Am J Physiol Heart Circ Physiol 289: H285–H294, 2005.
First published March 18, 2005; doi:10.1152/ajpheart.01291.2004.
A B56 regulatory subunit of protein phosphatase 2A localizes to nuclear
speckles in cardiomyocytes
Marisa S. Gigena,1 Akihiko Ito,2 Hiroshi Nojima,3 and Terry B. Rogers1
1
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine,
Baltimore, Maryland; and 2Department of Pathology, Medical School, and 3Department for Molecular
Genetics, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan
Submitted 22 December 2004; accepted in final form 7 March 2005
(PP2A) is widely distributed in many
cell types and accounts for a large portion of the serine/
threonine phosphatase activity in many tissues. It is now
appreciated that PP2A is not merely a housekeeping enzyme.
Rather, it is actively modulated and contributes to the control
and balance of a wide range of signaling pathways in a
cell-specific manner (24, 30, 33, 38). In cardiomyocytes, the
importance of PP2A as a regulator of Ca2⫹ signaling has been
documented in previous studies that included intracellular
application of the purified enzyme and the use of the inhibitor
calyculin A (4, 5). Furthermore, PP2A activation is seen after
stimulation of myocardial adenosine receptors, and this phosphatase is a modulator of p38-ERK cross talk in ventricular
cardiomyocytes (19, 20). One of the current challenges is to
understand how this abundant family of phosphatases mediates
such temporal and spatial signaling specificity in myocardial
tissues.
Molecular studies provide essential clues to how signaling
specificity of PP2A within cells is achieved. This phosphatase
exists as a heterotrimeric complex composed of a core enzyme,
containing a conserved catalytic (PP2Ac) and a structural/
scaffolding A subunit (PP2A/A), that is bound to a variety of
exchangeable regulatory or B subunits. There are at least 21
known PP2A B subunits that are grouped into three unrelated
gene families termed B (or PR55), B⬘ (or B56), and B⬙ (or
PR72) (33, 34, 38). Importantly, the B subunits can alter
substrate specificity of the core catalytic complex and govern
subcellular targeting as well (14, 22, 31). This molecular
scheme not only generates a family of diverse PP2A species
but also reveals that the properties of B subunits are crucial to
understand the physiological role of PP2A within cells, including cardiomyocytes. Although the genetic information of these
subunits is emerging (12, 21), there is little information on their
function within the cellular context.
Accordingly, the present study focused on the roles of B56
subunits highly expressed in cardiac cells. The main findings
are that small domains within the B56 proteins are responsible
for marked alterations in subcellular targeting in heart myocytes. B56␥1 is not only a nuclear protein, but, unexpectedly,
it targets to subnuclear organelles called nuclear speckles.
Finally, overexpression studies indicate that nuclear B56␥1 is
not associated with global changes in gene expression in the
heart, but, rather, it may regulate the dynamic assembly/
disassembly process of these macromolecular complexes.
PROTEIN PHOSPHATASE 2A
Address for reprint requests and other correspondence: T. B. Rogers, Dept.
of Biochemistry and Molecular Biology, Univ. of Maryland School of Medicine, 108 N. Greene St., Baltimore, MD 21201 (E-mail: trogers@som.
umaryland.edu).
http://www.ajpheart.org
METHODS
Antibodies. Two different anti-hemagglutin (HA) antibodies were
used, a mouse monoclonal from BAbCO (Richmond, CA) and a rabbit
polyclonal from Clontech (Palo Alto, CA). The polyclonal anti-HA
gave higher background staining in confocal images compared with
the monoclonal preparation but was used in studies when doubleimmunolabeling approaches were required. Antibodies against
PP2Ac, B56␣, and transcription enhancer factor (TEF) were purchased from Transduction Laboratories (Lexington, KY), and antibody against PP2A/A was purchased from Oxford Biomedical Research (Oxford, MI). The anti-SC35 antibody was a generous gift
from Dr. Joseph Gall (Department of Embryology, Carnegie Institution, Baltimore, MD). The production and purification of polyclonal
anti-B56␥ was previously described (12). Secondary peroxidaseconjugated mouse and rabbit antibodies were obtained from Jackson
ImmunoResearch, and Alexa Fluor 568 goat anti-rabbit IgG and
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.
0363-6135/05 $8.00 Copyright © 2005 the American Physiological Society
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Gigena, Marisa S., Akihiko Ito, Hiroshi Nojima, and Terry B.
Rogers. A B56 regulatory subunit of protein phosphatase 2A localizes
to nuclear speckles in cardiomyocytes. Am J Physiol Heart Circ
Physiol 289: H285–H294, 2005. First published March 18, 2005;
doi:10.1152/ajpheart.01291.2004.—Protein phosphatase 2A (PP2A)
is widely distributed in heart tissues, yet its precise cellular functions
are poorly understood. This study is based on the notion that PP2A
action is governed by interactions of the core enzyme with B targeting/regulatory subunits. The subcellular localizations of two B subunits, B56␣ and B56␥1, were assessed using adenovirus-driven expression of epitope-tagged (hemagglutinin, HA) in cultured neonatal
and adult rat ventricular myocytes. Confocal imaging revealed that
HA-B56␣ was excluded from the nucleus and decorated striated
structures, whereas HA-B56␥1 was principally found in the nucleus.
Precise immunolabeling studies showed that B56␥1 was concentrated
in intranuclear structures known as nuclear speckles, macromolecular
structures that accumulate transcription and splicing factors. Western
blot analyses revealed that overexpression of either B subunit had no
effect on the levels of other PP2A subunits in cultured neonatal
cardiac cells. However, overexpression of only B56␥1 increased
whole cell PP2A activity by 40% when measured in cell extracts.
Finally, B56␥1 did not alter global gene expression or expression of
hypertrophic gene markers such as ␣-skeletal actin. However, morphometric analyses of confocal images revealed that B56␥1 alters the
dynamic assembly/disassembly process of nuclear speckles in heart
cells. These studies provide new insight into mechanisms of PP2A
targeting in the subnuclear architecture in cardiomyocytes and into the
role of this phosphatase in nuclear signaling.
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between cotransfected pJM17 plasmid and the shuttle plasmids, pAdv/
4-HA-B56␣ or pAdv/4-HA-B56␥1, in HEK-293 cells by using methods previously described (39). The recombinant adenoviruses were
amplified, purified, and titered as described previously (16).
