Characterization and function of MYPT2, a target subunit of myosin phosphatase in heart.
ABSTRACT Characterization of cardiac MYPT2 (an isoform of the smooth muscle phosphatase [MP] target subunit, MYPT1) is described. Several features of MYPT2 and MYPT1 were similar, including: a specific interaction with the catalytic subunit of type 1 phosphatase, delta isoform (PP1cdelta); interaction of MYPT2 with the small heart-specific MP subunit; interaction of the C-terminal region of MYPT2 with the active form of RhoA; phosphorylation by Rho-kinase at an inhibitory site, Thr646 and thiophosphorylation at Thr646 inhibited activity of the MYPT2-PP1cdelta complex. MYPT2 activated PP1cdelta activity, using light chains from smooth and cardiac muscle, by reducing K(m) and increasing k(cat). The extent of activation (k(cat)) was greater than for MYPT1 and could reflect distinct N-terminal sequences in the two MYPT isoforms. Adenovirus-mediated gene transfer of MYPT2 and PP1cdelta reduced the phosphorylation level of cardiac light chains following stimulation with A23187. Overexpression of MYPT2 and PP1cdelta blocked the angiotensin II-induced sarcomere organization in cultured cardiomyocytes. Electron microscopy indicated locations of MYPTs, at, or close to, the Z-line, the A band and mitochondria. Similarity of the two MYPT isoforms suggests common enzymatic mechanisms and regulation. Cardiac myosin is a substrate for the MYPT2 holoenzyme, but the Z-line location raises the possibility of other substrates.
Characterization and function of MYPT2, a target subunit of myosin
phosphatase in heart
Ryuji Okamotoa,1, Takaaki Katoa,1, Akira Mizoguchib, Nobuaki Takahashid, Tetsuya Nakakukia,
Hideo Mizutania, Naoki Isakaa, Kyoko Imanaka-Yoshidac, Kozo Kaibuchie, Zhaojiang Luf,
Katsuhide Mabuchif, Terenc Taof, David J. Hartshorneg, Takeshi Nakanoa, Masaaki Itoa,⁎
aDepartment of Cardiology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan
bDepartment of Neural Regeneration and Cell Communication, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan
cDepartment of Pathology and Matrix Biology, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan
dPharmaceutical Research Laboratories, Pharmaceutical Division, Kirin Brewery co., Ltd., Takasaki, Gunma 370-1295, Japan
eDepartment of Cell Pharmacology, Graduate School of Medicine, Nagoya University, Nagoya, Aichi 466-8550, Japan
fMuscle Research Group, Boston Biomedical Research Institute, Boston, MA 02472, USA
gMuscle Biology Group, University of Arizona, Tucson, AZ 85721, USA
Received 27 October 2005; received in revised form 9 November 2005; accepted 9 November 2005
Available online 23 January 2006
Characterization of cardiac MYPT2 (an isoform of the smooth muscle phosphatase [MP] target subunit, MYPT1) is described. Several features
of MYPT2 and MYPT1 were similar, including: a specific interaction with the catalytic subunit of type 1 phosphatase, δ isoform (PP1cδ);
interaction of MYPT2 with the small heart-specific MP subunit; interaction of the C-terminal region of MYPT2 with the active form of RhoA;
phosphorylation by Rho-kinase at an inhibitory site, Thr646 and thiophosphorylation at Thr646 inhibited activity of the MYPT2–PP1cδ complex.
MYPT2 activated PP1cδ activity, using light chains from smooth and cardiac muscle, by reducing Kmand increasing kcat. The extent of activation
(kcat) was greater than for MYPT1 and could reflect distinct N-terminal sequences in the two MYPT isoforms. Adenovirus-mediated gene transfer
of MYPT2 and PP1cδ reduced the phosphorylation level of cardiac light chains following stimulation with A23187. Overexpression of MYPT2
and PP1cδ blocked the angiotensin II-induced sarcomere organization in cultured cardiomyocytes. Electron microscopy indicated locations of
MYPTs, at, or close to, the Z-line, the A band and mitochondria. Similarity of the two MYPT isoforms suggests common enzymatic mechanisms
and regulation. Cardiac myosin is a substrate for the MYPT2 holoenzyme, but the Z-line location raises the possibility of other substrates.
© 2005 Elsevier Inc. All rights reserved.
Keywords: Myosin phosphatase; RhoA; Rho-kinase; Myosin; Cardiomyocytes
Phosphorylation of the regulatory light chain of myosin II
(MLC) plays a pivotal role in many types of cells [1,2]. In
smooth muscle, phosphorylation of MLC at Ser19 by the Ca2+-
calmodulin-dependent myosin light chain kinase (MLCK)
initiates contraction by promoting cross-bridge cycling. Phos-
phorylation of MLC in nonmuscle cells also activates
actomyosin and this is involved in functions such as shape
change and motility and cytokinesis.
Despite several studies on smooth/nonmuscle myosin
phosphorylation, the phosphorylation of MLC in striated
muscles has received less attention. It is known, however, that
in striated muscle phosphorylation of MLC does not induce
contraction but appears to play a modulatory role . The
current hypothesis is that MLC phosphorylation in striated
muscle increases Ca2+-sensitivity, i.e. by inducing a shift to
lower [Ca2+]iin the force–Ca2+relationship . In cardiac
muscle, in addition to the effect on Ca2+-sensitivity, the
phosphorylation status of MLC and the regulated phosphory-
lation of MLC appear to be important in maintaining normal
Cellular Signalling 18 (2006) 1408–1416
⁎Corresponding author. Tel.: +81 59 231 5015; fax: +81 59 231 5201.
E-mail address: firstname.lastname@example.org (M. Ito).
1Authors contributed equally to this work.
0898-6568/$ - see front matter © 2005 Elsevier Inc. All rights reserved.
cardiac function over a long-term period . A spatial gradient
of MLC phosphorylation across the heart was observed and this
gradient might support cardiac torsion . In addition, the
phosphorylation of MLC in cardiac myocytes was demonstrated
to mediate sarcomere organization during cardiac hypertrophy
in vitro, indicating that abnormalities of MLC phosphorylation
might be linked to cardiac hypertrophy .
