirus gene for high-level transcription, and has a polyadenylation signal and transcription terminal sequence derived from the bovine growth hormone gene to enhance RNA stability. The neomycin resistance gene on the vector was used for later selection of geneticin (G418) -resistant stable cell lines.

Transfection of HSP-70 and HSF1 cDNAs into A-431 cells was conducted in a six-well plate using the calcium phosphate kit (Gibco BRL). Fifteen micrograms of either vector DNA or vector containing HSP-70 or HSF1 cDNA in HBSP solution containing 12 mM CaCl2 was added to each well of cells. Cells were incubated with the cDNAs or vector at 37°C for 16 h, washed twice with media, and incubated in regular media for the next 48 h. Subsequently, these cells were incubated with 200 µg/ml of geneticin (G418) for further selection of transfected cells. The surviving cells in media containing G418 were considered to be successfully transfected cells that contained the neomycin-resistant gene.Trypan blue exclusion assay indicated that these cDNA-transfected cells were viable.

Reverse transcription-polymerase chain reaction (RT-PCR)
The mRNA of HSP-70 and HSF1 was measured using a reverse transcriptase PCR method. Briefly, confluent monolayers of A-431 cells (1–5x106) were trypsinized and extracted with RNA SATA-60 kit (TEL-TEST `B', Inc., Friendswood, Tex.) according to the manufacturer's protocol. The total RNA amount and purity were assessed by measuring optical densities at 260/280 nm. One microgram of total RNA was used for reverse transcription. Three respective pairs of primers specific for HSF1, HSP-70, and ß-actin were designed and synthesized according to their sequences. All of the primers were 20 bases in length and contained 50% each of C+G and A+T (7). Thirty cycles (95°C for 1 min, 54°C for 1 min, and 72°C for 1.5 min) were chosen for all PCR reactions based on our previous experiments (7). After amplification, the same volume of each PCR product was loaded onto 1% agarose gels prepared with TBE buffer. The gel was stained with 2 µl ethidium bromide (100 mg/ml) and photographed.

Immunocytochemistry staining of intracellular HSP-70
A-431 cells were washed twice with phosphate-buffered saline (PBS) containing 1% bovine serum albumin (or fetal calf serum) and 0.1% sodium azide. The cells were fixed with PBS containing 4% paraformaldehyde on ice for 20 min and washed with buffer containing 0.1% sapollin. The cells were incubated with mouse monoclonal antibody against human HSP-70 (1:500; Amersham, Arlington Heights, Ill.) at 37°C for 30 min, washed twice with PBS, and then incubated with goat anti-mouse immunoglobulin G (IgG) fluorescein-conjugated antibody (1:1000) at 37°C for 30 min. The cells were washed twice with PBS and fixed with PBS containing 1% formalin. The cells were viewed under a fluorescence microscope.

Western blotting
Samples were isolated and purified according to a previously described method (42). Samples (35 µg protein) were separated by electrophoresis using 10% polyacrylamide gels. Proteins were electroblotted onto nitrocellulose membranes (Schleicher and Schuell, Keene, N.H.). The blots were incubated with mouse monoclonal antibody against human HSP-70 (Amersham) overnight at 4°C. After washing with PBS, the blots were incubated with rabbit anti-mouse IgG peroxidase-conjugated antibody (Sigma Chemical Co., St. Louis, Mo.) for 2 h at room temperature. The blots were washed and incubated with Western blot chemiluminescence reagent (DuPont-NEN, Boston, Mass.) for 1 min at room temperature and then exposed to X-ray film. HSP-70 levels were determined by laser densitometry.

