From the Centre de recherche en cancérologie de l'Université Laval, L'Hôtel-Dieu de Québec, Québec, Québec G1R 2J6, Canada
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ABSTRACT |
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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 Mammalian heat shock protein 27 (HSP27,1 also called HSP25)
belongs to the phylogenically conserved small heat shock protein (smHSP) family that includes Phosphorylation was shown in many studies to modulate the activity of
HSP27. The protein is phosphorylated on serine residues in a cascade of
protein kinases involving MAPKAP kinase 2/3 and the stress-activated
protein kinase SAPK2 (also called p38). The kinase cascade is
activated, and HSP27 is phosphorylated in cells in response to chemical
and physical stress such as heat shock, oxidative stress, or
hyperosmotic stress and also in response to various physiological
agonists involving serpentine, tyrosine kinase, or cytokine receptors
(11, 17-23). HSP27 shares with other proteins of the smHSP family a
common property to form large complexes in cells (1, 24-28). Sizes of
200-800 kDa have been reported for HSP27 (3, 29-31). The oligomeric
size of the proteins is highly dynamic, and many of the treatments that
induce HSP27 phosphorylation also induce changes in the size of the
native protein (3, 31-34). 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 (35-37). 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 (3,
9, 13, 38). 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. (39, 40). 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 (39-41).
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 (42-44). Monomers and dimers of HSP27 bind to granzyme A, a
protease involved in granule-mediated cell lysis (45). Furthermore, a
close relative of HSP27, MKBP, binds and modulates the activity of the
myotonic dystrophy protein kinase (46). 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.
Plasmids--
pSVHa27WT codes for wild type HaHSP27. It contains
the HaHSP27 sequences from pH8 (8) 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. pSVHa27 Cell Culture, Transfection, and Lysis--
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 (2). 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.
Glycerol Gradient Centrifugation--
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 (3). 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 µM
final 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
(48). The position of the 62-kDa firefly luciferase (produced in NIH3T3
cells transfected with pRSVLL/V (49)) 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 (19).
Glutaraldehyde Cross-linking--
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 mM
EDTA. 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 Screening and Analyses--
Two-hybrid assays were
performed essentially as described in the CLONTECH
Matchmaker 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 (50). 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 the
EcoRI 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) (50, 51). Colonies that arose on
Trp
Further two-hybrid assays were performed between pairs of GAL4
activation domain and LexA binding domain plasmids. GAL4 plasmids containing full-length HuHSP27, Modulation of HSP27 Size Distribution by
Phosphorylation--
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. 1A). 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. 1B). 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 (20, 22). 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. 3B). 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. 3C). 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. 3D). 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.
Domains Responsible for HSP27-HSP27 Interactions--
Information
on the nature of the intermolecular binding domains of HSP27 was
obtained in a two-hybrid screen made using full-length HaHSP27 or
HuHSP27 fused to the LexA DNA-binding domain as bait. The plasmid
libraries of fusions between the activation domain of Gal4 and cDNA
from either HeLa cells or human kidney were screened in a yeast
reporter system. In the HeLa cell library, 111 out of 193 clones that
were confirmed as real HSP27 interacting proteins were full-length or
N-terminal deletants of HuHSP27. The others corresponded to
unidentified gene products distinct from HSP27. In the kidney library,
55 out of 92 real interacting partners were identified as full or
partial length clones of HuHSP27. All the others were derived from the
homologous
To form multimeric structures, the C-terminal region of HSP27 must
contain either a multivalent binding domain or two distinct monovalent
binding domains. Another possibility is that a binding domain also
exists in the N-terminal domain. This possibility was directly tested
in the two-hybrid system. As shown in Table I, a peptide containing the
first 101 residues of HuHSP27 did interact with full-length HuHSP27.