Immunocytochemistry. Neonatal and adult rat cardiomyocytes were
grown on glass coverslips and transfected with HA-B56␣ or HAB56␥1 adenovirus as described. After 48 h of transfection, the cells
were fixed in 100% cold methanol for 15 min at ⫺20°C and rehydrated with PBS. Nonspecific sites were blocked with 5% normal goat
serum-3% BSA in PBS. Primary antibodies, as indicated, were incubated with the fixed cells overnight in PBS with 1% BSA at 4°C. After
three washes, the cells were incubated with appropriate fluorescently
labeled secondary antibodies for 2 h. Slides were prepared according
to the manufacturer’s instructions using the Anti-Fade kit (Molecular
Probes). Cells were visualized using a confocal laser microscope
(model 510; Carl Zeiss).
Immunoprecipitation. Cellular extracts from control and HA-B56transfected cells were incubated with protein G-Sepharose beads
(Sigma) for 2 h at 4°C. These precleared extracts were incubated with
anti-HA affinity matrix (BAbCO) at 4°C overnight. The matrix was
centrifuged and the supernatant was retained. The matrix was washed
three times in washing buffer [50 mM Tris 䡠 HCl (pH 7.4), 1 mM
EGTA, 1 mM EDTA, 0.1% ␤-mercaptoethanol, 150 mM NaCl, and
protease inhibitor cocktail (Sigma P8340; 1:400)]. Immunoprecipitated proteins were extracted from the pellets with SDS loading
buffer, resolved by SDS-PAGE, and analyzed using Western blotting
methods.
Western blotting. Proteins in cell extracts and immunoprecipitates
were resolved by SDS-PAGE and then transferred to polyvinylidene
difluoride membranes (Immobilon-P; Millipore). Membranes were
blocked with 5% dry milk-PBS and incubated with the indicated
antibodies overnight at 4°C. Blots were then incubated with antimouse or anti-rabbit IgG peroxidase-conjugated secondary antibodies
(Molecular Probes), and the proteins were detected using a chemiluminescent detection system (Pierce).
Nuclear Extract Analyses. Control and transfected neonatal rat
cardiomyocytes were washed with ice-cold PBS and incubated with
10 mM HEPES (pH 7.6), 20 mM KCl, 0.1 mM EDTA, 0.1 mM
EGTA, and protease inhibitor cocktail (Sigma; 1:400) on ice for 20
min. The cells were harvested and then centrifuged at 750 g at 4°C for
10 min. Nuclear extracts were obtained from the pellet with the use of
a NE-PER extraction kit (Pierce).
PP2A activity. PP2A activity assay was performed as previously
described (4). Briefly, cell extracts were incubated with 20,000
counts/min of [32P]RRATpVA in reaction buffer containing 20 mM
HEPES-NaOH (pH 7.5), 100 mM NaCl, and 0.02% ␤-mercaptoethanol in the presence or absence of 10 nM okadaic acid. After 15 min
at 30°C, the reaction was terminated by adding 500 ␮l of 100 mM
K2PO4 in 5% TCA. The 32P-labeled peptide and [32P]Pi released were
separated by applying the total reaction volume (550 ␮l) onto an ion
exchange column (1 ml Dowex 50WX8, 200 – 400 mesh, H form).
The [32P]Pi was eluted from the columns in 500 ␮l of H2O and
quantified using liquid scintillation counting. PP2A activity was
defined as the component of total phosphatase activity that was
inhibited by 10 nM okadaic acid.
Determination of size and number of speckles. A computer program
was written using IDL language version 5.5 (Research Systems) to
analyze confocal images of nuclear speckles. Individual “dots” within
immunofluorescent images were identified using an algorithm that
first found the location of the pixel of maximal intensity, f(x0, y0), and
then extracted line segments of 50 pixels in length starting at (x0, y0)
and extending in one of eight directions within the plane of the image.
A half-Gaussian function was mathematically fitted to each line
segment, and the point at which this fit fell to 10% of the peak was
identified as the extent of the dot in that direction. This fitting
procedure was carried out for each of the eight line segments,
resulting in identification of eight points representing the spatial
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Alexa Fluor 488 goat anti-mouse IgG were obtained from Molecular
Probes (Eugene, OR).
Cultured neonatal and adult rat ventricular cardiomyocytes. For
the culturing of neonatal rat ventricular cardiomyocytes, hearts were
removed from 1- to 2-day-old Sprague-Dawley rats. The ventricles
were separated from the atria and then digested in a digestion medium
(116 mM NaCl, 5.4 mM KCl, 20 mM HEPES, 5.5 mM D-glucose, 1.0
mM NaH2PO4, 0.8 mM MgSO4, and 15 ␮M phenol red, pH 7.35)
containing collagenase type II (75 U/ml; Worthington Biochemical)
and pancreatin (0.6 mg/ml; Sigma) for 30 min at 37°C. The supernatant was aspirated and discarded. The pellet was resuspended in
digestion medium for an additional 15 min. The supernatant was
removed and placed in a tube containing 1 ml of heat-inactivated
horse serum. These steps were repeated six to eight times until the
hearts were completely digested. All of the fractions were combined
and filtered through a sterile two-ply gauze that was prewetted with
plating medium (4:1 mixture of DMEM-M199 to which 5% fetal calf
serum, 10% heat-inactivated horse serum, 1 mM 5-bromo-2⬘-deoxyuridine, and 2% penicillin-streptomycin were added). The cells
were plated at a density of 350 –500 cells/mm2 and incubated at 37°C
in humidified air with 5% CO2. After 24 h, the cultures were irradiated
with gamma irradiation (2,500 rads) to eliminate fibroblast growth
(see Ref. 15). After a brief equilibration in the incubator, the plating
medium was aspirated and replaced with serum-free culture medium
[DMEM supplemented with 1% ITS⫹ (insulin, selenium, and transferring supplement; BD Biosciences) and 0.2% penicillin-streptomycin].
