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J. Biol. Chem., Vol. 277, Issue 22, 19913-19921, May 31, 2002

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Phosphorylation-dependent Cellular Localization and Thermoprotective Role of Heat Shock Protein 25 in Hippocampal Progenitor Cells^*

Dongho Geum, Gi Hoon Son, and Kyungjin Kim^§

From the School of Biological Sciences, Seoul National University, Seoul 151-742, Korea

Received for publication, May 15, 2001, and in revised form, March 5, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The present study examined phosphorylation-dependent cellular localization and the thermoprotective role of heat shock protein (HSP) 25 in hippocampal HiB5 cells. HSP25 was induced and phosphorylated by heat shock (at 43 °C for 3 h). HSP25, which was located in the cytoplasm in the normal condition, translocated into the nucleus after the heat shock. Transfection experiments with hsp27 mutants in which specific serine phosphorylation residues (Ser⁷⁸ and Ser⁸²) were substituted with alanines or aspartic acids showed that phosphorylation of HSP27 is accompanied by its nuclear translocation. Phosphorylation of mitogen-activated protein kinases (MAPKs) such as p38 MAPK and ERK was markedly increased by the heat shock, and SB203580 (a p38 MAPK kinase inhibitor) and/or PD098059 (a MEK inhibitor) inhibited the phosphorylation of HSP25, indicating that p38 MAPK and ERK are upstream regulators of HSP25 phosphorylation in the heat shock condition. In the absence of heat shock, actin filament stability was not affected by SB203580 and/or PD098059. Heat shock caused disruption of the actin filament and cell death when phosphorylation of HSP25 was inhibited by SB203580 and/or PD098059. In addition, actin filament was more stable in Asp^78,82-hsp27 (mimics the phosphorylated form) transfected HiB5 cells than in the normal and Ala^78,82-hsp27 (nonphosphorylative form) transfected cells. In accordance with actin filament stability, the survival rate against the heat shock increased markedly in Asp^15,78,82-hsp27 expressing HiB5 cells but decreased in Ala^15,78,82-hsp27 expressing cells. These results support the idea that phosphorylation of HSP25 is critical for the maintenance of actin filament and enhancement of thermoresistance. Interestingly, HSP25 was dephosphorylated and returned to cytoplasm in a recovery time-dependent manner. This phenomenon was accompanied by an increment of apoptotic cell death as determined by nuclear and DNA fragmentation and fluorescence-activated cell sorter analysis. These results suggest that nuclear-translocated HSP25 might function to protect nuclear structure, thereby preventing apoptotic cell death.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Temperature upshifts and a number of other stress conditions result in rapid production of important families of proteins called heat shock proteins (HSPs).¹ These families of proteins (HSP110, HSP90, HSP70, HSP60, and small HSP) are known to function as molecular chaperones that help other proteins to adopt a biologically active conformation (1). HSPs interact with nascent proteins that are not fully modified to maintain their folding/unfolding status and to regulate appropriate cellular compartmentalization. HSPs also recognize and bind to other proteins that are in a non-native conformation because of protein-denaturing environmental stress. HSPs either minimize aggregation of non-native proteins or target damaged proteins for hydrolysis and removal from the cell (2).

Among these HSP families, little is known about the small HSPs because of their wide range of molecular masses from 15 to 40 kDa and lower amino acid sequence homology. However, all members of small HSPs share a conserved C-terminal -crystalline domain, which is responsible for conferring their native structure (3). HSP25 is a mammalian homologue of the small HSP family and is also induced by various kinds of cellular stresses. Its expression is mainly regulated at the transcriptional level; it is induced upon binding of pre-existing heat shock transcription factors to the conserved heat shock elements (4). HSP25 is also regulated by post-translational modifications such as phosphorylation, deamidation, and acylation (5). The phosphorylation of HSP25 is catalyzed by MAPKAP kinase 2, a serine protein kinase, in a stress-dependent manner (6). However, the influence of the HSP25 phosphorylation on cellular stress responses such as thermoresistance is still controversial and not clearly defined (7, 8). HSP25 has a wide variety of different and seemingly unrelated cellular functions ranging from a molecular chaperone to a mediator of thermoresistance and chemoresistance (9, 10). It also inhibits actin polymerization, regulates apoptosis, and protects ribonucleic acids (11-13). However, the functional roles and regulatory mechanisms of HSP25 remain largely unknown.