Since the phosphorylation level of MLC is determined by the
balance of activities of MLCK and myosin phosphatase (MP),
the latter plays an important role in regulating cell functions
involving phosphorylation of myosin II. As an example, the
inhibition and activation of MP in smooth muscle induces Ca2+-
sensitization and Ca2+-desensitization of contractile behavior
Smooth muscle MP is composed of three subunits: a 38 kDa
catalytic subunit of type 1 protein phosphatase δ isoform
(PP1cδ), and two regulatory subunits of 110 kDa (MYPT1) and
20 kDa (M20) [10,11]. MYPT1, a target subunit of MP, has
several important functions including activation and regulation
of phosphatase activity. The interaction of MYPT1 with PP1cδ
increases phosphatase activity toward MLC, and phosphoryla-
tion of MYPT1 at Thr696 by Rho-kinase inhibits MP activity.
Considerably less is known about MP in cardiac/skeletal
muscle. Previously, an isoform of MYPT, MYPT2, was cloned
and this was predominantly expressed in striated muscle and
brain . Because of similarities in structure between MYPT1
and MYPT2 also served as a target subunit in MP of striated
muscle and brain. However, the properties of MYPT2 have not
been defined. In order to facilitate an understanding of the role
of MYPT2 in cardiac muscle, the biochemical properties,
regulation, localization and function of MYPT2 were examined.
2. Materials and methods
The following chemicals were used: [γ-32P]ATP (NEN Life Science
Products Inc.), ATP (Sigma), ATPγS and GTPγS (Roche Molecular
Biochemicals), Dulbecco's modified Eagle's medium (Sigma), Medium 199
(Gibco), microcystin-LR (Wako Pure Chemicals, Osaka, Japan), A23187
(Calbiochem) and angiotensin II (Sigma), anti-FLAG beads (Sigma), anti-HA
antibody (Y-11, Santa Cruz Biotechnology, CA) and anti-FLAG antibody (A-
2.2. Construction of expression vectors
The mammalian expression vectors encoding FLAG-tagged MYPT1 and
MYPT2 (pCMV-FLAG-MYPT1 and pCMV-FLAG-MYPT2, respectively)
were prepared by the subcloning of the full length human MYPT1 and
MYPT2 cDNAs, respectively, into pCMV FLAG tagged vector (pCMV-
Tag4B™; Stratagene). To construct these plasmids, the cDNAs coding the C-
terminal MYPT1 (MYPT1876–1030) and MYPT2 (MYPT2863–984) were
amplified by PCR in order to take off the terminal codon and the insertion of
restrictive enzyme sites. The primers used were: for MYPT1876–1030, 5′-CGC
GGG ATC CAA TAA GAA AGA AAC TC-3′ and 5′-CGG AAT TCT TTG
GAA AGT TTG CTTATA ACT C-3′(foward and reverse primers, respectively;
underlined residues show the BamHI and EcoRI sites, respectively); for
MYPT2863–984, 5′-TCC CCC GGG AGG CCC GCC TAG CC-3′ and 5′-CGG
AAT TCC TTG GAG AGT TTG CTG ATG AC-3′ (foward and reverse primers,
respectively; underlined residues show the SmaI and EcoRI sites, respectively).
The conditions used for the PCR amplification were 94 °C for 15 s, 58 °C for
30 s, and 68 °C for 1 min for 30 cycles using KOD plus™ DNA polymerase
(TOYOBO). The fragments were subcloned into pCR2.1 TOPO vector,
followed by digestion with BamHI+EcoRI (for MYPT1) or EcoRI+SmaI (for
MYPT2). The 5′ cDNAs encoding MYPT11–875and MYPT21–862were
obtained from the full MYPT1 and MYPT2 cDNAs digested by EcoRI+
BamHI and EcoRI+SmaI, respectively.Thenboth5′and3′cDNAsofMYPT1
and MYPT2 were subcloned into pCMV-Tag4B vector, which had been
fragment was sequenced to confirm that the PCR-amplified cDNAwas identical
to the original sequence.
Baculovirus encoding for MYPT1 (Bac-MYPT1) and MYPT2 (Bac-
MYPT2) were generated using the Bac-to-Bac baculovirus expression system
(Invitrogen). Human MYPT2 and MYPT1 cDNAs were inserted into pFAST
BAC vector at the EcoRI site and the integration of the transgene and Bacmid
DNA was determined by PCR and restriction analysis. Generation of MYPT2
and MYPT1 cDNA baculovirus was carried out through homologous
recombination between recombinant Bacmid DNA and the baculovirus genome
according to the protocol manual.
Recombinant adenoviruses expressing human MYPT2 (Ad-MYPT2) was
prepared with ADENO-QUEST (BD Biosciences). Human MYPT2 full cDNA
was subcloned into the pQBI-AdBM5 transfer vector. Cotransfection of QBI-
293A cells with viral DNA and the transfer vector was performed by the
precipitated calcium phosphate procedure, according to the manufacturer's
protocol. Plaques were picked up and amplified in QBI-293A cells. Screening
for positive recombinants was performed by Western blot using anti-MYPT2
antibodies . Similarly, the assembly and production of recombinant
adenoviruses expressing PP1cδ (Ad-PP1cδ) was made using the adeno-X
vectors obtained from Clontech, essentially following the manufacturer's
instructions (BD Biosciences). In brief, chicken PP1cδ full cDNA (the amino
acid sequence of chicken PP1cδ is identical to that of human) was subcloned
into the pShuttle vector and transferred into the adeno-X genome. The
recombinant adenovirus was packaged in HEK293 cells and amplified to obtain
high titer stocks. Adenovirus encoding β-galactosidase (Ad-βGal) was
purchased from BD Biosciences.