For culturing of adult rat ventricular myocyte cultures, acutely
dissociated cells were prepared from adult Sprague-Dawley rats
(250 –275 g) as previously described (5–7). After dissociation, ventricular cardiomyocytes were resuspended in NaHCO3-buffered medium 199 supplemented with 10⫺7 M insulin, 5 mM creatine, 2 mM
L-carnitine, 0.2% BSA, 5 mM taurine, 1% penicillin/streptomycin,
and 3 mM N-(2-mercaptopropionyl)glycine and were seeded onto
gelatin-coated glass coverslips. The cells were then placed in a 37°C
incubator with 5% CO2-95% air and allowed to equilibrate and settle
for 2 h. The medium was then changed to remove all nonattached
cells, and myocytes were returned to the incubator. The principles
governing the care and treatment of animals as expressed by the
American Physiological Society were followed at all times during this
study. In addition, the University of Maryland School of Medicine
Institutional Animal Care and Use Committee approved all of the
procedures used in this study.
Transgene experiments. The cultured cells, either adult or neonatal
day 1 cultures, were infected with recombinant adenoviruses, 50 –100
particles per cell, in serum-free DMEM medium for 2 h before culture
medium was added for a 2-day incubation. In all of the experiments
described, control cells were derived from cultures that had been
infected with nonrecombinant Ad-dl312 adenovirus in parallel. In all
cases, optimal conditions for transgene expression were confirmed by
appropriate Western blot analyses.
Preparation of cell extracts. Cellular extracts were prepared from
control (infected with Ad-dl312 adenovirus) and transfected cultured
neonatal myocytes in the following manner. After 48 h of transfection,
cultures were washed with phosphate-buffered saline (PBS) and
homogenized in ice-cold lysis buffer consisting of 20 mM Tris 䡠 HCl
(pH 7.4), 137 mM NaCl, 5 mM EDTA, protease inhibitor cocktail
(Sigma P8340; 1:400), and 0.05% digitonin. Samples were then
centrifuged for 2 min at 15,000 g to resolve soluble and particulate
fractions. Protein concentrations of these extracts were determined
with Bradford’s reagent (Bio-Rad) using BSA as a standard.
Construction of recombinant B56␣ and B56␥1 adenovirus. Expression plasmids encoding human B56␣ or B56␥1 tagged with (4⫻) HA
sequence were a generous gift from Dr. David M. Virshup and have
been described previously (22). Generation of recombinant adenoviruses expressing HA-tagged B56␣ or B56␥1 driven by the cytomegalovirus promoter were generated through homologous recombination
NUCLEAR B56␥ LOCALIZATION IN CARDIAC CELLS
extent of the dot. The area delimited by these eight points was taken
as the area of the dot after scaling by the pixel size, and the mean
amplitude was taken from the mean of the pixel values within the area.
The pixel values within this area were then set to zero, and the
procedure was repeated until the maximal intensity fell below a preset
threshold. The analysis thus provided the number of dots per nucleus
as well as the area and amplitude of each dot.
Statistical analysis. All data are reported as means (SD). The
statistical significance of differences between control and experimental groups were calculated using one-way analysis of variance
(ANOVA) followed by the Newman-Keuls test with the use of a
statistical software program (GB-STAT; Dynamic Microsystems). A
P value of ⬍0.05 was considered significant.
RESULTS
Thus, for identification of intracellular sites of these two
PP2A regulatory B subunits in heart cells, epitope-tagged B56
protein expression was driven by adenoviral constructs in
transfected cultured rat neonatal cardiomyocytes. Localization
of these ectopic proteins was determined using confocal immunofluorescent microscopy. As shown in Fig. 2A, HA-B56␣
and HA-B56␥1 were found in distinctly different subcellular
regions. B56␣ was excluded from the nucleus and displayed a
meshwork cytoplasmic distribution, frequently with a striated
pattern. In contrast, HA-B56␥1 was detected primarily in the
nucleus (Fig. 2A). This distinctive nuclear localization of
B56␥1 was confirmed in the images in Fig. 2B, where the
nuclei were imaged with 4⬘,6⬘-diaminidino-2-phenylindole. In
parallel experiments, Western blot analyses of subcellular
fractions from transfected myocytes revealed that the HAtagged protein was found in nuclear fractions (Fig. 3). HAB56␥1 also was detected in the cytosolic fraction (Fig. 3, lane
2). This may be expected because others have reported a
nonnuclear role for this subunit in other cell types (12, 13). It
is important to note that the gel lanes were loaded with equal
amounts of protein, not proportional quantities, so that the
fractional distribution between the two fractions is not represented in these blots. Together, these data support a targeting
role for the putative COOH-terminal NLS (see Fig. 1) and are
consistent with observations in NIH/3T3 and CV-1 cells
(22, 35).
The confocal images in Fig. 2 suggest that B56␥1 is not
uniformly distributed within the nucleus. This view was critically assessed in high-resolution confocal imaging studies
Fig. 1. Human B56␣ and B56␥1 subunits of
protein phosphatase 2A (PP2A) are highly
homologous proteins. The amino acid sequences, previously reported (NCBI accession
no. AAC37601 for B56␣ and AAC37603 for
B56␥1; Ref. 23), were aligned using ClustalW algorithm (36). Amino acid identities
are shown as dark gray boxes and similarities as light gray boxes, and gaps, introduced
to optimize alignment, are indicated by
dashes. The core regions are 80% homologous, whereas the diagram illustrates the
short divergent regions that are found in the
NH2- and COOH-terminal regions. These
sequences were analyzed in a range of databases for targeting or protein-binding motifs.
No known domains were identified except
for a monopartite nuclear localization signal
(NLS) present in the COOH-terminal region
of B56␥1, indicated by the open box.
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To understand the role of PP2A in heart cell function, we
must identify the subcellular binding locales of the B regulatory subunits. Figure 1 shows the sequence alignments of two
subunits highly expressed in the heart, human B56␣ and
B56␥1, emphasizing the 80% homology in the central region
and the divergent domains observed in the extreme NH2- and
COOH-terminal regions (22, 23). A prediction is that despite
their homologies, these subunits will partition to distinct subcellular sites. Analyses of either sequence with multiple databases failed to identify any consensus binding/targeting motifs,
for example, DNA binding, RNA binding, PDZ or PH domains, etc., that might provide clues to their subcellular targets.
One exception was the presence of a monopartite nuclear
localization signal (NLS) on the COOH-terminal region of the
B56␥1 subunit (see Fig. 1).