Hippocampus is a brain region most vulnerable to a variety of environmental stresses; various forms of stress lead to hippocampal cell damage and eventually cell death (14, 15). However, little is known about protective roles of HSPs in the hippocampus. In the present study, we attempted to elucidate the thermoprotective function of HSP25 in hippocampal HiB5 cells (16). We examined the signal transduction pathway of HSP25 phosphorylation, phosphorylation-dependent cellular localization, and actin stabilizing activity and the thermoprotective role of HSP25.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture-- HiB5 cells (kindly provided by Dr. R.D.G. McKay, National Institutes of Health) were maintained in Dulbecco's modified Eagle's medium with 4 mM glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin/streptomycin, and 10% fetal bovine serum under humidifying atmosphere containing 5% CO₂ at 32 °C for proliferation (16, 17). The medium was changed every 2 days. The photographs were taken under a phase contrast inverted microscope or a fluorescence microscope.

Cell Counting-- For cell counting, HiB5 cells (4 × 10⁴) were replated in 6-well tissue culture plates and grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. The cells were harvested at appropriate time points by trypsinization and stained with 0.4% trypan blue. The number of trypan blue-stained dead cells and trypan blue-exclusive viable cells were counted on a hemacytometer.

Immunocytochemistry-- The cells were grown on coverslips until 70% confluence, washed twice with Dulbecco's phosphate-buffered saline (D-PBS) and fixed in 2.5% paraformaldehyde. The fixed cells were then treated with 0.3% Triton X-100 in 3% fetal calf serum for 30 min. After washing with D-PBS, the cells were incubated with primary antibody for 1 h, rinsed with D-PBS two times, and treated with FITC- or TRITC-conjugated secondary antibody for 1 h in dark. For actin filament staining, the cells were treated with phalloidine-TRITC (1 µM) for 15 min. After washing with D-PBS, the cells were mounted with mounting solution containing 0.1 µM of DAPI.

Western Blot Analysis-- The cell extracts were resolved on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA) in a Bio-Rad Trans-Blot electrophoresis apparatus using Towbin's buffer (25 mM Tris, pH 8.3, 192 mM glycine, and 20% (v/v) methanol) (18). The blots were blocked in Tris-buffered saline (150 mM NaCl, 10 mM Tris-base, and 2 mM MgCl₂) containing 0.5% Tween 20 and 3% bovine serum albumin, and incubated with anti-HSP25, HSP70, phosphoserine, phospho-p38 MAPK, phospho-ERK1/2, or phospho-JNK/SAPK antibodies (1:3000) at room temperature for 1 h. The blots were then washed three times with Tris-buffered saline with 0.5% Tween 20. Primary antibody binding was subsequently detected by incubation with secondary antibodies linked to horseradish peroxidase (Jackson ImmunoResearch Laboratories, West Grove, PA). The blots were then washed four times as described above. Immunoreactive bands were visualized by Amersham Biosciences ECL reagents according to the manufacturer's instructions.

Immunoprecipitation-- The cytosolic and nuclear extracts were prepared by the method described by Andrew and Faller (19) with a minor modification. HiB5 cells in 10 cell culture dishes (100 mm in diameter) were scraped and pooled in cold D-PBS. The cells were pelleted at 2500 rpm for 30 s, resuspended in 1 ml of cold buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.2 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, and protease inhibitors such as 1 µg/ml aprotinin, 0.5 µg/ml leupeptin, and 0.7 µg/ml pepstatin), and incubated on ice for 10 min. The swollen cells were homogenized until 90% of cells were lysed (determined with trypan blue staining) and centrifuged at 12,000 rpm for 20 min. The supernatant was collected and used for cytosolic fraction. The pellet was resuspended in 1 ml of ice cold buffer C (20 mM HEPES, pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol, and protease inhibitors) and stirred for 30 min on ice for high salt extraction. The debris was removed by centrifugation at 12,000 rpm for 20 min. The supernatant was collected and used for nuclear fraction. The cytosolic and nuclear fractions were dialyzed against modified RIPA buffer (150 mM NaCl, 20 mM HEPES, 10 mM Na₄P₂O₇, 2 mM Na₃VO₄, 0.5 mM phenylmethylsulfonyl fluoride, and 1% Nonidet P-40). 1 mg of whole cell lysates or nuclear extracts were immunoprecipitated by incubation with anti-HSP25 or anti-phosphoserine antibody for 2 h at 4 °C, and the resulting immunoprecipitates were captured by 10% protein A-Sepharose (Amersham Biosciences) for 2 h. The immunoprecipitates were subsequently washed three times with modified RIPA buffer and subjected to Western blot analysis as described above.