The full length cDNA encoding for human HS-M21A (pCMV-HA-HS-
M21A)  was obtained by PCR amplification using human MYPT2 as
template using primer pairs 5′-GAA TTC GGA TGG ACA AAA ATG AGA
ATG-3′ and 5′-CGG AAT TCC TAC TTG GAC AGT TTG CTG-3′ (sense and
antisense primers, respectively; underlined residues show EcoRI sites). The
PCR conditions were the same as above. The cloned cDNA subcloned into
pCR2.1 TOPO vector were excised by digestion with EcoRI and the DNA
fragment was then ligated into the pCMV HA-tagged vector (Clontech). HA-
taggedwildtypeRhoA,HA-tagged dominant negativeRhoAN19andHA-tagged
constitutively active RhoAV14as described . The cDNA for myosin
regulatoryright chainof humancardiacventricle(MLC2v)was amplifiedfroma
human cardiac cDNA library (Lambda ZapII library, Stratagene) using the
primers based on the published cDNA sequence (GenBank™ accession no.
AF020768). The primer set used were: 5′-CAT GCC ATG GCA CCT AAG
AAA GCA AAG-3′ and 5′-GCG GAT CCC TAG TCC TTC TCT TCT CC-3′
(sense and antisense primers, respectively, underlined residues show the NcoI
site and the BamHI site, respectively). The conditions used for the PCR
amplification were the same as above and the cloned cDNA subcloned into
pCR2.1 TOPO vector was excised by digestion with NcoI and BamHI. The
DNA fragment was then ligated into pET3d vector (Novagen), which had been
previously digested with NcoI and BamHI and purified by agarose gel
electropheresis. The cloned cDNAwas sequenced and revealed to be identical to
the published cDNA data. Vectors for HA-tagged PP1cα, PP1cδ and PP1cγ
were kindly provided by Dr. L. Lim (Glaxo-IMCB Group, Institute of Mol. Cell
Biol., Singapore) .
2.3. Protein preparation
Baculovirus expressed recombinant MYPT1 and MYPT2 were purified as
follows. SF9 cells (BD Pharmingen) were infected with recombinant virus at a
multiplicity of infection of 10 and harvested 4 days after infection. Sf9 cells
collected from 300 ml of culture were homogenized in a buffer (60 ml) of
30 mM Tris–HCl, pH 7.5, 0.3 M NaCl, 1 mM EGTA, 5 mM EDTA, 1 mM
benzamidine, 1 μM (p-amidinophenyl)methanesulfonyl fluoride (aPMSF) and
1409 R. Okamoto et al. / Cellular Signalling 18 (2006) 1408–1416
0.1 mM diisopropyl fluorophosphate, then the homogenate was centrifuged at
20,000×g for 20 min. The supernatant was mixed with the same volume of
the buffer above without NaCl. The sample was then loaded onto a SP
Sepharose (1.0×11 cm)(Amersham Pharmacia) equilibrated with a buffer
containing 30 mM Tris–HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA and 1 mM
dithiothreitol. After washing the recombinant portein (MYPT1 or MYPT2)
was eluted with a linear gradient of 0.1 to 0.6 M NaCl. Fractions were pooled
and treated with 50 nM microcystin-LR, a specific protein phosphatase
inhibitor with irreversible effect, with mild rotation for 1 h at 4 °C to abolish
endogenous phosphatase activity completely. The sample was dialyzed against
the similar buffer containing 0.2 M NaCl and applied to Mono Q HR5/5
(Amersham Pharmacia). The column was washed and the recombinant protein
was eluted with a linear gradient of 0.2 to 0.7 M NaCl. The pooled fractions
were dialyzed against a buffer containing 30 mM Tris–HCl, pH 7.5, 0.2 M
NaCl and 1 mM dithiothreitol.
MLC2v was expressed in Escherichia coli BL21(DE3) cells. Colonies of the
cells containing the construct were used to inoculate initially a 5 ml culture
containing ampicillin and subsequently to a 4 l culture. The pellet of cells was
extracted with a buffer containing 25 mM Tris–HCl, pH 7.5, 2 M urea, 0.1 mM
aPMSF and 1 mM dithiothreitol (buffer A). The protein was purified using a Q
Sepharose FF (2.6×15 cm)(Amercham Pharmacia) followed by DEAE
Sepharose FF (1.0×10 cm)(Amersham Pharmacia). These ion-exchange
columns were equilibrated with buffer A and developed with linear gradient
of 0 to 0.5 M NaCl for a Q-Sepharose and0 to 0.3 M NaCl for DEAE FF. Finally
the elution was dialyzed against a buffer containing 25 mM Tris–HCl, pH 7.5,
30 mM NaCl.
Rho-kinase ,PP1cδ , GTPγS-GST-RhoA,smoothmuscleMLC
(sm-MLC) , smooth muscle myosin light chain kinase  and calmodulin
 were prepared as described.
2.4. Preparation of phosphoprotein
32P-labeled MLC2v was prepared with the same procedures as sm-MLC
except that phosphorylation was carried in a buffer containing30 mM Tris–HCl,
pH 7.5, 30 mM NaCl, 2 mM MgCl2, 1 mM CaCl2, 1 mM EGTA, 1 mM EDTA
and 1 mM dithiothreitol, 1 μM calmodulin and 0.45 μM rabbit skeletal
recombinant MLCK (kindly provided from Dr. J. Stull, University of Texas
Southwestern Medical Center). The levels of phosphorylation were 0.9 and
0.7 mol Pi/mol LC for sm-MLC and MLC2v, respectively.
2.5. Phosphatase assays
Assays were carried out as described previously  using32P-labeled sm-
dephosphorylation was initiated by the addition of phosphorylated MLC in a
mixture of PP1cδ and various amount of MYPT preinbubated for 4 min. To
determine the kinetic parameters, phosphatase activities were estimated with
variousamountsof32P-MLC. Forpracticalreasons,the highestconcentrationof
32P-sm-MLC and32P-MLC2v used were 10 μM and 3.3 μM, respectively.
To determine the effect of phosphorylation of MYPT2 by Rho-kinase,
thiophophorylated MYPT1 or MYPT2 was mixed and preincubated with PP1cδ
at 30 °C for 4 min to reconstitute the holoenzyme, then the phosphatase activity
was assayed using32P-sm-MLC as substrates. Thiophosphorylation of MYPT1
or MYPT2 by Rho-kinase was carried out at 30 °C for 1 h in 30 mM Tris–HCl,
pH 7.5, 200 mM NaCl, 2 mM MgCl2, 0.2 mg bovine serum albumin, 1 mM
EDTA, 6.2 nM MYPT, 2.4 nM Rho-kinase, 3.4 nM GTPγS-GST-RhoA and
100 μM ATPγS instead of ATP in a final volume of 50 μl.
