From the Department of Molecular Pathogenesis,
Division of Adult Diseases, Medical Research Institute, Tokyo Medical
and Dental University, Tokyo 101-0062, the § Department of
Cardiovascular Medicine, Graduate School of Medical Sciences, Kyushu
University, Fukuoka 812-8582, the ¶ Division of Molecular
Cardiology, Research Institution of Angiocardiology, Faculty of
Medicine, Kyushu University, Fukuoka 812-8582, and the
Etiology
and Pathogenesis Research Unit, Medical Research Institute, Tokyo
Medical and Dental University, Tokyo 101-0062, Japan
Received for publication, September 19, 2000, and in revised form, November 3, 2000
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ABSTRACT |
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Myosin light chain phosphatase consists of three
subunits, a 38-kDa catalytic subunit, a large 110-130-kDa myosin
binding subunit, and a small subunit of 20-21 kDa. The catalytic
subunit and the large subunit have been well characterized. The small subunit has been cloned and studied from smooth muscle, but little is
known about its function and specificity in the other muscles such as
cardiac muscle. In this study, cDNAs for heart-specific small
subunit isoforms, hHS-M21, were isolated and
characterized. Evidence was obtained from an analysis of genome to
suggest that the small subunit was the product of the same gene as the
large subunit. Using permeabilized renal artery preparation and
permeabilized cardiac myocytes, it was shown that the small subunit
increased sensitivity to Ca2+ in muscle contraction. It was
also shown using an overlay assay that hHS-M21 bound the
large subunit. Mapping experiments demonstrated that the binding domain
and the domain involved in the increasing Ca2+ sensitivity
mapped to the same N-terminal region of hHS-M21. These
observations suggest that the heart-specific small subunit hHS-M21 plays a regulatory role in cardiac muscle
contraction by its binding to the large subunit.
Ca2+ plays a central role in the regulation of muscle
contractile process mediated by interaction of myosin with actin. It is well known that phosphorylation status of myosin light chain
(MLC)1 is correlated with the
Ca2+ sensitivity of muscle contraction (1, 2). Two key
enzymes regulate the phosphorylation status of MLC; myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). The functional role of MLCK has been well investigated (3, 4), whereas that of MLCP
and related topics has only recently been expanding (5, 6).
A frequently used classification of protein phosphatases identifies two
classes, type 1 (PP1) and type 2 (PP2) (7). In general, PP1 holoenzymes
consist of a catalytic subunit (PP1c) and different regulatory subunits
that may target the phosphatase to particular substrates (8, 9). MLCP
is classified into PP1 (10) and composed of approximately 38-kDa PP1c
and regulatory subunits in the smooth muscle (11-13). There are two
different regulatory subunits, large and small, of about 110-130 kDa
(myosin binding subunit, MBS) and 20 kDa (sm-M20), respectively (5, 6).
MBS cDNAs have been cloned from various tissues of chicken, rat,
and human (5, 6), while cDNA for small subunit (sm-M20) has so far
been isolated only from chicken gizzard (14). Because MBS has a
regulatory role or a targeting function (5, 6), the gene for MBS was
designated as the myosin phosphatase
targeting subunit gene, MYPT1 (15). MBSs encoded
by MYPT1 from various species have a common structural
feature such as ankyrin repeats in the N-terminal half of the molecule,
and those from human and rat have leucine zipper motifs in the
C-terminal end. It has been reported that MBS binds to PP1c, myosin
heavy chain (MHC), and MLC to form an MLCP complex (12, 16-20) and
that both MHC and MLC are dephosphorylated in this MLCP complex
(16-18). The dephosphorylation of myosins by PP1c is enhanced by the
presence of MBS, and thereby, the function of MBS is to up-regulate the
activity of PP1c toward the myosins (16-18).
In contrast, only a limited amount of information is available for
sm-M20. It also contains C-terminal leucine zipper motifs and is
homologous to the C-terminal one-third of MBS (14). It has been
reported that sm-M20 interacts with myosin and the C-terminal end of
MBS (18, 19). In addition, we have recently demonstrated that sm-M20
increases the Ca2+ sensitivity of the contractile apparatus
in vascular smooth muscle and that this effect was conferred by the
N-terminal half of sm-M20 (21). However, the existence and function, if
any, of M20 subunit in other muscles than the smooth muscle remain unknown.
Several protein phosphatases in the cardiac muscle have been
characterized (14, 22, 23). There are, however, only a few reports
about the MLCP in the cardiac muscle (24, 25). In addition, it has been
considered that the Ca2+ sensitivity of contraction in the
striated muscle is mainly regulated by troponins (26), and the
significance of MLC phosphorylation in the Ca2+
sensitization of the striated muscle remains to be elucidated. However,
a recent report has demonstrated a possible involvement of MLC
phosphorylation in the development of cardiac hypertrophy (27).
Recently, another gene for MBS, MYPT2, was isolated by
Fujioka et al. (25). Toward the N terminus and C terminus,
MBS encoded by MYPT2 has seven ankyrin repeats and three
leucine zipper motifs, respectively, which are highly homologous to the
relevant sequences of MBS encoded by MYPT1. Because
MYPT2 is expressed preferentially in the striated muscles,
especially in the cardiac muscle, it was suggested that the function of
MYPT2 might be related with the regulation of MLC
phosphorylation in the heart (25). The functions of MLC and MLCP in the
cardiac muscle, then, should be unraveled for better understanding of
the regulation of muscle contractility in the heart.
We report here the isolation of cDNAs for heart-specific isoform of
M20 subunit (hHS-M21), which were obtained in the process of isolating novel genes being preferentially expressed in the cardiac
muscle. From an analogy of function for sm-M20 in the smooth muscle,
hHS-M21 was suggested to participate in the regulation of
MLC phosphorylation in the cardiac muscle. We, then, determined genomic
structure of the gene for hHS-M21 and analyzed the
expression profiles of hHS-M21. In addition, the role of
hHS-M21 in the Ca2+ sensitivity of muscle
contraction was investigated, along with its interaction with MBSs
encoded by MYPT1 and MYPT2.
Isolation of a Heart-specific cDNA--
To obtain
information on genes preferentially expressed in the cardiac muscle, a
normalized subtraction PCR-cDNA library between mRNAs of
cardiac and skeletal muscles was constructed using a PCR-Select
cDNA subtraction kit (CLONTECH). Randomly
selected clones from this library were determined for their sequences, and these sequences were compared with the primate nucleotide sequences
in the GenBankTM data base. Several cDNA fragments that were found
more than two times in the sequenced cDNA fragments and had no
identity in the data base except for human expressed sequence tags were
investigated for tissue specificity of gene expression by an RT-PCR
analysis. In the RT-PCR analysis, mRNAs from various human tissues
including fetal heart, adult heart, skeletal muscle, brain, kidney,
liver, uterus, lung, spleen, thymus, small intestine, and colon
(CLONTECH) were examined. A cDNA fragment (HS602) that was expressed only in the heart was investigated further.
