Phosphorylation-dependent structural alterations in the small hsp30 chaperone are associated with cellular recovery

Available online at www.sciencedirect.com R

Experimental Cell Research 286 (2003) 175–185

www.elsevier.com/locate/yexcr

Phosphorylation-dependent structural alterations in the small hsp30 chaperone are associated with cellular recovery Pasan Fernando,a,b Lynn A. Megeney,a and John J. Heikkilab,* a

Ottawa Health Research Institute, Ottawa General Hospital, Center for Molecular Medicine, Ottawa, Ontario, Canada K1H 8L6 b Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1 Received 13 November 2002, revised version received 29 January 2003

Abstract Small heat shock proteins (hsps) act as molecular chaperones by preventing the thermal aggregation and unfolding of cellular protein; however, the manner by which cells regulate chaperone activity remains unclear. In the present study, we examined the role of phosphorylation on the chaperone function of the Xenopus small hsp30. Both heat stress and sodium arsenite treatment in A6 cells resulted in a rapid activation of p38␣ and MAPKAPK-2. Surprisingly, the association of MAPKAPK-2 with hsp30 and its subsequent phosphorylation were more prevalent during recovery after heat stress. Treatment of A6 cells with SB203580, an inhibitor of the p38 MAP kinase pathway, resulted in a loss of hsp30 phosphorylation. Phosphorylation resulted in the formation of smaller multimeric hsp30 complexes and resulted in a significant loss of secondary structure. Consequently the phosphorylation-induced structural changes severely compromised the ability of hsp30 to prevent the heat-induced aggregation of citrate synthase and luciferase in vitro. We confirmed that the loss of chaperone activity was coincident with an attenuated binding of phosphorylated hsp30 with target proteins. Our data suggest that phosphorylation may be necessary to regulate the post-heat stress molecular chaperone activity of hsp30. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Small heat shock protein; hsp; Phosphorylation; Structure; MAPKAP kinase-2; p38; Chaperone; Cell stress; Signaling; Xenopus

Introduction Small heat shock proteins (hsps) are found in virtually all organisms and are distinct from the other two major hsp families, hsp70 and hsp90, by virtue of their size, pattern of expression, and cellular function. The primary characteristic of small hsps is their ability to act as molecular chaperones. In this manner, small hsps maintain the structure and solubility of other polypeptides by preventing their thermal aggregation as well as aiding in the refolding of unfolded proteins after heat or chemical denaturation [for review, see 1]. Interestingly, the structure of small hsps is often the key determinant in its function as a molecular chaperone. Most small hsps assemble into large polymeric units that range in size from 100 kDa up to 1 MDa, the formation of which * Corresponding author. Fax: ⫹1-519-746-0614. E-mail address: [email protected] (J. Heikkila).

facilitates their efficient chaperone activity. In addition, a flexible C-terminal tail is found on almost all small hsps that allows them to interact with cellular proteins and plays a key regulatory role in the maintenance of target protein solubility [2–5]. Cell stress not only activates the molecular chaperone response but also results in the activation of the mitogenactivated protein (MAP) kinases that include extracellular signal regulated kinase (ERK), stress-activated protein kinase-1/c-Jun N-terminal kinase (SAPK1/JNK), and SAPK2/ p38 kinase [reviewed in 6]. Of these, the JNK and p38 pathways are most potently activated by proinflammatory cytokines and environmental stresses. Indeed, the simulataneous activation of both the heat shock and the stressactivated signaling pathways emphasizes the likelihood that these two mechanisms act in concert to mount a protective response in mammalian cells. Several lines of evidence suggest an interaction of the p38 MAP kinase pathway with small hsps. The mammalian

0014-4827/03/$ – see front matter © 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0014-4827(03)00067-3

176

P. Fernando et al. / Experimental Cell Research 286 (2003) 175–185

small hsps, hsp25, and hsp27, are present under normal cellular conditions and are rapidly phosphorylated following exposure to various stresses [7–9]. Since these small hsps are readily phosphorylated, their mechanism(s) of activity may be regulated by stress-activated signaling cascades. Hsp27 is phosphorylated by mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK-2), a substrate of the ␣-isoform of p38 [10,11] and is known to regulate actin filament structure via the dissociation of highmolecular-weight multimers into monomers and dimers. Moreover, the monomeric form of hsp27 is better able to stabilize actin microfilaments in response to stress [8,9,12,13]. Clearly, phosphorylation enhances the function of cellular chaperones; however, the molecular details are poorly understood. Here, we report that MAPKAPK-2 directly phosphorylates the stress-induced small hsp isoform, hsp30C (referred to herein as hsp30), following both heat and chemical stress. Phosphorylated hsp30 was ineffective in its ability to chaperone target proteins. Importantly, we demonstrate that phosphorylation alters the secondary structure of hsp30 resulting in a loss of oligomerization. Finally, our data suggest that phosphorylation directs the release of hsp30 from denatured proteins and that this mechanism is associated with enhanced cellular recovery from stress.

conserved region of MAPKAPK-2 (Upstate, NY) were used in immunoblot analyses according to the manufacturers instructions and detection was carried out using an ECL chemiluminescence kit (Amersham) as described in [2]. Blots used for successive immunodetections were stripped and reprobed according to instructions of the ECL kit. Complete removal of the antibody was verified by predetection with ECL in each instance. Analysis of hsp30 phosphorylation state A6 cells were grown to confluence prior to heat treatment, at which point fresh L-15 media (as described above) containing 10 ␮Ci of [32P]orthophosphate was added. Cells were incubated at 35°C for various periods of time and in some instances a recovery period for up to 2 h at 22°C was included following heat treatment. In all cases, cells were washed twice with ice-cold phosphate buffered saline (PBS) containing 20 mM NaF and collected in ice-cold modified radioimmunoprecipitation (RIPA) buffer containing 50 mM Tris-HCl (pH 7.5), 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 20 mM NaF, and 20 mM Na3VO4 and 1 ␮g/␮l each of aprotinin, leupeptin, and pepstatin. Lysates were centrifuged at 13,000g for 15 min and the supernatant was used for immunoprecipitation with anti-hsp30 antibody. Radiolabeled hsp30 protein was visualized using a Storm 860 Phosphoimager (Molecular Dynamics).

