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Am J Physiol Heart Circ Physiol 289: H285-H294, 2005. First published March 18, 2005; doi:10.1152/ajpheart.01291.2004
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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

1Department 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


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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{alpha} and B56{gamma}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{alpha} was excluded from the nucleus and decorated striated structures, whereas HA-B56{gamma}1 was principally found in the nucleus. Precise immunolabeling studies showed that B56{gamma}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{gamma}1 increased whole cell PP2A activity by 40% when measured in cell extracts. Finally, B56{gamma}1 did not alter global gene expression or expression of hypertrophic gene markers such as {alpha}-skeletal actin. However, morphometric analyses of confocal images revealed that B56{gamma}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.

PROTEIN PHOSPHATASE 2A (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{gamma}1 is not only a nuclear protein, but, unexpectedly, it targets to subnuclear organelles called nuclear speckles. Finally, overexpression studies indicate that nuclear B56{gamma}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.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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 double-immunolabeling approaches were required. Antibodies against PP2Ac, B56{alpha}, 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{gamma} was previously described (12). Secondary peroxidase-conjugated mouse and rabbit antibodies were obtained from Jackson ImmunoResearch, and Alexa Fluor 568 goat anti-rabbit IgG and 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 µM 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·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{alpha} and B56{gamma}1 adenovirus. Expression plasmids encoding human B56{alpha} or B56{gamma}1 tagged with (4x) 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{alpha} or B56{gamma}1 driven by the cytomegalovirus promoter were generated through homologous recombination between cotransfected pJM17 plasmid and the shuttle plasmids, pAdv/4-HA-B56{alpha} or pAdv/4-HA-B56{gamma}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{alpha} or HA-B56{gamma}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-B56-transfected 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·HCl (pH 7.4), 1 mM EGTA, 1 mM EDTA, 0.1% {beta}-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 anti-mouse 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% {beta}-mercaptoethanol in the presence or absence of 10 nM okadaic acid. After 15 min at 30°C, the reaction was terminated by adding 500 µl of 100 mM K2PO4 in 5% TCA. The 32P-labeled peptide and [32P]Pi released were separated by applying the total reaction volume (550 µl) onto an ion exchange column (1 ml Dowex 50WX8, 200–400 mesh, H form). The [32P]Pi was eluted from the columns in 500 µl 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 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
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
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{alpha} and B56{gamma}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{gamma}1 subunit (see Fig. 1).



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  		Fig. 1. Human B56{alpha} and B56{gamma}1 subunits of protein phosphatase 2A (PP2A) are highly homologous proteins. The amino acid sequences, previously reported (NCBI accession no. AAC37601 for B56{alpha} and AAC37603 for B56{gamma}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{gamma}1, indicated by the open box.

 
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{alpha} and HA-B56{gamma}1 were found in distinctly different subcellular regions. B56{alpha} was excluded from the nucleus and displayed a meshwork cytoplasmic distribution, frequently with a striated pattern. In contrast, HA-B56{gamma}1 was detected primarily in the nucleus (Fig. 2A). This distinctive nuclear localization of B56{gamma}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 HA-tagged protein was found in nuclear fractions (Fig. 3). HA-B56{gamma}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).



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  		Fig. 2. Heterologous expression of human hemagglutinin (HA)-tagged B56{alpha} and B56{gamma}1 in cardiomyocytes. A: cultured rat neonatal cardiomyocytes were transfected with recombinant adenovirus constructs containing either HA-B56{alpha} or HA-B56{gamma}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{gamma}1 or HA-B56{alpha} adenovirus constructs (green). Cells also were labeled with nuclear stain 4',6'-diaminidino-2-phenylindole (blue).

 


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  		Fig. 3. Western blot analysis of heterologous expression of HA-B56{gamma}1. Rat neonatal cardiomyocytes were transfected with HA-B56{gamma}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 µg 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{gamma}1-transfected cells (lanes 2 and 4).

 
The confocal images in Fig. 2 suggest that B56{gamma}1 is not uniformly distributed within the nucleus. This view was critically assessed in high-resolution confocal imaging studies shown in Fig. 4. HA-B56{gamma}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{gamma}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{gamma} was imaged in nontransfected cultured neonatal cardiomyocytes with the use of a polyclonal antibody that recognizes the three alternatively spliced forms of B56{gamma}, including B56{gamma}1 (12), a similar dotted pattern was also observed (Fig. 4A).



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  		Fig. 4. B56{gamma}1 cosegregates in the nucleus with the PP2A A subunit (PP2A/A) and PP2Ac in a characteristic punctuate pattern. A: HA-B56{gamma}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 anti-B56{gamma} antibody to localize endogenous B56{gamma} within this compartment. Note that endogenous B56{gamma} also displays this characteristic punctuate pattern. B: photomicrographs of confocal immunofluorescent merged images of cells transfected with low titers of HA-B56{gamma}1 virus and double-labeled with anti-HA along with antibodies against PP2A subunits. Image at left shows colocalization of HA-B56{gamma}1 (green) with endogenous PP2A/A (red) displayed as yellow dots in the merged image. Image at right shows colocalization of HA-B56{gamma}1 (green) with endogenous PP2Ac (red) displayed as yellow dots in the merged image.

 
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{gamma}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{gamma}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 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{gamma}1 transfected cells (lane 4) but not from those of control cells (lane 2). The faint band of apparent 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{gamma}1 was bound to the catalytic subunit of PP2A (Fig. 5B).



