Proteomic Studies of PP2A-B56γ1 Phosphatase Complexes Reveal

Jul 31, 2007 - Synopsis. The paper describes the combination of affinity purification and LC−MS/MS to identify phosphorylation-regulated partners of...
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Proteomic Studies of PP2A-B56γ1 Phosphatase Complexes Reveal Phosphorylation-Regulated Partners in Cardiac Local Signaling Xing Wang Zhou,*,‡ Malkanthi Mudannayake,‡ Mariah Green,‡ Marisa S. Gigena,‡ Guanghui Wang,§ Rong-Fong Shen,§ and Terry B. Rogers*,‡ Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, and Proteomics Core Facility, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892 Received November 21, 2006

Defects of kinase-phosphatase signaling in cardiac myocytes contribute to human heart disease. The activity of one phosphatase, PP2A, is governed by B targeting subunits, including B56γ1, expressed in heart cells. As the role of PP2A/B56γ1 on the heart function remains largely unknown, this study sought to identify protein partners through unbiased, affinity purification-based proteomics combined with the functional validation. The results reveal multiple interactors that are localized in strategic cardiac sites to participate in Ca2+ homeostasis and gene expression, exemplified by the Ca pump, SERCA2a, and the splicing factor ASF/SF2. These results are corroborated by confocal imaging where adenovirally overexpressed B56γ1 is found in z-line/t-tubule region and nuclear speckles. Importantly, overexpression of B56γ1 in cultured myocytes dramatically impairs cell contractility. These results provide a global view of B56γ1-regulated local signaling and heart function. Keywords: B56γ1 • phosphatase • PP2A • proteomics • cardiac function • heart disease • signal transduction • splicing • EC coupling

Introduction Molecular defects in cardiac cell signal transduction have been linked to many animal models of heart failure and human heart disease.1,2 Protein phosphorylation is a well-recognized regulatory mechanism in these signaling pathways that relies on the balanced spatio-temporal control of specific protein kinases and opposing phosphatases. Although protein kinase cascades3 have been a major focus of the field thus far, protein phosphatases are emerging as equally important enzymes in cell signaling.4,5 Protein phosphatase 2A (PP2A) accounts for a large portion of serine/threonine phosphatase activity in most cells and plays important roles in modulating many cellular processes, including signal transduction, transcription, and development.6,7 PP2A is a heterotrimer that includes a dimeric core enzyme, comprised of catalytic, c (PP2A/c), and scaffolding, A (PP2A/A), subunits, associated with one of at least 20 exchangeable regulatory B subunits. Four unrelated gene families of PP2A B subunits have been identified to date, denoted PR55 (or B), B56 (or B′), PR72 (or B′′), and PR93/PR110 (or B′′′).8 B56 is the most complex of the four families, as its members are encoded by five distinct mammalian genes (R, β, γ, δ, and ) that * To whom correspondence should be addressed at Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Greene Street, Baltimore, MD 21201. E-mails: xzhou@ som.umaryland.edu (X.W.Z.) or [email protected] (T.B.R.); phone, 410-706-5734; fax, 410-706-6676. ‡ University of Maryland School of Medicine. § National Heart, Lung, and Blood Institute, National Institutes of Health. 10.1021/pr060619l CCC: $37.00

 2007 American Chemical Society

produce more than 13 splicing isoforms.9 One of these genes, B56γ,10 is preferentially expressed in cardiac and skeletal muscle and encodes at least three splice variants,11 including B56γ1, the isoform studied in the present paper. PP2A regulatory B subunits govern the subcellular localization and substrate preference of the PP2A holoenzyme and, in large part, are responsible for PP2A functional specificity. The five B56 family members (B56R-) have diverse functions, including binding to APC protein in HEK293 cells, which acts as a scaffold for β-catenin, axin, and GSK-3β.12 In fact, overexpression of B56R leads to inhibition of β-catenin signaling in oocytes.13 PP2A, when associated with members of the B family, BR and Bδ, dephosphorylates and inactivates extracellular signal-regulated kinases (ERKs) in neuronal PC12 cells.14 Also, B56δ, but not other PP2A regulatory subunits, influence transcription factors (HAND1 and HAND2) function during transcriptional regulation in HEK293 cells.15 Despite the research progress on PP2A/B subunit-mediated signal transduction in mammalian cells mentioned above, the molecular details of how PP2A, guided by B subunits, regulates cardiac local signaling and its impact on the heart function remain poorly understood. Accordingly, this study focused on B56γ1, a subunit preferentially expressed in heart.10,11 A limitation is that there are no consensus motifs within the B56γ1 sequence that would provide insight into its targeting function. Accordingly, we exploited an unbiased affinity purificationbased proteomic approach16,17 to identify the components of PP2A/B56γ1 signaling complexes. Through the use of high stringency criteria, a discrete set of phosphorylation-regulated Journal of Proteome Research 2007, 6, 3433-3442

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binding partners were identified. These results were further validated in phosphorylation studies in intact cardiac cells. This proteomic strategy has provided new insight into multiple signaling targets for PP2A that are central to cardiac cell function.