However, no interaction was detected when the peptide was co-expressed
with the C-terminal end. Similarly, an N-terminal peptide of
All of these data are consistent with a model in which HSP27 would
possess two homotypic interacting domains located in the N-terminal and
C-terminal region, respectively. A prediction of this model is that
deletion of either domain should produce a protein that forms smaller
multimers. Although the N-terminal domain of smHSP is poorly conserved,
a short conserved WDPF motif (see Fig. 6) is observed at the extreme N
terminus of most smHSP (26). A plasmid containing coding sequences of
HaHSP27 in which codons corresponding to residues
Arg5-Tyr23 were deleted (
A number of small deletions in the C-terminal domain were produced and
tested for expression in NIH-3T3 cells. The constructs produced
Triton-insoluble proteins that could not be analyzed further,
suggesting that a major structural domain responsible for the stability
or solubility of the protein is contained in these sequences. A fusion
protein was therefore engineered containing the N-terminal sequences
5-109 of HaHSP27 (NH) fused to luciferase (LUC). Luciferase is a
monomeric protein of about 62 kDa. NH-LUC (theoretical molecular mass
of 75 kDa) formed multimers of more than 350 kDa in NIH-3T3 cells. Upon
treatment with arsenite, the luciferase activity was shifted to
fractions corresponding to 75 kDa, consistent with a
phosphorylation-induced dissociation of the multimers into monomers
(Fig. 5). The results indicated that the
N-terminal end contains the phosphorylation-sensitive binding domain of
HSP27.
On the basis of the primary structure of smHSP from different
organisms including plant, yeast, bacteria and mammalian, the position
of introns in their respective genes, and the localization of well
conserved amino acid residues, it has been suggested that smHSP are
composed of two distinct domains (see Refs. 25 and 26 and Fig.
6). The C-terminal domain, also called
the -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
-crystallin domain. These dimers further multimerize through intermolecular interactions mediated by the phosphorylation-sensitive N-terminal domain.
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A- and
B-crystallins. HSP27 is expressed constitutively in many tissues and cell lines, and its expression increases to high levels after various types of stress (1).
By artificially manipulating the level of expression of HSP27, evidence
has been accumulated suggesting that the protein modulates cell
survival during stress (2-6), apoptosis mediated by the Fas/APO1
receptor (7), microfilament organization in response to growth factor
or stress (3, 8-13), and growth rate or differentiation in some cell
lines (14, 15). In endothelial cells where HSP27 is expressed
constitutively at high levels, strong evidence has been presented
suggesting that HSP27 is a mediator of vascular endothelial growth
factor-induced microfilament reorganization and cell motility (16).
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5-23 was prepared by deleting the fragment
MluI-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. 47). The expression construct was made in two steps. First, a
PvuII-SalI-MluI-BglII
polylinker was added at the HindIII-XhoI site of
the vector pCMVnlsLL/V (47), 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.
/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
-galactosidase expression by incubation at 30 °C with 0.1 M NaPO4, 10 mM KCl, 1 mM MgSO4, 0.27%
-mercaptoethanol, 0.33 mg/ml 5-bromo-4-chloro-3-indolyl-
-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 (52). 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
A- and
B-crystallin).
A- and
B-crystallin cDNA, and N-terminal deletion mutants of HuHSP27 and
B-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
B-crystallin were obtained after digesting their corresponding
full-length cDNA with HincII (yielding HuHSP27-(1-101) and
B-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
-galactosidase (blue colonies) within
5 h.
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Fig. 1.
Size distribution of endogenous HSP27 in
CCL39 cells. CCL39 cells were treated ( ) or not (
) with 200 µM arsenite for 2 h to induce phosphorylation of
HSP27. A, the cell extracts were fractionated by
centrifugation on glycerol gradient, and HSP27 in each fraction was
detected by immunoblot. Molecular mass markers are indicated at the
top: 20 S proteasome (700 kDa), 15 S proteasome (350 kDa),
firefly luciferase (62 kDa), p38/SAPK2 (38 kDa). B, the cell
extracts were incubated with increasing concentrations of
glutaraldehyde (0, 0.001, 0.002, 0.005, 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, and 0.3%), the cross-linked proteins were separated on
SDS-polyacrylamide gel electrophoresis, and HSP27 was detected by
immunoblot. Molecular weight markers are shown on the left:
thyroglobulin (cross-linked dimer, 670 kDa), thyroglobulin (335 kDa),
myosin (200 kDa),
-galactosidase (116 kDa), phosphorylase
(97 kDa), albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa).