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NUCLEAR B56␥ LOCALIZATION IN CARDIAC CELLS
shown in Fig. 4. HA-B56␥1 accumulated in a characteristic
punctate pattern in both neonatal and adult ventricular myocytes (Fig. 4A). Thus this distinct intranuclear localization is
independent of the developmental stage of the cardiac cells. A
similar pattern also was seen when HA-B56␥1 was expressed
in cultured mouse skeletal muscle cells (data not shown). It is
possible that this punctate distribution is an artifact of overexpression of the heterologous human protein. However, when
endogenous B56␥ was imaged in nontransfected cultured neonatal cardiomyocytes with the use of a polyclonal antibody that
recognizes the three alternatively spliced forms of B56␥, including B56␥1 (12), a similar dotted pattern was also observed
(Fig. 4A).
The known functions of B subunits require interactions with
their cognate binding partners, the heterodimeric complex
composed of PP2Ac and PP2A/A. Accordingly, a series of
experiments was performed to determine whether ectopically
expressed human HA-B56␥1 interacted with endogenous rat
PP2A proteins. Confocal immunofluorescent imaging of either
PP2A/A or PP2Ac (Fig. 4B) revealed that these proteins,
although found throughout the cells as expected, also were
localized in a punctuate pattern within the nuclei (red spots in
images). Double labeling revealed that both PP2A proteins
colocalized with HA-B56␥1 in the nucleus, shown as yellow
spots in Fig. 4B. In these experiments, low titers of adenovirus
vectors were used to achieve only partial transfection, thus
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Fig. 2. Heterologous expression of human
hemagglutinin (HA)-tagged B56␣ and B56␥1
in cardiomyocytes. A: cultured rat neonatal
cardiomyocytes were transfected with recombinant adenovirus constructs containing
either HA-B56␣ or HA-B56␥1 as indicated.
After 48 h, cells were fixed, labeled with
anti-HA antibody, and imaged using confocal fluorescent microscopy as described in
METHODS. The photomicrographic images include multiple cells within each field. B:
confocal images of immunolabeled neonatal
cardiomyocytes that were transfected with
either HA-B56␥1 or HA-B56␣ adenovirus
constructs (green). Cells also were labeled
with nuclear stain 4⬘,6⬘-diaminidino-2-phenylindole (blue).
Fig. 3. Western blot analysis of heterologous expression of HA-B56␥1. Rat
neonatal cardiomyocytes were transfected with HA-B56␥1 recombinant adenovirus or control virus (Ad-dl312). After 48 h, cells were harvested and
nuclear along with cytosolic fractions were prepared from both cultures as
described in METHODS. These fractions (20 ␮g protein/lane) were resolved by
SDS gel electrophoresis and developed using Western blot methods with
antibody against PP2A catalytic subunit (anti-PP2Ac) and anti-HA as indicated. The transcription enhancer factor (TEF) was used as a nuclear protein
marker as indicated. Shown are the cytosolic and nuclear fractions from control
(lanes 1 and 3) and Ad-HA-B56␥1-transfected cells (lanes 2 and 4).
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NUCLEAR B56␥ LOCALIZATION IN CARDIAC CELLS
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revealing the punctuate pattern for PP2Ac and PP2A/A in
nontransfected cells in the same field. Molecular studies were
performed to complement the imaging results. As shown in
Fig. 5A, in HA pull-down assays, PP2A/C immunoprecipitated
in extracts from HA-B56␥1 transfected cells (lane 4) but not
from those of control cells (lane 2). The faint band of apparent
Fig. 5. PP2Ac coimmunoprecipitates with HA-tagged B56␥1 when expressed
in cardiomyocytes. Rat neonatal myocytes were transfected with control or
HA-B56␥1 viruses for 48 h. A: whole cell extracts were prepared and subjected
to HA pull-down protocols as described in METHODS. Proteins (20 ␮g) in
supernatants (lanes 1 and 3) and immunoprecipitates (lanes 2 and 4) were
separated by SDS-PAGE and immunoblotted with anti-HA or anti-PP2Ac as
indicated. B: results of inverse experiments in which cell extracts were
subjected to immunoprecipitation with anti-PP2Ac and bound HA-B56␥1 and
PP2Ac were identified in Western blot analysis as indicated.
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Fig. 6. Effects of B56␥1 and B56␣ overexpression on endogenous PP2A
subunits. Rat neonatal cardiomyocytes were transfected with HA-B56␥1 or
HA-B56␣ recombinant adenoviruses or control virus (Ad-dl312) as indicated.
After 48 h, cells were harvested and a detergent extract was prepared and
analyzed using Western blot methods with antibodies as indicated. Shown are
the results from independent experiments from 3 different cultures for each
transfection condition. Note that the doublet that appears in anti-B56␥ blots
corresponds to endogenous rat B56␥1 (lower band) and human HA-B56␥1
(upper band).
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Fig. 4. B56␥1 cosegregates in the nucleus
with the PP2A A subunit (PP2A/A) and
PP2Ac in a characteristic punctuate pattern.
A: HA-B56␥1-transfected cultured neonatal
and adult rat cardiomyocytes were fixed and
immunostained with monoclonal anti-HA as
described in METHODS. Shown at left are
confocal photomicrographic images of nuclei from these cells as indicated. Image at
right displays a nucleus from a nontransfected cultured neonatal cell that was fixed
and immunostained with polyclonal antiB56␥ antibody to localize endogenous B56␥
within this compartment. Note that endogenous B56␥ also displays this characteristic
punctuate pattern. B: photomicrographs of
confocal immunofluorescent merged images
of cells transfected with low titers of HAB56␥1 virus and double-labeled with antiHA along with antibodies against PP2A subunits. Image at left shows colocalization of
HA-B56␥1 (green) with endogenous
PP2A/A (red) displayed as yellow dots in the
merged image. Image at right shows colocalization of HA-B56␥1 (green) with endogenous PP2Ac (red) displayed as yellow dots in
the merged image.