Activity assays of PKA, PKC, and PP
The samples were extracted from both cytosol and membrane-bound fractions according to the manufacturer's protocol provided in the assay kits (Gibco BRL). The cells were homogenized in extraction buffer without detergent and centrifuged at 100,000 g for 30 min. The supernatant contained the cytosolic enzyme and the pellet contained the membrane-bound enzyme. The amount of protein in the samples was measured with the Bio-Rad reagent (Bio-Rad laboratories, Richmond, Calif.). The activities of PKA, PKC, and PP were measured using commercial assay kits (Gibco BRL). The PKA activity was measured by phosphorylation of a specific substrate, Leu-Arg-Arg-Ala-Ser-Leu-Gly (45); the enzymatic activity of PKC was measured by phosphorylation of Ac-MBS(4–14), as described by Yasuda et al. (46); the PP assay (mainly PP-1 and PP-2A) was measured by the dephosphorylation of phosphorylase (47). {gamma}-[32P]ATP (6,000 Ci/mmol stock solution) was used in this study. The substrate for PKA and PKC was incubated with protein samples at 30°C for 5 min and then transferred to the phosphocellulose disc. The disc was washed twice for 3–5 min with phosphoric acid solution and dried. The radioactivity was measured with automatic liquid scintillation counter. For PP assay, 20 µl of each protein sample, assay buffer, and 32P-labeled phosphorylase were mixed and incubated at 30°C for 10 min. The reaction was stopped by adding 180 µl 20% trichloroacetic acid and centrifuged at 12,000 g for 3 min at 4°C. The radioactivity with 200 µl of the supernatant was measured. The activities of enzymes were calculated according to the manufacturer's protocol and expressed as percent of vector-transfected cells.

Chemicals
Chemicals used in this study were phorbol 12-myristate 13-acetate, staurosporine, okadaic acid (Sigma Co.), forskolin (Calbiochem, La Jolla, Calif.), and {gamma}-[32P]ATP (DuPont-NEN, Boston, Mass.).

Statistical analysis
All data are expressed as mean ±SEM. Analysis of variance, Newman-Keuls, and Student's t test were used to compare groups. P < 0.05 was the significance level used for the study.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Overexpression of HSP-70 induced by cDNA transfection in human epidermoid A-431 cells
HSP-70 cDNA was transfected into human epidermoid A-431 cells in order to investigate the effect of overexpression of HSP-70 on HSF1 activation. Vector-transfected cells were used as a control. To determine whether HSP-70 was overexpressed in A-431 cells, immunofluorescent staining was conducted in vector- and HSP-70 cDNA-transfected cells. Figure 1A shows that a basal level of HSP-70 was detected in the cytosol of vector-transfected cells. In cDNA-transfected cells, HSP-70 was also present in the cytosol, and the level was significantly greater than that in the vector-transfected cells. The increased HSP-70 protein was distributed mainly in the cytosol around the nucleus ( Fig. 1B). To further confirm the presence of HSP-70 overexpressing in cDNA-transfected cells, the levels of mRNA and protein of HSP-70 were measured by the RT-PCR method. The mRNA amount of HSP-70 in HSP-70 cDNA-transfected cells significantly increased when compared to that in vector-transfected cells ( Fig. 2A). The increase in HSP-70 mRNA was accompanied by an increase in the protein level of HSP-70 ( Fig. 2B) detected with Western blot analysis. These results indicate that HSP-70 cDNA transfection induces a significant increase in both gene transcription and translation of HSP-70.



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  		Figure 1. Immunofluorescent staining of intracellular HSP-70 in HSP-70 cDNA-transfected epidermoid A-431 cells. Immunofluorescent staining was performed using a monoclonal antibody against HSP-70 in vector- (A) and HSP-70 cDNA-transfected cells (B) as described in Materials and Methods. a) nucleus; b) cytosol.



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  		Figure 2. Confirmation of HSP-70 cDNA transfection into human epidermoid A-431 cells. Transfection was carried out as described in Materials and Methods. A) mRNA amounts of vector- (lane 2) and HSP-70 cDNA-transfected (lane 3) cells were measured with the RT-PCR method. Lane 1 displays {phi}X174 DNA/HaeIII markers. B) Protein amounts of HSP-70 in vector- and HSP-70 cDNA-transfected cells were detected with Western blot analysis.