Northern Blot Hybridization-- Total RNA was isolated by a single step acid-guanidinium-phenol-chloroform method with minor modifications (20). 20 µg of the total RNAs were denatured, resolved on a 1.2% formaldehyde denaturing gel, and transferred to Nylon membrane (Schleicher & Schuell). The membrane was prehybridized at 42 °C for 2 h and then hybridized for 16-24 h with ³²P-labeled hsp25 cDNA probe (kindly provided by Dr. Y. J. Lee, University of Pittsburgh). After autoradiography, the membranes were stripped and rehybridized with 18 S rRNA cDNA probe to ensure that equal amounts have been loaded.

Transfection Experiments-- HiB5 cells were replated in 12-well plates and grown to 70% confluence. The cells were washed twice with 1× D-PBS, received 800 µl of fresh medium, and were incubated at 32 °C for 10 min. Transfections were carried out by using Effectene^TM reagent procedure with 300 ng of Ala^78,82-hsp27, Ala^15,78,82-hsp27, Asp^78,82-hsp27, or Asp^15,78,82-hsp27 plasmids/dish according to the manufacturer's instructions. Stably transfected cell lines were established under 3 µg/ml puromysin for 2 months.

DNA Fragmentation Assays-- DNA fragmentation was analyzed essentially as described by Hockenbery et al. (21). Briefly, the cells were lysed for 20 min at 4 °C in a medium containing 5 mM Tris buffer, pH 7.4, 0.5% Triton X-100, and 20 mM EDTA. After centrifugation at 20,000 × g for 15 min, the nucleic acids were extracted with phenol-chloroform, precipitated in ethanol, and analyzed by gel electrophoresis (1.5% agarose). The gel was then incubated for at least 3 h at 37 °C in the presence of 20 µg/ml RNase A and stained with ethidium bromide.

Propidium Iodide Staining and Analysis of DNA Contents-- Dead floating cells and surviving cells dissociated by trypsinization were collected together by centrifugation at 800 × g. The cells were then fixed with 70% ethanol at 4 °C overnight. The fixed cells were washed with D-PBS and then resuspended in phosphate-buffered saline containing 500 µg/ml RNase A and 40 µg/ml propidium iodide (Sigma). The cells were incubated at 37 °C for 30 min and analyzed for DNA content by Becton Dickinson FACScan (BD immunocytometry systems, San Jose, CA) using LYSYSII software. Haploid cells were considered apoptotic.

Statistical Analyses-- The data from cell counting and flow cytometric analysis were statistically evaluated using Student's t test or one-way analysis of variance followed by Fisher's least significant difference test for a post-hoc comparison. Statistical significance was set at p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cellular Localization and Phosphorylation of HSP25 by Heat Shock-- At first, we examined the induction of HSP25 in HiB5 cells in response to heat shock (at 43 °C, for 3 h) by Northern blot and immunoblot hybridizations. The hsp25 mRNA level was up-regulated by 3-fold, and the protein level was up-regulated 5-fold by the heat shock (Fig. 1). Because cellular localization of HSP25 is known to be important for its function (22), we examined the cellular localization of HSP25 in HiB5 cells by immunocytochemistry. In the absence of heat shock, HSP25 was mainly located in the cytoplasm, but a large portion of HSP25 was translocated into the nucleus by 3 h of the heat shock (Fig. 2A). On the other hand, HSP70 was evenly located in both the cytoplasm and the nucleus under the normal condition, and its cellular localization was not affected by the heat shock (Fig. 2A). To confirm these findings, we isolated cytoplasmic and nuclear fractions and analyzed by immunoblot analysis with anti-HSP25 and anti-HSP70 antibodies, respectively. The level of HSP25 in the cytoplasmic fraction was much higher than that in the nuclear fraction under the normal condition, which was reversed in the heat shock condition (Fig. 2B). In the case of HSP70, however, the proportion between the cytoplasmic and nuclear fractions was not altered by the heat shock (Fig. 2B). Interestingly, nuclear translocation of HSP25 was dependent on the duration of the heat shock (Fig. 2C)

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Expression of HSP25 in response to heat shock. HiB5 cells were exposed to heat shock at 43 °C for 3 h. A, total RNAs were isolated, fractionated on a 1% formaldehyde denaturing gel, and transferred to a Nytran membrane. The blot was hybridized with -32P-labeled hsp25 cDNA probe. 18 S rRNA was used as an internal control. B, total protein was obtained by the Laemmli method and quantified by the Lowry method. The samples (20 µg/lane) were resolved by SDS-PAGE, and immunoblot hybridization was performed with anti-HSP25 antibody. CTL, control; HS, heat shock.