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Fig. 2.
Size distribution of HaHSP27-AA and
HaHSP27-EE mutants. NIH 3T3 cells were transfected with pSVHa27AA
( ) or pSVHa27EE (
). Cell extracts were fractionated by
centrifugation on glycerol gradient (A) or were incubated
(B) with increasing concentrations of glutaraldehyde (0, 0.001, 0.003, 0.01, 0,02, 0.05, and 0.1%), and the cross-linked
proteins were separated by SDS-polyacrylamide gel electrophoresis.
HSP27 was revealed by Western blot. Molecular mass markers are as
described in the legend to Fig. 1.
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Fig. 3.
Size 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 each fraction by Western blot.
-crystallin proteins (29
B-crystallin and 8
A-crystallin), confirming previous studies indicating that HSP27 can
form heterologous complexes with
-crystallins (53). In the HeLa cell
library, all HuHSP27 variants were deleted at the N terminus, and the
shortest clone started at residue 87; in the kidney library, all
N-terminal deletants started before residue 87, and the most severe
C-terminal deletant ended at residue 178. All
A-crystallin clones
were full-length; the shortest
B-crystallin clone was an N-terminal
deletant starting at residue 74 (corresponding to residue 102 in
HaHSP27 and 94 in HuHSP27). A C-terminal
B-crystallin deletant ended
at 162 (192 in HaHSP27; 184 in HuHSP27). This defined tentatively the
HuHSP27 peptide containing residues 94-178 (corresponding to HaHSP27
residues 102-186) as a domain sufficient for HSP27-HSP27 interactions.
Accordingly, we found that a C-terminal HuHSP27 peptide containing
residues 87-205 gave a positive signal in the two-hybrid system when
co-expressed with either full-length HuHSP27 or
A- or
B-crystallin. The HuHSP27 C-terminal peptide also interacted with
itself and with a C-terminal peptide of
B-crystallin-(74-175) but
not with the N-terminal residues 1-101 of HuHSP27 or 1-77 of
B-crystallin (Table I). This suggested
the involvement of a C-terminal-C-terminal interaction in smHSP
oligomerization.
Interacting domains in HSP27
A-crystallin,
and
B-crystallin were tested for interaction in the yeast two-hybrid
system. Positive (+) interactions were determined as described under
"Experimental Procedures."
B-crystallin did interact with full-length HuHSP27 and with
A-
and
B-crystallin but did not interact with the C-terminal peptide of
either HuHSP27 or
B-crystallin. The results suggested that the
N-terminal domain of smHSP also contains an intermolecular binding
domain, probably mediating N-terminal-N-terminal interactions. It was
not possible to test directly this interaction, since the fusion
protein made of the N-terminal end of HuHSP27 or
B-crystallin and
the transactivation domain yielded by itself a false positive signal.
5-23HSP27) was
expressed in NIH-3T3 cells and analyzed by chemical cross-linking and
ultracentrifugation.
5-23HSP27 sedimented as a discrete peak at the
top of the gradient and could not be significantly cross-linked in
species larger than dimers (Fig. 4).
Phosphorylation had little influence on the sedimentation profile or
the cross-linking properties of the protein, suggesting that the dimers
formed though C-terminal interactions are insensitive to serine 90 phosphorylation.
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Fig. 4.