Overexpression of HSP-70 inhibits HSF1 phosphorylation and translocation
We (31) and others (27–30) previously reported that HSF1 required phosphorylation before inducing HSP-70 production. To investigate the effect of overexpression of HSP-70 on HSF1 phosphorylation and translocation from the cytosol to the nucleus, the amounts of HSF1 were measured in both cytosolic and nuclear extracts by immunoblotting with a specific antibody against the HSF1. Cells were transfected with the empty vector, HSP-70 cDNA, HSF1 cDNA, or both. One band in the cytosol and two bands in the nucleus were observed in the vector-transfected cells ( Fig. 3). The upper band represents a phosphorylated form of HSF1 because it disappeared after treatment with phosphorylase (42). The lower band represents an unphosphorylated form of HSF1. In HSP-70 cDNA-transfected cells, the amount of unphosphorylated HSF1 in the cytosol was slightly greater than that in vector-transfected cells. However, the unphosphorylated HSF1 level in the nucleus of HSP-70 cDNA-transfected cells was lower than that in the vector-transfected cells and no phosphorylated HSF1 was found in the nucleus, suggesting that HSF1 translocation and phosphorylation are inhibited. Transfection of cells with the HSF1 cDNA increased the amounts of unphosphorylated HSF1 in both the cytosol and the nucleus, but the levels of phosphorylated HSF1 in the nucleus were not altered. In cells transfected with both cDNAs, an increase in cytosolic unphosphorylated HSF1 was found, but smaller amounts of unphosphorylated and phosphorylated HSF1 in the nucleus were detected when compared with that in HSF1 cDNA-transfected cells. These results are in agreement with those found in cells transfected with HSP-70 cDNA and reinforce the view that HSP-70 inhibits the HSF1 translocation and phosphorylation.



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  		Figure 3. Inhibition of HSF1 phosphorylation by overexpression of HSP-70. Cells were transfected with empty vector, HSP-70, HSF1, or both HSP-70 and HSF1 cDNAs. Nuclear and cytosolic proteins were both extracted. The amount of HSF1 in the nucleus and cytosol was measured using Western blots. Upper bands represent the phosphorylated form of HSF1 and lower bands represent the unphosphorylated form of HSF1.

The effect of overexpressed HSP-70 on activities of PKA, PKC, and PP
Protein phosphorylation generally occurs at serine, threonine, or tyrosine residues. Phosphorylation of these residues is accomplished by different enzymes. We previously reported that PMA (a PKC stimulator) increased HSF1 phosphorylation (31). To determine which enzyme is involved in the HSP-70-induced inhibitory effect on HSF1 phosphorylation, protein samples were extracted from vector-, HSP-70 cDNA-, or HSF1 cDNA-transfected cells. Serine phosphorylation was assessed with a monoclonal antibody against phosphoserine. Figure 4 shows that HSP-70 cDNA transfection reduced the amount of serine phosphorylation (lane 2). No changes in serine phosphorylation were found in the HSF1 cDNA-transfected cells (lane 3). Treatment of vector-transfected cells with 1 µM PMA, a specific PKC activator, increased serine phosphorylation (lane 4). PMA also slightly increased serine phosphorylation in HSP-70 cDNA-transfected cells (lane 5). Threonine phosphorylation was also assessed with a monoclonal antibody against phosphothreonine. No changes in threonine phosphorylation were found in vector-, HSP-70, or HSF1 cDNA-transfected cells (data not shown). These results suggest that overexpression of HSP-72 inhibits protein phosphorylation at serine residues.



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  		Figure 4. Attenuation of serine phosphorylation by overexpression of HSP-70. Cells were transfected with vector (lane 1), HSP-70 cDNA (lane 2), or HSF1 cDNA (lane 3), followed by treatment with 1 µM PMA for 10 min (lanes 4 and 5). Whole-cell proteins were separated electrophoretically. The amount of serine phosphorylation was determined with a monoclonal antibody against phosphoserine.

The enzymes involved in serine phosphorylation and dephosphorylation include PKC, PKA, and PP. To evaluate whether any of these enzymes was involved in decreasing HSF1 phosphorylation, their activities were measured in both vector- and HSP-70 cDNA-transfected cells. Figure 5 shows that HSP-70 cDNA transfection decreased PKA activity in the cytosolic and the membrane-bound fractions by 73 and 62%, respectively. PKC activity was reduced by 61% in the membrane-bound fraction but was not changed in the cytosolic fraction. The cytosolic and membrane-bound PP activities increased by 33 and 86%, respectively. These results indicate that overexpression of HSP-70 inhibits the activities of both PKA and PKC, whereas it stimulates the PP activity.



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  		Figure 5. Decrease in PKA and PKC activities and increase in PP activity by overexpression of HSP-70. Cells were transfected with vector or HSP-70 cDNA. The cytosolic and membrane-bound proteins were extracted and the activities of PKA, PKC, and PP were measured. The experiment was conducted four times independently. The data are presented as percent of enzymatic activities found in vector-transfected cells. *P < 0.05 vs. vector-transfected cells, determined by the Student's t test. Cytosol fraction ({square}); membrane-bound fraction ({blacksquare}).