Materials and methods Hsp30 in vitro kinase assays Tissue culture Xenopus A6 kidney epithelial cells (CCL-102) from the American Type Culture Collection were maintained at 22°C in L-15 media (Life Technologies, Inc.) supplemented with 10% fetal bovine serum and antibodies (50 units/ml penicillin and 50 ␮g/ml streptomycin) at 22°C. A6 cells do not express hsp30 message or protein under these conditions [14]. Hsp30 synthesis was induced by giving cells a 35°C heat stress for 2 h. In some instances, heat-stressed cells were placed at 22°C for various periods of time following heat shock treatment. Recovery at 22°C has been shown previously to facilitate maximal heat shock protein synthesis [15]. To examine the contribution of the p38 MAP kinase pathway to hsp30 phosphorylation, 20 ␮M of the pyridinyl imidazole inhibitor SB203580 (Sigma) was added to culture flasks 0.5 h prior to heat treatment. Western blot analysis Western blot analyses were performed on samples from gels that were transferred to polyvinylidene difluoride membrane (PVDF) (Millipore). Immunodetection for hsp30 was performed using a polyclonal anti-hsp30 antibody as previously described [2]. Pan-phosphoserine (Zymed, CA), panphosphotyrosine, pan-phosphothreonine, and p38␣ antibodies (Transduction Labs, KY) and an antibody against a

Histidine-tagged recombinant hsp30 was expressed and purified as previously described [2]. Kinase reactions consisted of 0.5 ␮g of hsp30 mixed with 5 ng of MAPKAPK-2 in kinase assay buffer containing 20 mM Mops (pH 7.2), 50 mM ␤-glycerophosphate, 0.1 mM EGTA, 1 mM DTT, 1 mM Na3VO4, and 1 mM NaF in a total volume of 20 ␮l supplemented with 1 ␮Ci/␮l [␥-32P]ATP. Reactions were carried out at 30°C for various periods of time and then terminated with the addition of SDS-sample buffer. Samples were separated by 12% SDS-PAGE and protein was visualized using Coomassie blue staining. Radiolabeled hsp30 was detected by phosphoimage analysis. In experiments where the oligomeric size of hsp30 was examined after in vitro phosphorylation, native pore exclusion limit electrophoresis was performed using a 5–17% acrylamide gradient. Samples from these gels were then transfered onto PVDF membranes. Phosphoimage analyses and immunodetection of hsp30 oligomers was performed as described above. Immunoprecipitation Protein was isolated from either unlabeled or [32P]orthophosphate-labeled A6 cells as described above and 100 ␮g of total protein was used for incubation with hsp30 antibody. Samples were incubated at 4°C for 16 h and

P. Fernando et al. / Experimental Cell Research 286 (2003) 175–185

then 25 ␮l of a suspension of protein A-Sepharose (Sigma) was added to the mixture and allowed to incubate for an additional 2 h. The Sepharose beads were washed three times with ice-cold RIPA buffer containing 20 ␮M NaF, and the supernatants were resolved using SDS-PAGE. Following electrophoresis, gels with immunoprecipitates from unlabeled A6 cells were subject to Western blotting and used for immunodetection and gels with immunoprecipitates from labeled cells were vacuum-dried and analyzed using a phosphoimager. Coimmunoprecipitations were carried out as described above using 350 ␮g of total protein. Immunoprecipitation/kinase analysis MAPKAPK-2 was immunoprecipitated from A6 cells treated with 50 ␮M sodium arsenite and lysates were subjected to kinase analysis as described above using 2.5 ␮g of myelin basic protein (MBP) (Upstate) as the target substrate. Samples were electrophoresed using 10% SDSPAGE. Gels were stained with Coomassie blue, and dried and phospho-labeled MBP was visualized by autoradiography. Thermal aggregation assays Thermal aggregation of citrate synthase (CS) or luciferase (LUC) was performed as previously described [2]. Briefly, a 750 nM (71 ng/␮l) monomer concentration of recombinant hsp30 was in vitro phosphorylated using active MAPKAPK-2. This reaction was then added to a thermal aggregation reaction containing 150 nM (0.8 ng/␮l) monomer concentrations of either citrate synthase or luciferase. Nonphosphorylated hsp30 mixed with an inactive MAPKAPK-2 was used as a control. Samples were incubated at 42°C for 1 h and thermal aggregation, as indicated by the relative amount of light scattering, was monitored at ␭320 nm. Circular dichroism Circular dichroism (CD) was performed in a Jasco J-715 spectropolarimeter using J-715 standard analysis software. Hsp30 samples (15 ␮M) were mixed in 10 mM phosphate buffer, pH 7.5, and held in a quartz 1-mm cuvette (Hellma, Concord, Ontario, Canada) kept at 22°C using a Peltier-type constant-temperature cell holder (Model PFD 3505, Jasco, Easton, MD) and measured at 260 –180 nm with a 5.0-nm bandwidth, 0.5 nm resolution, 100 mdeg sensitivity at a 0.25 s response, and at a rate of 100 nm/min with a total of 10 accumulations. For some samples, CD spectra were also gathered at 35 and 42°C under the same parameters as above. Data were normalized against a spectra of phosphate buffer alone and expressed as the mean residue ellipticity [⍜]. All spectra were normalized against a control spectrum of phosphate buffer alone.

