Title: HSP27 Multimerization Mediated by Phosphorylation-sensitive Intermolecular Interactions at the Amino Terminus
Abstract: Distinct biochemical activities have been reported for small and large molecular complexes of heat shock protein 27 (HSP27), respectively. Using glycerol gradient ultracentrifugation and chemical cross-linking, we show here that Chinese hamster HSP27 is expressed in cells as homotypic multimers ranging from dimers up to 700-kDa oligomers. Treatments with arsenite, which induces phosphorylation on Ser15 and Ser90, provoked a major change in the size distribution of the complexes that shifted from oligomers to dimers. Ser90 phosphorylation was sufficient and necessary for causing this change in structure. Dimer formation was severely inhibited by replacing Ser90 with Ala90 but not by replacing Ser15 with Ala15. Using the yeast two-hybrid system, two domains were identified that were responsible for HSP27 intermolecular interactions. One domain was insensitive to phosphorylation and corresponded to the C-terminal α-crystallin domain. The other domain was sensitive to serine 90 phosphorylation and was located in the N-terminal region of the protein. Fusion of this N-terminal domain to firefly luciferase conferred luciferase with the capacity to form multimers that dissociated into monomers upon phosphorylation. A deletion within this domain of residues Arg5–Tyr23, which contains a WDPF motif found in most proteins of the small heat shock protein family, yielded a protein that forms only phosphorylation-insensitive dimers. We propose that HSP27 forms stable dimers through the α-crystallin domain. These dimers further multimerize through intermolecular interactions mediated by the phosphorylation-sensitive N-terminal domain. Distinct biochemical activities have been reported for small and large molecular complexes of heat shock protein 27 (HSP27), respectively. Using glycerol gradient ultracentrifugation and chemical cross-linking, we show here that Chinese hamster HSP27 is expressed in cells as homotypic multimers ranging from dimers up to 700-kDa oligomers. Treatments with arsenite, which induces phosphorylation on Ser15 and Ser90, provoked a major change in the size distribution of the complexes that shifted from oligomers to dimers. Ser90 phosphorylation was sufficient and necessary for causing this change in structure. Dimer formation was severely inhibited by replacing Ser90 with Ala90 but not by replacing Ser15 with Ala15. Using the yeast two-hybrid system, two domains were identified that were responsible for HSP27 intermolecular interactions. One domain was insensitive to phosphorylation and corresponded to the C-terminal α-crystallin domain. The other domain was sensitive to serine 90 phosphorylation and was located in the N-terminal region of the protein. Fusion of this N-terminal domain to firefly luciferase conferred luciferase with the capacity to form multimers that dissociated into monomers upon phosphorylation. A deletion within this domain of residues Arg5–Tyr23, which contains a WDPF motif found in most proteins of the small heat shock protein family, yielded a protein that forms only phosphorylation-insensitive dimers. We propose that HSP27 forms stable dimers through the α-crystallin domain. These dimers further multimerize through intermolecular interactions mediated by the phosphorylation-sensitive N-terminal domain. mammalian heat shock protein 25/27 Chinese hamster HSP27 human HSP27 small heat shock protein Mammalian heat shock protein 27 (HSP27,1 also called HSP25) belongs to the phylogenically conserved small heat shock protein (smHSP) family that includes αA- and αB-crystallins. HSP27 is expressed constitutively in many tissues and cell lines, and its expression increases to high levels after various types of stress (1Arrigo A.P. Landry J. Morimoto R.I. Tissières A. Georgopoulos C. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994: 335-373Google Scholar). By artificially manipulating the level of expression of HSP27, evidence has been accumulated suggesting that the protein modulates cell survival during stress (2Landry J. Chrétien P. Lambert H. Hickey E. Weber L.A. J. Cell Biol. 1989; 109: 7-15Crossref PubMed Scopus (582) Google Scholar, 3Lavoie J.N. Lambert H. Hickey E. Weber L.A. Landry J. Mol. Cell. Biol. 1995; 15: 505-516Crossref PubMed Scopus (570) Google Scholar, 4Knauf U. Jakob U. Engel K. Buchner J. Gaestel M. EMBO J. 1994; 13: 54-60Crossref PubMed Scopus (124) Google Scholar, 5Mehlen P. Preville X. Chareyron P. Briolay J. Klemenz R. Arrigo A.P. J. 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Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994: 335-373Google Scholar, 24de Jong W.W. Hendriks W. Mulders J.W. Bloemendal H. Trends Biochem. Sci. 1989; 14: 365-368Abstract Full Text PDF PubMed Scopus (184) Google Scholar, 25de Jong W.W. Leunissen J.A. Leenen P.J. Zweers A. Versteeg M. J. Biol. Chem. 1988; 263: 5141-5149Abstract Full Text PDF PubMed Google Scholar, 26de Jong W.W. Leunissen J.A. Voorter C.E. Mol. Biol. Evol. 1993; 10: 103-126PubMed Google Scholar, 27Hickey E. Brandon S.E. Sadis S. Smale G. Weber L.A. Gene (Amst.). 1986; 43: 147-154Crossref PubMed Scopus (111) Google Scholar, 28van den Ijssel P.R. Smulders R.H. de Jong W.W. Bloemendal H. Ophthalmic Res. 1996; 28 Suppl. 1: 39-43Crossref PubMed Scopus (8) Google Scholar). Sizes of 200–800 kDa have been reported for HSP27 (3Lavoie J.N. Lambert H. Hickey E. Weber L.A. Landry J. Mol. Cell. Biol. 1995; 15: 505-516Crossref PubMed Scopus (570) Google Scholar, 29Arrigo A.P. Suhan J.P. Welch W.J. Mol. Cell. 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Mehlen P. Arrigo A.P. Cell Stress Chaperones. 1996; 1: 225-235Crossref PubMed Scopus (41) Google Scholar). It has been suggested that phosphorylation-induced changes in the ultrastructure may regulate the biochemical activities of HSP27. Two biochemical activities have been described for HSP27 in vitro. A first activity is restricted to monomeric HSP27. In solution, purified monomers behave as F-actin cap-binding proteins and inhibit actin polymerization (35Miron T. Wilchek M. Geiger B. Eur. J. Biochem. 1988; 178: 543-553Crossref PubMed Scopus (101) Google Scholar, 36Miron T. Vancompernolle K. Vandekerckhove J. Wilchek M. Geiger B. J. Cell Biol. 1991; 114: 255-261Crossref PubMed Scopus (389) Google Scholar, 37Benndorf R. Hayess K. Ryazantsev S. Wieske M. Behlke J. Lutsch G. J. Biol. Chem. 1994; 269: 20780-20784Abstract Full Text PDF PubMed Google Scholar). Only unphosphorylated HSP27 could block actin polymerization, hence providing a mechanism to explain the in vivo observations that phosphorylation of HSP27 during stress or growth factor stimulation regulates actin polymerization and modulates filament stability or reorganization (3Lavoie J.N. Lambert H. Hickey E. Weber L.A. Landry J. Mol. Cell. Biol. 1995; 15: 505-516Crossref PubMed Scopus (570) Google Scholar,9Lavoie J.N. Hickey E. Weber L.A. Landry J. J. Biol. Chem. 1993; 268: 24210-24214Abstract Full Text PDF PubMed Google Scholar, 13Piotrowicz R.S. Levin E.G. J. Biol. Chem. 1997; 272: 25920-25927Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 38Zhu Y. O'Neill S. Saklatvala J. Tassi L. Mendelsohn M.E. Blood. 1994; 84: 3715-3723Crossref PubMed Google Scholar). A second activity has been described for the oligomeric HSP27 complex. In vitro, high molecular weight recombinant HSP27 complexes can absorb heat-denatured proteins on their surface, preventing their aggregation and keeping them in a folding-competent state. The subsequent action of other chaperone proteins such as HSP70 leads to the renaturation of the unfolded proteins. (39Jakob U. Gaestel M. Engel K. Buchner J. J. Biol. Chem. 1993; 268: 1517-1520Abstract Full Text PDF PubMed Google Scholar, 40Ehrnsperger M. Graber S. Gaestel M. Buchner J. EMBO J. 1997; 16: 221-229Crossref PubMed Scopus (634) Google Scholar). Although not yet directly demonstrated, the mechanism proposed for this activity as well as studies performed with other smHSP suggested that the chaperone activity is limited to the oligomeric complexes (39Jakob U. Gaestel M. Engel K. Buchner J. J. Biol. Chem. 1993; 268: 1517-1520Abstract Full Text PDF PubMed Google Scholar, 40Ehrnsperger M. Graber S. Gaestel M. Buchner J. EMBO J. 1997; 16: 221-229Crossref