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NUCLEAR B56␥ LOCALIZATION IN CARDIAC CELLS
Western blot studies were used to determine whether increases in PP2Ac expression might explain this 39% stimulation of PP2A activity. As shown in Fig. 6, in HA-B56␥1transfected cells (lanes 7–9), the levels of core enzyme proteins, PP2Ac and PP2A/A, were unchanged relative to controls
(lanes 1–3). Furthermore, the overexpression of B56␥1 was
confirmed in HA-B56␥1 virus-treated cells (lanes 7–9). In
these blots (Fig. 6, bottom), the upper band of the doublet
corresponds to HA-tagged subunit. The precise level of overexpression is uncertain, because different preparations of polyclonal anti-B56␥ yielded different results. With another preparation, the overexpression appeared to be at least 10-fold. The
interpretation is that these polyclonal preparations have different relative affinities for endogenous rat B56␥1 compared with
heterologously expressed human HA-tagged human B56␥1.
Overexpression of B56␣ did not alter PP2Ac levels, as well
(lanes 4 – 6). Together, these data are consistent with the
conclusion that B56␥1 overexpression increases PP2A activity
through changes in substrate specificity rather than increases in
PP2Ac expression.
The distinct intranuclear targeting of B56␥1 is intriguing,
because it is now appreciated that the nucleus is a complex
organelle containing multiple dynamically organized subcompartments (1, 26, 40). To identify the compartment targeted by
Fig. 7. HA-B56␥1 accumulates into nuclear speckles. Neonatal rat cardiomyocytes grown on coverslips were fixed in 100% methanol after 48 h of transfection
with Ad-HA-B56␥1. Cells were stained with monoclonal anti-SC35 (green in merged image) and polyclonal anti-HA (red in merged image) as described in
METHODS. Fluorescent images were obtained with confocal microscopy as described in METHODS. Shown are separate and merged images of representative nuclei
that illustrate the marked colocalization of B56␥1 with SC35 (yellow in merged image).
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HA immunoreactivity seen in control cells (lane 2) results from
a slight cross-reactivity of the goat anti-rabbit IgG (secondary
in Western blotting) against mouse heavy chain IgG (used in
the immunoprecipitation step). This view is consistent with
other results where HA immunoreactivity is not seen in control
cell extracts that are probed with monoclonal anti-HA directly
(see Fig. 6 for example). Also, reciprocal immunoprecipitations with anti-PP2Ac confirmed that HA-B56␥1 was bound to
the catalytic subunit of PP2A (Fig. 5B).
It has been reported that one role for B regulatory subunits
is to alter PP2A catalytic activity (31). Accordingly, to begin to
assess the functional impact of HA-B56␥1 overexpression on
PP2A signaling, we analyzed extracts from transfected cells for
phosphatase activity in vitro. Although phosphorylase a has
been the pseudosubstrate of preference in many phosphatase
studies, a more specific pseudosubstrate for PP2A, RRATpVA
peptide, was used in the current study (4). PP2A activity
increased from 38 ⫾ 15 fmol phosphate䡠min⫺1 䡠mg protein⫺1
(n ⫽ 8) in control virus-treated cells to 58.9 ⫾ 15 fmol
phosphate䡠min⫺1 䡠mg protein⫺1 (n ⫽ 9, P ⬍ 0.01) in extracts
from HA-B56␥1-transfected cultures. These stimulatory effects were specific to B56␥1, because PP2A activity was not
significantly different in B56␣-overexpressing cells (26 ⫾ 9
fmol phosphate䡠min⫺1 䡠mg protein⫺1, n ⫽ 8, P ⫽ 0.15).
NUCLEAR B56␥ LOCALIZATION IN CARDIAC CELLS
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B56␥1, we compared the pattern of HA-B56␥1 accumulation
with that of other proteins known to reside in defined intranuclear regions. The observations from many images suggested
that HA-B56␥1 was excluded from large nuclear foci, perhaps
nucleoli (see Figs. 2 and 4). Thus we chose to examine its
potential colocalization with the splicing factor SC35, an
established marker for interchromatin granule clusters, also
known as nuclear speckles (26, 29). As shown in Fig. 7,
double-immunofluorescence confocal studies with polyclonal
anti-HA and monoclonal anti-SC35 showed that B56␥1 colocalizes with these nuclear speckles. Note that in these images,
the polyclonal anti-HA also decorated other nuclear regions in
addition to speckles. Because monoclonal anti-HA labeled
predominately speckles in parallel experiments (see Figs. 2 and
4), the significance of this non-speckle localization is not
known. Together, these results provide new insight into
how the PP2A core complex, which lacks nuclear targeting sequences, can be precisely localized into the nuclear
architecture.
Although controversial, speckles have been proposed as
storage sites for proteins and splicing factors involved in
transcription and pre-mRNA splicing (29). They are dynamic
compartments whose size and number are variable (26, 28).
For example, several previous studies in rapidly dividing cell
types revealed that the speckles are dynamic organelles, with
their abundance and size highly dependent on transcriptional
activity (2, 26). Thus a morphometric analysis algorithm was
developed to quantify speckles in confocal images as shown in
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Fig. 9. Inhibition of transcription evokes changes in dynamics of speckles in
cardiac cells. Cultured cardiomyocytes were treated with ␣-amanitin (25 ␮g/
ml) for a total of 3 h. At various time intervals, cells were fixed and stained
with anti-SC35 as described in METHODS to monitor the morphological changes
in nuclear speckles. A: shown are confocal immunofluorescent images of the
nuclei from representative cells showing nuclear speckles at various time
intervals of ␣-amanitin treatment as indicated. B: summary data are presented
in histograms that document the changes in size and number speckles over 3 h
measured as described in Fig. 8 and METHODS. These values are means (SD) of
10 –20 nuclei (for numbers of speckles) or 60 –70 speckles (for speckle size).
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Fig. 8. Morphometric analysis of nuclear
speckles in cardiac cells. A: nuclear speckles
were imaged in fixed neonatal cardiomyocytes by using immunofluorescent methods
with anti-SC35. The photomicrograph shows
a group of nuclei in the field to be analyzed.
B: individual “dots” within immunofluorescent images were identified using an algorithm, based on IDL language (version 5.5;
Research Systems), which found the location
of the pixel of maximal intensity in the
images as shown in the converted file. Thus
the number of speckles in each nuclei is
determined. C: for each dot, line segments
[f(x0, y0)] of 50 pixels in length were extracted starting at x0, y0 and extending in 1
of 8 directions within the plane of the image
as shown. D: a plot was made of pixel
fluorescence intensity vs. distance along
each line segment. Each function was mathematically fitted to a half-Gaussian function,
shown as a red line. The point at which this
fit fell to 10% of the peak identifies the
extent of the dot in that direction (indicated
with an arrow). Thus 8 points are identified
(shown as boxes in C), and the area delimited by them is used as a measure of the area
of the speckle.