Table 1. Proteomic Identification of PP2A/B56γ1 Protein Complexes

Materials and Methods

Calcium handling

Expression and Purification of GST-B56 γ1 Fusion Protein. The gene-encoding human B56γ1 was PCR-amplified and cloned into the pGEX4T-1 (Amersham) expression vector that adds a GST affinity-tag to the N-termini of expressed proteins. The fusion protein was expressed in Escherichia coli BL21-RIL (Stratagene). Briefly, the culture was incubated at 28 °C until it reached an absorbance of 0.6 at 600 nm and was then induced with 200 µM IPTG for 2 h. Following the culture and induction, cells were harvested by centrifugation at 7000g for 10 min. The GST-B56γ1 protein was purified using glutathioneSepharose 4B beads (Amersham) according to the manufacturer’s instructions. GST-B56γ1 was eluted from the column by the addition of 40 mM glutathione, 150 mM NaCl, and 50 mM Tris (pH 8) and then buffer-exchanged to 50 mM HEPES (pH 7.9) and 0.1 M NaCl. The Purification of PP2A/B56γ1 Complexes Using CrossLinked GST-B56γ1-Affi-Gel Beads. An affinity matrix, covalently cross-linked Affi-B56γ1 beads, was prepared by incubating 3.5 mg of purified fusion protein with 0.5 mL of AffiGel-15 for 4 h at 4 °C according to the manufacturer’s instructions. A control matrix, Affigel-GST, was prepared in identical manner with purified GST. Affi-B56γ1 beads were used in affinity pulldown protocols with mouse whole heart extracts (Affi-GST beads as control). The heart tissue extracts were prepared from 10 mice whole hearts that were freshly excised and immediately frozen in the liquid nitrogen. The frozen whole hearts were then ground with a mortar and pestle and homogenized in the lysis buffer containing 50 mM HEPES (pH 7.4), 150 mM NaCl, 2 mM DTT, 1% Triton X-100, and protease inhibitor cocktail (Sigma P8340). The crude extract was further homogenized on ice with 5 strokes with a Teflon glass homogenizer (Caframo, Ontario, Canada). The homogenate was filtered through two layers of cheesecloth, and then centrifuged at 100 000g for 1 h at 4 °C to obtain the supernatant extracts. The extract was precleared by incubation of 10 mL of 0.5% Triton solubilized mouse heart protein (5 mg/mL), which was diluted from the initial 1% Triton solubilized whole heart extracts, with Affi-GST beads (50 µL) for 1 h. Then, the Affi-B56γ1 beads (50 µL, ∼3 mg/mL) were added and incubated overnight at 4 °C while rotating. Following the incubation, the beads were collected by centrifugation and washed three times with incubation buffer. The bound PP2A/ B56γ1-interacting proteins were subsequently eluted in a denaturing solution of 8 M urea and 1 M NaCl. The eluted proteins were concentrated and buffer-exchanged to 50 mM NH4HCO3 with a 10 kDa cutoff Amicon concentrator. A small portion (5%) of the total eluted protein (20 µg) was used for SDS-PAGE, silver staining analysis, while the remaining samples were used for in-solution trypsin digestion and subsequent proteomic analysis as described below. LC-MS/MS Identification of PP2A/B56γ1 Protein Complexes. The PP2A/B56γ1-interacting partners eluted from AffiB56γ1 beads were identified using a shotgun proteomic method as we have described previously.18 The proteins were insolution-digested by trypsin (Promega), and the resultant peptides were subjected to mass spectrometry (MS) analysis 3434

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protein categorya PP2A subunit

Nuclear Speckles

Nuclear

Others

NCBI GI no.

peptides matchedb

8394024 8394027 2826866

11 8 10

Ca2+ ATPase, cardiac slow stitch 2 SERCA3a Sarcalumenin Calmodulin Phospholambanc Splicing factor, Arg/Ser-rich 2

6806903 1438541 34328417 6680832 12963503 6755478

7 2 3 2 n/a 2

Ribosomal protein S25 Ribosomal protein L22 Histone H1 Histone 1, H1c Histone 1, H1e 14-3-3 Zeta Polymerase I transcript release factor Y-box protein 3 Y-box transcription factor Y-box binding protein RNA binding protein MSY4 dbpA murine homologue Nucleolin Nuclease element binding protein Purine rich element binding protein SET translocation Peroxiredoxin 2 Cysteine and glycine-rich protein 3