The inhibitory effect of overexpressed HSP-70 on HSF1 phosphorylation was mediated by decreasing PKC activity and increasing PP activity
To further determine whether HSP-70-induced changes in the enzymatic activity of PKA, PKC, and PP were resposible for the down-regulation of HSF1 phosphorylation, we investigated the effect of PMA, forskolin (a PKA activator), and okadaic acid (a PP inhibitor) on HSF1 phosphorylation in HSP-70 cDNA-transfected cells. Both vector- and HSP-70 cDNA-transfected cells were incubated with PMA (1 µM, 30 min), forskolin (150 µM, 15 min), or okadaic acid (0.5 µM, 30 min). HSF1 phosphorylation levels were measured in the nucleus of vector- and HSP-70 cDNA-transfected cells by using immunoblotting method with a specific antibody against HSF1. Figure 6 shows that forskolin did not change the phosphorylated HSF1 level in the nucleus of either vector-transfected or HSP-70 cDNA-transfected cells, suggesting that the decrease in the PKA activity found in HSP-70 cDNA-transfected cells is not associated with HSF1 phosphorylation. However, PMA induced an increase in phosphorylated HSF1 in the nucleus of both vector-transfected and HSP-70 cDNA-transfected cells. Treatment with okadaic acid also induced an increase in phosphorylated HSF1 in the nucleus of vector- and HSP-70 cDNA-transfected cells, whereas the nuclear unphosphorylated HSF1 in these two types of cells was decreased (data not shown). The amounts of phosphorylated HSF1 increased more in vector-transfected cells than in HSP-70 cDNA-transfected cells ( Fig. 6). These results suggest that the decrease in PKC activity and the increase in protein phosphatase activity are involved in the regulation of HSF1 phosphorylation observed in HSP-70 overexpressing cells.



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  		Figure 6. The effect of stimulators of PKA and PKC and an inhibitor of PP on HSF1 phosphorylation in vector- and HSP-70 cDNA-transfected cells. Cells were treated with forskolin (150 µM, 15 min), PMA (1 µM, 30 min), or okadaic acid (0.5 µM, 30 min) after transfection with vector or HSP-70 cDNA. The level of HSF1 phosphorylation present in the nucleus of vector- and HSP-70 cDNA-transfected cells was measured using Western blots. The experiment was conducted four times independently. The data represent percent of the phosphorylated HSF1 level found in vector-transfected cells. *P < 0.05 vs. untreated vector-transfected cells; **P < 0.05 vs. untreated vector- and HSP-70 cDNA-transfected cells; ***P < 0.05 vs. untreated vector- and HSP-70 cDNA-transfected and respective treated vector-transfected cells.

To further determine whether the PMA-induced increase in HSF1 phosphorylation after HSP-70 cDNA transfection was specific, HSP-70 cDNA-transfected cells were treated with staurosporine (a PKC inhibitor) at 0.2 µM for 15 min prior to 1 µM PMA for 30 min. Figure 7 shows that treatment with staurosporine alone had no effect on HSF1 phosphorylation in these cells. However, the PMA-induced increase in the amount of phosphorylated HSF1 in the nucleus was blocked if cells had been pretreated with staurosporine. These results indicate that PKC is specifically involved in the HSP-70-mediated down-regulation of HSF1 phosphorylation.



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  		Figure 7. Staurosporine inhibits the PMA-induced phosphorylation of HSF1 in HSP-70 cDNA-transfected cells. HSP-70 cDNA-transfected cells were treated with staurosporine at 0.2 µM for 15 min prior to PMA (1 µM, 30 min). The nuclear proteins were extracted. HSF1 phosphorylation was measured by Western blot analysis. The experiment was conducted four times independently. The data represent percent of the phosphorylated HSF1 level found in vehicle-treated, HSP-70 cDNA-transfected cells. *P < 0.05 vs. vehicle-treated, HSP-70 cDNA-transfected cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study demonstrates that HSP-70 protein inhibits translocation of unphosphorylated HSF1 from the cytosol to the nucleus as well as the phosphorylation of HSF1 in the nucleus in human epidermoid A-431 cells. Together with the previous observation that HSP-70 inhibits the binding of HSF1 to the HSE (31), our data suggest that overexpression of HSP-70 leads to decreased HSP-70 gene transcription. The accumulated evidence suggests the presence of a negative feedback mechanism that prevents overproduction of HSP-70. This negative feedback mechanism also explains many well-established phenomena, including the inhibition of HSP-70 production after repetitive exposure of cells to heat stress (4, 14–17). In addition, the inhibitory effect of HSP-70 on the HSF1 phosphorylation in HSP-70 overexpressing cells is still present even if the cells express increased amounts of HSF1 ( Fig. 3, lane 4).