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NUCLEAR B56␥ LOCALIZATION IN CARDIAC CELLS
Fig. 8. This method allows for an estimation of speckle size
and population in large numbers of imaged nuclei. This morphometric approach was exploited to determine whether the
properties of speckles are dynamically controlled by transcriptional activity of cultured cardiomyocytes. As shown in the
confocal images in Fig. 9A, treatment of the cells with the
transcriptional inhibitor ␣-amanitin resulted in a time-dependent decrease in the number of speckles that was accompanied
by a marked increase in speckle size. The quantitative summary time course data in Fig. 9B reveal that speckle diameter
increased 1.9-fold and speckle number decreased by 69% after
3 h in ␣-amanitin.
Together, the data in Fig. 9 indicate that any broad change in
transcriptional activity evoked by B56␥1 overexpression in
cultured cardiac cells should be reflected in alterations in the
number and size of speckles (27). The results in Fig. 10 reveal
that overexpression of this B subunit had no effect on speckle
properties, providing evidence that generalized global changes
in transcriptional activity are not likely. This conclusion was
consistent with parallel dot blot studies that were designed to
DISCUSSION
The serine/threonine phosphatase PP2A is expressed in high
levels in diverse cell types and plays a central role in modulating many signaling pathways (24, 30, 33, 38). Several
groups have documented the importance of PP2A as a regulator of Ca2⫹ and intracellular signaling in intact cardiomyocytes
(4, 5, 19, 20). A central unanswered question is how a particular phosphatase with rather broad substrate specificity can
selectively mediate diverse signaling cascades. A motivating
premise for the present study is that PP2A actions are governed
through the interactions of the core enzyme with a range of B
targeting subunits. Accordingly, this study focused on the B56
family of subunits that are highly expressed in the heart (22,
23). The main findings are that small sequence changes in the
B56 proteins lead to marked alterations in subcellular targeting
in heart myocytes. Furthermore, B56␥1 is targeted to the
nucleus, where it is localized to subcompartments known as
nuclear speckles. Finally, nuclear B56␥1 is not associated with
global changes in gene expression but, rather, may be linked
Fig. 11. B56␥1 overexpression attenuates the
dynamic structural reorganization of nuclear
speckles. Control or HA-B56␥1-transfected cells
were treated with 25 ␮g/ml ␣-amanitin for a total
of 3 h. Cells were fixed and stained with antiSC35 as described in METHODS, and morphological changes were quantified using methods described in Fig. 8. A: summary data showing the
change in size of speckles over time [means
(SD), n ⫽ 10 –24 nuclei] for control (䊐) and
HA-B56␥1-treated cells (Œ). *P ⬍ 0.01, markedly significant differences at 2 and 3 h. B:
summary data showing a decrease in the number
of speckles per nucleus in response to ␣-amanitin treatment for control (䊐) and HA-B56␥1treated cells (Œ) [means (SD), n ⫽ 60 –70 speckles]. *P ⬍ 0.05, significant difference between
the 2 groups of cells at 3 h of treatment.
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Fig. 10. Overexpression of HA-B56␥1 does not alter the number or size of the
speckles. The morphological features of speckles in control or HA-B56␥1transfected cells were analyzed as described in Fig. 8 and METHODS. Histograms display summary results as means (SD) from control and HA-B56␥1transfected cells, respectively.
detect changes in mRNA levels of established markers of
cardiac stress and hypertrophy. In those analyses, no changes
in mRNA levels for either atrial natriuretic factor or ␣-skeletal
muscle actin were observed (data not shown).
It has been proposed that phosphatases are likely involved in
the dynamic relocation of enzymes and factors associated with
nuclear speckles (26, 29). Thus the impact of B56␥1 overexpression on the dynamics of nuclear reorganization following
inhibition of transcription was examined. As shown in the time
course results in Fig. 11, according to several quantitative
measures, the time-dependent effects of ␣-amanitin were significantly inhibited in HA-B56␥1 cells. First, the increase in
speckle size was markedly inhibited at 2 and 3 h (Fig. 11A).
For example, at 3 h the increase in speckle size was 85% in
control virus-treated cells and only 29% in transfected cells.
Furthermore, the decrease in speckle population observed after
transcription inhibition was significantly blunted in B56␥1overexpressing cells at 3 h (Fig. 11B). Longer incubations may
have revealed further effects, but these studies were not pursued because of apparent toxic effects of this drug on cultured
cardiocytes. Together, these data provide evidence that B56␥1
targeting of PP2A to speckles contributes to the regulation of
the dynamics of these subnuclear complexes.
NUCLEAR B56␥ LOCALIZATION IN CARDIAC CELLS
AJP-Heart Circ Physiol • VOL
including SC35, have been associated with human heart disease and transgenic mouse models of cardiomyopathy (3, 11).
However, there were no broad changes in transcription, as
assessed by the conserved size and number of nuclear speckles
in B56␥1-overexpressing cardiac cells. Also, there were no
hypertrophic responses (cell size, gene expression) in these
transfected cardiac cells. This may not be surprising given that
in SC35 splicing factor knockout mice, no global changes in
cardiac gene expression were observed (3). Thus, although
PP2A inhibitors such as okadaic acid have broad effects on
nuclear activity (1), the results presented suggest that the
PP2A-B56␥1 enzyme complex subserves a more focused nuclear function.
It is now recognized that the nucleus contains highly dynamic non-membrane-delimited macromolecular complexes,
including speckles, whose assembly and organization are dictated by the self-assembly of proteins. Although the molecular
details are not defined, this process is controlled, in part, by
phosphorylation/dephosphorylation cascades (1, 18, 26). Accordingly, a series of experiments was designed to determine
whether PP2A is a regulator of this dynamic assembly. We
have demonstrated that inhibition of transcription in cultured
cardiac cells evoked a pronounced reorganization of splicing
factors into large speckles, a process previously observed in
actively dividing cells (18). Importantly, B56␥1 overexpression resulted in a slowing in the reorganization of these large
structures when transcription was blocked. Although the precise steps in speckle protein clustering are not known, these
results are consistent with the observation that protein-protein
interactions are stabilized in speckles that have reduced levels
of phosphorylation (32).
