* Department of Molecular Biology, The First and § Second Departments of Internal Medicine, Yokohama City University
School of Medicine, Kanazawa-ku, Yokohama 236, Japan;
Institute of Molecular and Cellular Biosciences, the University of
Tokyo, Tokyo 113, Japan; and ¶ National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira,
Tokyo 187, Japan
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Abstract |
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Muscle cells are frequently subjected to severe conditions caused by heat, oxidative, and mechanical stresses. The small heat shock proteins (sHSPs)
such as B-crystallin and HSP27, which are highly expressed in muscle cells, have been suggested to play roles in maintaining myofibrillar integrity against such
stresses. Here, we identified a novel member of the
sHSP family that associates specifically with myotonic
dystrophy protein kinase (DMPK). This DMPK-binding protein, MKBP, shows a unique nature compared
with other known sHSPs: (a) In muscle cytosol, MKBP
exists as an oligomeric complex separate from the complex formed by
B-crystallin and HSP27. (b) The expression of MKBP is not induced by heat shock, although it shows the characteristic early response of
redistribution to the insoluble fraction like other
sHSPs. Immunohistochemical analysis of skeletal muscle cells shows that MKBP localizes to the cross sections of individual myofibrils at the Z-membrane as
well as the neuromuscular junction, where DMPK has
been suggested to be concentrated. In vitro, MKBP enhances the kinase activity of DMPK and protects it
from heat-induced inactivation. These results suggest
that MKBP constitutes a novel stress-responsive system
independent of other known sHSPs in muscle cells and
that DMPK may be involved in this system by being
activated by MKBP. Importantly, since the amount of
MKBP protein, but not that of other sHSP family
member proteins, is selectively upregulated in skeletal
muscle from DM patients, an interaction between
DMPK and MKBP may be involved in the pathogenesis of DM.
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Introduction |
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MYOTONIC dystrophy (DM)1 is an autosomal dominantly inherited disease characterized primarily
by myotonia and progressive muscle weakness,
although it is also associated with a range of other abnormalities, including cardiac conduction defects, cataracts, testicular and ovarian atrophy, diabetes, and mental dysfunction (Harper, 1989). In 1992, positional cloning studies
identified the responsible mutation as CTG repeat expansions in a gene encoding a putative serine/threonine kinase
(DMPK) (Brook et al., 1992
; Fu et al., 1992
; Mahadevan
et al., 1992
). However, unlike the case of Duchennne muscular dystrophy, this finding did not lead directly to accelerated progress in the study of the underlying molecular
mechanism of the disease because the mutation is located in the 3'-untranslated region of the gene, and thus DMPK,
if expressed, should work normally in DM patients. If one
assumes that a change in the function of DMPK is involved in the pathogenesis of DM, then a possible mechanism is that the CTG repeat expansions affect the expression level of DMPK by impairing a certain step in gene expression such as transcription, mRNA processing, or
translation (Taneja et al., 1995
). In fact, numerous studies
to quantify the levels of DMPK mRNA or protein in the
muscles of affected patients have been undertaken to validate this possibility. Although there are some conflicting
results (Sabouri et al., 1993
; Bhagwati et al., 1996
), most
studies have reported decreases in the level of DMPK
mRNA and protein, suggesting the involvement of DMPK
levels in the pathogenesis of DM (Fu et al., 1993
; Maeda
et al., 1995
; Wang et al., 1995
). Very recently, two papers
clearly demonstrated that CUG expansion results in the
nuclear retention of DMPK transcripts, which probably
leads to a reduction in DMPK protein (Davis et al., 1997
;
Hamshere et al., 1997
).
The demonstration that neither DMPK knockout nor
transgenic mice show the profound phenotypes seen in
DM patients suggests that the underlying molecular mechanism of DM is not simply a lack of or excess amounts of
the DMPK protein (Jansen et al., 1996; Reddy et al.,
1996
). However, it is important that mice lacking DMPK
demonstrate late onset myopathy with weakness and some
abnormalities in the skeletal muscle fibers (Jansen et al., 1996
; Reddy et al., 1996
), whereas mice that overexpress
DMPK develop cardiomyopathy (Jansen et al., 1996
).
These results suggest that the DMPK protein plays an essential role in maintaining muscle structure and function
and support the possibility that changes in DMPK levels
contribute to the pathology of DM. These results raise the
question of how a lack or excess of DMPK protein causes muscle disorder.