To isolate full-length cDNA clones encoding the HS602 gene, a human
heart cDNA library in Determination of Genomic Organization of MYPT2--
Several
clones containing a part of MYPT2 were isolated from a human
genomic DNA library in EMBL3 (CLONTECH) using the
longest cDNA clone for the HS602 gene, 602-7, as a
probe. Isolated genomic DNA clones were subcloned into pBluescript KS
( RT-PCR Analyses of MYPT2 and MYPT1--
Tissue-specific
expression patterns of hHS-M21 gene (corresponding to
HS602 cDNAs) and that of MBS gene (MYPT2)
were investigated by RT-PCR analyses using mRNAs from human
tissues. The primers used for the RT-PCR analyses had the following
sequences: 602F (5'-TAGAGAAGGACAGTCAGAGG-3') and 602-700R
(5'-TGGGAACTGGTGTAGAGTGA-3') for hHS-M21
(HS602), and MYPT2LAF (5'-CTACTCCTGTGCTCTCCATT-3') and
602-5R (5'-GCTGTCCCTTTCTTTCCTCA-3') for MBS (MYPT2). The
RT-PCR products were electrophoresed in a 0.9% agarose gel. For
comparison of the mRNA expression levels of MYPT1 and
MYPT2, RT-PCR analyses were performed for mRNAs from six
human tissues, heart, skeletal muscle, brain, uterus, lung, and small
intestine. The sequences of primers used were as follows; MYPT1-o-40F
(5'-AACGAGCAGCTGAAACGCTG-3') and MYPT1-o-200R
(5'-TTGGCGTAATTGATGTCGGC-3') for the MYPT1, and MYPT2-o-20F
(5'-ACTGGAGCACCTAGGAGGGAA-3') and MYPT2-o-490R
(5'-TCTGCAAGGTCAGAGGGAAC-3') for the MYPT2. The
RT-PCR products were electrophoresed in a 1.4% agarose gel.
Expression and Purification of Recombinant Proteins--
Various
fragments of the hHS-M21 cDNA were expressed as
hexahistidine-tagged fusion proteins in a prokaryote expression system using pQE30 (Qiagen). Recombinant MBS proteins encoded by
MYPT1 and MYPT2 were obtained as flag-tagged
fusion proteins using a pQE30-flag vector in which the hexahistidine
tag was replaced by a flag tag. The pQE30-flag vector was constructed
as follows. At first, PCR amplification with primers,
5'-CCTTTCGTCTTCACCTCGAG-3' (sense) and
5'-GGATCCCTTATCGTCATCGTCCTTGTAATCTCTCATAGTTAATTTCTCCTC-3' (antisense, containing a flag sequence), was performed using pQE30 as a
template. The PCR product was then cloned into pCR2.1 (Invitrogen) to
be confirmed for the sequences. The insert was excised by digestion with EcoRI and BamHI and cloned back into the
EcoRI-BamHI-cleaved pQE30 to obtain
pQE30-flag.
The recombinant hHS-M21 proteins generated were
hHS-M21 A and hHS-M21 B (two constructs for
major isoforms; corresponding to exons 16-23 and 25 or exons 16-24 of
MYPT2, respectively), hHS-M21 t-1 (deletion of
the leucine zipper motifs; corresponding to exons 16-23),
hHS-M21 t-2 (N-terminal half of hHS-M21; exons 16-20), hHS-M21 t-3 (deletion of N-terminal 56 residues
and leucine zipper motifs; exons 19-23), t-4 (C-terminal half of
hHS-M21 A; exons 21-23 and 25), t-5 (C-terminal half of
hHS-M21 B; exons 21-24), hHS-M21 o-1
(N-terminal 56 residues of hHS-M21; exons 16-18), and
hHS-M21 o-2 (deletion of N-terminal 56 residues and C-terminal half of hHS-M21; exons 19 and 20). On the other
hand, MYPT1- or MYPT2-coded MBSs were expressed
as divided into three parts. The recombinant MBS proteins generated
were MYPT1 A (N-terminal one-third of MYPT1-MBS; aa 1-344),
MYPT1 M (middle one-third part of MYPT1-MBS; aa
345-688), MYPT1 P (C-terminal one-third of MYPT1-MBS; aa
689-1030), MYPT2 A (N-terminal one-third of MYPT2-MBS; aa
1-328), MYPT2 M (middle one-third part of
MYPT2-MBS; aa 329-656), and MYPT2 P (C-terminal one-third
of MYPT2-MBS; aa 657-982). The cDNAs for
hHS-M21 and MBSs encoded by MYPT1 and
MYPT2 were obtained by the RT-PCR from human heart cDNA.
The amplicons were once cloned into pCR2.1 for sequence confirmation
and the insert cDNAs were excised by digestion with
BamHI and SalI (for all constructs of hHS-M21 and MYPT2, and MYPT1 A and MYPT1 M) or
with SphI and SalI (for MYPT1 P). These
restriction sites were included in the design of sense and antisense
primers of which sequences are available upon request. The excised DNA
fragments were cloned into pQE30 (hHS-M21) or pQE30-flag
(MYPT1 and MYPT2). The recombinant constructs were used to transform Escherichia coli M15[pREP4], and
the expression of recombinant proteins in the pQE system (Qiagen) was
performed according to the manufacturer's instructions. After the
induced expression of recombinant proteins, the E. coli cell
pellets were washed with ice-cold phosphate-buffered saline (PBS) (116 mM NaCl, 10 mM Na2HPO4, and 3 mM K2HPO4 (pH 7.4)) and stored at
The preparation of recombinant proteins from the cell pellets was done
as follows. After thawing at 4 °C, cells were sonicated in 6 M urea, 5 mM imidazole, 500 mM
NaCl, 5 mM phenylmethanesulfonyl fluoride, 5 mM
N-ethylmaleimide, and 20 mM Tris-HCl (pH 7.5)
(buffer A) and centrifuged at 10,000 × g for 15 min at
4 °C. Supernatants containing recombinant MBS proteins were stored
at Far Western Analysis--
Equal amounts of recombinant MBS
proteins were applied to SDS-PAGE in 12% acrylamide gels, and
transferred to PVDF membranes (Millipore). The membranes were blocked
with 5% nonfat dry milk, incubated for 4 h at room temperature
with hexahistidine-tagged hHS-M21 recombinant proteins (A,
B, t-1, t-2, t-3, t-4, t-5, o-1, and o-2) solubilized at a
concentration of 10 µg/ml in Tris-buffered saline (TBS), washed once
with TBS, and once with PBS. Subsequently, the membranes were soaked in
100 ml of 0.5% formaldehyde solution in PBS, then in 2% glycine/PBS.