28372479 6677775 558679 9845257 13430890 1841387 6679567 10185723 2745892 532211 7385223 1160331 31543315 6756033 6755252 13591862 31560539 7304987

2 2 3 3 3 2 10 3 4 2 2 2 5 4 3 2 3 4

protein name Catalytic subunit A subunit SERCA2a

a Category classification based on published literatures. b Only peptides with Mascot score larger than 40 are included. c Identified by Western blot.

using Thermo LTQ ion trap mass spectrometers. To increase coverage of the protein identification spectrum, we analyzed the sample digests twice by LC-MS/MS. Tandem MS-based protein identification was performed by searching NCBI database using an in-house version of Mascot MS/MS ion search program (Matrix Sciences Ltd., London, U.K.) or the Biowork 3.1 (Finnigan, San Jose, CA). The Mascot search generates a list of significant “hit” proteins (proteins with at least one peptide ion scoring higher than 40 were regarded as significant “hits” by Mascot (p < 0.05)). These significant “hit” proteins were further verified by manual inspection of MS/MS spectra and evaluated according to the following criteria for positive identification: (a) only hit peptide ions scoring higher than 40 were counted as true matched peptides, and (b) only proteins with at least two matched peptides of distinct sequence identified were accepted as positive identification. In addition, proteins found in both the bait and GST-control beads were removed, and predicted or unnamed proteins identified by Mascot were not included in our identification table (Table 1). The criteria for Biowork 3.1 search has been described previously.18 Cardiac Myocyte Culture, Adenoviral Transfection, and the Preparation of Cell/Tissue Extracts. Neonatal rat ventricular myocytes were routinely prepared from 1-day-old rats and cultured according to Gigena et al.19 Isolation and culturing of adult rat ventricular myocytes has also been previously described.20,21 All animal protocols were approved by the Animal Use and Care Committee of the University of Maryland. Two adenoviruses were used for infection of cardiac myocytes in this paper: (1) control adenovirus, Ad-dl312, a replication deficient human adenovirus type 5 mutant; and (2) AdHA-B56γ1, adenovirus encoding the coding region of HAtagged human B56γ1 (a gift from Dr. David Virshup22). The

Local Signaling of Protein Phosphatase PP2A Gamma 1

adenoviral infection of neonatal and adult rat cardiac myocytes and subsequent culture were performed as described previously.19 Cellular extracts were prepared from virus-transfected culture myocytes. Briefly, transfected and cultured myocytes were washed with PBS buffer and homogenized in ice-cold lysis buffer consisting of 50 mM HEPES (pH 7.4), 150 mM NaCl, 2 mM DTT, 1% Triton X-100, protease inhibitor cocktail (Sigma P8340), and phosphorylation-blocking cocktail (100 nM staurosporine, 250 nM okadaic acid, 20 mM NaF, 1 mM EGTA, and EDTA). Samples were then centrifuged at 15 000g for 10 min at 4 °C to obtain the supernatant extracts. Western-Blotting and Immunocytochemistry. For Western blot analysis, the samples were solubilized in loading buffer for 10-15 min at 95 °C and run using 10% reducing NuPAGE Bis-Tris gels (Invitrogen) and MOPS or MES running buffer (Invitrogen). PAGE gels were then transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore). Blotting was performed according to the protocol described previously.23 Primary antibodies used were anti-HA (monoclonal, BabCo), anti-PP2A C subunit (monoclonal, BD Biosciences), PP2A A subunit (polyclonal, Oxford Biomedical Research), anti-SF2/ ASF (monoclonal, Zymed laboratories), anti-phospholamban (PLB) (Monoclonal, Upstate), and anti-phospho-PLB (Ser-16) and anti-phospho-PLB (Thr-17) (polyclonal, Badrilla). Antimouse IgG and anti-rabbit IgG secondary antibodies conjugated to horseradish peroxidase were used where appropriate. Western blot signals were quantified using NucleoVision imaging station (Nucleotech, San Carlos, CA). The immunocytochemistry experiment for validating PP2A/B56γ1 localization in cultured adult rat myocytes was performed as described previously.19 Anti-HA (monoclonal, BabCo) and anti-DHPR primary antibodies (gift from M. Hosey, Chicago, IL) were used for immunocytochemistry. The secondary antibody was Alexa Fluor-488 goat anti-mouse IgG (Molecular Probes). Contractility Measurement. Contractility studies were carried out on three sets of 24 h cultured adult rat ventricular myocytes. Three hours after plating on glass coverslips, myocytes were incubated with either Ad-dl312 (control virus) or Ad-HA-B56γ1 virus at 50 plaque forming units/cell. Another group of cells were left untreated and served as an additional control in the analysis. After 24 h, the infected adult rat cultured myocytes were placed in a temperature-controlled flow chamber fitted with platinum electrodes mounted on the stage on an inverted phase microscope, superfused with 0.9 mM Ca2+ in Ringer’s Potassium Bicarbonate solution (100 mM NaCl, 5 mM KCl, 25 mM HEPES, 1 mM MgSO4, and 1 mM Na2HPO4, pH 7.40, 35 °C), and field-stimulated at 1 Hz at a voltage 10% above threshold. Contraction amplitude (sarcomere length of a single cell over time) was recorded online using an optical video analysis system (IonOptix). Statistical Analyses. Prism GraphPad software was used for statistical analyses. Two-tailed Student’s t tests were used for analysis of Western blot quantification. A p-value of less than 0.05 was considered statistically significant.