Heat shock proteins (HSPs), whose synthesis is induced
by heat or other stresses, are divided into several groups
on the basis of their molecular masses (Linquist and Craig,
1988). HSPs with low molecular masses of 15-30 kD, observed in most species, have been shown to form large aggregates in the cytosol and are called small heat shock proteins (sHSP) (Caspers and Bhat, 1995
). So far, four
mammalian sHSPs, HSP27,2
A- and
B-crystallin, and
p20, have been identified (Hickey and Weber, 1982
; Ingolia and Craig, 1982
; Klemenz et al., 1991
; Head et al., 1994
;
Kato et al., 1994
), and some have been shown to work as
molecular chaperones (Jakob et al., 1993
; Rao et al., 1994
)
and confer thermotolerance when overexpressed in mammalian cells (Lavoie et al., 1993a
,b, 1995; Iwaki et al., 1994
;
Mehlen et al., 1995
). Recent results on HSP27 and
B-crystallin suggest that sHSPs confer stress resistance by
stabilizing the actin cytoskeleton (Lavoie et al., 1993b
,
1995
; Iwaki et al., 1994
). Interestingly, three of these proteins, HSP27,
B-crystallin, and p20, are highly expressed
in muscle cells and together form large oligomeric complexes (Kato et al., 1994
). These results suggest that sHSPs
may play important roles in the stress resistance of muscle
cells, which are often subjected to severe conditions.
In this study, we searched for DMPK-binding proteins in a human skeletal muscle cDNA library with the expectation that such binding proteins would provide clues to the physiological function of DMPK and thus suggest a molecular basis for the pathogenesis of DM. Here, we report that a novel member of the small heat shock protein family, designated MKBP (myotonic dystrophy protein kinase binding protein), specifically associates with DMPK. It activates DMPK kinase activity in vitro and protects it from heat-induced inactivation. Importantly, the expression of MKBP in skeletal muscles from DM patients is selectively upregulated, suggesting that a protein interaction between DMPK and MKBP is involved in the pathogenesis of DM. Based on the results presented here, we propose that DMPK is involved in the stress-responsive system in muscle cells.
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Materials and Methods |
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cDNA Cloning
A 2.0-kb EcoRI-BamHI fragment derived from DMPK/SRD (Sasagawa
et al., 1994) was cloned in-frame with the GAL4-DNA-binding domain
into pGBT9 (CLONTECH Laboratories, Inc., Palo Alto, CA). The resultant plasmid (DMPK [1-541]/pGBT) was sequentially transformed with 1 × 106 cDNAs from a human skeletal muscle library (CLONTECH
Laboratories, Inc.) into yeast HF7C. The transformants were plated onto
SD medium lacking histidine, tryptophan, leucine, and uracil, and containing 20 mM 3-aminotriazole (Sigma Chemical Co., St. Louis, MO). The plates were incubated at 30°C for 6 d and His+ colonies were assayed for
-galactosidase activity by a filter assay to identify true positive colonies.
The cDNAs coding human
B-crystallin and HSP27 were cloned by PCR
from the same library described above using appropriate synthetic oligonucleotide primers. The validity of the PCR products was established by
sequencing.
Antibodies
Anti-MKBP polyclonal antisera, c-2 and MKC148, were generated against
the glutathione-S-transferase (GST)-MKBP fusion protein and a synthetic polypeptide encoding amino acid residues 148-167 (RGGRHLDTEVNEVYISLLPA) of MKBP, respectively. Before use, anti-GST IgG
in the c-2 antiserum was depleted by incubation with purified GST immobilized on glutathione beads (Pharmacia Biotech, Piscataway, NJ). Anti-
human B-crystallin antiserum, anti-human HSP27 monoclonal antibody,
and anti-mouse HSP25 antiserum were purchased from StressGen Biotechnologies Corp. (Victoria, British Columbia, Canada).
Electrophoresis and Western Blot Analysis
Tissue samples were homogenized and sonicated in 2% SDS, 1 mM
EDTA, 70 mM Tris-HCl, pH 6.7, and 10% glycerol and heated at 100°C
for 5 min. Protein concentration was determined with DC Protein Assay
kit (Bio-Rad Laboratories, Inc., Hercules, CA) using BSA as a protein
standard. 2-Mercaptoethanol and Bromo Phenol blue were added to a final concentration of 125 mM and 0.02%, respectively, and the samples
were denatured again at 100°C for 5 min. Protein samples were electrophoresed on 12% polyacrylamide gels in the presence of SDS. For two-
dimensional PAGE, tissues were solubilized in 8 M urea, 0.5% NP-40, and
5% 2-mercaptoethanol, and electrophoresed as described (Suzuki et al.,
1995). Protein transfer for immunostaining was performed as described
(Suzuki et al., 1995
). All immunoblots were visualized by chemiluminescence ECL (Amersham Corp., Arlington Heights, IL).