After washing twice with PBS and twice with TBS, the membranes were
incubated with horseradish peroxidase-conjugated rabbit
anti-His6 tag polyclonal antibody (Santa Cruz) for 1 h. The immunocomplex was detected by enhanced chemiluminescence
(Pierce). The experiments were repeated at least three times to confirm
the binding results.
Permeabilization of Porcine Renal Artery and Rat Cardiac Myocytes
and Force Measurements--
Measurements of alteration in
Ca2+ sensitivity of muscle contraction using permeabilized
porcine renal artery were performed as described previously (21, 29,
30). In brief, renal arterial rings were permeabilized with 1% Triton
X-100 in the Ca2+-free cytoplasmic substitution solution
(CSS) (10 mM EGTA, 100 mM potassium
methanesulfonate, 3.38 mM MgCl2, 2.2 mM Na2ATP, 10 mM creatine
phosphatase, 2 µM calmodulin, 50 units/ml creatine phosphokinase, and 20 mM Tris-maleate (pH 6.8)) at
24-25 °C for 20 min. The recombinant hHS-M21 proteins
were dissolved in 100 mM potassium methane sulfonate, 20 mM Tris-maleate (pH 6.8), and applied directly in CSS. The
permeabilized arterial rings were activated by various concentrations
of Ca2+ from 0.05 µM to 10 µM,
and the developed force was monitored. Ca2+-CSS containing
the indicated concentration of free Ca2+ was prepared by
adding appropriate amount of CaCl2, using a
EGTA-Ca2+ binding constant of 106/M
(31).
The measurements of alteration in Ca2+ sensitivity using
permeabilized rat cardiac myocytes were done as reported previously (32). Single cardiac cells were prepared from rat left ventricular myocardium (33) and permeabilized with 2.5% Other Procedures--
Nucleotide sequences were determined using
dye terminator cycle sequencing pre-mix kit (Amersham Pharmacia
Biotech) and ABI373A automated DNA sequencer. SDS-PAGE and the
immunoblotting technique were carried out according to the standard
procedures (36, 37). Concentration of protein was measured by the
Bradford method (38) with bovine serum albumin (Pierce) as standard.
Data Analysis--
The extent of force development was expressed
as a percentage, assigning values in Ca2+-free buffer
(resting state) and in 10 µM Ca2+ buffer
(maximum contraction) to be 0% and 100%, respectively. The
EC50 value, a concentration of Ca2+ required to
induce the 50% force of the maximum response, was determined by
fitting the Ca2+ concentration-tension response curves to a
four-parameter logistic model (39). The measured values were expressed
as means ± S.E. Effects of recombinant hHS-M21
proteins on Ca2+-induced force were statistically analyzed
by analysis of variance. The obtained EC50 values from
pCa-tension relations were compared in the absence or
presence of the hHS-M21 fragments using Student's t test for paired values, and the P values of
less than 0.05 were considered to be statistically significant.
Isolation of hHS-M21 cDNAs--
We constructed a
normalized subtraction PCR-cDNA library between the mRNAs from
cardiac and skeletal muscles to obtain heart-specific cDNA
fragments. Randomly selected 1,021 clones were sequenced, and the
sequence data were compared with the GenBankTM data base. It was
revealed that 243 (23.8%) clones were not matched to the known human
genes in the data base except for expressed sequence tags. Forty-seven
cDNA fragments from unmatched genes were tested for their
expression in various human tissues by the RT-PCR analysis. Among them,
three cDNA fragments were expressed only in the heart (data not
shown), and one of these heart-specific clones, which were 209 bp of
length, was designated as HS602.
The HS602 fragment was used as a probe to isolate cDNA
clones from a human heart cDNA library. Restriction mapping and end sequencing of the isolated cDNAs showed that there were at least three types of cDNAs different by a small deletion or insertion. Representative clones of each type, 602-4, 602-6, and 602-7, were completely determined for their sequences, and it was revealed that
602-6 and 602-7 had an insertion of 181 bp and a deletion of 3 bp,
respectively, as compared with 602-4 (Fig.
1). From an estimation by the numbers of
isolated cDNA clones corresponding to these three types, it was
suggested that two of them, 602-4 type and 602-6 type, were major
isoforms and another type, 602-7 type, was a minor isoform.
Full sequences of HS602 cDNAs for two major isoforms
were determined from the isolated overlapping clones. These two
isoforms span a total of 2,046 and 2,227 bp with the open reading frame predicting to encode for 208 and 224 amino acid residues, respectively (Fig. 2). The full sequence included 827 bp in the 5'-untranslated region and 595 or 731 bp in the
3'-untranslated region for two different termination codons in the
HS602 cDNAs.
Characteristic feature of the predicted proteins was the presence of
leucine zipper motifs at the C-terminal end. Although the long isoform,
hHS-M21 B, had different C-terminal structures by the
181-bp insertion from the short isoform, hHS-M21 A, leucine zipper motifs were found in both isoforms (Fig. 2). A data base search
revealed that the amino acid sequence of hHS-M21 A was 76.8% identical to the 20-kDa smooth muscle small subunit (sm-M20) of
chicken myosin phosphatase (14) and 50.9% identical to C-terminal part
of MBS (MYPT1) of human myosin phosphatase (15). Of
particular interest was that the C-terminal half sequence of
hHS-M21 A was 92.4% and 69.6% identical to the relevant
sequence of sm-M20 and MBS (MYPT1), respectively. It was,
then, suggested that the HS602 gene encoded for a
heart-specific 21-kDa small subunit (hHS-M21) of human
myosin phosphatase, and that the hHS-M21 had two major isoforms; the short type (hHS-M21 A) and the long type
(hHS-M21 B). To our surprise, hHS-M21 A
cDNA sequence was identical to 3'-terminal one-third sequence of
MYPT2 cDNA for another MBS (25), except that 5'-sided
355-bp sequence of hHS-M21 cDNA was lacked from the
MYPT2 cDNA. In addition, the initiation codon of
hHS-M21 was corresponding to codon 775 of MYPT2,
i.e. aa 1-208 of hHS-M21 A was exactly
identical to aa 775-982 of MBS encoded by MYPT2. These
observations strongly suggested that the MYPT2 gene was a
multi-functional gene encoding for both the striated muscle type MBS
and heart-specific M21. To obtain a structural evidence of
this multicoding feature, we determined the genomic organization of the
human MYPT2 gene.
Genomic Organization of Human MYPT2 Gene--
To determine
exon-intron organization of the human MYPT2 gene, a human
genomic DNA library was screened with 602-7 as a probe. Several
different genomic clones were obtained and EcoRI or
XhoI fragments hybridized to 602-7 were subcloned for the
sequencing analyses from primers designed in the MYPT2
cDNA sequence. Representative genomic clones, 602G2, 602G4, 602G3,
and 602G1, are shown in Fig. 3 along with
their subclones, 602gs2, 602gs7, 602gs4, 602gs3, 602gs1, 602gs11, and
602gs10, containing exons 14-18, 20, 21, and 23-25. Because the
screening of genomic DNA library has failed to isolate clones
corresponding to the other exons, the remaining genomic organization
was determined by sequencing of overlapping LA-PCR products (Fig.