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Cellular localization of HSP25 and HSP70 after 3 h heat shock at 43 °C. HiB5 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum at 37 °C with 5% CO2 and then heat shocked at 43 °C for 3 h. A, three hours after the heat shock (HS), the cells were fixed in 2.5% paraformaldehyde and immunostained with anti-HSP25 antibody followed by FITC (green)-conjugated secondary antibody and anti-HSP70 antibody followed by TRITC (red)-conjugated secondary antibody, respectively. The nucleus was stained with DAPI (1 µM) in blue. B, cytoplasmic and nuclear proteins were prepared and quantified by the Lowry method. The samples (20 µg/lane) were resolved by SDS-PAGE, and immunoblot hybridization was performed with anti-HSP25 and anti-HSP70 antibodies, respectively. C, HiB5 cells were heat shocked for 1, 2, and 3 h, then fixed in 2.5% paraformaldehyde, and immunostained with anti-HSP25 antibody followed by FITC (green)-conjugated secondary antibody. C, cytoplasmic fraction; N, nuclear fraction; CTL, control; HS, heat shock.

It has been well known that phosphorylation is important for HSP25 function (8, 23). However, it is yet controversial whether phosphorylation is crucial for the cellular distribution of HSP25 in HiB5 cells as shown in many different cell lines (24-26). HSP25 is phosphorylated at two serine residues, and thus we examined the phosphorylation state of HSP25 in the heat shock conditions. We immunoprecipitated nuclear extracts with anti-HSP25 antibody followed by immunoblot analysis with anti-phosphoserine antibody and vice versa. The level of serine-phosphorylated HSP25 in the nucleus was increased profoundly under heat shock conditions (Fig. 3).

View larger version (47K): [in this window] [in a new window]   Fig. 3.   Serine phosphorylation of HSP25 by heat shock. HiB5 cells were exposed to heat shock at 43 °C for 3 h. The nuclear extracts were immunoprecipitated with anti-phosphoserine (A) or anti-HSP25 (B) antibodies and resolved by SDS-PAGE. Immunoblot hybridization was performed with anti-HSP25 (A) or anti-phosphoserine (B) antibodies, respectively. CTL, control; HS, heat shock; IP, immunoprecipitation; IB, immunoblot.

Construction of Mutant hsp27s (Ala^78,82-hsp27 and Asp^78,82-hsp27) and the Cellular Localization of Mutant HSP27s-- To further delineate the involvement of HSP25 phosphorylation for its nuclear translocation, we analyzed cellular localization of mutant HSP27s. We used human HSP27, a human homologue of murine HSP25, to distinguish it from the endogenous murine HSP25 in HiB5 cells. Human HSP27 is known to be phosphorylated at three serine residues (Ser¹⁵, Ser⁷⁸, and Ser⁸²), and Ser-78 and Ser-82 are the major phosphorylation sites (27). We introduced mutations to HSP27 at these two serine residues with alanines (Ala^78,82-HSP27, nonphosphorylative form) or aspartic acids (Asp^78,82-HSP27, mimics the phosphorylated form) and transiently transfected each to HiB5 cells (Fig. 4A). We then examined cellular localization of the transfected Ala^78,82-HSP27 and Asp^78,82-HSP27 by immunocytochemistry with anti-HSP27 antibody that has no cross-reactivity with murine HSP25. Ala^78,82-HSP27 was located in the cytoplasm even after the heat shock (Fig. 4B). On the contrary, a majority of Asp^78,82-HSP27 was constitutively located in the nucleus even in the absence of heat shock (Fig. 4C). These results clearly indicate that phosphorylation of HSP27 at Ser⁷⁸ and Ser⁸² is important for its nuclear localization.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Cloning of mutant hsp27s (Ala78,82-hsp27 and Asp78,82-hsp27) and the cellular localization of mutant HSP27s in normal and heat shock conditions. A, human hsp27 was point mutated at two serine residues (Ser78 and Ser82 in amino acid sequence) to alanines (Ala78,82-hsp27) or aspartic acids (Asp78,82-hsp27) using PCR and inserted into pRetroTet-On vector. HiB5 cells were transfected with the mutant hsp27 plasmids, which were overexpressed with doxicyclin (2 µg/ml) treatment. B, cells transfected with Ala78,82-hsp27 were stained with human monoclonal anti-HSP27 antibody followed by FITC-conjugated secondary antibody in both normal and heat shock conditions. C, cells transfected with Asp-hsp27 were also immunostained as mentioned above. The nucleus was stained with DAPI. CTL, control; HS, heat shock.