Northern Blot Analysis
Multiple-tissue Northern blot membranes (CLONTECH Laboratories,
Inc.) were used to analyze the tissue distribution of MKBP. Total RNA
preparations from C2C12 cells were obtained using a Quick Prep Total
RNA Extraction kit (Pharmacia Biotech). RNAs were separated in 1%
agarose gels and blotted onto Hybond-N nylon membranes. Membranes
were probed with a cDNA fragment containing the full-length coding region of each sHSP. Membrane-washing conditions were as follows: For
multiple-tissue Northern blot, four times in 2× SSC, 0.05% SDS at room
temperature for 10 min followed by two times in 0.1× SSC, 0.1% SDS at
50°C for 40 min; for the analysis of the total RNA from C2C12 cells, three
times in 2× SSC, 0.1% SDS at room temperature for 10 min followed by
two times in 0.2× SSC, 0.1% SDS at 47°C for 30 min. Autoradiography
was carried out at 70°C using Fuji x-ray films (Tokyo, Japan) with an intensifying screen.
Assays for Protein-Protein Interaction Analysis
For two-hybrid analysis, vectors were constructed by subcloning the corresponding regions of the cDNA into pGBT9 or pGAD10 (CLONTECH
Laboratories, Inc.), except for constructs for DMPK (KD) and protein kinase C (PKC)- (KD), which were prepared in pAS2 (CLONTECH Laboratories, Inc.). Interactions were confirmed by observing growth on histidine-lacking plates and by the acquisition of
-galactosidase activity. For
the immunoprecipitation assay, COS1 cells transiently transfected with
the appropriate expression vectors were suspended in lysis buffer containing 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM EGTA, 2 mM EGTA,
10 mM MgCl2, 1 mg/ml BSA, 10% glycerol, 2 mM Na3VO4, 20 mM NaF,
2 mM pyrophosphate, 1 mM DTT, 10 µg/ml leupeptin, and 2 mM PMSF
and homogenized mildly in a Dounce homogenizer. The resultant extract
was clarified by centrifugation (15,000 rpm, 30 min) and then mixed with
anti-T7 tag monoclonal antibody (Novagen, Inc., Madison, WI) immobilized on protein G-Sepharose 4B (Pharmacia Biotech). After incubation for 2 h at 4°C, the resin-bound complexes were washed with the same lysis
buffer, and the bound proteins were analyzed by 10% SDS-PAGE followed by immunoblotting. Blot overlay assays were performed essentially
as described previously (Suzuki et al., 1995
). Briefly, purified recombinant
DMPK expressed in Escherichia coli using the pET-15b expression vector
(Novagen, Inc.) (0.5 µg) was blotted onto polyvinylidene difluoride membranes. The membrane was blocked with 5% (wt/vol) skimmed milk powder in PBS and incubated with GST or GST-MKBP (30 µg/ml) in buffer
A (20 mM Tris-HCl, pH7.5, 100 mM NaCl, 10 mM MgCl2, 1 mg/ml BSA,
0.5% Triton X-100, 0.5 mM DTT, and 5 µg/ml leupeptin) at room temperature for 2 h. The bound proteins were detected with anti-GST antibody.
Protein Kinase Assay
The kinase reaction was carried out in 20 µl of kinase assay buffer (50 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM MgCl2,, and 0.01% leupeptin)
containing 10 µM [-32P]ATP (185 GBq/mmol) with or without exogenous substrates or additives. When using PKC
, which was expressed in
and purified from SF21 insect cells, 0.5 mM CaCl2, 25 µg/ml phosphatidyl
serine, and 50 ng/ml TPA were added, and the amount of [
-32P]ATP was
lowered to 1.85 GBq/mmol. After incubation for 30 min at 30°C (10 min in
the case of PKC
), the reaction mixture was boiled in SDS sample buffer
and analyzed in 12% SDS-PAGE and autoradiography. To monitor the
chaperone-like activity of MKBP, reaction mixtures without ATP were incubated for 15 min (11 min for PKC
) at 43°C. After brief cooling, the kinase reaction was started by the addition of [
-32P]ATP as described
above. In this assay, recombinant sHSPs free from the carrier protein GST
were used. These were prepared by subcloning the cDNA fragment covering the complete open reading frame of each sHSP into pGEX-6P (Pharmacia Biotech). The GST fusion proteins obtained were purified on glutathione Sepharose 4B, and the sHSPs were specifically eluted by
digesting the linker site with PreScission Protease (Pharmacia Biotech)
according to the manufacturer's directions. Because this protease is a recombinant protein fused to GST, it is trapped by the glutathione resin and does not contaminate the sHSP preparations.
Cell Cultures
Primary cultures of cardiac myocytes were prepared from ventricles of
1-d-old ICR mice as described previously (Nyui et al., 1997). To prepare
the soluble fraction, cells from a 60-mm culture dish were suspended in
300 µl of PBS containing 1 mM EGTA, 1 mM DTT, 10 µg/ml leupeptin, 5 µg/ml aprotinin, and 1 mM PMSF, sonicated at 0°C for 30 s, and centrifuged at 130,000 g for 40 min. The C2C12 mouse myoblast cell line was
kindly provided by Shosei Yoshida (National Institute of Neuroscience,
NCNP, Kodaira, Tokyo, Japan). C2C12 cells were maintained in DME
supplemented with 10% FCS. To induce differentiation, the cells were
cultured on collagen-coated tissue culture plates (Corning, Tokyo, Japan)
and then switched to a serum-free ITS medium consisting of DME supplemented with insulin (10 µg/ml), transferrin (5 µg/ml), and sodium selenite (10 nmol). For heat treatment, differentiated C2C12 cells (3 d in ITS medium) were exposed to transitory heatshock by floating on a water bath at
44°C for 15 min. The cells were recovered after appropriate times at 37°C.