To demonstrate the phosphorylation of MYPT1 and MYPT2, phosphory-
lation of MP was carried out under similar conditions in the presence of 1 μM
microcystine-LR using ATP in place of ATPγS. After incubation for 30 min, the
reaction was terminated by addition of Laemmli sample buffer and immediately
boiled for 3 min.
32P-labeled MLC2v. For the activation of PP1c with MYPT,
2.6. Cell culture and DNA transfection
COS7 cells were grown by standard procedures. Subconfluent COS7 cells
were transfected with HA- or FLAG-tagged DNA constructs (1 μg/ml) with
Lipofectamine Plus (Invitrogen). After 24 h incubation, COS-7 cells coexpres-
sing FLAG-MYPT and HA-tagged DNA constructs (HA-PP1c, HA-HS-M21A
or HA-RhoA) were lysed in a buffer containing 20 mM Tris–HCl, pH7.4,
150 mM NaCl, 10 mM Na4P2O7, 10 mM NaF, 2 mM Na3VO4, 4 mM EDTA,
10% Glycerol, 0.1% sodium deoxycholate, 1% Nonidet P-40, 1× protease
inhibitor mixture (Roche) by shearing a 27-gauge needle and subjected to a
10-min high speed spin (15,000 rpm). The supernatant was incubated with anti-
FLAG-conjugated agarose beads (Sigma) for 4 h at 4 °C. After an extensive
wash, the immunoprecipitates were resolved by SDS-polyacrylamide gel
electropheresis (PAGE) and subjected to Western blots using antibodies as
Primary cultures of cardiac myocytes were prepared from Sprague–Dawley
rat pups (Japan SLC, Hamamatsu, Japan) as described . The experimental
procedures were approved by the Animal Investigation Committee of the Mie
University Graduate School of Medicine. Following incubation for 24 h in
serum-containing medium, the cardiomyocytes were incubated for 24 h in
serum-free medium. The cells were infected with adenoviruses (Ad-MYPT2,
Ad-PP1cδ and Ad-β-gal) at a multiplicity of infection (m.o.i.) of 5. The cells
were further incubated for 48 h in serum-free medium prior to stimulation with
100 nM angiotensin II or 30 μM A23187.
2.7. Urea gel analysis
After the treatment of subconfluent myocytes with A23187 for 30 min,
reactions were terminated by addition of ice-cold trichroloacetic acid (final 10%
wt/vol) and the samples were processed as described previously . The
extracts were subjected to glycerol-urea PAGE following immunoblotting using
an antibody against cardiac MLC2 antibody (BioCytex, Marseille). Immunos-
tained proteins were visualized with the ECL method.
2.8. Western blot analysis
Protein-matched samples were separated by SDS-PAGE and transferred to
PVDF membrane (Millipore). The primary antibodies used were rabbit
polyclonal anti-MYPT2 and anti-leucine zipper (LZ) antibodies . Immuno-
detection was accomplished using appropriate horseradish peroxidase-linked
secondary antibodies (Amersham) (1/2000). The blots were exposed to films
(Fuji RX) for various times (from 30 to 180 s) to obtain a linear response with
the ECL method and band density was estimated by densitometry (Atto).
Immunoprecipitation of myosin phosphatase from rat heart was as follows:
The hearts were homogenized with lysis buffer (50 mM Tris–HCl, pH 7.4, 1%
NP40, 150 mM NaCl, 0.5% sodium deoxycholate, 1 mM diisofluorophosphate
and 1× protease inhibitor mixture). The supernatant after centrifugation at
15,000 rpm for 10 min was collected and incubated with anti-PP1cδ antibody
 for 3 h, followed by the additional incubation with protein A-Sepharose
(Amersham Pharmacia) for 30 min at 4 °C. The immunoprecipitates were
washed three times with buffer (50 mM Tris–HCl, pH 7.4, 500 mM NaCl, 0.1%
NP40 and 0.05% sodium deoxycholate) and followed by additional wash (three
times) with the same buffer without NaCl. The immunoprecipitated proteins
were eluted with Laemmli SDS sample buffer and immediately boiled for 3 min.
2.10. Immunofluorescence and immunoelectron microscopy
Myocytes on collagen type I-coated coverslips were fixed in 4%
paraformaldehyde in phosphate-buffered saline, permabilized for 5 min in
0.1% Triton X-100 and stained with rhodamine-conjugated phalloidin.
Immunoelectron microscopy, using the silver-enhanced immunogold
method, was performed as previously described . The 10-μm-thick sections
from adult mice (C57BL6/J) (Japan SLC, Hamamatsu, Japan) hearts were
incubated with the primary antibody rabbit anti-LZ antibody (Fujioka et al.
), followed by incubation with a secondary antibody coupled with 1.4-nm
gold particles (Nanoprobes Inc.). The sample-bound gold particles were silver-
enhanced by the HQ-silver kit (Nanoprobes Inc.) at 18 °C for 12 min. The
samples were again washed and postfixed with 0.5% osmium oxide in a buffer
1410 R. Okamoto et al. / Cellular Signalling 18 (2006) 1408–1416
containing100mM cacodylate buffer,pH 7.3.Theyweredehydratedbypassage
through a graded series of ethanol (50%, 70%, 90%, and 100%) and propylene
oxide, and embedded in epoxy resin. From this sample, ultrathin sections were
cut, stained with uranyl acetate and lead citrate, and then observed with an
electron microscope (JEM-1010EX; JEOL).
2.11. Other procedures
Protein concentrations were determined by the BioRad or the BCA
procedure (Pierce) using bovine serum albumin as a standard. All values are
expressed as mean±standard errors of the mean (S.E.M.). Data were analyzed
by Student's t-test. A level of Pb0.05 was considered statistically significant.