177

Fig. 1. Hsp30 protein is phosphorylated in vivo. A6 cells were incubated with [␥-32P]ATP and maintained at either 22°C or heat stressed at 35°C for 2 h. Some samples were allowed to recover at 22°C for either 1 or 2 h after heat shock and lysed as described under Materials and methods. Approximately 100 ␮g of total protein from lysates was used to immunoprecipitate hsp30 using an anti-hsp30 antibody. Samples were separated by 12% SDS-PAGE and gels were stained with Coomassie blue and vacuum-dried (bottom panel). Radiolabeled hsp30 was detected using a phosphoimager (top panel). The position of phosphorylated hsp30 is indicated on the left.

Results Hsp30 is phosphorylated on serine residues Xenopus kidney epithelial cells (A6 cells) were incubated at 35°C for 2 h in the presence of [32P]orthophosphate and collected at the indicated time points (Fig. 1). Hsp30 was present in all heat-stressed samples but not in cells maintained at the normal growth temperature of 22°C. Cells expressing hsp30 protein had also incorporated radiolabeled phosphate, thus indicating that hsp30 was targeted for phosphorylation by endogenous kinases upon exposure to heat stress. Several reports have demonstrated that small hsps are phosphorylated on serine residues. A sequence of amino acids highly similar to the recognition motif of mammalian mitogen-activated protein kinase-activated protein kinase-2 was found between T97 and H106 of the hsp30 amino acid sequence (Fig. 2A). MAPKAPK-2 is a serine/threonine kinase shown to catalyze phosphorylation of human hsp27 and mouse hsp25 [10,16,17]. Proteins containing the MAPKAPK-2 recognition site have an arginine residue Nterminal to the phosphorylation site (position n-3) [18]. An arginine is located in a similar position on the hsp30 protein. A bulky hydrophobic residue, found N-terminal to the phosphorylation site (position n-5), may also be necessary for efficient phosphorylation; however, a less bulky residue at the same position is found on hsp30 (Fig. 2A). Serine phosphorylation of hsp30 during and after heat stress was determined using a pan-phosphoserine-specific antibody. Although hsp30 accumulated during heat exposure, it was not readily phosphorylated until the cells were allowed to recover from stress (Fig. 2B, top panel). Additionally, hsp30 phosphorylation was not evident in transiently transfected HeLa cells maintained under normal growth conditions (data not shown). The doublet observed

Fig. 2. Hsp30 is a substrate for MAPKAPK-2. (A) The amino acid sequence of Xenopus hsp30 protein is shown with a MAPKAPK-2 consensus sequence highlighted in bold type. Below is a comparison of the mammalian MAPKAPK-2 consensus sequence (Stokoe et al., 1993) with the corresponding amino acids from Xenopus hsp30. Xaa denote any amino acid and asteriks denote the putative site of phosphorylation. (B) A6 cells were either maintained at 22°C or incubated at 35°C for 0.5 to 2 h. Alternatively, cells were incubated for 2 h at 35°C and then allowed to recover at 22°C for 0.5 to 2 h. Immunoprecipitation using an hsp30 antibody was carried out with approximately 200␮g of A6 cell total protein lysate. Samples were electrophoresed and Western-transferred followed by immunodection with a pan-serine antibody (top panel). The blot was stripped and reprobed using an anti-hsp30 antibody (bottom panel). The position of hsp30 is shown at the right. (C) In vitro phosphorylation of recombinant hsp30. Approximately 5 ng of MAPKAPK-2 and 0.5 ␮g of recombinant hsp30 were mixed with [␥-32P]ATP and reacted at 30°C (hsp30 ⫹ MAPKAPK-2 30°C) for selected periods of time in an in vitro kinase reaction as described under Materials and methods. As a comparison, similar reactions were assembled with either hsp30 or MAPKAPK-2 alone and left as control (con) or reacted at 30°C. Samples were electrophoresed and gels were stained with Coomassie blue and dried. The incorporation of radiolabeled 32P was determined by phosphoimage analysis. The position of recombinant hsp30 is indicated on the right and the relative positions of the 45 and 31 kDa molecular mass markers are indicated on the left. (D) Coimmunoprecipitation of MAPKAPK-2 with hsp30 following heat stress. A6 cells were heat treated at 35°C for 2 h. A control group was maintained at 22°C. Cells were either lysed immediately after heat treatment or lysed after recovering for 1 or 2 h at 22°C as described-under Materials and methods. Approximately 350 ␮g of total protein from lysates was used to immunoprecipitate hsp30 using an anti-hsp30 antibody. MAPKAPK-2 protein was detected with an anti-MAPKAPK-2 antibody (upper panel). Membranes were stripped and reprobed with an anti-hsp30 antibody (middle panel). Equal loading was assessed using an antibody against the nonphosphorylated form of p38 (bottom panel).

P. Fernando et al. / Experimental Cell Research 286 (2003) 175–185

179

Fig. 3. Phosphorylation of hsp30 involves the p38 MAPK pathway. A6 cells were heat-shocked (hs) either with or without SB203580 for indicated periods of time and were collected either immediately or after a 2 h recovery (rec) period (hs/rec) as indicated under Materials and methods. Phosphorylated p38 was detected with a phospho-specific-p38 antibody (top panel). The same blots were used to detect levels of hsp30 protein using an anti-hsp30 antibody (middle panel). A pan-serine antibody was used in a corresponding immunoblot to verify the phosphorylation of hsp30 protein (bottom panel).

may represent additional hsp30 family members that are detected by the anti-hsp30 antibody. Phosphorylation of tyrosine or threonine residues using either a pan-tyrosine or a pan-threonine antibody was not detected (data not shown). These results clearly demonstrate that heat shock results in the phosphorylation of hsp30. Importantly, phosphorylation occurs after removal of the heat stress, when cells recover from heat-induced damage. Furthermore, the results indicate that hsp30 phosphorylation is targeted by a phosphoserine-specific kinase. Unlike the mammalian hsp 25/27, hsp30 is not constitutively present. Therefore phosphorylation of hsp30 may have a unique temporal and contextdependent function.

heat-stressed cells after 2 h. However, the highest detectable levels were found in cells given a 2 h heat shock followed by a 2 h recovery. Both hsp30 and MAPKAPK-2 were absent in lysates incubated either with protein A beads alone or with MyoD as a nonspecific antibody (not shown). Interestingly, we have observed that the inhibition of p38 phosphorylation by the p38-specific inhibitor SB203580 leads to an attenuated MAPKAPK-2:hsp30 association (not shown). These results agree with our previous results (Fig. 2B) and demonstrate that MAPKAPK-2 associates with hsp30. Importantly, the MAPKAPK-2:hsp30 association becomes more evident once the stress is removed and cells are allowed to recover.