PubMed Scopus (634) Google Scholar, 41Leroux M.R. Melki R. Gordon B. Batelier G. Candido E.P. J. Biol. Chem. 1997; 272: 24646-24656Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Both the chaperone and actin modulation activities could explain the protective action of HSP27 in vivo. However, specific chaperone functions regulating the activity or the stability of specific target proteins may also contribute to the homeostatic functions of HSP27. For example, oligomeric HSP27 binds to activated protein kinase B, a protein that lies in a survival pathway during stress (42Konishi H. Matsuzaki H. Tanaka M. Takemura Y. Kuroda S. Ono Y. Kikkawa U. FEBS Lett. 1997; 410: 493-498Crossref PubMed Scopus (237) Google Scholar, 43Downward J. Curr. Opin. Cell Biol. 1998; 10: 262-267Crossref PubMed Scopus (1188) Google Scholar, 44Franke T.F. Kaplan D.R. Cantley L.C. Cell. 1997; 88: 435-437Abstract Full Text Full Text PDF PubMed Scopus (1522) Google Scholar). Monomers and dimers of HSP27 bind to granzyme A, a protease involved in granule-mediated cell lysis (45Beresford P.J. Jaju M. Friedman R.S. Yoon M.J. Lieberman J. J. Immunol. 1998; 161: 161-167PubMed Google Scholar). Furthermore, a close relative of HSP27, MKBP, binds and modulates the activity of the myotonic dystrophy protein kinase (46Suzuki A. Sugiyama Y. Hayashi Y. Nyu-i N. Yoshida M. Nonaka I. Ishiura S. Arahata K. Ohno S. J. Cell Biol. 1998; 140: 1113-1124Crossref PubMed Scopus (126) Google Scholar). The role of HSP27 phosphorylation and multimeric state in modulating these interactions is unknown. In the present study, we studied the relationships between the phosphorylation and the oligomerization properties of HSP27. Chinese hamster HSP27 (HaHSP27) is phosphorylated on two serine residues, Ser15 and Ser90. We show here that phosphorylation on Ser90 is sufficient and necessary to cause HSP27 to shift from a 700-kDa multimeric structure to dimers. Two homotypic binding domains were identified in HSP27. A first one, located within residues 95–186 of HaHSP27 (87–178 in HuHSP27), mediates dimerization and is insensitive to phosphorylation. A second one includes a small conserved stretch in the extreme amino terminus and is destabilized by phosphorylation of Ser90. We show that the N-terminal domain of HSP27 is sufficient to confer firefly luciferase with a phosphorylation-sensitive multimerization capacity. pSVHa27WT codes for wild type HaHSP27. It contains the HaHSP27 sequences from pH8 (8Lavoie J.N. Gingras-Breton G. Tanguay R.M. Landry J. J. Biol. Chem. 1993; 268: 3420-3429Abstract Full Text PDF PubMed Google Scholar) inserted at the HindIII site of the vector pSVT7. Other HaHSP27 constructs were made from a derivative of pSVHa27WT, pSVHa27Mlu, in which a MluI restriction site was created by introducing a silent mutation in the third codon of HaHSP27. pSVHa27AA, pSVHa27EE, pSVHa27SA, pSVHa27AS, pSVHa27AE, and pSVHa27EA express phosphorylation site mutants of HaHSP27, the last two letters in the names corresponding to the replacement amino acid residues at position 15 and 90, respectively. Mutations were introduced in pSVHa27Mlu by polymerase chain reaction using specific synthetic oligonucleotide primers replacing the serine codon AGC by the alanine codon GCC or the glutamate codon GAA. pSVHa27Δ5–23 was prepared by deleting the fragmentMluI–KpnI in pSVHa27Mlu, yielding a protein lacking residues 5–23. pCMVHa5–109.Luc expresses a fusion protein made from residues 5–109 of HaHSP27 and the cytoplasmic firefly luciferase (L550V conversion in the peroxisomal localization signal; Ref. 47Michels A.A. Nguyen V.T. Konings A.W. Kampinga H.H. Bensaude O. Eur. J. Biochem. 1995; 234: 382-389Crossref PubMed Scopus (71) Google Scholar). The expression construct was made in two steps. First, aPvuII–SalI–MluI–BglII polylinker was added at the HindIII–XhoI site of the vector pCMVnlsLL/V (47Michels A.A. Nguyen V.T. Konings A.W. Kampinga H.H. Bensaude O. Eur. J. Biochem. 