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  		Fig. 5. PP2Ac coimmunoprecipitates with HA-tagged B56{gamma}1 when expressed in cardiomyocytes. Rat neonatal myocytes were transfected with control or HA-B56{gamma}1 viruses for 48 h. A: whole cell extracts were prepared and subjected to HA pull-down protocols as described in METHODS. Proteins (20 µg) 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{gamma}1 and PP2Ac were identified in Western blot analysis as indicated.

 


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  		Fig. 6. Effects of B56{gamma}1 and B56{alpha} overexpression on endogenous PP2A subunits. Rat neonatal cardiomyocytes were transfected with HA-B56{gamma}1 or HA-B56{alpha} 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{gamma} blots corresponds to endogenous rat B56{gamma}1 (lower band) and human HA-B56{gamma}1 (upper band).

 
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{gamma}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·min–1·mg protein–1 (n = 8) in control virus-treated cells to 58.9 ± 15 fmol phosphate·min–1·mg protein–1 (n = 9, P < 0.01) in extracts from HA-B56{gamma}1-transfected cultures. These stimulatory effects were specific to B56{gamma}1, because PP2A activity was not significantly different in B56{alpha}-overexpressing cells (26 ± 9 fmol phosphate·min–1·mg protein–1, n = 8, P = 0.15).

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{gamma}1-transfected 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{gamma}1 was confirmed in HA-B56{gamma}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{gamma} 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{gamma}1 compared with heterologously expressed human HA-tagged human B56{gamma}1. Overexpression of B56{alpha} did not alter PP2Ac levels, as well (lanes 4–6). Together, these data are consistent with the conclusion that B56{gamma}1 overexpression increases PP2A activity through changes in substrate specificity rather than increases in PP2Ac expression.

The distinct intranuclear targeting of B56{gamma}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 B56{gamma}1, we compared the pattern of HA-B56{gamma}1 accumulation with that of other proteins known to reside in defined intranuclear regions. The observations from many images suggested that HA-B56{gamma}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{gamma}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.



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  		Fig. 7. HA-B56{gamma}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{gamma}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{gamma}1 with SC35 (yellow in merged image).

 
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 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 {alpha}-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 {alpha}-amanitin.



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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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  		Fig. 9. Inhibition of transcription evokes changes in dynamics of speckles in cardiac cells. Cultured cardiomyocytes were treated with {alpha}-amanitin (25 µg/ 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 {alpha}-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).

 
Together, the data in Fig. 9 indicate that any broad change in transcriptional activity evoked by B56{gamma}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 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 {alpha}-skeletal muscle actin were observed (data not shown).



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  		Fig. 10. Overexpression of HA-B56{gamma}1 does not alter the number or size of the speckles. The morphological features of speckles in control or HA-B56{gamma}1-transfected cells were analyzed as described in Fig. 8 and METHODS. Histograms display summary results as means (SD) from control and HA-B56{gamma}1-transfected cells, respectively.

 
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{gamma}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 {alpha}-amanitin were significantly inhibited in HA-B56{gamma}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{gamma}1-overexpressing 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{gamma}1 targeting of PP2A to speckles contributes to the regulation of the dynamics of these subnuclear complexes.



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  		Fig. 11. B56{gamma}1 overexpression attenuates the dynamic structural reorganization of nuclear speckles. Control or HA-B56{gamma}1-transfected cells were treated with 25 µg/ml {alpha}-amanitin for a total of 3 h. Cells were fixed and stained with anti-SC35 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 ({square}) and HA-B56{gamma}1-treated cells ({blacktriangleup}). *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 {alpha}-amanitin treatment for control ({square}) and HA-B56{gamma}1-treated cells ({blacktriangleup}) [means (SD), n = 60–70 speckles]. *P < 0.05, significant difference between the 2 groups of cells at 3 h of treatment.

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
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{gamma}1 is targeted to the nucleus, where it is localized to subcompartments known as nuclear speckles. Finally, nuclear B56{gamma}1 is not associated with global changes in gene expression but, rather, may be linked with assembly/disassembly of these macromolecular complexes.

An important finding was that B56{gamma}1 was targeted to the nucleus. Thus a focus of these studies was to determine whether expression of B56{gamma}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{gamma}1 expression. Confocal immunofluorescent imaging revealed that human HA-tagged B56{gamma}1 colocalized with endogenous (rat) PP2Ac and PP2A/A in nuclear compartments. Complementary immunoprecipitation studies confirmed the HA-B56{gamma}1/PP2Ac interaction. It also was important to note that PP2A activity was increased in B56{gamma}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{gamma}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{gamma} also displayed the same pattern. This targeting was not an artifact of the epitope tag, because the highly homologous HA-B56{alpha} protein displayed a completely different localization. In fact, in other studies we have reported that B56{alpha} 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 high-resolution, 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{gamma}1 is challenging. Although B56{gamma}1 contains a putative nuclear localization sequence, it lacks an arginine/serine-rich sequence, a consensus RNA-recognition 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{gamma}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, 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{gamma}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{gamma}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{gamma}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{gamma}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.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants AG-14637 and P01-HL-70709 (to T. B. Rogers).


   ACKNOWLEDGMENTS
 
We thank Dr. Michael Klein for valuable assistance in developing the IDL program for analyses of nuclear speckle morphology.


   FOOTNOTES
 

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{at}som.umaryland.edu)

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.


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