Results Although the B56γ1-PP2A heterotrimeric complex segregates to discrete subcellular locales in cardiac myocytes, a lack of canonical protein binding motifs within B56γ1 has prevented the identification of its associated protein binding partners.19 Accordingly, we exploited an unbiased affinity-based proteomic strategy to address this issue. First, GST-B56γ1 chimeric protein

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Figure 1. Preparation and characterization of Affi-B56γ1 affinity matrix. (A) The heterotrimeric PP2A complex is depicted which contains the catalytic subunit, PP2A/c (red). The scaffolding protein, PP2A/A (gray), and the targeting subunit B56γ1 (yellow). The cDNA encoding B56γ1 was cloned into the pGEX4T-1 expression vector that adds the GST affinity-tag to the N-terminus as shown. The corresponding fusion protein was expressed in E. coli BL21-RIL cells and then purified with glutathione sepharose 4B beads. (B) Shown is a Coomassie-stained SDS gel of the purified fusion protein which was then covalently cross-linked to Affi-gel-15 beads. (C) Affi-B56γ1 beads were incubated with 0.5% Triton extracts from mouse hearts overnight at 4 °C. Following centrifugation and washing, bound proteins were eluted in SDS sample buffer and resolved by SDS electrophoresis. The blots were developed in the presence of both anti-PP2A/A and anti-PP2A/c. Shown are the analyses of these subunits in supernatant (S) and pull-down fractions (P) in control beads (AffiGST) and B56γ1 affinity matrix, Affi-B56γ1.

was expressed in E. coli and subsequently purified on glutathione beads (Figures 1A,B). The purified B56γ1 fusion protein was covalently cross-linked to Affi-gel beads as this matrix provides a low nonspecific protein background.24 It was important to know if the affinity matrix was indeed bioactive. Accordingly, pull-down experiments with cardiac cell extracts revealed that Affigel-B56γ1 could bind to the established partners in the trimeric PP2A complex, the catalytic subunit, PP2A/c, and the scaffolding subunit, PP2A/A (Figure 1C). Importantly, negative control experiments showed that Affi-GST beads failed to bind these PP2A subunits (Figure 1C, lane 2). Journal of Proteome Research • Vol. 6, No. 9, 2007 3435

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Figure 2. Schematic diagram of the affinity purification-based proteomic strategy. The schematic flow chart (A) depicts the affinitypurification-based proteomic strategy used to identify binding partners of PP2A/B56γ1 in cardiomyocytes. The Affi-B56γ1 beads (see Figure 1) were used to pull down PP2A/B56γ1-associated binding proteins from mouse heart extracts (see Materials and Methods for details), while Affi-GST beads were used as a negative control in parallel experiments. A fraction of purified proteins (5%) was analyzed for specificity with SDS gel electrophoresis and silver staining. The affinity-purified proteins (95%) were then reduced, alkylated, and in-solution trypsin-digested, and the resultant peptides were subjected to shotgun proteomic analysis by LC-MS/MS. (B) Analysis of the affinity-based purified proteins in silver-stained SDS gels of both B56γ1 affinity matrix and control matrix (Affi-GST).

The Affi-B56γ1 beads were exploited in an affinity-based method to isolate proteins from mouse heart as depicted in the schematic flow diagram in Figure 2A. A 1% Triton extract from mouse hearts was incubated with Affi-B56γ1. The beads were extensively washed with 0.5% Triton buffer, and the bound proteins were eluted in a denaturing buffer as described in Materials and Methods. This procedure resulted in 20 µg of affinity-purified protein. The specificity of this approach was evaluated on silver stained SDS-PAGE gels. As shown in Figure 2B, Affi-B56γ1 beads specifically pulled-down multiple interacting proteins from mouse heart lysate. In contrast, few bands were detected in control Affi-GST bead fractions collected in parallel experiments (Figure 2B, control lane). LC-MS/MS-Based Proteomic Analysis. A LC-MS/MS shotgun proteomic approach was used for the analysis of PP2A/ B56γ1 protein complexes eluted from Affi-B56γ1 bound heart extracts. The resulting eluates were trypsin-digested and subjected to LC-MS/MS analysis. The peptide digests from all PP2A/B56γ1 protein complexes were separated on-line by LC and sequentially introduced into the mass spectrometer. Peptides were identified by searching the uninterpreted product ion spectra against NCBI database using Mascot program (Matrix Sciences Ltd., London, U.K.) or the Biowork 3.1 (Finnigan, San Jose, CA). Figure 3 illustrates the examples of peptide analyses that contributed to the identification of two proteins (SERCA2a and ASF/SF2) in LC-MS/MS experiments. 3436