After washing with cold PBS, the cells were scraped and total RNA was
extracted.
Treatment of Rat Hindlimb Muscle and Preparation of Tissue Extracts
SD rats (11-wk-old) were killed under ether anesthesia, and the hindlimb
muscles (gastrocnemius) were immediately removed and subjected to
heat treatment for 20 min as described previously (Kato et al., 1994). The
tissues were frozen in liquid nitrogen and stored at
80°C. The tissues
were ground into powder in liquid nitrogen and homogenized in 10 vol
(vol/wt) of extraction buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM
MgCl2, 2 mM EGTA, 1 mM DTT, 10 µg/ml leupeptin, 5 µg/ml aprotinin,
and 1 mM PMSF). The homogenates were centrifuged at 130,000 g for 40 min at 4°C, and the supernatants were used for gel filtration analysis or protein analysis. Pellets were solubilized in the same volume of SDS solubilizing buffer as the extraction buffer used above.
Gel Filtration
50 µl of mouse cardiomyocyte or rat hindlimb muscle extract was loaded
onto a prepacked column of Superose 12 (PC3.2/30) set on the SMART
System (Pharmacia Biotech) and equilibrated with the appropriate extraction buffer for each sample at 4°C. Blue dextran (>2,000 kD), bovine
-globulin (154 kD), BSA (67 kD), chicken ovalbumin (45 kD), and
lysozyme (14 kD) were used as molecular mass standards.
Immunohistochemistry
Human limb muscle specimens (mostly from biceps bracii, and, in some
cases, rectus femoris) were obtained for diagnostic purposes with informed consent. The procedures for immunohistochemical observation
were described previously (Hayashi et al., 1993).
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Results |
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The DMPK Kinase Domain Binds Specifically to the
-Crystallin Domain of MKBP, a Novel Member of the
sHSP Family
To obtain clues as to the physiological function of myotonic dystrophy protein kinase (DMPK), we searched for
DMPK-binding proteins in a human skeletal muscle
cDNA library using a yeast two-hybrid system. As shown
in Fig. 1 a, we used the first 541 amino acids of DMPK,
DMPK (1-541), which contains the NH2-terminal kinase
domain and the -helical coiled-coil domain (Sasagawa et al., 1994
), as a probe to identify a cDNA clone (D2-1) (Fig.
1 b). The clone also interacted with the first 358 residues of
DMPK, DMPK (KD), suggesting that the protein encoded
by D2-1 interacts with the kinase domain of DMPK (Fig.
1 b). This clone with a 0.9-kb insert encodes a novel protein that shows striking similarity to members of the sHSP
family (Fig. 2; the partial sequence of D2-1 has already
been reported as one of the eye muscle autoantigens in
thyroid-associated ophthalmopathy) (Elisei et al., 1993
).
Based on the size of the mRNA (see below) and a comparison with other sHSPs, we identified a methionine codon
preceded by a Kozak's consensus sequence located 23 amino acid residues downstream of the cloning junction.
Thus this protein, which we named MKBP, contains 182 amino acid residues and has an estimated molecular mass
of 20,295 kD. As shown in Fig. 2, MKBP represents the
most diverged member of this family, showing more than
30% overall sequence identity to each known human
sHSP (Caspers and Bhat, 1995
). The greatest similarity
(42% or greater identity) occurs in the
-crystallin domain, which is shared by all sHSPs and is thought to be important for their molecular chaperone activity (Jakob et al.,
1993
; Rao et al., 1994
). Further analysis using an MKBP
mutant, MKBP (64-182), showed that the interaction between DMPK and MKBP is mediated by this
-crystallin
domain (Fig. 1 b). In addition, the other members of the
sHSP family abundant in skeletal muscle,
B-crystallin
and HSP27, do not interact with DMPK (Fig. 1 b). Therefore, the interaction between DMPK and MKBP is not
due to some nonspecific binding activity of MKBP as a
molecular chaperone. Moreover, MKBP does not interact
with the kinase domain (1352-2420) of PKC-
, PKC-
(KD), confirming that the observed binding of MKBP is
specific for DMPK (Fig. 1 b).