3).
Sequencing analyses of genomic subclones and LA-PCR products have
revealed that the MYPT2 gene consists of 25 exons as shown in Fig. 3 and Table I. The
hHS-M21 B cDNA was the product of alternative splicing
at exon 24, and the 3-bp deletion in cDNA clone 602-7 was suggested
to be a differential splicing product to a minor acceptor site in exon
22 (Table I). It also was revealed that the ankyrin repeats in the
N-terminal part of MBS (MYPT2) molecule were encoded by
exons 1-7, and two mutually exclusive leucine zipper motifs at the
C-terminal part of the hHS-M21 A and B cDNAs were
encoded by exon 25 and 24, respectively (Fig. 3). Quite interestingly,
the 5' sequence (1-355 bp) of hHS-M21 cDNA (exon 1 of
hHS-M21) was found in intron 13 (1 kbp upstream of the exon
14) of the MYPT2 gene and exons 14-25 were exactly matched
to the hHS-M21 sequences (Fig. 3). In addition, several cis-elements that were observed in promoter regions of
heart-specific genes, GATA binding site, MEF-2-like binding motif,
E-box, AP2 and M-CAT, were found in upstream of the hHS-M21
coding exon 1 (data not shown). These results indicate that the
hHS-M21 cDNAs are transcribed from a heart-specific
promoter in intron 13 of the MYPT2 gene.
Expression of MYPT1, MYPT2, and hHS-M21 in Human
Tissues--
Expression of MYPT2 (this means MBS coding
region of the MYPT2 gene here) and hHS-M21 in
various human tissues was investigated using the RT-PCR analysis and
compared with that of MYPT1 (MBS coding region of the
MYPT1 gene). As shown in Fig.
4A, hHS-M21 mRNA was expressed only in the heart, while MYPT2
expression was detected in several tissues, preferentially in heart,
skeletal muscle, and brain. It was confirmed by the RT-PCR analysis
that exon 24 of the MYPT2 gene was alternatively spliced in
encoding for the MBS molecule (Fig. 4A), as is the case for
encoding for the hHS-M21 molecule (Fig. 1). Expression of
mRNA skipped for exon 24 was relatively abundant as compared with
mRNA utilizing exon 24 in most tissues, whereas similar quantities
of both type mRNA were found in the skeletal muscle (Fig.
4A).
A competitive RT-PCR analysis showed the different expression level of
MYPT1 and MYPT2 in human tissues (Fig.
4B). The expression of MYPT2 mRNA was higher
than that of MYPT1 in the striated muscles, such as heart
and skeletal muscle. In contrast, the amount of MYPT1
mRNA was more abundant than that of MYPT2 mRNA in
brain and other tissues including lung, uterus, and small intestine. It
should be noted here that the expression level of MYPT1
mRNA was relatively constant but somewhat different in tissues
tested here (Fig. 4B).
Effect of hHS-M21 Fragments on the Ca2+
Sensitivity of Contraction in Permeabilized Porcine Renal Artery and
Rat Cardiac Myocytes--
To analyze the function of
hHS-M21 in muscle contraction, various recombinant
hHS-M21 proteins were prepared (Fig.
5A) and used in permeabilized
cell assays. Fig. 5 (B and C) shows the effect of
hHS-M21 proteins on the Ca2+-induced
contraction in 1% Triton X-permeabilized porcine renal artery and
2.5%
In permeabilized porcine renal artery, application of 3 µM recombinant proteins, hHS-M21 A,
hHS-M21 B, and hHS-M21 t-1, caused augmentation
of the Ca2+-induced contractions, and the
[Ca2+]i force relation curves were
shifted to left as compared with that in the controls where no
recombinant proteins were added (Fig. 5B). The
EC50 value of Ca2+ in the control assay was
434.8 ± 17.4 nM (n = 3). In the
presence of 3 µM recombinant proteins,
hHS-M21 A, hHS-M21 B, and hHS-M21 t-1, the EC50 values were significantly lowered to
86.9 ± 2.0 nM, 94.0 ± 7.1 nM, and
118.4 ± 2.4 nM, respectively (for each case,
n = 3, p < 0.001) (Table
II).
In permeabilized rat cardiac myocytes, the
[Ca2+]i force relationships
obtained in the presence of these recombinant proteins were shifted
leftward (Fig. 5C), as observed in the permeabilized porcine
renal artery. Although the EC50 value obtained in the control assay was 957.6 ± 169.3 nM (n = 5), it was significantly decreased to 318.9 ± 31.7 nM (n = 5, p < 0.01),
261.2 ± 31.6 nM (n = 5, p < 0.01), and 555.7 ± 90.1 nM
(n = 5, p < 0.05) in the presence of 1 µM recombinant protein, hHS-M21 A,
hHS-M21 B, and hHS-M21 t-1, respectively.
These results indicated that the exogenously added hHS-M21
proteins increase the Ca2+ sensitivity of the contractile
apparatus in the permeabilized smooth muscle (porcine renal artery) and
cardiac muscle (rat cardiac myocytes). The effect of
hHS-M21 was prominent in the smooth muscle, but it was
significantly observed also in the cardiac muscle. Because the presence
or absence of C-terminal leucine zipper motifs showed a little but with
no statistically significant difference in the effect of muscle
contraction, it was suggested that the main functional domain of
hHS-M21 was not located in the C-terminal leucine zipper motifs.
Interaction of hHS-M21 with MYPT1 and MYPT2--
As
demonstrated in the previous section, hHS-M21 showed a
prominent effect in the smooth muscle and, to less extent, in the cardiac muscle. This phenomenon might be curious, because the hHS-M21 gene is expressed only in the cardiac muscle and
not in the smooth muscle. However, it can be explained if
hHS-M21 exhibits its function mainly through interaction
with MBS encoded by MYPT1, and not with MBS encoded by
MYPT2. To investigate the interaction of hHS-M21
with MBSs encoded by MYPT1 and MYPT2, an overlay
assay was used to evaluate the binding affinity between them. Both
isoforms of full-length hHS-M21 (A and B) showed a binding
ability to C-terminal one-third of MYPT1-MBS (MYPT1 P),
while its binding to the corresponding part of MYPT2-MBS
(MYPT2 P) was extremely low (Fig.
6A). In addition, the
hHS-M21 proteins did not bind to N-terminal two-thirds of MBS encoded by either MYPT1 or MYPT2 (Fig.
6A).
To further map the binding domain of hHS-M21 to the
C-terminal one-third of MYPT1-MBS or MYPT2-MBS,
various hHS-M21 fragments were tested in the overlay assay.