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In this case, automated collision-induced dissociation (CID) of the doubly charged precursor peptides with m/z 811.30 and 876.60 produced the MS/MS spectra shown in Figure 3. The MS/MS data were then formatted for query against NCBI protein database, which matched to sequences in two proteins, SERCA2a and ASF/SF2. The amino acid sequences, VGEATETALTCLVEK and DAEDAMDAMDGAVLDGR, derived from the protein SERCA2a and ASF/SF2, respectively, are consistent with the spectra and account for all the major “y” and “b” fragment ions shown in Figure 3 (fragmentation ions are labeled according to Biemann’s nomenclature25). In control experiments, LCMS/MS data obtained from extracts from Affi-GST control beads yielded only background noise with no conclusive protein identification except keratin, hemoglobin, and actin. A group of identified proteins was generated by including only those with at least two matching peptides (see Materials and Methods). As a result of this stringency, a list of more than 20 proteins was identified as associated partners (see Table 1). These proteins can be classified into several main categories, including the expected PP2A subunits, PP2A/A and PP2A/c (see Figure 1A); calcium handling proteins such as SERCA2a, sarcalumenin, and calmodulin; nuclear proteins including polymerase I transcript release factor, Y-box proteins, and nucleolin; proteins localized to nuclear speckles such as ASF/ SF2, ribosomal protein, and histones;26 and other proteins (peroxiredoxin, cysteine, and glycine-rich protein).

Local Signaling of Protein Phosphatase PP2A Gamma 1

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Figure 3. Examples of the LC-MS/MS identification of PP2A/B56γ1-interacting proteins: SERCA2a and ASF/SF2. (A). Shown is the MS/MS spectrum of [M + 2H]2+ m/z 811.30 that corresponds to the peptide VGEATETALTCLVEK assigned to the protein SERCA2a. (B). MS/MS spectrum of [M + 2H]2+ m/z 876.60 corresponding to the peptide DAEDAMDAMDGAVLDGR assigned to the protein ASF/SF2. Fragmentation ion assignments are based on nomenclature according to Biemann.25

Subcellular Localization of PP2A/B56γ1 in Cardiac Myocytes. If the group of associated binding partners identified in the proteomic analyses above is physiologically relevant, then they should co-segregate with B56γ1 in cardiomyocytes. Since specific B56γ1 antibodies are not available, HA-tagged B56γ1 was expressed in cultured adult ventricular myocytes, and its subcellular distribution was determined through immunofluorescent labeling of the epitope combined with laser scanning confocal microscopy. Although labeling of endogenous B56 protein would be an optimal approach, our previous studies have shown this adenovirus-based method leads to a 2-fold increase in total B56γ1 protein expression in cardiac myocytes.19 As shown in the whole cell image in Figure 4A, HAB56γ1 is highly enriched in the nucleus, consistent with a monopartite nuclear localization sequence motif (KIRRT) in the C-terminal domain.19 High-resolution images (Figure 4A, left images) revealed that B56γ1 distributes in a distinct nuclear punctuate pattern. We have recently reported that this pattern is a well-documented, specific, characteristic targeting of B56γ1 to subnuclear organelles in both neonatal and adult cardiac myocytes known as nuclear speckles.19,27 The lower nuclear image reveals that B56γ1 is localized to other nuclear regions beyond nuclear speckles.19 Taken together, this distinctive nuclear localization is consistent with the affinity proteomic results which identified 17 nuclear proteins including 7 found in nuclear speckles (Table 1). The whole cell image in Figure 4A reveals that B56γ1 also organizes in a banded pattern throughout the cell. Since this