Signal Transduction Pathway of HSP25 Phosphorylation: Involvement of p38 MAPK and ERK-- It has been reported that HSP25 is phosphorylated by MAPKAP kinase 2 in mouse and Chinese hamster cells and that MAPKAP kinase 2 is phosphorylated by members of MAPK (6, 28). In an attempt to determine which kind of MAPK is involved in the phosphorylation of HSP25 in HiB5 cells in response to heat shock, we examined phosphorylation of three MAPKs: p38 MAPK, JNK/SAPK, and ERK. The phosphorylation of p38 MAPK and ERK was markedly induced by the heat shock, whereas that of JNK/SAPK was not altered (Fig. 5A). To confirm these findings, we inhibited p38 MAPK and ERK signal cascades with their specific inhibitors SB203580 and PD098059, respectively, and examined whether HSP25 is phosphorylated. The heat shock-induced phosphorylation of HSP25 was markedly inhibited by PD098059 but slightly by SB203580 (Fig. 5B). Treatment with SB203580 plus PD098059 clearly blocked the phosphorylation. It appears that p38 MAPK and ERK signal transduction pathways are involved in the phosphorylation of HSP25.

View larger version (38K): [in this window] [in a new window]   Fig. 5.   p38 MAPK and ERK are upstream regulators of HSP25 phosphorylation. A, samples (20 µg/lane) of total proteins were resolved by SDS-PAGE, and immunoblot hybridization was performed with anti-phospho-p38 MAPK, anti-phospho-SAPK/JNK, and anti-phospho-ERK antibodies, respectively. SB203580 (10 µM) and PD098059 (10 µM) were added 1 h before heat shock. B and C, 30 min after the heat shock, the nuclear proteins were extracted and immunoprecipitated with anti-phosphoserine antibodies, and immunoblot hybridization was performed with anti-HSP25 (B) and vise versa (C). CTL, control; HS, heat shock; VEH, vehicle; SB, SB203580; PD, PD098059; IP, immunoprecipitation; IB, immunoblot.

Importance of HSP25 Phosphorylation in Actin Filament Stability and Thermoprotection-- One of the major functions of HSP25 is that it binds F-actin and inhibits actin polymerization. It has been known that phosphorylation causes release of the protein from F-actin (29). In an attempt to examine whether the phosphorylation-dependent actin binding activity of HSP25 correlates with actin filament stability and thermoprotection, we inhibited phosphorylation with SB203580 and/or PD098059 and examined actin filament stability and cell survival under the heat shock condition. Actin stress fibers were stable both in normal and heat shock conditions. Inhibition of the phosphorylation could not affect actin filament in normal condition. However, actin filament was severely disrupted by heat shock in the SB203580- and/or PD098059-treated groups, suggesting that the phosphorylation of HSP25 is critical for actin filament stability in response to heat shock (Fig. 6).

View larger version (48K): [in this window] [in a new window]   Fig. 6.   Effects of SB203580 and/or PD098059 on the actin filament stability. Vehicle or SB203580 (10 µM) and/or PD098059 (10 µM) were added 1 h before heat shock. 3 h after the heat shock, the cells were immunostained with anti-HSP25 antibody followed by FITC-conjugated secondary antibody. Actin cytoskeleton was stained with phalloidine-TRITC. CTL, control; HS, heat shock; VEH, vehicle; SB, SB203580; PD, PD098059.

Consistent with actin filament stabilities, the cell death rate (6.9 ± 1.1% in cells treated with vehicle, determined by trypan blue staining) was also not significantly altered by treatment with SB203580 and/or PD098059 in normal condition. Heat shock markedly induced cell death by 3-fold (22.2 ± 5.1% in vehicle-treated cells) without altering the morphology. When the phosphorylation was inhibited, most cells were shrunken to a round form by the heat shock, and 53.8 ± 6.1% of the cells died (Fig. 7).

View larger version (47K): [in this window] [in a new window]   Fig. 7.   Effects of SB203580 and/or PD098059 on the thermoprotective role of HSP25. HiB5 cells were pretreated with vehicle or SB203580 and/or PD098059 1 h before heat shock. A, 3 h after the heat shock, the cells were fixed in 2.5% paraformaldehyde and stained with hematoxilin and eosin staining solutions. The photographs were taken under phase contrast inverted microscope (×100). B, the cells were stained with trypan blue (0.4%), and the dead cell population was counted by hemacytometer under light microscope. The data are expressed as percentages of the total cells counted. *, p < 0.05 (versus vehicle in control); , p < 0.05 (versus vehicle in heat shock group). CTL, control; HS, heat shock; VEH, vehicle; SB, SB203580; PD, PD098059.