The fragments containing the N-terminal 56 residues of
hHS-M21 (t-1, t-2, and o-1) bound to the
MYPT1-MBS with high affinity and to the MYPT2-MBS
with extremely low affinity, as the full-length hHS-M21
proteins did. In contrast, the C-terminal hHS-M21 fragments
(t-3, t-4, and t-5) showed only a scarce binding to MBSs (Fig.
6B) and no binding was observed with the middle part of
hHS-M21 (o-2). These results also demonstrated that the
C-terminal leucine zipper motifs of the hHS-M21 proteins were dispensable for the sufficient interaction with MBS encoded by
MYPT1.
Effect of N- or C-terminal Fragments of hHS-M21 on
Ca2+ Sensitivity--
Because the hHS-M21
protein showed a high binding affinity to MYPT1-MBS via its
N-terminal 56 residues and because the exogenously added full-length
hHS-M21 proteins enhanced the Ca2+-induced
muscle contraction, the effects by hHS-M21 fragments were
investigated to map the main functional enhancing domain of
hHS-M21. To determine the enhancing domain, we used the
permeabilized porcine renal artery assay, because the effect of
hHS-M21 was prominent in this assay as compared with that
in the rat cardiomyocytes.
The [Ca2+]i force relationship
obtained in the presence of 3 µM hHS-M21
recombinants, t-2 and o-1, was shifted leftward (data not shown). The
EC50 values obtained in the presence of hHS-M21
t-2 and o-1 were significantly small as compared with that in the
control (n = 3, p < 0.001 and
p < 0.01, respectively), although the EC50
value for hHS-M21 t-2 was smaller than that for
hHS-M21 o-1 (Table II). On the other hand, the
[Ca2+]i force relationship in the
presence of 3 µM hHS-M21 recombinants, t-3,
t-4, t-5, and o-2, overlapped with that in the control (data not shown)
and the EC50 values were not significantly different from
the control value (Table II). Therefore, the main active domain of
hHS-M21 was mapped in the similar region as the binding
domain, although the enhancing activity of o-1 (residues 1-56) was
less than that of t-2 (residues 1-110).
In the present study, we isolated and characterized cDNA
clones for the human myosin light chain phosphatase (MLCP) small subunit (hHS-M21) that was expressed only in the heart.
Genomic organization of the human MYPT2 gene was determined,
and it was revealed that one of the large subunits
(MYPT2-MBS) and the small subunit of MLCP in the heart are
the products of the same gene. In addition, we investigated the
function of hHS-M21 as measured by the Ca2+
sensitivity of contraction in the permeabilized porcine renal artery
and rat cardiac myocytes. Moreover, the interaction of the
hHS-M21 with MBSs encoded by MYPT1 and
MYPT2 was investigated by the overlay assay using
recombinant proteins. We found that: 1) hHS-M21 induced an
additional contraction at a constant Ca2+ concentration and
shifted the [Ca2+]i force
relationship to the left; 2) the fragments containing the N-terminal
half conferred this action of hHS-M21, while the deletion
of the N-terminal 56 residues completely abolished the action; 3)
hHS-M21 bound to the C-terminal one-third of MBS encoded by
MYPT1 with high affinity, and only to a little extent, bound to the same region of MBS encoded by MYPT2; and 4) the
binding domain of hHS-M21 to MYPT1-MBS was
mapped to the same region as the main effective domain for the
Ca2+ sensitivity on the Ca2+-induced
contraction in permeabilized porcine renal artery.
The MLCP holoenzyme is composed of three subunits; catalytic subunit
and small and large regulatory subunits (5, 6). The MYPT2
gene locates on chromosome 1q32 and encodes for MBS of an approximately
125-kDa protein in the cardiac muscle, of which sequence is 61%
identical to that of the human MYPT1 gene expressed in the
smooth muscle (25). Unexpectedly, the C-terminal region (residues
775-982) of the MYPT2-MBS was 100% identical to the
full-length amino acid sequence of the hHS-M21 A, one of the two major isoforms of human hHS-M21, and moreover,
residues 775-954 of the MYPT2-MBS were 100% identical to
residues 1-180 of the hHS-M21 B, another isoform of human
hHS-M21. These observations led us to speculate that the
hHS-M21 isoforms may be products of the MYPT2
gene. To confirm this speculation, we carried out determination of
genomic organization of the MYPT2 gene and investigated their expressions by the RT-PCR analyses. It was revealed that sequence
of 5'-untranslated region of the hHS-M21 cDNA was
present in intron 13 of MYPT2, and that the two major
isoforms of human hHS-M21 were derived from the alternative
splicing of exon 24 of MYPT2 (Figs. 3 and 4). These results
have demonstrated that the C-terminal 208 (or 224) residues of
MYPT2-MBS are expressed in the heart as a separate protein,
termed as hHS-M21, of which mRNAs are transcribed from
a heart-specific promoter located within an intron of the
MYPT2 gene. We also have confirmed that the
hHS-M21 protein is expressed in the human heart by a
Western blot analysis using rabbit antisera raised against the
recombinant hHS-M21 A protein (data not shown).
It has recently been suggested that the sm-M20 subunit from chicken
gizzard may be produced from an avian orthologue of MYPT2 (40), while the sm-M20 subunit has not been isolated yet from human
tissue. However, the C-terminal 120 residues of chicken sm-M20 (residue
67-186) showed 91% identity to the C-terminal 120 residues of
hHS-M21, whereas the homology in N-terminal residues between chicken sm-M20 and hHS-M21 were only about 50%. It
is of interest that intron 18 of the human MYPT2 gene is
long (more than 40 kbp) as is the case with intron 13 (about 20 kbp)
where hHS-M21 exon 1 exists and that the high sequence
similarity of hHS-M21 with chicken sm-M20 was found after
the sequence of exon 19. If mRNA of human sm-M20 subunit were
transcribed from intron 18 of the MYPT2 gene, it would be a
very unique gene that encodes for three different MLCP regulatory
subunits: MBS, hHS-M21, and sm-M20.
It might be unusual for one gene to produce two or more proteins with
different function in the same tissue by utilizing different promoters,
but MLCK gene is known to generate two different proteins in the smooth
muscle. The C-terminal 154 residues of the smooth muscle MLCK is
expressed as an independent protein, telokin (41, 42), and the telokin
cDNA is transcribed from a promoter within an intron of the MLCK
gene (42, 43). Telokin binds specifically to dephosphorylated MLC of
smooth muscle and inhibits MLC phosphorylation by MLCK (44, 45). In
addition, telokin induces Ca2+ desensitization by enhancing
the MLCP activity in the smooth muscle (46). The function of
hHS-M21 appears opposite to that of telokin, but the
production of hHS-M21 from the MYPT2 gene is
analogous to the situation of telokin produced by the MLCK gene.