Recombinant hsp30 is phosphorylated by MAPKAPK-2

Hsp30 phosphorylation is dependent upon p38 activation

Efforts to specifically identify serine kinases responsible for the phosphorylation of hsp30 were directed at the serine/ threonine kinase MAPKAPK-2. In vitro kinase assays were performed using recombinant hsp30 as the substrate protein and MAPKAPK-2 as the phospho-active enzyme (Fig. 2C). Neither MAPKAPK-2 nor hsp30 autophosphorylate when incubated alone; however, when reacted together at 30°C, hsp30 is phosphorylated in a time-dependent manner. In addition, kinase analyses undertaken with mutated hsp30 (S104A) resulted in a loss of phosphorylation (not shown). Thus hsp30 is specifically phosphorylated by MAPKAPK-2 in vitro. A6 cells were used to determine the phosphorylation of hsp30 by MAPKAPK-2 in vivo. Cells were heat treated under the same conditions as those found to induce phosphorylation of hsp30 (see Fig. 2B). A band that coprecipitated at approximately 46 kDa, corresponding to MAPKAPK-2, was identified in these samples (Fig. 2D). A small amount of MAPKAPK-2 coprecipitated with hsp30 in

As MAPKAPK-2 is found downstream of both p38 and mitogen-activated protein kinase kinase 3/6 (MKK3/6) [17,19,20], inhibition of these signaling components should result in attenuated levels of phosphorylated hsp30. To test this hypothesis, we used the pyridinylimidazole drug SB203580, a specific inhibitor of both p38␣ and p38␤ activity, to examine the downstream consequences of p38␣ inactivation [21,22]. Under control conditions, a basal level of p38␣ activation was present in A6 cells (Fig. 3, top panel). This level increased upon heat shock and subsequent heat shock/recovery treatments. Incubation of cells with SB203580 markedly reduced p38␣ phosphorylation; however, the total amount of p38 protein did not change (not shown). Hsp30 phosphorylation was completely abolished when cells were incubated with SB203580 (Fig. 3, bottom panel). In vivo labeling using [32P]orthophosphate confirmed these results (not shown). Previous experiments from our lab have demonstrated an accumulation of hsp30 after 12 h with sodium arsenite exposure [23]. In more recent

180

P. Fernando et al. / Experimental Cell Research 286 (2003) 175–185

experiments, we have observed that the p38 MAPK pathway is readily primed within 15 min of exposure to sodium arsenite, well before hsp30 and phosphorylated hsp30 are present (data not shown). Taken together, our results suggest that hsp30 phosphorylation is a process that is most significant after recovery from cellular stress. Since hsp30 behaves as a molecular chaperone, phosphorylation is likely to affect its activity. Unlike the mammalian hsp25/27 chaperones; however, hsp30 is not present constitutively. Therefore, the stress-induced expression of hsp30 may allow this chaperone to have very different cellular roles when phosphorylated relative to the mammalian hsp25/27 chaperones. Phosphorylation drastically alters hsp30 secondary structure and reduces its chaperone ability Posttranslational modifications such as phosphorylation often result in changes to the size and structure of proteins, which consequently affects their function. Recently, it was shown by size-exclusion chromatography that recombinant hsp30 exists as a large 800- to 900-kDa multimeric complex [2]. To examine the oligomeric state of phosphorylated hsp30, native pore-exclusion limit electrophoresis followed by Western blotting and immunodetection with an antihsp30 antibody was employed. As protein migration relies exclusively on size and shape in pore exclusion limit electrophoresis, this technique in conjunction with Western blotting has been described as a useful method to compare small hsp oligomers [24]. Our analysis revealed the presence of a large hsp30 complex migrating just below the 669-kDa marker in all samples (Fig. 4A). Three smaller bands at approximately 400, 140, and 67 kDa in relation to the molecular weight marker were also detected. These same bands were observed with unphosphorylated hsp30; however, phosphorylation resulted in the formation of several additional bands between 500 and 400 kDa, and bands at approximately 300, 280, 210, 170, 90, and 85 kDa relative to the molecular weight marker. These bands were most prominent after 15 min of incubation with MAPKAPK-2 and decreased in intensity with further kinase activity. Subsequently, new hsp30 bands appeared (Fig. 4A, compare 15 min with both 30 and 60 min of phosphorylation). Similar results were obtained in both the presence and the absence of protease inhibitors (not shown). These results demonstrate that changes occur to the native oligomeric structure of hsp30 once phosphorylated, resulting in the formation of smaller complexes. The circular dichroism spectra of hsp30 between 208 and 210 nm was larger in intensity than at 222 nm, indicating an ␣-helical and ␤-sheet-like structure (Fig. 4B). Surprisingly, when hsp30 was phosphorylated, a considerable decrease in molar ellipticity was observed between 200 and 210 nm. These results indicated a very significant decrease in secondary structure. Similar profiles were observed when these analyses were performed at 35 and 42°C (not shown). Phosphorylation-induced alterations in hsp30 oligomeric structure may alter chaperone activity. We have previously