In summary, B56␥1 may be regarded as a targeting protein
that tethers PP2A activity to nuclear speckles. It will be
important in future studies to identify the specific domains that
contain the localization signal and to elucidate its protein
binding partners in cardiac nuclear speckles.
ACKNOWLEDGMENTS
We thank Dr. Michael Klein for valuable assistance in developing the IDL
program for analyses of nuclear speckle morphology.
GRANTS
This work was supported by National Institutes of Health Grants AG-14637
and P01-HL-70709 (to T. B. Rogers).
REFERENCES
1. Bollen M and Beullens M. Signaling by protein phosphatases in the
nucleus. Trends Cell Biol 12: 138 –145, 2002.
2. De Koninck P and Schulman H. Sensitivity of CaM kinase II to the
frequency of Ca2⫹ oscillations. Science 279: 227–230, 1998.
3. Ding JH, Xu X, Yang D, Chu PH, Dalton ND, Ye Z, Yeakley JM,
Cheng H, Xiao RP, Ross J, Chen J, and Fu XD. Dilated cardiomyopathy
caused by tissue-specific ablation of SC35 in the heart. EMBO J 23:
885– 896, 2004.
4. DuBell WH, Gigena MS, Guatimosim S, Long XL, Lederer WJ, and
Rogers TB. Effects of the PP1/PP2A inhibitor calyculin A on the ECcoupling cascade in murine ventricular myocytes. Am J Physiol Heart Circ
Physiol 282: H38 –H48, 2002.
5. DuBell WH, Lederer WJ, and Rogers TB. Dynamic modulation of
cardiac excitation-contraction coupling by protein phosphatases. J Physiol
493: 793– 800, 1996.
6. DuBell WH, Lewartowski B, Spurgeon HA, Silverman HS, and
Lakatta EG. Repletion of sarcoplasmic reticulum Ca after ryanodine in
289 • JULY 2005 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.32.247 on June 16, 2017
with assembly/disassembly of these macromolecular complexes.
An important finding was that B56␥1 was targeted to the
nucleus. Thus a focus of these studies was to determine
whether expression of B56␥1 in the nucleus was biologically
relevant. Because most of the known actions of B subunits are
mediated through their interactions with PP2A core enzyme, a
dimeric complex of PP2Ac and PP2A/A (30, 34, 38), several
experiments were designed to identify such complexes following adenovirus-driven B56␥1 expression. Confocal immunofluorescent imaging revealed that human HA-tagged B56␥1
colocalized with endogenous (rat) PP2Ac and PP2A/A in
nuclear compartments. Complementary immunoprecipitation
studies confirmed the HA-B56␥1/PP2Ac interaction. It also
was important to note that PP2A activity was increased in
B56␥1-overexpressing cells in the absence of possible underlying molecular changes, such as an increase in PP2Ac expression (Fig. 5) or changes in COOH-terminal methylation (unpublished results). Thus the increases in phosphatase activity
are consistent with other reports that B subunits can alter PP2A
substrate specificity (9, 37).
An important new finding is that B56␥1 is spatially coassembled with intranuclear structures known as speckles. The
biological relevance of this conclusion was underscored in
confocal images in which endogenous B56␥ also displayed the
same pattern. This targeting was not an artifact of the epitope
tag, because the highly homologous HA-B56␣ protein displayed a completely different localization. In fact, in other
studies we have reported that B56␣ is localized to sarcomeric
structures in adult cardiomyocytes (10). These results seem to
conflict with previous studies reporting that PP2A is principally nucleoplasmic (22, 35, 37). However, it is important to
note that the speckle localizations observed for PP2A/A and
PP2Ac in Fig. 4 are likely a threshold effect in such highresolution, low-gain confocal images. In fact, at higher gain,
PP2A appears distributed throughout the nuclei of cardiac cells
(data not shown). Importantly, until this study, there was little
information on how the PP2A core enzyme, lacking its own
nuclear localization sequences, could be targeted to specific
locales within the subnuclear architecture (for a review, see
Ref. 1).
Nuclear speckles are dynamic macromolecular complexes
whose function, although controversial, is likely related to
storage and/or activation sites of components of RNA splicing
(17, 26). Given that these morphologically defined structures
are composed of some 150 known proteins (25), identification
of the binding partners for B56␥1 is challenging. Although
B56␥1 contains a putative nuclear localization sequence, it
lacks an arginine/serine-rich sequence, a consensus RNArecognition domain, or a recently discovered speckle targeting
domain that are identified motifs for known speckle-associated
proteins (8). Although amino acid sequence motifs responsible
for nuclear transport are well defined, little is known about
motifs that specify intranuclear targeting. It will be important
to identify the peptide domains that localize this B subunit to
these strategic nuclear sites in cardiomyocytes.
The distinct nuclear targeting of B56␥1 combined with the
observation that reversible phosphorylation of splicing/spliceosome proteins is an important regulatory mechanism in speckles suggest that this PP2A complex may regulate gene expression (1, 26). Further splicing factors found in nuclear speckles,
H293
H294
7.
8.
9.
10.
11.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
rat ventricular myocytes. Am J Physiol Heart Circ Physiol 265: H604 –
H615, 1993.
DuBell WH, Wright PA, Lederer WJ, and Rogers TB. Effect of
immunosuppressant FK506 on excitation-contraction coupling and outward K⫹ currents in rat ventricular myocytes. J Physiol 501: 509 –516,
1997.
Eilbracht JE and Schmidt-Zachmann MS. Identification of a sequence
element directing a protein to nuclear speckles. Proc Natl Acad Sci USA
98: 3849 –3854, 2001.
Evans DR and Hemmings BA. Mutation of the C-terminal leucine
residue of PP2Ac inhibits PR55/B subunit binding and confers supersensitivity to microtubule destabilization in Saccharomyces cerevisiae. Mol
Gen Genet 264: 425– 432, 2000.
Gigena MS and Rogers TB. Localization of PP2A targeting subunits,
B56␣ and B56␥1, to cardiac sarcomere regions. Biophys J 82: 595a, 2002.