Although MYPT2 mRNA of 11.4 kbp was abundantly found in
the heart and skeletal muscle (25), the MYPT2 protein
(MYPT2-MBS) was reported to be expressed only in the heart
and brain (25), in contrast to MYPT1-MBS, which is widely
distributed among chicken or human tissues except for skeletal muscle
(25, 47). In this study, we investigated the expression of
MYPT2-MBS and hHS-M21 in various human tissues
at the mRNA level by the RT-PCR analysis (Fig. 4). The amount of
MYPT2 mRNA was slightly more abundant than that of
MYPT1 mRNA in the cardiac and skeletal muscles, while the MYPT1 mRNA was much abundant as compared with
MYPT2 mRNA in the brain and smooth muscle tissues (Fig.
4B). These findings were consistent with that the
MYPT1-MBS was present in most tissues except for skeletal
muscle, and that it appeared more abundant in the smooth and cardiac
muscles than in the other tissues (47). Although both MBSs encoded by
MYPT1 and MYPT2 increase the activity of MLCP,
MYPT1-MBS was a more efficient activator than
MYPT2-MBS, because MYPT2-MBS required about
10-fold higher concentration to achieve the same extent of activation
as MYPT1-MBS for which maximum activation was found at
approximately equimolar ratio of MYPT1-MBS and catalytic
subunit (25). These observations are in good agreement with the present
findings that MYPT1-MBS, and not MYPT2-MBS, was a
main regulatory/target subunit bound by hHS-M21 to modulate
the MLCP activity in the cardiac muscle, as in the smooth muscle.
The function of MYPT2-MBS is yet unclear. It was observed in
this study that hHS-M21 proteins bound to the C-terminal
one-third of MYPT2-MBS to a lesser extent (Fig. 6). This
binding appears not to be background, because hHS-M21
proteins did not bind at all to the N-terminal two-thirds of
MYPT2-MBS under the same conditions (Fig. 6A) and
several truncated hHS-M21 mutants, t-1, t-2, and t-4,
showed a weak binding to the C-terminal MYPT2-MBS (Fig.
6B). It also may be worth noting that a weak binding of the
hHS-M21 t-4 protein to MYPT2-MBS was observed to
a similar level as that to MYPT1-MBS. Although we could not
demonstrate the Ca2+-sensitizing effect of
hHS-M21 t-4 in the smooth muscle (Table II), we cannot
exclude the possibility that there might be other function(s) than the
Ca2+ sensitization of contraction, which is conferred by
interaction between the C-terminal half of hHS-M21 and
MYPT2-MBS, in the cardiac muscle. Further studies will be
required to elucidate the function of MYPT2-MBS.
The C terminus of MYPT1-MBS was reported to contain binding
sites for sm-M20 (12, 18, 19), Rho A (48), arachidonic acid (49), and
acidic phospholipids (50). We have demonstrated here that
hHS-M21 interacts with the C-terminal one-third of
MYPT1-MBS and increases the Ca2+ sensitivity of
muscle contraction. There might be no evidence for the direct
involvement of hHS-M21 in the Ca2+
sensitization effect. However, it was reported that the
Ca2+-calmodulin MLCK complex induced an increase in
Ca2+ sensitivity in rat single skinned cardiac cells (51),
and the movements of tension/pCa relationships were in
similar extent to that observed in this study (Fig. 5C). It
is therefore likely that the increase in the Ca2+
sensitivity by hHS-M21 is a reflection of the increased MLC
phosphorylation. In addition, the binding domain to
MYPT1-MBS and the main activating domain in Ca2+
sensitization were mapped to the N-terminal half region of the hHS-M21 in this study. It is suggested, then, that the
exogenously applied recombinant hHS-M21 proteins may bind
to endogenous MYPT1-MBS and exhibit the inhibitory action on
the MLCP activity.
On the other hand, the N-terminal 56 residues of hHS-M21
(representative of hHS-M21 o-1) were sufficient for full
activity of binding to the C-terminal one-third of MYPT1-MBS
(Fig. 6), but they were not enough to confer full activity in
increasing the Ca2+ sensitivity as demonstrated by
hHS-M21 t-2 (residues 1-110) (Table II). However, neither
binding activity nor Ca2+ sensitization effect was found
with hHS-M21 o-2 that encompassed residues 57- 110 (Fig. 6
and Table II). These results suggest that the main binding domain and
the main active domain of hHS-M21 are overlapped
considerably in the N-terminal 56 residues and that the main active
domain is extended to C-terminal side of 57th residue but not exceeds
the 110th residue. It is noteworthy that the main active domain of
sm-M20 was mapped in the N-terminal half region (21). Because the
C-terminal halves of hHS-M21 and sm-M20 have virtually
identical amino acid sequences and no enhancing function of
Ca2+ sensitivity, these observations suggest that the
C-terminal halves of MLCP small subunits are dispensable for their
functions in regulation of muscle contraction. In turn, these findings
indicated that the N-terminal halves of hHS-M21 and sm-M20
exhibit their functions despite the low similarity in amino acid
sequences. It will be interesting to determine which motifs in the
N-terminal half of MLCP subunit would confer the function. Further
investigations including site-directed mutagenesis of MLCP small
subunit genes will be needed to demonstrate the functional motif(s).
The effect of hHS-M21 on the Ca2+ sensitivity
was prominent in the smooth muscle as compared with in the cardiac
muscle. The apparent difference in the efficiency of Ca2+
sensitization by hHS-M21 in these muscles may be due to the
difference in the amount of recombinant proteins used in the assays.
This possibility is unlikely because the increases in Ca2+
sensitivity with the different amounts of hHS-M21 were
constant within a range from 1 to 10 µM in the assay with
the porcine renal artery and within a range from 1 to 3 µM in the assay with rat cardiac myocytes (data not
shown). The other possibilities for the difference are the species
difference, porcine versus rat, in the assay system and the
different expression or activity level of MYPT1-MBS between
the smooth and cardiac muscles because the expression of
MYPT1-MBS was a little more abundant in the smooth muscle
than in the cardiac muscle. It also is possible that the contraction of
tissue (porcine renal artery) can be measured more prominently than
that of single cells (rat cardiac myocytes), because the contraction
power from tissue is a summation of that from single cells. In support
of the last possibility, the effect of MLCK on force development found
in demembraned heart muscle strips (52, 53) was stronger than that in
single-skinned cardiac cells (51).
In the cardiac muscle, it has been poorly understood about the role of
MLC phosphorylation. However, a recent report has focused on the
relevance of MLC phosphorylation system in the cardiac hypertrophy
(27), and our observations highlight the role of MLCP system in the
cardiac muscle contraction via identification and functional analysis
of the heart-specific MLCP subunit. This is the first report
demonstrating that hHS-M21, a heart-specific MLCP small
subunit, plays a role in the regulation of
Ca2+-dependent contraction in the cardiac
muscle via binding to C-terminal one-third of MYPT1-MBS.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
gt11 (CLONTECH) was
screened using HS602 fragment as a probe according to the standard
methods (28). The cDNA inserts from positive recombinant phages
were subcloned into pBluescript KS (
) (Invitrogen) for sequence determination.