pattern is reminiscent of sarcomeres, that include M-lines and Z-discs, double immunolabeling studies were performed to determine which myocardial structures are decorated by B56γ1. As shown in Figure 4A, the merging of confocal images demonstrates that B56γ1 co-localizes with staining for the L-type calcium channel, or dihydropyridine receptor (DHPR), an established marker for the junction of t-tubule/Z-disc structures at the light microscopy level of resolution. Control experiments were performed to confirm the specificity of the HA-B56γ1 staining. As shown in Figure 4B, when B56γ1expressing cells were stained in an identical manner except that anti-HA was preblocked by incubation with HA peptide, no fluorescent labeling was seen under identical imaging conditions. Other controls revealed no fluorescent labeling of cells infected with control virus and processed in an identical manner (data not shown). Taken together, the targeting of B56γ1 to the region of t-tubule/junctional sarcoplasmic reticulum agrees with SERCA2a localization seen in confocal imaging studies with ventricular cardiac myocytes.28 While compelling proof of colocalizations would require electron microscopic analysis of immunolabeled proteins, these results are consistent with SERCA2a as an associated binding partner of B56γ1 in intact cardiac myocytes. Validation of Mass Spectrometry (MS)-Based Proteomic Identification. If PP2A/B56γ1 is targeted to protein complexes defined in Table 1, a prediction is that overexpression of B56γ1 should alter the phosphorylation state of several of these proteins. Accordingly, to further validate the MS-based proJournal of Proteome Research • Vol. 6, No. 9, 2007 3437

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Figure 4. The subcellular localization of adenovirally overexpressed B56γ1 in adult rat cardiac myocytes. Cultures of adult rat ventricular myocytes were transfected with Ad-HA-B56 γ1 and, after 24 h, were developed for immunofluorescence imaging with anti-HA as described in Materials and Methods. (A) The central confocal image depicts a whole cell that reveals that B56γ1 is primarily in the nucleus while is also seen in a striated pattern throughout the cell. The high-resolution images of nuclei on left show that B subunit is localized to nuclear speckles (upper image) as well as other subnuclear regions (lower image). Shown at left is confocal anti-HA immunofluorescent image which shows that B56γ1 was localized not only to nuclei but also in striated structures as well. At right are images from confocal double immunolabeling experiment where cells were labeled for HA-B56γ1 (green) or an established marker for t-tube region in cells, anti-L type Ca2+ channel, or dihydropyridine receptor (DHPR) (red). The merged image shows that B56γ1 colocalized with DHPR along the Z-line/t-tubule region (yellow). (B) Control experiments were performed by processing HA-B56γ1-expressing cells in an identical manner except the primary antibody, anti-HA, preincubated with 1 µM HA peptide for 1 h prior to labeling. The upper panels show a transmitted light (left) and immunofluorescent confocal image of an experimental cell displaying HA-B56γ1 labeling. The lower panels show an identical experiment except cells were incubated with preblocked anti-HA during processing.

teomic results, protein phosphorylation studies were performed on proteins that are central to cardiac cell function in Table 1. Adenoviral-driven overexpression of B56γ1 was achieved in cultured cardiac myocytes as previously described.19 In the first set of experiments, Western blot analyses were used to assess the phosphorylation state of the identified nuclear specklelocalized ASF/SF2, a prototypical serine/arginine(SR)-rich RNAbinding protein.29 This RNA-binding protein is known to be a key regulator of pre-mRNA splicing during heart functional remodeling,30 and frequently exists in a highly phosphorylated state (hyper-ASF/SF2) in a range of cells.31 The Western blot in Figure 5A reveals that in control cardiac myocytes ASF/SF2 migrates as a broad band, an observation reported by others to reflect the multiple phosphorylated forms of the protein.32 When B56γ1 is overexpressed, there is a shift in the distribution of ASF/SF2 from the hyperphosphorylated state to faster migrating hypo-phosphorylated forms of the protein. This shift 3438

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is most clearly illustrated in the densitometric scans shown in Figure 5B. These results reveal that overexpression of B56γ1 does indeed lead to a shift in the phosphorylation state of ASF/ SF2 in heart cells supporting the biological relevance of the interactions discovered in the proteomic studies. Another binding partner identified through proteomic analysis with high statistical probability of 10 matching peptides was the cardiac sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) (see Table 1). This finding raised the question of how PP2A/B56γ1 might regulate phosphorylation of a SERCA2a-containing complex in heart. While SERCA2a itself is not known to be phosphorylated, the activity of this Ca2+ pump is controlled by phosphorylation-dephosphorylation cycles of Ser16 and Thr17 in the associated regulatory protein, phospholamban (PLB).33-36 Therefore, it is possible that PP2A/B56γ1 regulates the phosphorylation state of PLB rather than SERCA2a. While such a notion is confounded by the observation that the