Hein S, Arnon E, Kostin S, Schonburg M, Elsasser A, Polyakova V,
Bauer EP, Klovekorn WP, and Schaper J. Progression from compensated hypertrophy to failure in the pressure-overloaded human heart:
structural deterioration and compensatory mechanisms. Circulation 107:
984 –991, 2003.
Ito A, Kataoka TR, Watanabe M, Mazur W, Sabe H, Kitamura Y, and
Nojima H. A truncated isoform of the PP2A B56 subunit promotes cell
motility through paxillin phosphorylation. EMBO J 19: 562–571, 2000.
Ito A, Koma YI, Sohda M, Watabe K, Nagano T, Misumi Y, Nojima
H, and Kitamura Y. Localization of the PP2A B56␥ regulatory subunit
at the Golgi complex. Am J Pathol 162: 479 – 489, 2003.
Kamibayashi C, Estes R, Lickteig RL, Yang SI, Craft C, and Mumby
MC. Comparison of heterotrimeric protein phosphatase 2A containing
different B subunits. J Biol Chem 269: 20139 –20148, 1994.
Kohomoto O, Levi AJ, and Bridge JH. Relation between reverse
sodium-calcium exchange and sarcoplasmic reticulum calcium release in
guinea pig ventricular cells. Circ Res 74: 550 –554, 1994.
Kohout TA, O’Brian JR, Gaa ST, Lederer WJ, and Rogers TB. A
novel adenovirus component system that transfects cultured cardiac cells
with high efficiency. Circ Res 78: 971–977, 1996.
Lamond AI and Earnshaw WC. Structure and function in the nucleus.
Science 280: 547–553, 1998.
Lamond AI and Spector DL. Nuclear speckles: a model for nuclear
organelles. Nat Rev Mol Cell Biol 4: 605– 612, 2003.
Liu Q and Hofmann PA. Modulation of protein phosphatase 2a by
adenosine A1 receptors in cardiomyocytes: role for p38 MAPK. Am J
Physiol Heart Circ Physiol 285: H97–H103, 2003.
Liu Q and Hofmann PA. Protein phosphatase 2A-mediated cross-talk
between p38 MAPK and ERK in apoptosis of cardiac myocytes. Am J
Physiol Heart Circ Physiol 286: H2204 –H2212, 2004.
McCright B, Brothman AR, and Virshup DM. Assignment of human
protein phosphatase 2A regulatory subunit genes b56␣, b56␤, b56␥, b56␦,
and b56⑀ (PPP2R5A–PPP2R5E), highly expressed in muscle and brain, to
chromosome regions 1q41, 11q12, 3p21, 6p21.1, and 7p112 3 p12.
Genomics 36: 168 –170, 1996.
McCright B, Rivers AM, Audlin S, and Virshup DM. The B56 family
of protein phosphatase 2A (PP2A) regulatory subunits encodes differen-
AJP-Heart Circ Physiol • VOL
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
tiation-induced phosphoproteins that target PP2A to both nucleus and
cytoplasm. J Biol Chem 271: 22081–22089, 1996.
McCright B and Virshup DM. Identification of a new family of protein
phosphatase 2A regulatory subunits. J Biol Chem 270: 26123–26128,
1995.
Millward TA, Zolnierowicz S, and Hemmings BA. Regulation of
protein kinase cascades by protein phosphatase 2A. Trends Biochem Sci
24: 186 –191, 1999.
Mintz PJ, Patterson SD, Neuwald AF, Spahr CS, and Spector DL.
Purification and biochemical characterization of interchromation granule
clusters. EMBO J 18: 4308 – 4320, 2002.
Misteli T. Protein dynamics: implications for nuclear architecture and
gene expression. Science 291: 843– 847, 2001.
Misteli T, Caceres JF, and Spector DL. The dynamics of pre-mRNA
splicing factor in living cells. Nature 387: 523–527, 1997.
Misteli T and Spector DL. Serine/threonine phosphatase 1 modulates the
subnuclear distribution of pre-mRNA splicing factors. Mol Biol Cell 7:
1559 –1572, 1996.
Misteli T and Spector DL. The cellular organization of gene expression.
Curr Opin Cell Biol 10: 323–331, 2001.
Mumby MC and Walter G. Protein serine/threonine phosphatases:
structure, regulation and functions in cell growth. Physiol Rev 73: 673–
699, 1995.
Price NE and Mumby MC. Effects of regulatory subunits on the kinetics
of protein phosphatase 2A. Biochemistry 39: 11312–11318, 2000.
Sacco-Bubulya P and Spector DL. Disassembly of interchromatin granule clusters alters the coordination of transcription and pre-mRNA splicing. J Cell Biol 156: 425– 436, 2002.
Shenolikar S. Protein serine/threonine phosphatases–new avenues for cell
regulation. Annu Rev Cell Biol 10: 55– 86, 1994.
Sontag E. Protein phosphatase 2A: the Trojan horse of cellular signaling.
Cell Signal 13: 7–16, 2001.
Tehrani MA, Mumby MC, and Kamibayashi C. Identification of a
novel protein phosphatase 2A regulatory subunit highly expressed in
muscle. J Biol Chem 271: 5164 –5170, 1996.
Thompson JD, Higgins DG, and Gibson TJ. CLUSTAL W: improving
the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix
choice. Nucleic Acids Res 22: 4673– 4680, 1994.
Turowski P, Fernandez A, Favre B, Lamb NJ, and Hemmings BA.
Differential methylation and altered conformation of cytoplasmic and
nuclear forms of protein phosphatase 2A during cell cycle progression.
J Cell Biol 129: 397– 410, 1995.
Virshup DM. Protein phosphatase 2A: a panoply of enzymes. Curr Opin
Cell Biol 12: 180 –185, 2000.
Wang Y, Krushel LA, and Edelman GM. Targeted DNA recombination
in vivo using an adenovirus carrying the cre recombinase gene. Proc Natl
Acad Sci USA 93: 3932–3936, 1996.
Zeng C, Kim E, Warren SL, and Berget SM. Dynamic relocation of
transcription and splicing factors dependent upon transcriptional activity.
EMBO J 16: 1401–1412, 1997.
289 • JULY 2005 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.32.247 on June 16, 2017
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NUCLEAR B56␥ LOCALIZATION IN CARDIAC CELLS