) and the subclones hybridized to 602-7 were sequenced. These
subclones were revealed to contain exons 14-18, 20, 21, and 23-25 of
MYPT2. To obtain sequence information for the remaining
regions, a long amplification PCR (LA-PCR) method was performed using
an LA-PCR kit (Takara) according to the manufacturer's instructions.
The primer pairs used in the LA-PCR experiments were as follows;
MYPT2LA1F (5'-GCAACTCGAGCCCCAACAGTAATTT-3') and MYPT2LA1AR
(5'-CGTTGTCTTGCTGGTTTACATTGGC-3'), MYPT2LA1PF
(5'-GGACATGGTGAAGTTTCTGGTGGA-3') and MYPT2LA1R
(5'-CACATCCTCTATTTTCCCACTGTTG-3'), MYPT2LA2F
(5'-GAAGAAGAGCAGCAGATGTTGCAG-3') and 602-MYPT2R
(5'-CCCACTCTGAATCTTGCTGT-3'), 602-MYPT2F (5'-ACAGCAAGATTCAGAGTGGG-3') and MYPT2LA3R-2 (5'-GAGCTTCCGGGGTGCTAGTGAAC-3'), MYPT2LA4F
(5'-CTCTTCAAGCCCTCGAATTTCTGC-3') and MYPT2LA4-1R
(5'-CAAAGCAGAGCCCCACAAAT-3'), MYPT2LAF (5'-CTACTCCTGTGCTCTCCATT-3') and
HSpromR5 (5'-CTTGCCATTTCTGTCCTGTC-3'), 602-EX6F
(5'-GCTTTCAAGGATGTCTGTGAC-3') and 602-EX7R
(5'-CGACATGCATTCACTCACAAG-3'), 602-1000LAF (5'-GCATGAAAGACTTTCTAGGT-3') and 602-LA4RR (5'-ATGCTCAGGGGTAGTTTGCGCC-3'), and 602-EX9F
(5'-TGTGAGTTCTAACGCGAACG-3') and 602-EX11R
(5'-AGAGATGAAATGGGGTGGAG-3'). The LA-PCR products were
electrophoresed in 0.6-0.8% agarose gels and extracted from the gels
using a Gel Band Purification kit (Amersham Pharmacia Biotech). The
purified DNA fragments were sequenced from multiple scanning primers
designed in exons of MYPT2 to determine the sequences of
each exon and adjacent introns.
80 °C until preparation of recombinant proteins.
80 °C until use in binding assay. On the other hand, the
supernatants containing recombinant hHS-M21 proteins were
loaded into columns in which the resin (Novagen) charged with
Ni2+ and equilibrated with buffer A was bedded. After the
columns were washed extensively with 6 M urea, 60 mM imidazole, 500 mM NaCl, and 20 mM Tris-HCl (pH 7.5), bound proteins were eluted by 6 M urea, 400 mM imidazole, 500 mM
NaCl, and 20 mM Tris-HCl (pH 7.5). The eluates were
dialyzed against 0.1% acetic acid (pH 4.0), and the dialysates were
clarified by centrifugation at 10,000 × g for 15 min
at 4 °C. The supernatants were freeze-dried and dissolved in 0.1%
acetic acid (pH 4.0) for concentration. The recombinant
hHS-M21 proteins were further purified by using an HPLC
system consisting of a pump (LC-10AD; Shimazu), a column (TSKgel
G2000SWXL; Toyo-soda), and an UV detector (SPD-10AVP; Shimazu).
-escin for 10 min in a
relaxing solution (110 mM potassium methane sulfonate, 5 mM magnesium methane sulfonate, 5 mM
Na2ATP, 10 mM creatine phosphate, 4 mM EGTA, and 20 mM PIPES-KOH (pH 7.1)) at
25 °C. In experiments for pCa-tension relation, skinned
cells were treated first with 30 µM ryanodine and 30 mM caffeine in pCa 6 solution for 4 min to
impair the Ca2+ releasing function of the sarcoplasmic
reticulum (34) so that the effect of the released Ca2+ from
the sarcoplasmic reticulum on pCa-tension relation could be
neglected. Ryanodine and caffeine was then washed out with the relaxing
solution and the cells were relaxed completely. After this procedure,
the striation uniformity remained unchanged, and the initial resting
sarcomere length was set at 2.03 ± 0.03 µm. The permeabilized
cells were then activated by various concentrations of Ca2+
cumulatively from 0.05 to 10 µM, and the developed force
was monitored as sarcomere length during the activation. The sarcomere length did not vary at >pCa 6, varied by less than 0.2 µm
at pCa 6, and varied by more than 0.2 µm at
<pCa 6 due to a significant internal shortening with
obscured striation pattern. After the cell was relaxed completely with
the relaxing solution, however, sarcomere length was returned to the
initial value. At 10-min intervals, the second activation was repeated
in the absence (control) or presence of hHS-M21 recombinant
proteins (A, B, or t-1) solubilized in 20 mM PIPES-KOH (pH
7.1). If the sarcomere length at pCa 6 varied more than 0.2 µm, or the sarcomere length after the first activation varied more
than 5% from the initial one, the records were discarded (35). Using
the measured values of cell width and length, each maximum tension was
expressed as the force/cross-sectional area based on the assumption of
cylindrical cell geometry, because cell depth could not be measured due
to technical difficulties. The activating solutions of the desired
Ca2+ concentrations (0.05-10 µM) were
prepared by adding the appropriate amounts of calcium methane sulfonate
to the relaxing solution.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic representation of
hHS-M21 cDNAs and isolated cDNA clones from a human
heart cDNA library. Upper part
represents the full cDNA structure, where thin and
thick bars indicate the noncoding and coding
regions, respectively. Positions of initiation and termination codons
are indicated. A dotted box represents the HS602
cDNA fragment initially isolated from the subtraction PCR-cDNA
library. An open box indicates the insertion of
181bp. Lower part demonstrates the covered region
by isolated cDNAs and their lengths are indicated in
parentheses.
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Fig. 2.
Nucleotide and predicted amino acid sequence
of hHS-M21 A and hHS-M21 B. A complete
sequence was obtained by combining the sequences of isolated clones
shown in Fig. 1. The hHS-M21 cDNAs consisted of two
major isoforms, representative of cDNA clones 602-4 and 602-6. A, complete sequences of hHS-M21 A cDNA.
B, sequence of hHS-M21 B cDNA, where only
the differences from hHS-M21 A cDNA are shown. Stop
codons are indicated by asterisks. The large isoform
(hHS-M21 B) differed from the small isoform
(hHS-M21 A) by the presence of 181-bp insert within a 3'
coding region, and the termination codon of the large isoform was found
within the insertion. A vertical line in
A indicates position of the insertion. Leucine zipper motifs
are underlined, which have completely different sequences
between the two isoforms.