Local Signaling of Protein Phosphatase PP2A Gamma 1

Figure 5. Western Blot analysis of phosphorylation status of ASF/ SF2 in B56γ1-overexpressing cardiac myocytes. Cultured neonatal rat cardiomyocytes were transfected with either adenovirus harboring HA-B56γ1 cDNA (Ad-B56γ1) or control virus with no insert. After 48 h, cultures were lysed and the proteins resolved on SDS gels in panel A. The upper blot was developed with antiHA to show specificity of epitope-tagged B56γ1 expression, while the lower blot shows the same samples developed with antiASF/SF2. The positions of hyper- and hypo-phosphorylated forms are indicated on right side. (B) The densitometric scans of the ASF/SF2 bands are shown with the positions of phosphorylated forms indicated on graph.

affinity-based proteomic analyses failed to detect PLB, it is likely that the 10 kDa cutoff concentrators used during the proteomic sample preparation failed to retain the 6 kDa PLB. This view was supported by Western blot analyses of the filtrate and retentate using anti-PLB antibodies (data not shown). To directly test the PLB dephosphorylation hypothesis, the levels of Ser16 and Thr17 phosphorylation were assessed in Western blots of homogenates of adult rat cardiomyocytes overexpressing B56γ1. As shown in Figure 6A, although the levels of total PLB and pThr17 were unaltered, phosphorylation of Ser16 was reduced by about 50% in the B56γ1 cells compared to control cultures. The summary results from three experiments are depicted in the histogram in Figure 6B. These results reveal that overexpression of B56γ1 reduces steady-state phosphorylation of PLB. They provide independent molecular information supporting the proteomic-based conclusion that B56γ1 has a biologically relevant association with a SERCA2a-containing macromolecular complex. Overexpression of B56γ1 in Adult Rat Myocytes Reduces Contractility. The proteomic and phosphorylation results combined with the subcellular distribution of B56γ1 to ttubule/Z-disc regions (Figure 4) suggest that B56γ1-targeted PP2A should regulate excitation-contraction (EC) coupling in cardiac myocytes. To test this notion, the contractile properties of cultured cardiac myocytes overexpressing B56γ1 were assessed by optical videomicroscopy. As shown in Figure 7, time course tracings of contraction in a field-stimulated cultured cell infected with control virus had maximal cell shortening of

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Figure 6. Western blot analysis of PLB phosphorylation in B56γ1overexpressing cardiac myocytes. Protein extracts of adult rat cardiomyocytes overexpressing B56γ1 or treated with control virus were resolved on 10% reducing NuPAGE Bis-Tris gels using MES buffer, and the blots were incubated with an antibody directed against PLB (total PLB) or with phosphorylation-specific antibodies against PLB phosphorylated at Ser16 (PLB-pS16) or Thr17 (PLB-pT17). (A) Representative Western blot extracts from either control or B56γ1 cultures as indicated. (B) The summary data from multiple experiments which was quantified by measurement of optical density of bands (arbitrary units) in scanned gels for both control cells (open bars) and B56γ1 cells (filled bars). The data are the means of 3 separate experiments ( SEM; * represents statistically different from control cell value, p < 0.05.

Figure 7. Contractile response of cultured rat ventricular myocytes overexpressing HA-B56 γ1. Cells were cultured on glass coverslips and treated with adenovirus constructs as described in Materials and Methods. Coverslips were mounted in flow bath on stage of microscope and field-stimulated at 1 Hz. Contractility was monitored with an IonOptix videomicroscopy system. Shown are time course tracings of the contractions of representative control and B56γ1-transfected cells in the initial steadystate, after 5 min of stimulation, and 3 min following the application of 1 µM isoproterenol (+Iso). The magnitude of twitch is expressed as % resting length (%RL). The striking result is that in the steady-state; there is a 70% reduction in fractional shortening in the B56γ1-cells compared to control (see left side traces). Interestingly, despite the low initial contractile state, the B56γ1 cells have a very large positive inotropic response to the β-adrenergic agonist, isoproterenol, restoring shortening to levels seen in control cells in isoproterenol (traces on right side).

about 5% of resting length, consistent with previous results with freshly isolated rodent ventricular myocytes,21 and nearly identical to the contractile shortening seen in parallel cultures where cells were untreated (see Table 2). In contrast, with B56γ1 cells, maximal fractional shortening in the steady-state is dramatically reduced by at least 65% as shown in lower tracing. The summary results are presented in Table 2, which demonstrates that maximal fractional shortening was dramatiJournal of Proteome Research • Vol. 6, No. 9, 2007 3439

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Table 2. Contractile Properties of Field-Stimulated Cultured Adult Ventricular Myocytes Overexpressing B56γ1 peak twitch contractions (% resting length)

Control Virus Cells B56γ1 Cells Untreated Cells

sarcomere length (µM)

steady state

isoproterenol (1 µM, 3 min)

1.71 ( 0.06 (n ) 7) 1.73 ( 0.02 (n ) 7) 1.80 ( 0.02 (n ) 2)