To further delineate the importance of HSP25 phosphorylation on actin filament stability, we examined the effect of heat shock or cytochalasin D, an actin filament disruptor, on cellular morphology in the cells transfected with Ala^78,82-hsp27 and Asp^78,82-hsp27. Actin filament in control or Asp^78,82-hsp27-transfected HiB5 cells was stable despite heat shock; however, a majority of Ala^78,82-hsp27-transfected cells showed shrunken morphology with disruption of actin filament. Cytochalasin D disrupted actin filament in the normal and Ala^78,82-hsp27 transfected HiB5 cells but not in the Asp^78,82-hsp27 transfected cells (Fig. 8). It seems likely then that phosphorylation of HSP27 at Ser⁷⁸ and Ser⁸² is essential for the stability of actin filaments.

View larger version (55K): [in this window] [in a new window]   Fig. 8.   Role of HSP25 phosphorylation in the actin filament stability against heat shock or cytochalasin D treatment. Ala78,82-hsp27 and Asp78,82-hsp27 were used to transfect HiB5 cells and overexpressed with doxicyclin (2 µg/ml) for 24 h. After heat shock for 3 h or treatment with cytochalasin D (5 µg/ml) for 1 h, endogenous HSP25 (control and untransfected cells) or transfected HSP27 (mutant hsp27-expressing cells) was immunostained with anti-HSP25 or anti-HSP27 antibodies followed by FITC-conjugated secondary antibody. Actin filament was stained with phalloidine-TRITC. CTL, control; HS, heat shock.

To confirm the importance of HSP27 phosphorylation in thermoresistance, we mutated all three serine phosphorylation sites (Ser¹⁵, Ser⁷⁸, and Ser⁸²) with alanines or aspartic acids and then established two stable HiB5 cell lines (Ala^15,78,82-hsp27 and Asp^15,78,82-hsp27). In the absence of heat shock, about 5.2 ± 1.3% of the cells died in normal as well as mutant HiB5 cells (Ala^15,78,82-hsp27 and Asp^15,78,82-hsp27) determined by trypan blue staining (Fig. 9). In the heat shock condition, however, 17.9 ± 1.8%, 30.4 ± 1.5%, and 8.2 ± 1.5% of cells died in normal, Ala^15,78,82-hsp27, and Asp^15,78,82-hsp27 cells, respectively. These results clearly suggest that HSP27 phosphorylation is important for maintenance of the actin stress fibers in response to heat shock and thus enhances thermoresistance.

View larger version (14K): [in this window] [in a new window]   Fig. 9.   Survival rate of Ala15,78,82-hsp27 and Asp15,78,82-hsp27 transfected HiB5 cells against heat shock. Hsp27 genes mutated at three points (Ala15,78,82-hsp27 and Asp15,78,82-hsp27) were transfected into the HiB5 cells and selected with 3 µg/ml puromysin for 2 months. The stable Ala15,78,82-hsp27 and Asp15,78,82-hsp27 transfected HiB5 cells were heat shocked for 3 h at 43 °C. The cells were stained with trypan blue (0.4%), and the dead cell population was counted by hemacytometer under a light microscope. The data are expressed as percentages of total cells counted. *, p < 0.05 (versus control); +, p < 0.01 (versus control in heat shock group). CTL, control; HS, heat shock.

Cellular Localization of HSP25 and Apoptotic Cell Death during Recovery Period-- Finally, we examined cellular level, phosphorylation, and localization of HSP25 during the recovery period. The heat shock-induced rise in HSP25 gradually decreased in time; however, HSP25 was rapidly dephosphorylated to basal level within 6 h (Fig. 10A). During the recovery period, most of the nuclear-translocated HSP25 returned to the cytoplasm gradually and redistributed in the cytoplasm within 9 h (Fig. 10B). Based on recent reports that HSP25 prevents apoptosis (30), we also investigated apoptotic cell death of HiB5 cells in normal, heat shock, and heat shock recovery conditions. DNA contents determined by fluorescence-activated cell sorter analysis showed that the haploid apoptotic cells were 1.4 ± 0.4% in normal and 1.6 ± 0.2% in heat shock conditions. However, the apoptotic cells increased to 7.3 ± 0.8% at 6 h of recovery, indicating an increment of apoptosis during the recovery period (Fig. 11A). One of the apoptotic cell death markers, DNA fragmentation, occurred during the recovery period at 32 °C but not in normal or heat shock conditions (Fig. 11B). Nuclear fragmentation determined by DAPI staining also supported the above results (Fig. 11C). These results clearly indicate that apoptotic cell death occurred during the recovery period.