demonstrated that hsp30 functions effectively in the prevention of heat-induced aggregation of a series of target proteins including citrate synthase, luciferase, aldolase, and malate dehydrogenase [2,25]. We used a similar assay to examine the ability of phosphorylated hsp30 to prevent heat-induced aggregation of proteins. In this assay, we incubated hsp30 protein, after in vitro phosphorylation by MAPKAPK-2, with either citrate synthase or luciferase and monitored heat-induced aggregation as indicated by the relative change in absorbance at 320 nm. For comparison, we mixed hsp30, including the components of the phosphorylation buffer, with either CS or LUC and similarly measured heat-induced absorbance. Unphosphorylated hsp30 prevented CS aggregation by more than 95% when heated together at 42°C for 60 min (Fig. 4C). In contrast, phosphorylated hsp30 prevented aggregation of CS by only 30% after 60 min. A similar pattern was observed when LUC was used as the target protein (Fig. 4D). These results demonstrate that in vitro, the chaperone ability of hsp30 is severely weakened when this protein is phosphorylated. Reduced binding of phosphorylated hsp30 to denatured proteins An in vitro binding assay was used to show that hsp30 interacts with luciferase (LUC) after a 20-min heat shock (Fig. 5A). Additional heating for 45 min resulted in a greater amount of luciferase that coprecipitated with hsp30 (compare lane a with b). Undenatured luciferase did not coprecipitate with hsp30 (hsp30⫹LUC unheated lane). These results suggest that the interaction of luciferase and hsp30 occurs when luciferase is heat-destabilized. In contrast, phosphorylated hsp30 that was heat-treated for 45 min with luciferase resulted in a significant reduction in the amount of coprecipitated luciferase (compare lanes c and d with lane b). The relevance of phosphorylation is illustrated with the use of varying amounts of hsp30 (in vitro phosphorylation for either 20 or 60 min) in that a more abundant amount of phosphorylated hsp30 resulted in a minimal interaction with luciferase after heat shock (compare lane c with d). In order to understand the interaction of phosphorylated hsp30 with cellular proteins, a series of experiments were performed with A6 cells. Previous experiments using 35Smethionine-treated A6 cells (heat shock vs. non-heat shock) followed by hsp30 immunoprecipitation have demonstrated that hsp30 interacts with several cytosolic proteins. In particular, we determined that heat shock treatment of A6 cells resulted in an actin-hsp30 interaction and that this interaction was enhanced when cells were given a period of recovery following heat stress (P. Fernando and J.J. Heikkila unpublished data). Therefore, we examined the effect of phosphorylated and unphosphorylated hsp30 on hsp30-actin association. To this end, we chose to inhibit hsp30 phosphorylation by incubating cells in SB203580, as previously characterized (see Fig. 3). Actin did not interact with hsp30

P. Fernando et al. / Experimental Cell Research 286 (2003) 175–185

181

Fig. 4. Phosporylation-induced changes in the oligomeric size and chaperone properties of recombinant hsp30. (A) Recombinant hsp30 (0.25 ␮g) was left either unphosphorylated (hsp30 unphos) at 30°C for 60 min or phosphorylated in vitro (hsp30 phos) at 30°C for selected periods of time with MAPKAP kinase 2 as described under Materials and methods. The samples were analyzed by native pore-exclusion limit electrophoresis (5–17% acrylamide) followed by Western blot analysis using an anti-hsp30 antibody. The position of the largest hsp30 complex is indicated by an asterisk. Arrows indicate the position of smaller bands whose relative levels are enhanced by phosphorylation. Molecular mass standards in kilodaltons are indicated at the left of the figure. (B) Secondary structure of hsp30. Recombinant hsp30 and hsp30 phosphorylated by MAPKAPK-2 in vitro (phos-hsp30) were analyzed by circular dichroism as described under Materials and methods. In both instances, a 15 ␮M final concentration of recombinant hsp30 was analyzed. (C) Aggregation of citrate synthase. Approximately 0.1 ␮M citrate synthase was incubated alone (■) or with either 0.5 ␮M hsp30 (F) or 0.5 ␮M hsp30 phosphorylated in vitro with MAPKAPK-2 (Œ) at 42°C. Equivalent molar ratios were used in (D), using luciferase as the target protein. (䊐) Luciferase alone, (E) hsp30 ⫹ luciferase, (‚) phosporylated hsp30 ⫹ luciferase. (E) In vitro phosphorylation of hsp30. The level of phosphorylation of recombinant hsp30 was assessed by SDS-PAGE/autoradiography prior to its use in aggregation assays. C and D represent phosphorylated hsp30 used for panels C and D, respectively. Unphosphorylated hsp30 (con) is also shown. In both panels C and D, thermal aggregation was determined by light-scattering assays as described under Materials and methods. Data were calculated as a percentage of the maximum aggregation of CS or luciferase after 60 min and expressed as the mean ⫾ SD.

immediately following a 2 h heat shock but an association was observed in cells given a 2 h heat shock followed by a 2 h recovery (Fig. 5B, top panel). Cells allowed to recover expressed more hsp30 than heat shock alone (Fig. 5B, middle panel). When SB203580 was added to cells to prevent hsp30 phosphorylation, we observed an increase in the amount of actin that co-precipitated with hsp30. SB203580 alone had no effect on the accumulation of hsp30. Similar results were observed with actin immunoprecipitation and hsp30 immunodetection (data not shown). To verify these observations, we performed the corollary experiment whereby the p38 pathway was hyperstimulated

after heat shock. Since hsp30 is only present with stress, A6 cells were given a 2 h heat shock/2 h recovery to allow for hsp30 accumulation. Following this treatment, a 15-min exposure to sodium arsenite served to hyperstimulate the p38 pathway and therefore phosphorylate hsp30. This resulted in an accumulation of hsp30 that was comparable to the previous 2 h heat shock/2 h recovery treatments (Fig. 5B, middle panel, ⫹NaAs lane). In addition, hsp30 was strongly phosphorylated in this sample (Fig. 5B, bottom panel, ⫹NaAs lane). However, unlike the previous heat shock/recovery treatments, actin did not coprecipitate with hsp30 (Fig. 5B, top panel, ⫹NaAs lane).