3.1. Interaction of MYPT2 with PP1c and HS-M21A
To examine the interaction of MYPT2 with the catalytic
subunit of type 1 phosphatase (PP1c), the FLAG-tagged MYPT
vectors were co-transfected with each type of HA-tagged PP1c
vector (i.e. PP1cα, -δ or -γ) into COS cells, followed by
immunoprecipitation with the anti-FLAG antibody. As shown
in Fig. 1A, MYPT1 interacted only with PP1cδ, which is the
subunit identified in the MP holoenzyme. Using this assay
MYPT2 also was found to form a specific complex with PP1cδ.
In addition, a weak interaction was occasionally observed
between PP1cα and MYPT2 (data not shown). Conversely,
MYPT2 was immunoprecipitated from mouse heart homoge-
nate with anti-PP1cδ antibody (Fig. 1B), indicating that
MYPT2 could form complex with PP1cδ in heart.
M20 is the other regulatory subunit of smooth muscle MP.
As the existence of a heart-specific isoform of M20, termed HS-
M21, had been reported , we investigated whether MYPT2
can associate with HS-M21A, one isoform of HS-M21, using co-
transfection of HA-tagged HS-M21A and FLAG-tagged MYPT.
As shown in Fig. 1C, HS-M21A was immunoprecipitated with
MYPT2, indicating an interaction of HS-M21A with MYPT2.
Of interest was the finding that HS-M21 and MYPT1 also
interacted (Fig. 1C), although this was slightly weaker than with
3.2. Interaction of MYPT2 with RhoA
As MYPT1 is one of the target molecules for activated
RhoA, we investigated whether MYPT2 also could interact with
RhoA. HA-tagged RhoA and FLAG-tagged MYPT2 plasmids
were co-transfected into COS cells and the MYPTs were
(+) (-)(-) (+)(+)
Fig. 1. Interaction of MYPT2 with PP1c, HS-M21A and RhoA. (A) Interaction of MYPT2 with three different types of PP1c. FLAG-tagged MYPT1 or-MYPT2 were
co-expressed without or with HA-tagged PP1cα, δ or γ in COS-7 cells. Protein immunoprecipitated (IP) were analyzed by Western blot (WB) with anti-LZ antibody
(upper panel) and anti-HA antibody (middle panel) for associated PP1c isoform. Overexpressed PP1c present in total cell lysate were also shown (lower panel). (B)
Interaction of MYPT2 with PP1c in rat heart tissue. Protein was immunoprecipitated from homogenized rat heart using beads only (–), beads plus normal rabbit IgG
and beads plus anti-PP1c antibody respectively. Crude protein was shown as positive control of WB. (C) Interaction of MYPT2 with HS-M21A. Experimental
conditions were almost same as those in Panel A except that HA-tagged HS-M21A instead of PP1c isoforms was co-expressed with each MYPT. (D) Interaction of
MYPT2 with RhoA. FLAG-tagged MYPTs were co-expressed without or with RhoA-WT (wild type), RhoA-Val14 (active form), or RhoA-Asn19 (dominant negative
form) in COS-7 cells. Protein immunoprecipitated (IP) with mouseanti-FLAG beads wereanalyzed by anti-LZ antibody (upper panel) andby anti-HA antibody(lower
panel) for the association with RhoA.
1411R. Okamoto et al. / Cellular Signalling 18 (2006) 1408–1416
immunoprecipitated by anti-FLAG antibody beads. As shown
in Fig. 1D, only the active form of RhoA (RhoA-Val14) was
immunoprecipitated with MYPT2, indicating a specific inter-
action between MYPT2 and activated RhoA, i.e. GTP-RhoA. A
control study showing the interaction of MYPT1 and RhoA-
Val14 was consistent with previous results . Using the yeast
two-hybrid system, it was determined that residues 813–982 of
MYPT2 were involved in the RhoA binding site (data not
3.3. Effects of MYPT2 on phosphatase activity
The effects of baculovirus-expressed MYPT2 on phospha-
tase activity were investigated and compared to MYPT1. As
shown in Fig. 2, MYPT2 increased the activity of PP1cδ to a
maximum of about 35-fold with sm-MLC as substrate. In
contrast, MYPT1 increased the activity only about 15-fold,
indicating that MYPT2 was a more efficient activator than
MYPT1. Maximum activation occurred at about a 3–4 fold
molar excess (MYPTs to PP1cδ). At a molar stoichiometry the
activation levels were approximately 21- and 7.5-fold, for
MYPT2 and MYPT1, respectively.
Kinetic parameters were determined for PP1cδ and each
MYPT. A 4-fold molar excess with respect to PP1cδ was used
for both MYPT1 and MYPT2. The results are shown in Table 1.
MYPT2 induced a decrease in Kmto about one-half and an
increase in kcatof about 11-fold with cardiac MLC as substrate.
Similar kinetic properties for MYPT2 also were obtained with
sm-MLC as substrate. For MYPT1 a similar reduction in Km
was observed while the increase in kcat was less (for both
substrates: see Table 1). Thus, the mechanism for activation of
PP1cδ by MYPT is due to both a reduction in Kmand an
increase in kcat. It is interesting that with MYPT2 the increase in
kcat, particularly with MLC2v as substrate, is greater than that
obtained with MYPT1.
3.4. Phosphorylation of MYPT2 by Rho-kinase and its effect on
As the region around the inhibitory phosphorylation site
(Thr696 in human MYPT1)  is highly conserved in
MYPT2, phosphorylation of MTPT2 at Thr646 (the equivalent
site to Thr696 in MYPT1) by Rho-kinase was investigated. The
site-specific and phosphorylation-dependent antibody for
Thr696 in MYPT1 (Anti-pMYPT1Thr696)  was used. As
shown in Fig. 3A, the positive reaction with Anti-
pMYPT1Thr696for MYPT2 phosphorylated by Rho-kinase
indicated that Thr646 was phosphorylated. Phosphorylation of
MYPT2 at Thr646 by Rho-kinase also was confirmed in COS7
cells co-transfected with plasmids encoding MYPT2 and a
constitutively active fragment of Rho-kinase (data not shown).