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The physical association between DMPK and MKBP was independently confirmed by an immunoprecipitation assay on extracts of COS1 cells transiently transfected with expression vectors encoding T7 epitope-tagged DMPK and wild-type MKBP (Fig. 1 c). MKBP immunoprecipitated with the anti-T7 antibody only when T7-DMPK (the full-length isoform corresponding to DMPK-2 in Fig. 1 a) as well as T7-DMPK (1-541) were coexpressed in the cell. Furthermore, an in vitro solid-phase binding assay (overlay) using purified recombinant DMPK (1-541) and MKBP proteins clearly demonstrated a direct interaction between DMPK and MKBP. As shown in Fig. 1 d, GST-MKBP, but not GST, binds to histidine-tagged DMPK (1-541) blotted onto a membrane. The binding affinity did not depend very much on the presence of ATP.
MKBP Is the Fourth Member of the sHSP Family That Is Highly Expressed in Muscle Cells
Northern blot analysis of mRNA from human tissues showed a single transcript of ~1.0 kb expressed highly in skeletal and cardiac muscle as well as ubiquitously at lower levels (Fig. 3 a).
Antiserum (c-2) against recombinant MKBP detected a
protein of 22 kD in skeletal and cardiac muscle (Fig. 3 b).
Another anti-MKBP antibody (MKC148) raised against
the COOH-terminal sequence specific for MKBP (amino
acid residues 148-167) also detected the same band (Fig. 3 c).
Because MKBP and B-crystallin show similar mobilities on one-dimensional SDS-PAGE, we also performed two-dimensional PAGE analysis and confirmed the expression
of the MKBP protein in human skeletal muscle as well as
the specificity of c-2 (Fig. 3 d). c-2 detected spots consistent with the predicted pI of 4.98 for MKBP (open triangle
in lower panel), whereas anti-
B-crystalline antiserum reacted only with spots corresponding to the pI of 6.92 for
B-crystalline (Fig. 3 d, open triangle in upper panel). Using these specific antibodies and each purified recombinant protein as a standard, we estimated the protein concentration of MKBP and
B-crystallin in skeletal muscle
cell. MKBP was found to present at high levels (0.65 µg/
mg protein) in rat hindlimb muscle (gastrocnemius) extracts, comparable to the concentration of
B-crystallin
(0.72 µg/mg protein).
MKBP Exists in an Oligomeric Complex Separate from Other sHSPs in Muscle Cells
Since sHSPs expressed in muscle cells have been shown to
associate with one another to form a large oligomeric complex (Kato et al., 1992, 1994
), we tested the possibility that
MKBP is also a component of this complex. Soluble proteins extracted from primary cultures of mouse cardiomyocytes or rat hindlimb skeletal muscle were subjected to
gel filtration and analyzed by immunoblotting. In both cell
types, MKBP was found to exist in an oligomeric complex (~150 kD) separate from the complex containing
B-crystallin and HSP27 (>500 kD) (Fig. 4 a). This result was also
supported by yeast two-hybrid assays monitoring the interactions between the three members of the sHSP family
(Fig. 4 b); strong homo- and heterooligomeric
B-crystallin and HSP27 activities were observed in this assay, consistent with the previous biochemical analysis (Kato et al.,
1992
). On the other hand, MKBP interacted with neither
B-crystallin nor HSP27, whereas it showed homophilic
binding activity. Therefore, we conclude that MKBP works
independently of the known sHSPs in muscle cells by
forming a separate oligomeric complex. Interestingly, the
apparent molecular masses of MKBP in skeletal muscle
and cardiomyocytes differ slightly. This suggests the possibility of different MKBP complex compositions in these cells. Although Kato et al. have demonstrated that p20 in
soluble rat diaphragm extracts exists mainly in two forms
of oligomeric complexes with apparent molecular masses
larger than 500 kD and lower than 67 kD but none of
~150 kD (Kato et al., 1994
), there remains the possibility
that some fraction of p20 associates with MKBP.
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Heat Shock Does Not Induce the Accumulation of MKBP mRNA, but Causes an Early Redistribution into the Insoluble Fraction
Cells once exposed to environmental stress have been suggested to acquire an increased capacity to survive subsequent stress by accumulating HSPs (Linquist and Craig,
1988). In many cell lines, the expression of sHSPs has also
been shown to be induced by heat and other stresses, suggesting that they also contribute to the acquisition of stress
resistance in cells (Klementz et al., 1991; Head et al.,
1994
). In a mouse myoblast cell line, C2C12, which expresses at least three members of the mammalian sHSP
family, HSP27,
B-crystallin, and MKBP, we also observed the dramatic accumulation of the mRNA for
HSP27 and
B-crystallin within 1 h after heat treatment
(44°C 15 min). However, the expression of MKBP was not
induced at all, even after 8 h, and the levels did not change
even when more severe heat treatments were applied (44°C 1 h or 46°C 15 min) (Fig. 5 a; data not shown).