182

P. Fernando et al. / Experimental Cell Research 286 (2003) 175–185

Fig. 5. Inability of phosphorylated hsp30 to bind target proteins. (A). In vitro binding assay. Recombinant hsp30 was phosphorylated (phos-hsp30) in vitro by MAPKAPK-2 for either 20 min (a and c) or 60 min (b and d) and then heated with luciferase (LUC) in a 5:1 (hsp30:LUC) molar ratio. Coimmunoprecipitation of LUC with hsp30 was assessed as described under Materials and methods. LUC alone and an unheated mixture of hsp30 and LUC were used as controls. The position of LUC and hsp30 are indicated at the left of the figure. (B). Association of actin with hsp30. A6 cells were given a 2 h heat shock (hs) or a 2 h heat shock followed by a 2 h recovery (hs/rec). SB203580 was added to cells during the recovery period. In another group, cells were given a 2 h heat shock/2 h recovery followed by a 50 ␮M sodium arsenite (NaAs) treatment for 15 min. In all samples, hsp30 was immunoprecipitated as described under Materials and methods and the coprecipitation of actin was determined by immunobloting with an anti-actin antibody (top panel). Phosphorylation of hsp30 was determined using a pan-serine antibody on successive immunoblots (bottom panel).

Discussion The phosphorylation of hsp30 resulted in three related events. First, phosphorylation induced a destabilization of hsp30 multimeric complexes. This was followed by an attenuated binding of hsp30 for denatured target proteins and a subsequent loss of chaperone activity. These phos-

phorylation-induced effects seem to defeat the role of hsp30 as a chaperone. Unlike the mammalian hsp25/27, the Xenopus hsp30 chaperone is not present constitutively and only accumulates with exposure to stress. As such, regulation of chaperone activity by phosphorylation may have a unique role in Xenopus that is different from molecular chaperones from other organisms.

P. Fernando et al. / Experimental Cell Research 286 (2003) 175–185

Recombinant hsp30 (hsp30) exists predominantly as a large multimer of greater than 900 kDa [2]. In the present study, we have shown that in vitro, phosphorylation destabilizes the hsp30 multimeric complex. Changes to the oligomeric structure of small hsps via phosphorylation have been well documented although the precise mechanism effecting the change has remained unclear [8,12,26,27]. Oligomeric disruption of small hsps may facilitate their translocation to constricted areas of the cell [26]. Conceivably, hsp30 may also be translocated upon phosphorylation. A specific phosphatase may then dephosphorylate hsp30, allowing it to reassemble into a higher order, functional complex. Indeed, previous studies have demonstrated that some shsp chaperones may translocate to the nucleus [28 –30]. Furthermore, intranuclear localization of phosphorylated hsp25 was recently demonstrated in myoblasts following stress treatment [31]. During heat stress, proteins begin to unfold and denature. Hsp30 plays a critical role in the cellular protection from stress since it directly associates with unfolded proteins and maintains them in a foldable state [2,25]. Interestingly phosphorylated hsp30 was unable to prevent thermal-induced aggregation of citrate synthase and luciferase. This loss in chaperone function was explained by the inability of phosphorylated hsp30 to bind target proteins. Phosphorylationinduced structural changes in hsp30 may have created either a steric hindrance at available binding sites or a chargeinduced reconformation of hsp30 itself. This was indeed the case for molecularly mimicked phospho-hsp27 where both the secondary structure and the quartenary confirmation were altered [26]. Recently, we have demonstrated that the replacement of charged aspartate residues with neutral glycine residues considerably altered the secondary structure hsp30 and its ability to respond as a molecular chaperone [32]. The cellular stress response leads to the recruitment of molecular chaperones to aid in maintaining protein solubility and structure as well as assist in the refolding of unfolded proteins. Interestingly, the MAPK signaling cascade is also activated in the response to stress. It is possible that this is an initial reaction in the acquisition of cellular thermotolerance. Our results show a rapid and robust activation of p38␣ upon stress induction, even in the absence of hsp30. The early activation of p38␣ may reflect a cellular priming mechanism that enables cells to respond quickly to stress once enough hsp30 has accumulated. Several reports have indicated that a rapid phosphorylation of small hsps is required for their function in the ability to promote cellular survival [33–35]. Interestingly, attenuated activation of the p38 cascade has been noted in thermotolerant cells, thus implying that cellular priming has already taken place [33,35]. Undoubtedly, p38 may also phosphorylate other unidentified downstream targets that play a role in the cellular response to heat shock [36]. In our experiments, the use of SB203580 has helped to define the signaling cascade leading to the regu-

183

lation of small hsp30 chaperone activity. This inhibitor readily affects the enzymatic activity of p38␣; however, we have also observed an attenuation of p38␣ phosphorylation upon SB203580 inhibition. Interestingly, a reduction in p38␣ phosphorylation by SB203580 has also been reported in both amphibian cardiomyocytes [37] and human fibroblasts [38]. Since SB203580 binds both active and inactive p38␣ with similar affinities [39], it is possible that an interaction with inactive p38␣ alters the p38␣ structure such that it is less accessible to phosphorylation by the upstream activators MKK3/6. Nevertheless, our results clearly demonstrate a role for the p38 MAPK pathway in regulating small hsp30 chaperone activity. Xenopus hsp30 and mammalian hsp25/27 share similar functions in their actions as molecular chaperones. In addition, MAPKAPK-2 can phosphorylate hsp30, hsp25, and hsp27, further demonstrating similar means of chaperone regulation. The primary difference, however, is that hsp30 is not constitutively present but only appears following heat or other stress-related cellular insults. Therefore, we propose that during cellular recovery from stress in Xenopus, the role of phosphorylation is to accelerate the release of tightly bound hsp30 particles from its targets by inducing a change in the oligomeric assembly of hsp30. Thus, chaperones such as hsp/hsc70 are permitted access to the target proteins in order to assist in their refolding and reactivation. Observations from other groups demonstrating the ability of small hsps to network with cellular chaperones support our model [40 – 42]. However, unlike other molecular chaperones, our data indicate that for hsp30, the p38 MAPK pathway in Xenopus may be more important during the stress-related cellular recovery period rather than during the stress-related cellular insult in regulating chaperone activity.