Fig. 2. Activation of MP activity by MYPT2. Each sample at the indicated molar
ratio of MYPT2 (○) or MYPT1 (●) with respect to PP1cδ was incubated at
30 °C for 4 min, and the phosphatase activity of the mixture was assayed
using 5 μM32P-sm-MLC as substrate. The results are presented as the relative
activity with respect to PP1cδ alone which was expressed as 1. n=3 or 4.
The effects of MYPT2 and MYPT1 on the kinetics parameters
MLC2v ;(cardiac MLC) Smooth MLC
A molar ration of 4:1 (recombinant MYPTs/PP1cδ) was used. The values are
mean±S.E. (nN3). kcatwas calculated using a molecular mass of 38 kDa for
WB: Anti-LZ (MYPT)
Phosphatase activity (%)
(-) (+)(-) (+)
Fig. 3. Phosphorylation of MYPT2 by Rho-kinase and its effect on MP activity.
(A) MYPT2 was incubated with or without Rho-kinase as described under
Materials and methods. The samples were analyzed by Western blot (WB) using
anti-pMYPT1Thr696and anti-LZ antibodies. (B) Effects of MYPT thiopho-
sphorylation by Rho-kinase on MP activity. MYPT thiophosphorylated or
nonphosphorylated (preincubation without Rho-kinase) was mixed with PP1cδ,
then their phosphatase activity were determined as described under Materials
andmethods. MPactivity, priorto thiophosphorylation of MYPT,was expressed
as 100%. n=4.
1412 R. Okamoto et al. / Cellular Signalling 18 (2006) 1408–1416
The effect of phosphorylation of MYPT2 by Rho-kinase on
phosphatase activity was investigated. As shown in Fig. 3B,
thiophosphorylation of MYPT2 by Rho-kinase inhibited
phosphatase activity of MYPT2–PP1cδ, an effect similar to
that obtained with MYPT1–PP1cδ. The activity of thiopho-
sphorylated MYPT2 plus PP1cδ was 9.4±0.3% of the control
(non-thiophosphorylated MYPT2) level. Inhibition with
MYPT1 thiophosphorylated by Rho-kinase is consistent with
previous results [25,26].
3.5. Effect of MYPT2 and PP1cδ on the phosphorylation level
of cardiac MLC
To investigate the effect of MYPT2 and/or PP1cδ on the
level of cardiac MLC phosphorylation cultured rat neonatal
cardiomyocytes were transfected with adenoviruses encod-
ing human MYPT2 (Ad-MYPT2) and/or PP1cδ (Ad-PP1cδ)
48 h prior to stimulation with A23187 (3×10−5M). Stimu-
lation with A23187 significantly increased the phosphoryla-
tion level of MLC in control cardiomyocytes (Fig. 4)
transfected with adenovirus encoding β-galactosidase (Ad-
βGal) (33.6±3.8% and 49.9±4.8% without and with A23187
stimulation, respectively). The phosphorylation level of
cardiac MLC stimulated by A23187 in the cardiomyocytes
in which both Ad-MYPT2 and Ad-PP1cδ were overexpressed
was significantly lower (19.5±6.5%) than in control Ad-βGal
transfected cells. Overexpression of MYPT2 or PP1cδ had no
significant effect on the level of MLC phosphorylation
(compared to control Ad-βGal transfection plus A23187).
3.6. Effect of MYPT2 and PP1cδ on angiotensin II-induced
To investigate the influence of overexpression of MYPT2
and PP1cδ the adenoviral vectors were applied to cultured
cardiomyocytes. More than 95% of cardiomyocytes were
positive for MYPT2 or PP1cδ expression after incubation
under the experimental condition (data not shown). Before the
addition of angiotensin II (Ang II) a defined sarcomere pattern
was not observed, as indicated by punctuate staining with
rhodamine-phalloidin (Fig. 5, panel a). Thirty minutes after the
addition of Ang II a distinct sarcomere organization was
observed with parallel-aligned myofibrils (Fig. 5, panel b).
Pretreatment of the cardiomyocyte with the adenoviral vectors
(Ad-MYPT2 and Ad-PP1cδ) almost completely eliminated the
Ang II-induced sarcomere organization. Under these conditions
the actin filaments showed an irregular organization (Fig. 5,
3.7. Subcellular localization of MYPTs
We investigated the distribution of the MYPTs in mouse
heart. Initially a Western blot, using the anti-LZ antibody, was
carried out on cardiac muscle to identify the isoforms present.
The major isoform was MYPT2 (Fig. 6A) and a doublet of
MYPT1 also was observed. A faint band at approximately
85 kDa (indicated by asterisk) may correspond to a related
member of the MYPT family, i.e. MBS85 . The ratios of
MYPT2/MYPT1/MBS85 were 10:2.7:1.0, respectively.
* * *
Phosphorylated MLC/ total MLC
Ad-βgalAd-βgal Ad-PP1cδ Ad-MYPT2 Ad-PP1cδ
Fig. 4. Effects of MYPT2 and/or PP1cδ on the phosphorylation level of cultured
cardiomyocytes. Upper panel, representative Western blots for nonphosphory-
lated MLC2v (nonP-MLC) and phosphorylated MLC2v (P-MLC). Lower panel,
densitometrical data showing the relative amounts of phosphorylated MLV2v to
total MLC2v are summarized. Cultured neonatal rat ventricular myocytes were
stimulated with 30 μM A23187 for 30 min after 48-h incubation with Ad-βgal,
or Ad-MYPT2 and/or Ad-PP1cδ. Phosphorylated MLC2v were separated by
urea PAGE and detected by immunobloting. n=4. *, **Pb0.05.
Fig. 5. Effect of Ad-MYPT2 and Ad-PP1cδ on Ang II-induced sarcomeric organization. Cardiac myocytes on collagen-coated glass coverslips were incubated with
Ad-βgal (panels a and b) or both Ad-MYPT2 and Ad-PP1cδ (panel c) for 48 h, followed by treatment with Ang II (100 nmol/L) for 30 min (panel b and c). Cells were
stained with rhodamine-phalloidin. Scale bar=20 μm for all panels.