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In addition to transcriptional upregulation, heat shock has been suggested to induce an immediate redistribution of preexisting sHSPs from the cytosolic fraction to the insoluble fraction. As shown in Fig. 5 b, MKBP also showed a similar response to heat shock. At physiological temperature, almost 50% of the MKBP in the skeletal muscle of the rat hindlimb was extracted in the soluble fraction, whereas after heat shock treatment (44 or 46°C 20 min), increased amounts of MKBP were found in the insoluble fraction (Fig. 5 b). These results suggest that MKBP not only shows sequence homology with other known sHSPs but also, like the others, behaves as a stress responsive protein.
MKBP Localizes to Z-Membranes and the Neuromuscular Junction in Human Skeletal Muscle
Fig. 6 shows the subcellular immunolocalization of MKBP
in human skeletal muscle. In cross section, anti-MKBP antiserum, MKC148, shows an antigen-specific, dotlike staining pattern that may correspond to individual cross sections of myofibril fibers (Fig. 6, a-c). On the other hand, in
longitudinal section, the staining pattern shows an ordered
striation identified as the Z-line from the overlapping pattern produced by desmin staining (Fig. 6, d-f). In addition to the predominant staining in the cytoplasm, MKBP also
localizes to the neuromuscular junction identified by
rhodamine-labeled -bungarotoxin (Fig. 6, g-i). Based on
the recent demonstration that DMPK localizes predominantly to the neuromuscular junction (Maeda et al., 1995
;
Whiting et al., 1995
), these results support the notion that
DMPK and a fraction of MKBP colocalize endogenously at the neuromuscular junction in skeletal muscle cells.
MKBP Activates the Kinase Activity of DMPK and Confers Thermoresistance
To assess the physiological significance of the interaction
between DMPK and MKBP, we performed an in vitro kinase assay using recombinant of DMPK (1-541) and
sHSPs (Fig. 1 d). For these assays, we excised the carrier
protein, GST, from each GST fusion protein of sHSP to
eliminate possible steric hindrance (Fig. 7 a). Interestingly, gel filtration analysis revealed that, in contrast with the situation in vivo (Fig. 4), these purified sHSPs all exist in oligomeric complexes with apparent molecular sizes ranging
from 150 to 200 kD under the assay conditions used
(MKBP and HSP27, ~150 kD; B-crystalline, ~200 kD,
data not shown). As shown in Fig. 7 b, recombinant
DMPK (1-541) shows autophosphorylation activity as reported previously (Dunne et al., 1994
); however, it does
not phosphorylate MKBP, suggesting that MKBP is not a
downstream target of DMPK. On the other hand, the data
in Fig. 7 b also suggest that when sHSPs coexist in the reaction mixture, DMPK autophosphorylation increases, and that this effect is greatest when MKBP is in the solution (3.4-fold for MKBP, 1.8-fold for HSP27, and 2.4-fold
for
B-crystallin). This enhancement of DMPK kinase activity was further confirmed using myelin basic protein
(MBP) as a substrate (James et al., 1996
). In contrast with
GST or BSA (data not shown), the existence of very low
concentrations of sHSPs (25 µg/ml) in the solution increases the phosphorylation of MBP by DMPK two- to
threefold, and among the sHSPs examined, MKBP was
again found to be the most effective (Fig. 7, c and d). Since
MKBP does not affect MBP phosphorylation by PKC
(Fig. 7, e and f), the observed effect of MKBP is suggested to be specific for DMPK. Fig. 7 g shows a Lineweaver-Burk plot derived from the substrate dose dependence
data for MBP phosphorylation by DMPK. It shows that
MKBP increases not only the maximal rate, Vmax, of phosphorylation, but also the Michaelis constant, Km, excluding
the possibility that MKBP increases MBP phosphorylation by mediating the interaction between DMPK and MBP (if
so, Km should be lowered). Rather, MKBP lowers the affinity of DMPK for MBP, suggesting that it alters the recognition of DMPK by affecting the tertiary structure of
the kinase domain.
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Because HSP27, as well as B-crystallin, has been
shown to have chaperone activity, we next examined the
possibility that MKBP also has a chaperone-like activity to
suppress the heat-induced inactivation of DMPK (Fig. 8).
While the kinase activity of His-DMPK (1-541) decreases
to less than 4.3% after incubation at 43°C for 15 min,
26.4% of the activity remains when 50 µg/ml MKBP is
present with the kinase during the heat treatment. Since
this difference (larger than sixfold) cannot be explained by
the enhancement of the remaining DMPK activity (Fig. 7,
c and d), these data suggest that MKBP suppresses the
heat-induced inactivation of DMPK by binding to its kinase domain. Similar but smaller effects were also observed in the case of HSP27 and
B-crystallin. However, MKBP hardly inhibits the inactivation of PKC
, suggesting again that the effect is specific for DMPK.