View larger version (54K): [in this window] [in a new window]   Fig. 10.   Expression, dephosphorylation, and cellular localization of HSP25 during the recovery period. HiB5 cells were heat shocked at 43 °C for 3 h and recovered at 32 °C permissive temperature for 3, 6, and 9 h. A, at each indicated time point, the total protein was obtained, and immunoblot hybridization was performed with anti-HSP25 antibody (upper panel). The phosphorylation state of HSP25 was determined by immunoprecipitation with anti-phosphoserine antibody followed by immunoblot analysis using anti-HSP25 antibody (lower panel). B, at each time point indicated, immunocytochemistry was performed with anti-HSP25 antibody as described for Fig. 2C. Re, recovery; CTL, control; HS, heat shock.

View larger version (48K): [in this window] [in a new window]   Fig. 11.   Proportion of apoptotic cell death during recovery period. HiB5 cells were heat shocked at 43 °C for 3 h and recovered at 32 °C permissive temperature for 3, 6, and 9 h. A, apoptic cell death population was examined by flow cytometry. The cells were harvested at each time point indicated, fixed in 70% ethanol, and stained with propidium iodide (50 µg/ml). DNA content was analyzed with fluorescence associated flow cytometry. The cells containing haploid DNA were regarded apoptotic. The data are expressed as percentages of total cells counted. *, p < 0.05 (versus control), **, p < 0.01 (versus control). B, DNA fragmentation was examined in each time point indicated. The cells were lysed with 5 mM Tris buffer, pH 7.4, 0.5% Triton X-100, and 20 mM EDTA for 20 min at 4 °C. After centrifugation at 12,000 × g for 15 min, the nucleic acids were extracted with phenol-chloroform and precipitated in ethanol. DNA was electrophoresed on a 1.5% agarose gel, incubated at 37 °C for 3 h in the presence of 20 µg/ml RNase A, and stained with ethidium bromide. C, cells were fixed in 2.5% paraformaldehyde and stained with DAPI (1 µM) at each time point indicated. Re, recovery; CTL, control; HS, heat shock.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

HSP25 is a cytoplasmic protein but redistributes in the perinuclear region or inside the nucleus in stress- and cell type-dependent manners (8, 25, 31-33). In the present study, we show that HSP25 is located mainly in the cytoplasm in the normal condition, but a large portion translocated into the nucleus in response to heat shock in HiB5 hippocampal cells. This nuclear translocation of HSP25 is accompanied by its phosphorylation. Involvement of the phosphorylation of HSP25 in its nuclear translocation is still controversial (8, 26), but our study with the mutants Ala^78,82-hsp27 and Asp^78,82-hsp27 clearly shows that phosphorylation at Ser⁷⁸ and Ser⁸² is important for its nuclear translocation. HSP27 has three serine phosphorylation sites, and Ser⁷⁸ and Ser⁸² are more susceptible for phosphorylation than Ser¹⁵ (28). Therefore, it is reasonable that phosphorylation at these two sites is sufficient for the nuclear translocation.

The phosphorylation of HSP25 is dependent on MAPKAP kinase 2/3, which is phosphorylated and activated by various MAPKs such as p38 MAPK, JNK/SAPK, and ERK in a cell type-specific manner (34-36). Among these MAPKs, phosphorylation of p38 MAPK and ERK was increased by heat shock in hippocampal HiB5 cells, and inhibition of their phosphorylation by SB203580 and/or PD098059 reduced the phosphorylation of HSP25. These results clearly indicate that p38 MAPK and ERK are upstream regulators of HSP25 phosphorylation in the heat shock condition.

Interestingly, we found that the inhibition of HSP25 phosphorylation resulted in the destruction of actin filaments and shrinkage of HiB5 cells to death in the heat shock condition. In the normal condition, however, SB203580 and/or PD098059 have no effect on morphology and survival of HiB5 cells. These findings further indicate that phosphorylation of HSP25 is important for stability of actin filaments and thermoresistance. Actin filament stability is dependent on polymerization and depolymerization of actin monomers. Actin polymerization/depolymerization is regulated by the concentration of free actin monomers, the number of nucleation sites, GDP-GTP exchange on G-actin, and the affinity of G-actin for the barbed ends (37-39). One of the important features of HSP25 is the actin capping activity, thereby inhibiting actin polymerization (12, 29). Phosphorylation of HSP25, however, causes a conformational change leading to its dissociation from the barbed ends of actin filaments. Therefore, phosphorylation of HSP25 could regulate spatial organization of F-actin by freeing the barbed ends of microfilaments for the addition of monomers. Considering the facts mentioned above, it seems likely that the destruction of actin filaments observed after the heat shock and treatments with SB203580 and PD098095 results from inhibition of HSP25 phosphorylation.