Acknowledgments This work was supported by grants from the Natural Sciences and Engineering Research Council to J.J.H. and the Heart and Stroke Foundation of Canada to L.A.M. J.J.H. is a recipient of a Canada Research Chair in Stress Protein Gene Research. L.A.M. is a CIHR Scholar. P.F. is supported by a Post-Doctoral Fellowship from the Heart and Stroke Foundation of Canada.

References [1] R.V. van Montfort, C. Slingsby, E. Vierling, Structure and function of the small heat shock protein/␣-crystallin family of molecular chaperones, Adv. Protein Chem. 59 (2002) 105–156. [2] P. Fernando, J.J. Heikkila, Functional characterization of Xenopus small heat shock protein, Hsp30C: the carboxyl end is required for stability and chaperone activity, Cell Stress Chaperones 5 (2000) 148 –159. [3] U. Jakob, M. Gaestel, K. Engel, J. Buchner, Small heat shock proteins are molecular chaperones, J. Biol. Chem. 268 (1993) 1517–1520.

184

P. Fernando et al. / Experimental Cell Research 286 (2003) 175–185

[4] J. Carver, G. Espsito, G. Schwedersky, M. Gaestel, 1H NMR spectroscopy reveals that mouse Hsp25 has a flexible C-terminal extension of 18 amino acids, FEBS Lett. 369 (1995) 305–310. [5] D.A. Haley, M.P. Bova, Q.L. Huang, H.S. McHaourab, P.L. Stewart, Small heat-shock protein structures reveal a continuum from symmetric to variable assemblies, J. Mol. Biol. 298 (2000) 261–272. [6] J.M. Kyriakis, J. Avurch, Sounding the alarm: protein kinase cascades activated by stress and inflammation, J. Biol. Chem. 271 (1996) 24313–24316. [7] L. Takemoto, T. Emmons, J. Horwitz, The C-terminal region of the ␣-crystallin: involvement in protection against heat-induced denaturation, Biochem. J. 294 (1993) 435– 438. [8] J.N. Lavoie, H. Lambert, E. Hickey, L.A. Weber, J. Landry, Modulation of cellular thermoresistance and actin filament stability accompanies phosphorylation-induced changes in the oligomeric structure of heat shock protein 27, Mol. Cell. Biol. 15 (1995) 505–516. [9] J. Guay, H. Lambert, G. Gingras-Breton, J.N. Lavoie, J. Huot, Regulation of actin filament dynamics by p38 map kinase-mediated phosphorylation of heat shock protein 27, J. Cell Sci. 110 (1997) 357–368. [10] J. Rouse, P. Cohen, S. Trigon, M. Morange, A. Alonso-Llamazares, D. Zamanillo, T. Hunt, A.R. Nebreda, A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins, Cell 78 (1994) 1027–1037. [11] J. Huot, H. Lambert, J.N. Lavoie, A. Guimond, F. Houle, J. Landry, Characterization of 45-kDa/54-kDa HSP27 kinase, a stress-sensitive kinase which may activate the phosphorylation-dependent protective function of mammalian 27-kDa heat-shock protein HSP27, Eur. J. Biochem. 227 (1995) 416 – 427. [12] H. Lambert, S.J. Charette, A.F. Bernier, A. Guimond, J. Landry, HSP27 multimerization mediated by phosphorylation-sensitive intermolecular interactions at the amino terminus, J. Biol. Chem. 274 (1999) 9378 –9385. [13] J. Landry, J. Huot, Modulation of actin dynamics during stress and physiological stimulation by a signaling pathway involving p38 MAP kinase and heat-shock protein 27, Biochem. Cell. Biol. 73 (1995) 703–707. [14] N.W. Ohan, Y. Tam, P. Fernando, J.J. Heikkila, Characterization of a novel group of basic small heat shock proteins in Xenopus laevis A6 kidney epithelial cells, Biochem. Cell Biol. 76 (1998) 665– 671. [15] D. Phang, E.M. Joyce, J.J. Heikkila, Heat shock-induced acquisition of thermotolerance at the levels of cell survival and translation in Xenopus A6 kidney epithelial cells, Biochem. Cell Biol. 77 (1999) 141–151. [16] J. Landry, H. Lambert, M. Zhou, J.N. Lavoie, E. Hickey, L.A. Weber, C.W. Anderson, Human HSP27 is phosphorylated at serines 78 and 82 by heat shock and mitogen-activated kinases that recognize the same amino acid motif as S6 kinase II, J. Biol. Chem. 267 (1992) 794 – 803. [17] D. Stokoe, K. Engel, D.G. Campbell, P. Cohen, M. Gaestel, Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins, FEBS Lett. 313 (1992a) 307–313. [18] D. Stokoe, B. Caudwell, P.T. Cohen, P. Cohen, The substrate specificity and structure of mitogen-activated protein (MAP) kinase-activated protein kinase-2, Biochem. J. 296 (1993) 843– 849. [19] D. Stokoe, D.G. Campbell, S. Nakielny, H. Hidaka, S.J. Leevers, C. Marshall, P. Cohen, MAPKAP kinase-2; a novel protein kinase activated by mitogen-activated protein kinase, EMBO J. 11 (1992b) 3985–3994. [20] J. Han, J.D. Lee, Y. Jiang, Z. Li, L. Feng, R.J. Ulevitch, Characterization of the structure and function of a novel MAP kinase kinase (MKK6), J. Biol. Chem. 271 (1996) 2886 –2891. [21] A. Cuenda, P. Cohen, V. Buee-Scherrer, M. Goedert, Activation of stress-activated protein kinase-3 (SAPK3) by cytokines and cellular