1995; 234: 382-389Crossref PubMed Scopus (71) Google Scholar), thereby removing the NLS site. This new vector named pCMVLuc yields the expression of a cytoplasmic luciferase with 12 amino acids added at the N terminus. Then, the fragment MluI–HincII from pSVHa27Mlu was inserted at the SalI–BglII restriction sites of pCMVLuc, resulting in a final expression construct coding for the fusion protein Met-Asn-Ser-Try(HaHSP27)5–109-Leu-Leu-Glu-Asn-(luciferase)4–549-Val. CCL39 and NIH3T3 cells were maintained at 37 °C in a 5% CO2 humidified atmosphere, in Dulbecco's modified Eagle's medium containing 2.2 g/liter NaHCO3 and 4.5 g/liter glucose and supplemented with 5% fetal calf serum or 10% calf serum, respectively. NIH3T3 cells were plated 24 h before transfection at a concentration of 5000–16,000 cells/cm2 in a 75-cm2 culture flask. Transfection by calcium phosphate precipitation was done as described before using 10 μg of plasmid/flask (2Landry J. Chrétien P. Lambert H. Hickey E. Weber L.A. J. Cell Biol. 1989; 109: 7-15Crossref PubMed Scopus (582) Google Scholar). 50 μm chloroquine was added for the first 5 h of transfection. The cells were used 48–72 h after transfection. When indicated, the cells were then treated for 2 h with 200 μm arsenite to induce phosphorylation of the expressed proteins. After treatment, the cells were lysed by brief sonication in 25 mm HEPES buffer, pH 7.4, containing 3.33% glycerol, 1 mm EDTA, 1 mm dithiothreitol, and 0.1 mm phenylmethylsulfonyl fluoride at 4 °C. The lysate was cleared by centrifugation at 17,000 × g for 5 min at 4 °C. The supernatant was used directly for glycerol gradient centrifugation or glutaraldehyde cross-linking. The cell lysates (0.5 ml) were loaded on top of a 12.6-ml linear gradient of glycerol (10–40%) made in 25 mm HEPES buffer, pH 7.4, containing 1 mm EDTA and 1 mm dithiothreitol. The tubes were centrifuged for 18 h at 30000 rpm in a SW40 rotor (Beckman) at 4 ° C. The gradient were fractionated in 44 fractions. Aliquots were diluted in Tris/glycine/SDS buffer (25 mm Tris, 192 mm glycine, 0.01% SDS) and dot-blotted on nitrocellulose membrane. HSP27 was revealed by immunoblotting using an antibody against the HaHSP27 C-terminal AGKSEQSGAK peptide (3Lavoie J.N. Lambert H. Hickey E. Weber L.A. Landry J. Mol. Cell. Biol. 1995; 15: 505-516Crossref PubMed Scopus (570) Google Scholar). Antigen-antibody complexes were detected with a 125I-labeled goat anti-rabbit IgG. Detection and quantification were done using a Storm imaging system from Molecular Dynamics, Inc. (Sunnyvale, CA). Varying amounts of supernatant were also analyzed to confirm the linearity of the detection method. Luciferase was revealed from its activity. Aliquots (10 μl) of each fraction were diluted in 300 μl of 25 mm glycyl/glycine buffer, pH 7.8, containing 10 mm MgSO4, 2 mm ATP, and 1 mm dithiothreitol. Luciferase activity was measured in a Berthold Lumat 9501 luminometer for 30 s after the addition 100 μl of the substrate d-luciferin (50 μmfinal concentration). The gradient was calibrated from the positions of known protein complexes. The positions of the 20 S (700 kDa) and 15 S (340 kDa) proteasomes were determined by Western blot using an anti-HC8 antibody and confirmed by pore exclusion electrophoresis on native gel (48Yang Y. Fruh K. Ahn K. Peterson P.A. J. Biol. Chem. 1995; 270: 27687-27694Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). The position of the 62-kDa firefly luciferase (produced in NIH3T3 cells transfected with pRSVLL/V (49de Wet J.R. Wood K.V. DeLuca M. Helinski D.R. Subramani S. Mol. Cell. Biol. 1987; 7: 725-737Crossref PubMed Scopus (2480) Google Scholar)) was determined by measuring luciferase activity. The position of p38/stress-activated protein kinase 2 (38 kDa) was determined using an antibody against the C-terminal sequence PPLQEEMES of murine p38 (19Guay J. Lambert H. Gingras-Breton G. Lavoie J.N. Huot J. Landry J. J. Cell Sci. 1997; 110: 357-368Crossref PubMed Google Scholar). The cell lysates were mixed with one volume of 0–0.8% glutaraldehyde in water. After incubation for 30 min at 30 °C, the reaction was stopped by adding one volume of 1 m TRIS-HCl containing 10% SDS and 10 mmEDTA. Aliquots were analyzed by electrophoresis on a 3–10% SDS-polyacrylamide gel. Crossed-linked HSP27 species were detected by immunoblotting with antibody to HSP27. Two-hybrid assays