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Fig. 3.
Genomic organization of MYPT2
gene. Parts of genomic MYPT2 gene were isolated
in several phage clones, and the remaining regions that were not
covered by phage clones were amplified from genomic DNA by the LA-PCR
method to determine the exon/intron organizations. Four representative
phage clones 426G1, 426G2, 426G3, and 426G4 are indicated along with
their subclones 426gs1, 2, 3, 4, 7, 10, and 11, used for determination
of organization for exons 14-18, 20, 21, and 23-25. The other parts
of genomic organization were determined by sequencing the LA-PCR
products as shown in the lower part. Positions of
the LA-PCR primers are indicated along with their names. Protein coding
region of MYPT2 was separated into 25 exons.
Dotted boxes represent ankyrin repeats in N
terminus of MYPT2, which corresponded to exons 1-7.
Solid boxes indicate leucine zipper motifs in C
terminus encoded by exons 24 or 25. Splicing patterns of
hHS-M21 cDNAs are indicated, where exon 24 is
alternatively spliced.
Sequences of exon/intron boundaries in the human MYPT2 gene
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Fig. 4.
RT-PCR analyses of MYPT1,
MYPT2, and hHS-M21 in various human
tissues. A, RT reaction was performed using total RNA
from fetal heart, adult heart, skeletal muscle, brain, kidney, liver,
uterus, lung, spleen, thymus, small intestine, and colon, followed by
PCR to detect expression of MYPT2, hHS-M21, and
glyceraldehyde phosphodehydrogenase. Two bands of MYPT2
(1376 and 1195 bp) correspond to insertion and deletion of
MYPT2 exon 24, respectively. B, comparison of
mRNA expression of MYPT1 and MYPT2 in various
human tissues. The RT reaction was performed using total RNA from adult
heart, skeletal muscle, brain, uterus, lung, and small intestine. Upper
band (486 bp) and lower band (179 bp) represent MYPT2 and
MYPT1, respectively. Glyceraldehyde phosphodehydrogenase
(GAPDH) represents the external control showing that similar
amounts of cDNAs were analyzed. All the RT-PCR analyses were done
within the course of exponential amplification.
-escin-permeabilized rat cardiac myocytes, respectively. In
these protocols, contractions were monitored by stepwise increases in
Ca2+ concentration in the presence of 3 µM
recombinant hHS-M21 proteins in permeabilized porcine renal
artery and 1 µM recombinant hHS-M21 proteins
in permeabilized rat cardiac myocytes. In the Ca2+-free
buffer, application of hHS-M21, even up to 10 µM, was not able to produce any significant tension
development in both permeabilized porcine renal artery and rat cardiac
myocytes (data not shown). Then, the observed contraction reflected the
Ca2+ sensitization effects by the hHS-M21
proteins.
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Fig. 5.
Schematic representation of recombinant
hHS-M21 proteins and their effects on
[Ca2+]i force relationship
of Ca2+-induced contraction. A, wild types
and mutants of hHS-M21 proteins are schematically indicated
along with their covering residues. Dotted and
open box indicate leucine zipper motifs of
hHS-M21 A and hHS-M21 B, respectively.
B and C show the
[Ca2+]i force relation curves of
contractions induced by increment of Ca2+ levels in absence
(control, open circles) or presence of
recombinant proteins, hHS-M21 A (closed
circles), hHS-M21 B (open
triangles), and hHS-M21 t-1 (closed
squares). Force development was expressed as percentage of
that obtained at 10 µM Ca2+. Data are
represented by means ± S.E. (n = 3-5).
B, in 1% Triton X-permeabilized porcine renal artery.
Amount of applied recombinant hHS-M21 proteins was 3 µM in each case. C, in 2.5% -escin rat
cardiac myocytes. Amount of applied hHS-M21 proteins was 1 µM in each case.
EC50 values for Ca2+ in the absence or presence of
hHS-M21
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Fig. 6.
Interaction of hHS-M21 subunit
with MYPT1 and MYPT2.
A, recombinant MBS proteins were separated in SDS-PAGE
(12%), transferred to PVDF membranes, and immunoblotted with anti-flag
antibody, or incubated with recombinant hHS-M21 A or
hHS-M21 B proteins (10 µg/ml), which were detected by
anti-His6-tag polyclonal antibody. Positions of marker
proteins, ovalbumin (54 kDa), carbonic anhydrase (35 kDa), and soybean
trypsin inhibitor (29 kDa), are indicated. Lane
1, MYPT1 A; lane 2, MYPT2 A;
lane 3, MYPT1 M; lane 4,
MYPT2 M; lane 5, MYPT1 P; lane
6, MYPT2 P. B, recombinant MYPT-MBSs
proteins were resolved in SDS-PAGE (12%) and blotted to PVDF membrane
as in A. The membranes were incubated with
hHS-M21 t-1, t-2, t-3, t-4, t-5, o-1, or o-2 (10 µg/ml).
Lane 1, MYPT1 P; lane 2,
MYPT2 P. Representative results in repeated experiments are shown in
both A and B.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Drs. M. Shimoda, M. Kuroha, T. Hojyo, and M. Ikenaga for help in the purification of recombinant proteins by HPLC systems.
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FOOTNOTES |
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* This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture, Japan; a research grant from the Ministry of Health and Welfare, Japan; and a grant from the Atsuko Ouchi Memorial Fund.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB050641 (for hHS-M21 A) and AB050642 (for hHS-M21 B).
** To whom correspondence should be addressed: Dept. of Molecular Pathogenesis, Div. of Adult Diseases, Medical Research Inst., Tokyo Medical and Dental University, 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan. Tel.: 81-3-5280-8056; Fax: 81-3-5280-8055; E-mail: akitis@mri.tmd.ac.jp.
Published, JBC Papers in Press, November 6, 2000, DOI 10.1074/jbc.M008566200
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ABBREVIATIONS |
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The abbreviations used are: MLC, myosin light chain; MLCK, myosin light chain kinase; MLCP, myosin light chain phosphatase; PP1, protein phosphatase type 1; PP2, protein phosphatase type 2; PP1c, catalytic subunit of PP1; MBS, myosin binding subunit; sm-M20, smooth muscle 20-kDa small subunit of MLCP; MYPT, myosin phosphatase target (or targeting); MHC, myosin heavy chain; hHS-M21, human heart-specific small regulatory subunit of MLCP; PCR, polymerase chain reaction; RT, reverse transcription; LA, long amplification; PBS, phosphate-buffered saline; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; CSS, cytoplasmic substitution solution; PIPES, piperazine-1,4-bis(2-ethanesulfonic acid); aa, amino acid(s); bp, base pair(s); kbp, kilobase pair(s); PVDF, polyvinylidene difluoride.
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