Size distribution of an
Arg5-Tyr23 deletion mutant of HSP27. NIH
3T3 cells were transfected with pSVHa27 5-23 and treated (
) or
not (
) with 200 µM arsenite for 2 h. The cell
extracts were fractionated by glycerol gradient centrifugation
(A) or incubated with varying concentrations of
glutaraldehyde (0, 0.005, 0.01, 0.02, 0.04, 0.1, and 0.2%) and
analyzed by immunoblot after SDS-polyacrylamide gel electrophoresis
(B).
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Fig. 5.
Size distribution of a N-terminal
HSP27-luciferase fusion protein. NIH 3T3 cells transfected with
pCMVLuc ( ) and left untreated or with pCMVHa5-109.Luc and treated
(
) or not (
) with 500 µM arsenite for 1 h.
Cell extracts were fractionated by glycerol gradient centrifugation,
and the position of the expressed proteins was revealed by measuring
the luciferase activity in each fraction.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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-crystallin domain, is well conserved between the various
species and constitutes the signature for proteins of this family. In
contrast, the N-terminal domain is very weakly conserved except near
the N terminus, where a similar hydrophobic motif is often observed.
The rest of the N-terminal domain is highly variable both in length and
in composition and probably forms at its end a connecting peptide to
the C-terminal domain. Most smHSP end with a C-terminal tail of varying
length. Alignments of mammalian HSP27 with other smHSP easily identify the two major domains. The C-terminal domain of HuHSP27 extends from
residue 88 to 183 (from 96 to 191 in HaHSP27). In rodent HSP27, it has
been determined by two-dimensional NMR that the last 18 residues of the
C terminus form a highly flexible tail. The last 10 residues form a
flexible peptide in
A- and
B-crystallins (54, 55). Human and
rodent HSP27 are almost identical except for the presence of a shorter
connecting peptide between the N- and C-terminal domains in the human
protein. Both rodent and human HSP27 contain a site of phosphorylation
located at serine 15 in the conserved hydrophobic motif. HuHSP27 has
two sites of phosphorylation in the connecting peptide (serines 78 and
82). In rodents, the residue corresponding to serine 78 is replaced by
an asparagine residue.
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Fig. 6.
Conserved domains in small heat shock protein
family members. A, the sequences of a few selected
members of the small heat shock protein family were aligned to
illustrate the position of the WDPF motif and -crystallin domain
(26). B, conservation of the WDPF motif. The sequences used
are from HaHSP27 (National Center for Biotechnology Information (NCBI)
accession no. 109436), HuHSP27 (NCBI accession no. 32478), human
B-crystallin (NCBI accession no. 2144811), pea HSP18.1 (NCBI
accession no. 123555), rice HSP16.9 (NCBI accession no. 169799),
Stigmatella aurantiaca sp21 (NCBI accession no. 548963),
Methanococcus jannaschii HSP16.5 (NCBI accession no.
2495337), and S. cerevisiae HSP26 (NCBI accession no.
123568).
This study indicated that HSP27 exists in cell mainly as oligomers,
which, after cross-linking and denaturation, distribute on an SDS gel
in a regular ladder-like pattern of species whose masses are multiples
of 28 kDa. This suggests that the structure is homotypic,
i.e. formed only of HSP27. The results do not exclude the
possibility that HSP27 can interact with other proteins in the cells,
but they suggest that such an association is not stoichiometric. The
most abundant form of HSP27 oligomers as revealed by
ultracentrifugation has a molecular mass of about 700 kDa, which
corresponds also to the size of the major cross-linked species obtained
at high concentrations of glutaraldehyde. Some dimeric and monomeric
species, however, appeared to resist cross-linking, and species with
molecular mass lower than 700 kDa distributed along the glycerol
gradient, suggesting that the structure might be highly dynamic, small
species being in equilibrium with bigger ones. Such a high dynamic
property has also been described for the structure of A-crystallin,
in which exchange of subunits between different oligomers occurs at
high rates (56). Our results showed that upon phosphorylation of serine
90 but not of serine 15, most HSP27 complexes reduced also to dimers.