To confirm the importance of HSP25 phosphorylation on actin filament stability more precisely, we examined the actin filament stability in Ala^78,82-hsp27- and Asp^78,82-hsp27-transfected HiB5 cells in combination with heat shock or cytochalasin D treatment. Consistent with the results from SB203580- and/or PD098059-treated cells, the majority of Ala^78,82-hsp27-expressing cells showed disruption of actin filament, although a significant portion of cells remained actin filament. Phosphorylation of endogenous HSP25 and expression of Asp^78,82-hsp27 successfully protected actin filament structure against heat shock. Cytochalasin D, an actin filament disruptor, interacts with both F-actin and G-actin and reduces actin polymerization activities (40). Cytochalasin D disrupted actin filaments in both normal and Ala^78,82-HSP27-expressing HiB5 cells, but not in Asp^78,82-HSP27-expressing cells (Fig. 8). These results clearly support the idea that phosphorylation of HSP25 is important for the stability of actin filament and enhances thermoresistance against heat shock. In support of the idea mentioned above, Asp^15,78,82-hsp27-transfected HiB5 cells are more protective against the heat shock. On the contrary, more cells died by the heat shock in Ala^15,78,82-hsp27 transfected cells, because this nonphosphorylative form binds to the barbed end of actin filaments and thus inhibits actin filament polymerization.

One of the unique and important findings of our study is the nuclear translocation of HSP25 in response to heat shock accompanied by phosphorylation and redistribution to the cytoplasm with decreased phosphorylation during recovery accompanied by apoptosis. The reasons why HSP25 is translocated into the nucleus and functional role of HSP25 in the nucleus under stress conditions such as heat shock remain unknown. Our result that HSP25 time-dependently relocates into the cytoplasm during the recovery period, however, gives an insight into the functional role of HSP25 in the nucleus. During the recovery period, HiB5 cells underwent apoptosis along with nucleus and DNA fragmentation. Recently, it has been reported that HSP15, a new member of small HSPs, binds to nucleic acids, which is different from the known functions of HSPs such as molecular chaperone or protease activities (41). It is then presumed that HSP25 in the nucleus may protect the nucleic acids from heat shock induced-DNA fragmentation. Although there is no direct evidence, the simultaneous events of HSP25 release from the nucleus and nuclear breakdown surmise the idea that HSP25 might protect nuclear lamina and thus prevent nuclear breakdown during the heat shock process. It is well known that apoptosis can occur by oxidative stress (42). Our preliminary data that increased the level of manganese-superoxide dismutase and dichlorofluoroscein-stained cells (data not shown) by the heat shock may suggest that thermal stress might evoke apoptotic cell death as oxidative stress. Further studies on the mechanism of action are needed.

In conclusion, HSP25 is phosphorylated by heat shock via the p38 MAPK and ERK signal transduction pathways. The phosphorylated HSP25 in the cytoplasm protects the actin microfilaments and thus enhances thermoresistance against heat stress. A large portion of the phosphorylated HSP25 translocates into the nucleus, where it may attenuate DNA fragmentation and nuclear breakdown.

	FOOTNOTES

^* This work was supported by grants from the Korea Ministry of Science and Technology through the Korean Brain Science and a grant from the National Research Laboratory (2000-N-NL-01-C-149) and Basic Research Program of the Korea Science & Engineering Foundation.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.

Supported by Brain Korea 21 Research Fellowship from the Korea Ministry of Education.

^§ To whom correspondence should be addressed. Tel.: 82-2-880-6694; Fax: 82-2-884-6560, E-mail: kyungjin@snu.ac.kr.

Published, JBC Papers in Press, March 22, 2002, DOI 10.1074/jbc.M104396200

	ABBREVIATIONS

The abbreviations used are: HSP, heat shock protein; MAPK, mitogen-activated protein kinase; MAPKAP, MAPK-activated protein; ERK, extracellular signal-regulated kinase; D-PBS, Dulbecco's phosphate-buffered saline; FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine isothiocyanate; JNK, c-Jun N-terminal kinase, SAPK, stress-activated protein kinase; DAPI, 4,6-diamidino-2-phenylindole.

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