were performed essentially as described in the CLONTECHMatchmaker Library user manual. Full-length Chinese hamster or human HSP27 cDNAs were cloned in the pBTM116 (TRP1) vector to produce the bait fusion protein LexA-HaHSP27 or LexA-HuHSP27 (50Bartel P.L. Chien C. Sternglanz R. Fields S. Hartley D.A. Cellular Interactions in Development: A Practival Approach. IRL Press, Oxford1993: 153-179Google Scholar). The LexA-HaHSP27 construct was used to screen a HeLa cell cDNA library constructed at the EcoRI–XhoI site of the GAL4 activation domain plasmid pGADGH (LEU2) (CLONTECH). The LexA-HuHSP27 protein was used to screen a human kidney cDNA library constructed at theEcoRI site of the GAL4 activation domain plasmid pGAD10 (LEU2) (CLONTECH). Screening was performed by sequential transformation of bait and library vectors in the Saccharomyces cerevisiae reporter strain L40 (MATa trp1 leu2 his3 LYS2::lexA-HIS3 URA3::lexA-lacZ) (50Bartel P.L. Chien C. Sternglanz R. Fields S. Hartley D.A. Cellular Interactions in Development: A Practival Approach. IRL Press, Oxford1993: 153-179Google Scholar, 51Hollenberg S.M. Sternglanz R. Cheng P.F. Weintraub H. Mol. Cell. Biol. 1995; 15: 3813-3822Crossref PubMed Scopus (585) Google Scholar). Colonies that arose on Trp−/Leu−/His−-selective plates were replica-plated, and one set was transferred to filter disks (Whatman Inc., Clifton, NJ) lysed by freezing, and tested for β-galactosidase expression by incubation at 30 °C with 0.1m NaPO4, 10 mm KCl, 1 mm MgSO4, 0.27% β-mercaptoethanol, 0.33 mg/ml 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside, pH 7.0. LEU2 plasmids were isolated from the blue colonies and retested by co-transfection with Ras(V12) or lamin C fused to LexA to eliminate false positive clones (52Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1663) Google Scholar). Inserts from the pGADGH or pGAD10 plasmids of the remaining true positive clones were sequenced to identify sequences of proteins belonging to members of the smHSP family (HSP27 and αA- and αB-crystallin). Further two-hybrid assays were performed between pairs of GAL4 activation domain and LexA binding domain plasmids. GAL4 plasmids containing full-length HuHSP27, αA- and αB-crystallin cDNA, and N-terminal deletion mutants of HuHSP27 and αB-crystallin were as obtained from the screenings described above. LexA plasmids were constructed by recloning the insert of the GAL4 plasmids into pBMT116. Additional GAL4 and LexA C-terminal deletion mutants of HuHSP27 and αB-crystallin were obtained after digesting their corresponding full-length cDNA with HincII (yielding HuHSP27-(1–101) and αB-crystallin-(1–77)) and recloning in pBTM116 and pGADGH. All DNA constructs were confirmed by DNA sequencing. Interactions were determined by co-transfecting the pBTM116 and pGADGH or pGAD10 constructs in pairs in L40. Positive interactions were determined by the ability of the transformed yeast to grow on Trp−/Leu−/His− media and for their capacity to express β-galactosidase (blue colonies) within 5 h. CCL39 cell extracts were fractionated by ultracentrifugation on glycerol gradient, and each fraction was analyzed for the presence of HSP27 by immunoblotting. HSP27 sedimented as complexes of heterogeneous sizes distributed between the top of the gradient and fractions corresponding to a molecular mass of about 700 kDa (Fig. 1 A). To better assess the nature of the HSP27 complex, increasing concentrations of glutaraldehyde was added to the cell extracts, and the cross-linked products were analyzed by SDS-polyacrylamide gel electrophoresis and HSP27 immunoblotting (Fig. 1 B). With increasing concentrations of glutaraldehyde, HSP27 was cross-linked progressively in species showing a uniform ladder distribution of sizes with apparent molecular masses in multiples of 28 ± 1 kDa (determined by linear regression analysis of the position of the cross-linked products). These data indicate that no other proteins were associated stoichiometrically with HSP27 and, thus, that in situ the HSP27 complex is mainly a homopolymer. Some 700-kDa species, dimers, and monomers resisted cross-linking even at the highest concentration of