Phosphorylation of serine 90 may shift the equilibrium for the
formation of HSP27 complexes toward smaller species, perhaps by
destabilizing the association of the dimers into higher molecular weight structures. Since mostly dimers were seen after phosphorylation, one could conclude that the dimer is the building block of the HSP27
complex. This conclusion is in line with that of Dudich et
al. (57), who showed that the temperature dependence of the excessive heat capacity of the denaturation of HSP27 corresponds to a
protein with a molecular mass of 50 kDa, suggesting that the dimer
structure is a minimum cooperative subunit of the protein. The
conclusion is also in agreement with the crystallographic data recently
obtained for a distantly related bacterial smHSP, MjHSP16.5 (58). The
analysis revealed a 24-subunit hollow spherical complex made of three
asymmetric units of eight subunits, in which the strongest contacts
occur between dimers. Although many of the residues involved in subunit
contact of MjHSP16.5 are not conserved in HSP27, many of the structural
features such as the basic symmetry may be conserved. A 24-mer of HSP27
would yield a size of 648 kDa, in close agreement with our estimated
size of about 700 kDa.
Our data suggest that HSP27 oligomerizes through the interaction of two
distinct binding domains. Both domains appear to act as independent
binding modules that are required for oligomerization up to high
molecular weight complexes. Analyses in the two-hybrid interaction
assay system localized one of the binding domains between residues 94 and 178 of HuHSP27 (corresponding to HaHSP27 residues 102-186). This
region corresponds almost exactly to the -crystallin domain. It
starts 6 residues after the position corresponding to intron I in
-crystallins and ends 7 residues before the beginning of the
C-terminal flexible tail found in HSP27 (55). Although there is little
known about the structure of HSP27, in several studies a role for this
conserved domain as the building block of the quaternary structure of
other members of this family has been suggested. In particular, the
sequence extending between Tyr109 and Leu120 in
A-crystallin (Tyr133-Leu144 in HuHSP27;
Tyr141-Leu152 in HaHSP27) was suggested to be
the region where major intermolecular interactions occur (59). This
sequence, which is very well conserved in mammalian smHSP (7 of 11 of
these residues are conserved between
A-crystallin and HSP27), forms
a
-strand conformation in close proximity to the equivalent strand
from opposing molecules. It also corresponds to the region of most
extensive subunit contact in the recently crystallized MjHSP16.5 (58).
The resemblance in primary structure between HSP27 and MjHSP16.5 makes
it likely that the
-crystallin domain of HSP27 has also multiple
subunit-subunit interacting surfaces that could mediate the formation
of high order molecular complexes. Our data obtained with the
N-terminal deletant of HSP27 suggest that by itself the C-terminal
domain can only form stable dimers when expressed in vivo.
However, N-terminal deletants of HSP27 or
B-crystallin purified from
bacteria formed large molecular species after in vitro
renaturation (60). We conclude that in the normal cell environment, the
higher molecular weight multimers formed through C-terminal
interactions are not stable and require to be stabilized by the
N-terminal domain.
Indeed, our results indicated that the N terminus is also essential for
the formation of HSP27 oligomers. We showed that this region,
corresponding to exon I in -crystallin, behaves as an independent
structural domain, conferring upon fusion to luciferase the capacity to
multimerize in a phosphorylation-sensitive manner. A role for the N
terminus in providing essential stabilizing forces to smHSP oligomers
has been suggested before (61, 62). Recombinant
A-crystallin in
which the complete N-terminal domain was deleted formed dimers or
tetramers upon renaturation (60, 63). Similarly, deletions of the first
15 residues in Caenorhabditis elegans HSP16.2 reduced considerably the size of the oligomers and its chaperone activity (41). The structure of smHSP appears to contain a large central cavity in which the rather hydrophobic N terminus is buried, being semiprotected from the environment but still keeping access to
solvent (41, 58, 64). From our results, it could be proposed that
N-terminal-N-terminal domain interactions in this central cavity
stabilize the supramolecular organization of the dimers formed by the
-crystallin domain in the same way as they result in the
multimerization of luciferase. Phosphorylation of serine 90 somehow
weakens these N-terminal interactions, resulting in the dissociation of
HSP27 into dimers. Serine 90 is located in a highly variable region
suggested to form a connecting peptide between the
-crystallin and
the N-terminal domains (25). It is possible that phosphorylation of
serine 90 somehow modifies the structure of this connecting arm, which,
for example, by changing the orientation of the two domains relative to
each other would prevent the N terminus from gaining access to the
central cavity and therefore prevent interactions and destabilize the oligomer.