1413R. Okamoto et al. / Cellular Signalling 18 (2006) 1408–1416
Immunoelectron microscopy studies using the anti-LZ
antibody and colloidal gold-labeled secondary antibodies
revealed two major locations for MYPTs. These were at, or
close to, the Z-line (41% of the signal) and the A band (33% of
the signal). Localization with mitochondria (24% of the signal)
also was observed. The distribution for the A-band and
mitochondria was irregular and particularly with respect to the
A band did not show a preference for localization on the thick
filaments. Tubules, plasma membrane, nuclei and intercalated
disc contained a negligible number of gold particles.
MYPT2 was originally cloned from a human brain cDNA
library screened with a cDNA fragment of rat MYPT1 and
shown to be an isoform of MYPT1 encoded by a different gene
. Overall identity between MYPT1 and MYPT2 is 61%, but
there are three regions more highly conserved, namely the N-
terminal ankyrin repeats, a central sequence spanning the
inhibitory phosphorylation site and the C-terminal leucine
zipper motif [10–12]. MYPT2 is expressed preferentially in
heart, skeletal muscle and brain, and thus was considered to be
the target subunit of MP in striated muscle . Isolation and
characterization of intact MP from heart or skeletal muscle
proved difficult because of lability to proteolysis of MYPT2 and
its low abundance. Thus MYPT2 was characterized using
recombinant proteins and cDNA transfection.
In this study the following properties of MYPT2 were shown
to be similar to MYPT1: 1) MYPT2 interacts specifically with
PP1cδ and HS-M21; 2) interaction of MYPT2 with PP1cδ
enhances its phosphatase activity toward MLC2v and sm-MLC;
3) MYPT2 binds to the active form of RhoA; 4) MYPT2 is
phosphorylated by Rho-kinase, notably at the putative inhibi-
tory phosphorylation site, with associated inhibition of the
A critical region of MYPT1 (and other PP1c-target subunits)
involved in the PP1cδ interaction is the PP1c-binding motif,
RVxF . This lies at the N-terminal edge of the ankyrin
repeats in both MYPT1 (residues 35–38) and MYPT2 (residues
53–56 ). This motif acts as the primary interaction site but
flanking structures also are involved in binding to PP1cδ. As
shown by several studies on MYPT2 (for reviews [10,11]) the
N-terminal sequence is implicated in activation of PP1cδ (kcat)
and the ankyrin repeats also contribute to binding with PP1cδ.
From the recently solved crystal structure of PP1cδ and an N-
terminal fragment of MYPT1 , it was shown that the N-
terminal sequence of MYPT1 wraps around PP1cδ to reach the
hydrophobic groove of the catalytic cleft. The C-terminal
sequence incorporates the two groups of ankyrin repeats and
repeats 1, 5, 6 and 7 were involved in interaction with PP1cδ.
Also the C-terminal region of PP1cδ is important in interaction
with the ankyrin repeats and may determine specificityof PP1cδ
for MYPT1. Comparison of the sequence of MYPT1 and
MYPT2 shows that the N-terminal sequences (1–35 and 1–52,
Fig. 6. Immunoelectron microscopy of MYPTs. (A) Western blot analysis of mouse heart by anti-LZ antibody. Asterisk indicates the band cross-reacted with the
molecular mass of 85 kDa. (B) Immunoelectron microscopic analysis of MYPTs in mouse heart. Mouse heart tissue was stained with an anti-LZ antibody (MYPT2+
MYPT1) followed by immunogold-conjugated second antibody as described under Materials and methods. Panels indicated anti-LZ antibody has decorated both the
sides of the Z bands (arrow heads). Magnification of the boxed area in panel b is shown in panel α. Bars, 1 μm (panel a) and 500 nm (panel b).
1414 R. Okamoto et al. / Cellular Signalling 18 (2006) 1408–1416
respectively) are more unlike (36% identity) that other regions
of the two molecules, but the ankyrin repeats are conserved
(75% identity). Thus, the difference in effects on kcatcould
reflect the two distinct N-terminal regions and the similarity in
the shifts in Kmin the conserved ankyrin repeats. The ankyrin
repeats are also thought to bind substrate [11,12]. It is
interesting that the overall capacity for dephosphorylation of
MLC is higher for MYPT2–PP1cδ, compared to MYPT1–
PP1cδ. Previously it was shown that MYPT1–PP1cδ did not
effectively dephosphorylate skeletal muscle MLC . An
earlier result from our laboratory  suggested that MYPT2–
PP1cδ was less effective with sm-MLC than MYPT1–PP1cδ.
This discrepancy probably was due to the use of GST-fusion
truncation mutants for both MYPT1 and MYPT2, rather than
the non-GST baculovirus full-length molecules used above.
Also it was shown that a GST-fusion mutant had reduced
relaxing ability, compared to the non-GST mutant, in
permeabilized smooth muscle, probably due to steric hindrance
by the GST moiety .
In smooth muscle MP, a smaller regulatory subunit, M20,
interacts with the C-terminal region of MYPT1 [10,11]. Similar
to smooth muscle MP (MYPT1-type MP), this study revealed
the possible interaction of MYPT2 with HS-M21A, at least
equal to or stronger than MYPT1. These results are different
from the previous result using Far Western analysis indicated
that HS-M21preferentially interacted with MYPT1 and bound
to the C-terminal one-third of MYPT2 to a lesser extent .
The reasons for this discrepancy are not clear though the
different assay systems might be one of the reasons. In MYPT1-
type MP the binding of M20 to MYPT1 does not affect
phosphatase activity [10,11]. Although it had suggested that
isolated HS-M21recombinant increased sensitivity to Ca2+in
permeabilized renal artery and cardiac myocytes , the
functions of HS-M21 and its interaction with MYPT2 still
remain to be investigated.
The active GTP-bound form of RhoA was shown to bind to
the C-terminal region of MYPT1 (residues 930–1030) . In
this study MYPT2 also interacts with activated RhoA and the
binding region in MYPT2 was located in the C-terminal region
(residues 813–982, as identified by the yeast two hybrid
system). The functional significance of MYPT2 binding to
activated RhoA remains to be determined, but targeting of Rho-
kinase to its substrate, MYPT1/2, should be considered.