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MKBP Is Selectively Upregulated in DM Patients
In a first attempt to address the question of whether the
interaction between DMPK and MKBP is involved in the
pathogenesis of DM, we analyzed the amount of MKBP
protein in skeletal muscle from DM patients. As demonstrated clearly in Fig. 9, the amount of MKBP is commonly
upregulated in these muscles compared with skeletal muscle from normal subjects, whereas no changes in the amounts of B-crystallin and HSP27 are observed. In addition, no change in the amount of MKBP was detected in
the case of polymyositis (PM), indicating that the change
in the MKBP level does not result from muscle degeneration itself. Consistent with these results, immunostaining
of DM muscle with MKC148 sometimes shows increased cytosolic background staining in addition to dotlike staining (Fig. 6, b and g). In some cases, the dotlike staining is
enlarged (Fig. 6 b).
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Discussion |
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Since muscle cells are frequently subjected to severe conditions caused by heat, oxidative, and mechanical stresses,
especially during exercise (Brooks et al., 1971a,b; Davis et
al., 1982
), there must be some mechanisms to protect cells
from damage. Several lines of evidence suggest that
sHSPs, which are constitutively highly expressed in muscle
cells, play roles in maintaining myofibrillar integrity
against such stresses. Atomi et al. (1991)
suggested that
B-crystallin localizes to Z-bands, and its dramatic decrease is a characteristic feature of disuse atrophy of the
slow muscle fibers. In addition, HSP27 and
B-crystallin
have been suggested to bind to actin filaments (Miron et al.,
1991
; Bennardini et al., 1992
) and confer thermoresistance
by affecting their structure and stability (Lavoie et al.,
1993a
,b; 1995; Iwaki et al., 1994
).
In this work, we identified a novel member of the sHSP
family, MKBP, which is also constitutively expressed in
skeletal and cardiac muscles. We demonstrated that MKBP
not only shows strong homology with other sHSPs but also
has features characteristic of sHSPs (Figs. 4 and 5). MKBP
shows homooligomeric activity and forms aggregates in
muscle cytosol. Furthermore, it redistributes to the insoluble fraction in response to heat shock, suggesting that it is
one of the stress responsive proteins found in muscle cells.
However, the present results also indicate that MKBP is a
unique member of the sHSP family since it forms an independent complex distinct from the complex formed by
other sHSPs such as HSP27 and B-crystallin. In addition,
its expression is not induced by heat treatment. Based on
these results, we conclude that MKBP is involved in a
novel stress responsive system distinct from the known
system composed of HSP27,
B-crystallin, and p20 (Kato et al., 1994
), and that both systems may work independently to confer stress resistance to muscle cells. The intracellular localization of MKBP at the Z-membranes and
neuromuscular junction suggests a role for the protein in
protecting the components of these muscle-specific structures.
Various stresses other than heat, such as heavy metal exposure, hypertonicity, and the stimulation with tumor necrosis factor , have been shown to induce the expression
of HSP27 and
B-crystallin (Head et al., 1994
). Therefore,
there may be some stress stimulations that induce the expression of MKBP. In fact, we observed that the protein levels of MKBP, but not
B-crystallin, increase in response to
stretch stimulation in primary cultures of neonatal mouse
cardiomyocytes (Suzuki, A., Y. Sugiyama, N. Nyu-i, and S. Ohno, unpublished results). MKBP might play a role in a
system by which muscle cells respond to mechanical stress.
We identified MKBP by searching for DMPK binding
proteins. Three independent experiments established the
specific and direct interaction between DMPK and MKBP.
The localization of MKBP at the neuromuscular junction
where DMPK is also suggested to be concentrated (Fig. 6,
h-j) (Maeda et al., 1995; Whiting et al., 1995
), as well as
the selective change in the amount of MKBP in skeletal
muscle from DM patients (Fig. 9), further suggest an intrinsic interaction between these proteins. We also found
that MKBP is not significantly phosphorylated by DMPK
but rather activates the kinase activity of DMPK two- to
threefold and protects it from heat-induced inactivation.
An analysis of the kinetic parameters of DMPK also suggests the possibility that MKBP binding changes the tertiary structure of the kinase domain of DMPK (Fig. 7 g). These results, although indirect, suggest that MKBP can
work as a kind of molecular chaperone for DMPK at least
in vitro. Recently, a growing number of intracellular signaling molecules, including various kinases, receptors, and
transcription factors, have been found to be constitutively
associated with chaperone molecules such as HSP90 and
immunophilins (Rutherford and Zuker, 1994
; Pennisi, 1996
). Based on these observations, the regulated folding
or assembly of signaling molecules by chaperone molecules
is beginning to be recognized as a general mechanism by
which signal transduction pathways are controlled. With regard to sHSP, HSP27 was very recently shown to bind and
activate PKB/Akt/RAC-protein kinase in response to heat
or oxidative stress (Konishi et al., 1997
). Taken together, it
seems reasonable to speculate that one of the physiological functions of MKBP may be to act as a molecular chaperone specific for DMPK that stabilizes and protects its
kinase activity. Furthermore, if there are some stress stimulations that regulate the MKBP expression level, MKBP
might act as a signal transducer activating DMPK in response to such stimulations (for example, stretch stimulation as stated above). The data in Figs. 7 and 8 show that
HSP27 and
B-crystallin also have a similar, although
smaller, effect on DMPK. However, because several observations in vivo suggest the specificity of the interaction
between DMPK and MKBP (Figs. 1 and 9), we speculate
that there are unknown factors that make MKBP the main
sHSP affecting the activity of DMPK in vivo. In this sense,
it should be noted that the size of the oligomeric structure
of recombinant MKBP is similar to that observed in vivo (Fig. 4), while those of HSP27 and
B-crystallin are different (data not shown).