HSP-70 has been shown to affect the function of Ca2+/Na+ exchanger (4), steroid receptors (48), Ras, Raf, and pp60v-src kinase (49, 50). Our data show that HSP-70 decreases the amounts of phosphorylated HSF1 by altering the balance between protein kinase and protein phosphatase activities. The inhibition of HSF1 phosphorylation by HSP-70 is associated primarily with decreased phosphorylation at serine residues. A search for altered phosphorylation patterns at threonine residues gave equivocal results (data not shown). We therefore conducted experiments to investigate whether the activities of enzymes involved in phosphorylation and dephosphorylation of proteins are altered in HSP-70 overexpressing cells. Specifically, we investigated the activities of serine/threonine kinases PKA and PKC. Decreased phosphorylation of HSF1 at serine residues by HSP-70 is supported by the fact that immunoblots of electrophoretically separated proteins from HSP-70 cDNA-transfected cells, but not from vector- and HSF1 cDNA-transfected cells with an anti-phosphoserine antibody, revealed the decreased intensity of various bands ( Fig. 4). The inhibitory effect of HSP-70 can be overridden by PMA, because treatment of HSP-70 cDNA-transfected cells with the protein kinase C activator PMA resulted in increased serine phosphorylation. The PMA data along with the fact that the membrane-bound PKC activity in HSP-70 overexpressing cells was decreased by 60% ( Fig. 5) provide additional support that PKC is associated with decreased phosphorylation of the serine residues.

Although HSP-70 cDNA-transfected cells exhibited decreased PKA activity, we do not think that PKA is involved in the process of down-regulating HSF1 phosphorylation. This assumption is based on the finding that the PKA stimulator forskolin did not alter the level of the HSF1 phosphorylation in HSP-70 cDNA-transfected cells. In other studies (51), we found that HSP-70 inhibits phosphorylation of cytosolic proteins at tyrosine residues. Therefore, HSP-70 is likely to exert an indiscriminate down-regulating effect on the activities of various protein kinases. This view is corroborated by a recent report that indicates a reduction of the stress kinases Jun amino-terminal kinase and p38 kinase (52). The inhibition of HSF1 phosphorylation by HSP-70 apparently represents part of a gen~eralized effect on the phosphorylation pattern of cytosolic and nuclear proteins.

In contrast to the reduction of the protein kinase activity, the activity of protein serine/threonine phosphatases PP-1 and PP-2A in HSP-70 overexpressing A-431 cells was increased. The overall effect on these cells resulting from this shift of protein kinases-protein phosphatases balance activity is not clear. However, in T lymphocytes, this shift has been implicated in the induction of the biochemical and ultrastructural events that characterize apoptosis (53). We have recently shown that HSP-70 overexpression elevates PP-1 and PP-2A activities and promotes TCR/CD3- and FAS/Apo-1/CD95-mediated apoptotic cell death (51). On the contrary, overexpression of HSP-70 in neonatal dorsal root ganglion does not alter the apoptosis induced by serum-free medium (54). In another report, it was shown that HSP-70 and HSP-27 decrease the apoptosis of monoblastoid U937 cells and murine fibrosarcoma Wehi-s cells that was induced by actinomycin-D, camptothecin, and etoposide (55). The discrepancy between the studies mentioned above may be attributed to differences in the cell types or stimuli used to induce apoptotic cell death.

It is believed that the well-balanced ratio between HSF1 phosphorylation and dephosphorylation maintains the basal level of HSP-70 and serves various functions, including protein folding, unfolding, aggregation, and translocation (1, 5, 6). The fact that overexpression of HSP-70 increased PP activity and the PP inhibitor okadaic acid increased HSF1 translocation and phosphorylation in the nucleus suggests that dephosphorylation of HSF1 occurs. The physiological significance of HSF1 phosphorylation is the initiation of HSP-70 production, thereby enhancing cell survival despite detrimental assaults. HSF1 dephosphorylation is thus thought to contribute to the maintainance of HSP-70 cell homeostasis and ensures only a transient increase in the number of HSP-70 responding to stressors.