MYPT1 is phosphorylated by Rho-kinase and this event
leads to the inhibition of MP activity [25,26]. The region
(residues 663–708) around the inhibitory phosphorylation site
(Thr696) in MYPT1 is highly conserved in MYPT2 (91%).
Thus, it was not unexpected that the specific antibody for
phosphorylated Thr696 in MYPT1 also detected phosphoryla-
tion by Rho-kinase of the equivalent site in MYPT2 (Thr646).
As a consequence of phosphorylation at this site the activity of
MYPT2–PP1cδ was inhibited and this has not been previously
shown. MBS85, a third form of MYPT family, contains similar
structures, including ankyrin repeats, a phosphorylation-depen-
dent inhibitory region and a leucine zipper motif, also was
inhibited by phosphorylation at the conserved inhibitory site
. Thus, inhibition via phosphorylation at a conserved site
appears to be a mechanism common to several members of the
Several lines of evidence indicated that agonist stimulation
induces Ca2+sensitization of vascular contraction though both
MYPT1 phosphorylation by Rho-kinase and CPI-17 phosphor-
ylation by Rho-kinase and/or PKC [10,11]. CPI-17 is a
phosphorylation-dependent inhibitory protein for the MP
holoenzyme . Phosphorylation of CPI-17 at Thr38 by
several kinases including PKC and Rho-kinase enhances its
inhibitory potency [10,11]. CPI-17 phosphorylated by Rho-
kinase showed a dose-dependent inhibition of phosphatase
activity of MYPT2–PP1cδ with a slightly higher IC50value that
observed with MYPT1–PP1cδ (data not shown). These in vitro
data indicate the similar molecular properties between MYPT2
and MYPT1, though the difference in the sensitivity of CPI-17
toward MYPT2- and MYPT1-associated PP1cδ could reflect
some distinct conformational changes in PP1cδ induced by
either MYPT1 or MYPT2. As CPI-17 expressed mainly in
smooth muscle and brain and its message is scarcely found in
heart , the inhibition of cardiac MP by CPI-17 is unlikely.
Therefore, RhoA/Rho-kinase-mediated inhibition of MP is
thought to be primarily mediated by the phosphorylation of
MYPT2 by Rho-kinase.
Many reports have documented connections between RhoA
signaling and cardiac MLC phosphorylation. Recently α-
adrenergic receptor stimulation was reported to result in
phosphorylation of MYPT and MLC2v and an increase in
myocyte Ca2+sensitivity of tension that all depend on Rho-
kinase activation . In addition, the pattern of MYPT
phosphorylation in unstimulated heart was inversely related to
the spatial gradient of MLC2v phosphorylation level . In
failing heart, an increased phosphorylation level of cardiac
MLC was observed with upregulation of RhoA/Rho-kinase
signaling and administration of a Rho-kinase inhibitor sup-
pressed MLC phosphorylation . Activation of RhoA/Rho-
kinase signaling also regulated myofibril formation and
organization in neonatal rat ventricular myocytes and MLC
phosphorylation was critical to this process . Since Rho-
kinase cannot directly phosphorylate MLC2v (Okamoto and
Ito, unpublished data), RhoA/Rho-kinase-mediated increase in
cardiac MLC phosphorylation could reflect inhibition of MP.
The biochemical studies reported here suggest that MYPT2
acts as a target subunit for cardiac muscle MP. However, it was
important to obtain supporting data in a more physiological
setting and for this the adenoviral transfections of cardiomyo-
cytes were used. Stimulation of cardiomyocytes by A23187
increased MLC phosphorylation and this was markedly reduced
by overexpression of MYPT2 and PP1cδ, suggesting that
cardiac MLC in vivo are dephosphorylated by the MYPT2–
PP1cδ complex. Since the phosphorylation level of MLC has
been implicated in Ca2+sensitization of contraction , cardiac
torsion  and sarcomere organization during hypertrophy ,
it follows that the MYPT2-associated MP also is critical in these
processes. Our data support the hypothesis that the level of
myosin phosphorylation is critical to sarcomere organization.
Notably that increase in MP activity following overexpression
of MYPT2 and PP1cδ, blocked the Ang II-induced sarcomere
1415 R. Okamoto et al. / Cellular Signalling 18 (2006) 1408–1416
organization in cultured cardiomyocytes. The phenotype of
cardiac-specific MYPT2 transgenic mice currently is being
investigated to elucidate in-vivo function of MYPT2.
The immunolocalization studies suggested that MYPT was
localized at or close to the Z-line, A band and mitochondria. The
major isoform in heart is MYPT2 and although MYPT1 and
MBS85 also are present the dominant signals in localization
would be due to MYPT2. The localization of MYPTs at the A
band is reasonable since phosphorylated myosin is an accepted
substrate for myosin phosphatase. Any functional significance
associated with localization of MYPTs at mitochondria is not
known. However, the Z-line localization of MYPTs in cardiac
muscle was not expected. An interesting and emerging idea is
that the Z-line does not merely serve as a static structural unit
but in addition acts as a center for several signaling molecules
and mechanisms that could involve cardiac hypertrophy,
myopathies and heart failure . For example, both PKC
and PKA are anchored at the Z-line  and calcineurin is
bound via calsarcin-1 . To the number of Z-line components
should now be added MYPT2. One potential binding site for
MYPT2 is non-muscle myosin II, which also is located in
striated muscle Z-lines . In addition, MYPT2 itself could act
as an interactive platform in that several proteins bind to
MYPT2, including: RhoA ; cGMP-dependent protein
kinase Iα; myosin phosphatase–RhoA interacting protein
; and interleukin-16 precursor proteins . Thus, path-
ways or mechanisms that involve MYPT2 could form part of
the dynamic role of the Z-lines in intracellular signaling and
This work was supported in part by grants-in-aid for
Scientific Research from the Ministry of Education, Science,
Technology, Sports and Culture, Japan (to M.I.), and by Grant
HL23615 (D.J.H.) and P01 AR41637 (T.T.) from the National
Institute of Health. We also thank Dr. Hiroki Aoki and Dr.
Nobuyuki Moriki for helpful comments.
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