glutaraldehyde. At this concentration, glutaraldehyde started to produce a general cross-linking of all proteins as revealed from the fainting of all protein bands on the Coomassie-stained gel (data not shown). These properties agreed with the sedimentation profile data and suggested that HSP27 was expressed in cells as large polymers of about 700 kDa in equilibrium with smaller species, mainly monomers and dimers. HaHSP27 is phosphorylated on Ser15 and Ser90 by MAPKAP kinase 2, a serine kinase activated by the stress sensitive SAPK2/p38 kinase (20Rouse J. Cohen P. Trigon S. Morange M. Alonso-Llamazares A. Zamanillo D. Hunt T. Nebreda A.R. Cell. 1994; 78: 1027-1037Abstract Full Text PDF PubMed Scopus (1503) Google Scholar, 22Huot J. Lambert H. Lavoie J.N. Guimond A. Houle F. Landry J. Eur. J. Biochem. 1995; 227: 416-427Crossref PubMed Scopus (173) Google Scholar). To analyze changes in the size distribution of HSP27 upon phosphorylation, CCL39 cells were exposed to arsenite for 2 h before extraction. Such treatment induced almost complete phosphorylation of HSP27 (data not shown) and a dramatic change in the size distribution profile of HSP27. After treatment, essentially all HSP27 was recovered in the first few fractions at the top of the glycerol gradient, between stress-activated protein kinase 2 (38 kDa) and firefly luciferase (62 kDa), and most of it could not be cross-linked in larger species than dimers (Fig. 1). To confirm that phosphorylation was directly involved in modulating the supramolecular organization of HSP27, phosphorylation mutants of HaHSP27 were prepared by replacing Ser15 and Ser90 with alanine to mimic nonphosphorylatable serine residues (HaHSP27-AA) or with glutamate to mimic constitutively phosphorylated residues (HaHSP27-EE). These constructs were expressed in NIH 3T3 cells, and their size distribution was investigated as above. NIH 3T3 cells were chosen as recipient cells because they express negligible amounts of endogenous HSP27. As expected, HaHSP27-AA had a sedimentation profile and cross-linking pattern similar to those obtained with wild type HSP27 in untreated cells (Fig.2) but did not shift to smaller sizes upon treatments that induce phosphorylation (not shown). In contrast, HaHSP27-EE was found almost exclusively as dimers and monomers in both control (Fig. 2) and treated cells (not shown). The relative importance of each site of phosphorylation was investigated by expressing single site mutants. HaHSP27, in which serine 15 was changed for alanine (HaHSP27-AS), behaved as the wild type protein (Fig.3, B versus A). It distributed mainly as large multimers in control cells. After stress, it was found essentially as dimers and monomers (Fig. 3 B). The data indicated that phosphorylation of serine 90 was sufficient and that phosphorylation of serine 15 was not required to cause the ultrastructural changes in HaHSP27. In contrast, HaHSP27 in which serine 90 was converted to alanine (HaHSP27-SA) had a normal size distribution in control cells and did not produce dimers or monomers after phosphorylation (Fig. 3 C). This indicated that phosphorylation of serine 15 was not sufficient and that phosphorylation of serine 90 was necessary for the production of small molecular weight species. The results were confirmed by analyzing HaHSP27-EA and HaHSP27-AE double mutants. As expected, HaHSP27-EA was found mostly in large oligomeric complexes, whereas HaHSP27-AE was mostly found at the top of the gradient (Fig. 3 D). We reproducibly observed that phosphorylated HaHSP27-SA and HaHSP27-EA yielded a peak slightly smaller than control HSP27. There is therefore a possibility that phosphorylation of serine 15 might affect the stability of the very high molecular weight species, causing a size shift in the 500–800-kDa range.Figure 3Size distribution of individual phosphorylation site mutants of HSP27. NIH 3T3 cells were transfected with pSVHa27WT (A), pSVHa27AS (B), pSVHa27SA (C), pSVHa27EA (▪ in D) or pSVHa27AE (■ in D) and treated (●) or not (▪, ■) with 200 μm arsenite for 2 h. Cell extracts were fractionated by centrifugation on glycerol gradient, and HSP27 concentration was determined in