Other heat shock proteins have been reported to regulate the protein phosphorylation–dephosphorylation process. HSP-90, HSP-59, and HSP-70 are known to be involved in stabilizing the steroid/thyroid receptors in a dephosphorylated form in the cytoplasm (48). HSP-90 and HSP-50 are also known to regulate the activity of kinases such as Ras, Raf, and pp60v-src kinase (49, 50). The mechanism whereby HSP-70 reduces PK activity and increases PP activity needs to be elucidated. We have shown that in HSP-70 overexpressing cells, a new steady state of Na+/Ca2+ exchanger activity is established and Ca2+-related machinery is altered (4). It is possible that HSP-70 exerts its action on these enzymes either directly by altering their Km and Vmax or indirectly through other, undetermined processes. Because PKC and PP are Ca2+ dependent, it is possible that the altered Ca2+ homeostasis in HSP-70 overexpressing cells may contribute at least partially to the PKC and PP activities.

Data presented in this study suggest the existence of a mechanism whereby the levels of HSP-70 produced in response to stressors are autoregulated ( Fig. 8). Stressors such as environmental, pathological, and physiological stressors cause an immediate increase in [Ca2+]i as a result of Ca2+ influx (step 1, 56), which activates PKC activity and inhibits PP activity (step 2). The activation of PKC and inhibition of PP activate HSF1 phosphorylation (step 3) and increase binding HSF1 to HSE (step 4), leading to increased HSP-70 expression (step 5). This overexpression of HSP-70 is capable of inhibiting the increase in [Ca2+]i (step 6; 51, 57) and subsequent steps.



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  		Figure 8. Proposed autoregulation of HSP-70 through PKC and PP. Stressors cause an immediate increase in cytosolic free Ca2+ concentration [Ca2+]i (step 1), which activates PKC activity and inhibits PP activity (step 2). This leads to HSF1 phosphorylation (step 3) and binding to HSE (step 4), which induces an increase in HSP-70 production (step 5). This overexpression of HSP-70 is capable of inhibiting the increase in [Ca2+]i (step 6) and subsequent steps.

Increased levels of various types of HSPs have been found in cells and tissues from patients with autoimmune (58) and degenerative diseases (59). Reduction of protein kinase activity and the elevation of protein phosphatase activity are expected on the basis of findings in this study. It becomes very critical to balance the ratio of protein phosphorylation and dephosphorylation under these pathological situations. Therefore, methods that can be used to maintain a balance between protein phosphorylation and dephosphorylation may provide a tool in controlling the disease process.

In summary, overexpression of HSP-70 in HSP-70 cDNA-transfected cells decreased unphosphorylated HSF1 translocation, inhibited nuclear HSF1 phosphorylation at serine residues, reduced PKA and PKC activities, and increased PP activity. The inhibition of HSF1 phosphorylation was reversed by treatment with PMA or okadaic acid. In conclusion, HSP-70 down-regulates HSF1 phosphorylation in the nucleus at serine residues by activating PP activity and inhibiting PKC activity.


   ACKNOWLEDGMENTS
 
The authors thank Drs. R. I. Morimoto and Carl Wu for providing human HSP-70 and HSF1 genes. This work was supported by DOD RAD II STO C. The views presented in this paper are those of the authors; no endorsement by the Department of the Army or the Department of Defense has been given or should be inferred.


   FOOTNOTES
 
1 Correspondence: Department of Clinical Physiology, Division of Medicine, Bldg. 40, Walter Reed Army Institute of Research, Washington, DC 20307–5100, USA.

2 Abbreviations: PBS, phosphate-buffered saline; HSP-70, heat shock protein 70 kDa; HSF, heat shock factor; HSE, heat shock elements; PKA, protein kinase A; PP, protein phosphatase; Ig, immunoglobulin; RT-PCR, reverse transcription-polymerase chain reaction.

Received for publication November 2, 1997. Accepted for publication December 11, 1997.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Morimoto, R., Tissieres, A., and Georgopoulos, C. (1994) Progress and perspectives on the biology of heat shock proteins and molecular chaperones. In The Biology of Heat Shock Proteins and Molecular Chaperones (Morimoto, R. I., et al., eds) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. pp. 1–30
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