The only relatively well conserved region in the N-terminal domain of
distantly related smHSP is located at the extreme N terminus (26, 65).
In Fig. 6, we refer to this region as the WDPF domain. The archetype
pattern is found in the myxobacterium smHSP SP21, which contains two
WDPF motifs separated by 8 residues. Although important variations in
sequence are observed between species, some acidic (D or E), proline,
and aromatic residues (W or F) appear to have been conserved even in
the most distantly related species. We have shown that deletion of
residues 5-23 within the WDPF domain of HaHSP27 results in the
formation of dimers that were insensitive to phosphorylation. Mutations
of conserved phenylanaline residues in the WDPF domain of
B-crystallin also abolish its chaperone activity (66). These results
suggest that this domain may be directly involved in the stabilization of the HSP27 oligomer.
The role of serine 15 phosphorylation remains to be determined. As
compared with phosphorylation of serine 90 or to deletion of the WDPF
domain, phosphorylation of serine 15 did not cause a major effect on
the structure of HSP27. However, a subtle effect on the stability of
the higher molecular weight oligomers was observed and cannot be
excluded completely without more investigations. Another possibility is
that serine 15 modulates the interaction of HSP27 with other proteins.
As stated before, the WDPF domain has been suggested to play a role in
the chaperone function of -crystalline (66). It is possible that
phosphorylation of serine 15 regulates the interaction of this domain
with unfolded proteins, which in the case of
-crystallin would occur
in the central cavity where the N terminus also appears to be located
(67). A similar situation occurs in the GroEL-GroES chaperone, which
changes the hydrophobic characteristics of its cavity during the
binding and refolding process (68, 69). Alternatively, phosphorylation of serine 15 may regulate the interaction of HSP27 with other proteins
such as protein kinase B, granzyme A, or F-actin (35-37, 42, 45). In
the case of actin, both the phosphorylation and the oligomeric state of
HSP27 has been shown to modulate the interaction. An intriguing
possibility is that upon phosphorylation of serine 90 and dissociation
of the oligomer, the N-terminal domain becomes exposed and thus
available for interaction with F-actin. The described effects of
phosphorylation on the activities of HSP27 at the level of actin
filament polymerization (37) suggest the additional possibility that
once the oligomer is broken up following phosphorylation of serine 90, phosphorylation of serine 15 modulates the association of HSP27 with actin.
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ACKNOWLEDGEMENTS |
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We thank Olivier Bensaude of the École Normale Supérieure de Paris for useful discussions and the gift of the luciferase pCMVnlsLL/V plasmid.
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FOOTNOTES |
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* This study was supported by Medical Research Council Grant MT-7088.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 studentships from the Cancer Research Society, Inc.
and the FCAR/FRSQ Santé, Québec.
§ To whom correspondence should be addressed. Centre de recherche en cancérologie de l'Université Laval, L'Hôtel-Dieu de Québec, 11 côte du Palais, Québec, Québec G1R 2J6, Canada. Tel.: 418-691-5555; Fax: 418-691-5439; E-mail: jacques.landry{at}med.ulaval.ca.
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ABBREVIATIONS |
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The abbreviations used are: HSP27, mammalian heat shock protein 25/27; HaHSP27, Chinese hamster HSP27; HuHSP27, human HSP27; smHSP, small heat shock protein.
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REFERENCES |
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