Although CTG repeat expansions in the 3'-untranslated
region of the DMPK gene are responsible for DM, the molecular basis of the pathogenesis of the disease, including
even the involvement of the DMPK protein, remains controversial. However, recent studies on DMPK knockout
mice have demonstrated that DMPK plays an essential role in maintaining muscle structure and function, and
thus its absence does contribute to some aspects of the disease (Reddy et al., 1996). Therefore, the following hypothesis based on the present results may provide a partial explanation for the molecular basis of the disease: DMPK is
involved in the stress-responsive system by being activated
by MKBP, and its absence or a reduction in its level impairs its activity to protect cells against stresses. This
would result in the accumulation of cellular damage leading to the gradual muscle degeneration observed in the
late stages in knockout mice (Reddy et al., 1996
) and in
DM patients. In this context, the selective upregulation of
MKBP observed in patients (Fig. 9) might be explained as
a sign of a feedback mechanism trying to compensate for
the reduction in DMPK.
To our knowledge, this is the first report suggesting a
correlation between a specific muscular dystrophy and the
stress-responsive system of muscle cells. Although characterized by skeletal muscle wasting and myotonia, DM is a
multisystem disorder with diverged phenotypes (Harper,
1989). The tissue distributions of DMPK and MKBP transcripts correlate well (Fig. 3; Jansen et al., 1992
), and although their levels are very low, they are expressed ubiquitously. Therefore, the pathological mechanism we have
hypothesized might also work in tissues other than muscle.
Of course, some of the diverged phenotypes may arise via
other unknown mechanisms not related to the function of
DMPK. To understand the molecular basis of this complex
disease, much further study is needed. Also, with respect
to our hypothetical picture, a great many gaps, including the identity of the specific substrate of DMPK, remain to
be filled. However, the finding of a stress-responsive protein that activates DMPK provides a new opportunity to
clarify the currently obscure DMPK signaling pathway. In
addition, it provides novel and important insight into the
molecular basis underlying some of the essential phenotypes of this disease.
Very recently, during the course of analyzing the promoter region of the human B-crystallin gene located on
chromosome 11q22, Iwaki et al. (1997)
identified a gene
encoding a novel member of the sHSP family (HSPB2) in
a head-to-head orientation with the
B-crystallin gene
with an intermediate region of ~1 kb. Interestingly, this
HSPB2 is identical to MKBP. They also demonstrated
that, in contrast to
B-crystallin, the HSPB2 mRNA is not
detectable in rat lens by Northern blot. Together with the
present results, this finding raises intriguing questions as to
how the expression of both genes is regulated and how
they have evolved.
![]() |
Footnotes |
---|
Received for publication 23 June 1997 and in revised form 24 November 1997.
Please address all correspondence to Dr. Atsushi Suzuki or Dr. Shigeo Ohno, Department of Molecular Biology, Yokohama City University School of Medicine, Kanazawa-ku, Yokohama 236, Japan. Tel.: 81-045-787-2596. Fax: 81-045-785-4140. E-mail: abell{at}med.yokohama-cu.ac.jp (Dr. Suzuki) or ohnos{at}med.yokohama-cu.ac.jp (Dr. Ohno).We thank S. Yoshida and Y. Nabeshima for providing us with the C2C12 cell line; H. Sugita, E. Ozawa, and R. Matsuda for stimulating discussion; and M. Okamoto for technical assistance. We are grateful to A. Iwaki and Y. Fukumaki for communication of their results before publication.
This work was supported by a grant [8A-1] from the National Center of Neurology and Psychiatry of the Ministry of Health and Welfare, Japan.
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Abbreviations used in this paper |
---|
DM, myotonic dystrophy; DMPK, myotonic dystrophy protein kinase; GST, glutathione-S-transferase; MBP, myelin basic protein; MKBP, myotonic dystrophy protein kinase binding protein; PKC, protein kinase C; sHSP, small heat shock protein; TPA, 12-O-tetradecanoyl phorbol-13-acetate.
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