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

stresses is mediated via SAPKK3 (MKK6); comparison of the specificities of SAPK3 and SAPK2 (RK/p38), EMBO J. 16 (1997) 295– 305. H. Enslen, J. Raingeaud, R.J. Davis, Selective activation of p38 mitogen-activated protein (MAP) kinase isoforms by the MAP kinase kinases MKK3 and MKK6, J. Biol. Chem. 273 (1998) 1741– 1748. S. Darasch, D.D. Mosser, N.C. Bols, J.J. Heikkila, Heat shock gene expression in Xenopus laevis A6 cells in response to heat shock and sodium arsenite treatments, Biochem. Cell Biol. 66 (1988) 862– 870. G.J. Lee, E. Vierling, Expression, purification and molecular chaperone activity of plant recombinant small heat shock proteins, Methods Enzymol. 290 (1998) 350 –365. R. Abdulle, A. Mohindra, P. Fernando, J.J. Heikkila, Xenopus small heat shock proteins, Hsp30C and Hsp30D, maintain heat- and chemically denatured luciferase in a folding-competent state, Cell Stress Chaperones 7 (2002) 6 –16. T. Rogalla, M. Ehrnsperger, X. Preville, A. Kotlyarov, G. Lutsch, C. Ducasse, C. Paul, M. Wieske, A.P. Arrigo, J. Buchner, M. Gaestel, Regulation of Hsp27 oligomerization, chaperone function, and protective activity against oxidative stress/tumor necrosis factor alpha by phosphorylation, J. Biol. Chem. 274 (1999) 18947–18956. R. Benndorf, K. Hayess, S. Ryazantsev, M. Wieske, J. Behlke, G. Lutsch, Phosphorylation and supramolecular organization of murine small heat shock protein HSP25 abolish its actin polymerizationinhibiting activity, J. Biol. Chem. 269 (1994) 20780 –20784. A.P. Arrigo, Small stress proteins: chaperones that act as regulators of intracellular redox state and programmed cell death, Biol. Chem. 379 (1998) 19 –26. X Preville, H Schultz, U Knauf, M Gaestel, AP Arrigo, Analysis of the role of Hsp25 phosphorylation reveals the importance of the oligomerization state of this small heat shock protein in its protective function against TNFalpha- and hydrogen peroxide-induced cell death, J. Cell. Biochem. 69 (1998) 436 – 452. S.A. Loktionova, O.P. Ilyinskaya, V.L. Gabai, A.E. Kabakov, Distinct effects of heat shock and ATP depletion on distribution and isoform patterns of human Hsp27 in endothelial cells, FEBS Lett. 392 (1996) 100 –104. A.L. Bryanstev, S.A. Loktionova, O.P. Ilyinskaya, E.M. Tararak, H.H. Kampinga, A.E. Kabakov, Distribution, phosphorylation and activities of hsp25 in heat-stressed H9c2 myoblasts: a functional link to cytoprotection, Cell Stress Chaperones 7 (2002) 146 –155. P. Fernando, R. Abdulle, A. Mohindra, J. Guillemette, J. Heikkila, Mutation or deletion of the C-terminal tail affects the function and structure of Xenopus laevis small heat shock protein, hsp30, Comp. Biochem. Physiol. B. 133 (2002) 95. J. Landry, P. Chretien, A. Laszlo, H. Lambert, Phosphorylation of Hsp27 during development and decay of thermotolerance in Chinese hamster cells, J. Cell. Physiol. 147 (1991) 93–101. P. Mehlen, A. Mehlen, D. Guillet, X. Preville, A.P. Arrigo, Tumor necrosis factor-alpha induces changes in the phosphorylation, cellular localization, and oligomerization of human hsp27, a stress protein that confers cellular resistance to this cytokine, J. Cell. Biochem. 58 (1995) 248 –259. S. Dorion, J. Berube, J. Huot, J. Landry, A short lived protein involved in the heat shock sensing mechanism responsible for stressactivated protein kinase 2 (SAPK2/p38) activation, J. Biol. Chem. 274 (1999) 37591–37597. J.J. Cotto, M. Kline, R.I. Morimoto, Activation of heat shock factor 1 DNA binding precedes stress-induced serine phosphorylation. Evidence for a multistep pathway of regulation, J. Biol. Chem. 271 (1996) 3355–3358. I-K.S. Aggeli, C. Gaitanaki, A. Lazou, I. Beis, Hyperosmotic and thermal stresses activate p38-MAPK in the perfused amphibian heart, J. Exp. Biol. 205 (2002) 443– 454.

P. Fernando et al. / Experimental Cell Research 286 (2003) 175–185 [38] S. Bordin, D. Whitfield, Proliferating fibroblasts respond to collagenous Clq with phosphorylation of p38 mitogen-activated protein kinase and apoptotic features, J. Immunol. 170 (2003) 667– 671. [39] J.C. Lee, S. Kumar, D.E. Griswold, D.C. Underwood, B.J. Votta, J.L. Adams, Inhibition of p38 MAP kinase as a therapeutic strategy, Immunopharmacology 47 (2000) 185–201. [40] G.J. Lee, A.M. Roseman, H.R. Saibil, E. Vierling, A small heat shock protein stably binds heat-denatured model substrates and can main-

185

tain a substrate in a folding-competent state, EMBO J. 16 (1997) 659 – 671. [41] M. Ehrnsperger, S. Graber, M. Gaestel, J. Buchner, Binding of nonnative protein to Hsp25 during heat shock creates a reservoir of folding intermediates for reactivation, EMBO J. 16 (1997) 221– 229. [42] J.R. Glover, S. Lindquist, Hsp104, Hsp70, and Hsp40: a novel chaperone system that rescues previously aggregated proteins, Cell 94 (1998) 73– 82.