5.18 ( 0.96 (n ) 7) 1.73 ( 0.48 (n ) 7) 6.50 ( 0.9 (n ) 2)

11.64 ( 0.71 (n ) 7) 11.32 ( 0.95 (n ) 7) 15.45 ( 2.7 (n ) 2)

cally reduced for B56γ1 cells compared to either untreated or control virus-treated cells. Further, other control experiments revealed that this decrease in fractional shortening of B56γ1 cells was not due to increased contraction in basal state (i.e., diastolic contraction). This is demonstrated by optical analyses in Table 2 which showed that diastolic sarcomere length was identical (1.7 µm) in control virus and Ad-B56γ1-treated cells. If this functional response is caused by B56γ1-mediated “over-targeting” of PP2A to specific subcellular sites that control EC coupling, it may be possible to override the EC coupling dysfunction by robust stimulation of endogenous kinases. Accordingly, isoproterenol, a well-established β-adrenergic agonist and activator of PKA in heart cells,37 was applied to the cells. Consistent with this view, B56γ1 cells mounted a strong positive contractile response to β-adrenergic stimulation, restoring twitch amplitude to a level identical to that seen in both sets of isoproterenol-treated control cells. The summary data for this response are also presented in Table 2.

Discussion An emerging theme in the biology of the ubiquitous phosphatase, PP2A, is that its cellular function is governed, in part, by a B subunit-mediated targeting to specific macromolecular complexes.38-41 One such subunit in the B′ family, B56γ1, is preferentially expressed in heart,10,11 yet the role of the PP2A/ B56γ1 heterotrimer in cardiac signaling remains largely unknown. This study exploited an unbiased affinity-based proteomic approach combined with high stringency selection to identify a discrete set of associated binding partners for B56γ1 in cardiac cells. The identification of several nuclear proteins along with a sarcoplasmic reticulum Ca2+ pump complex is supported by subcellular localization studies which reveal that adenovirally overexpressed B56γ1 is principally a nuclear protein with some targeting to Z-line structures. Finally, the validity of the proteomic results was tested in a series of adenoviral-driven overexpression experiments with cardiac cells, which revealed that B56γ1 regulated the phosphorylation state of several associated proteins that are critical to cardiac cell function. Thus, the analysis of these identified proteins provides a global view of multiple PP2A/B56γ1-mediated dephosphorylation events that underlie cardiac cell signaling and function. Although the proteomics approach used in this study is potentially a powerful one, it was crucial to validate the results by independent methods. Importantly, the expected binding partners for B56γ1, PP2A/A, and PP2A/c appeared on the restricted list of identified proteins with very high probability, based on 8 and 11 matching peptides, respectively. The high stringency criteria also selected 17 nuclear proteins, which is 3440

Journal of Proteome Research • Vol. 6, No. 9, 2007

notable as the nuclear proteins comprise only a small percentage of total myocardial proteins. These results are consistent with a nuclear localization sequence in the C-terminal region of B56γ1 and the observation that this B subunit is principally a nuclear protein in cardiac myocytes (Figure 4). Thus, taken together, these findings support the conclusion that these identified binding partners are physiologically relevant. Phosphatases are much more important nuclear signaling enzymes than previously conceived.42 Yet, relatively little is known about the specific action of nuclear PP2A especially in cardiac cells. The results reported here of the proteomic analysis of PP2A/B56γ1 complexes reveal a number of associated nuclear proteins related to regulation of transcription and translation. For example, the identification of several RNA binding proteins, including Y-box proteins,43 dbpA (a member of the Y box family of proteins),44 MSY4,45 and nucleolin,46 suggests that PP2A may be involved in activation or repression of transcription. It is intriguing that most of these identified PP2A/B56γ1-interacting nuclear proteins are regulated by phosphorylation, such as Y-Box proteins,47 nucleolin,46 and histone H1.48,49 Interestingly, some of these identified proteins may be part of macromolecular complexes. For example, nucleolin and Y-Box protein are associated together to facilitate JNK-mediated interleukin-2 mRNA stabilization during T-cell activation.50 Further, more precise analyses with nuclear subproteomes will be required to identify these PP2A/B56γ1interacting protein networks in the nucleus. Since we have recently reported that B56γ1 is localized in nuclear speckles,19 an important result here was the identification of a cohort of nuclear speckle-localized associated proteins. Nuclear speckles are dynamic macromolecular suborganelles that contain splicing factors, along with nuclear ribonucleoproteins (RNPs), and serve as storage and/or activation sites for components of pre-mRNA splicing.27,51 Further, alternative splicing is controlled in part by cycles of protein phosphorylation and dephosphorylation.51 An important finding is that PP2A/B56γ1 is directly linked to this process, since an alternative splicing factor, the prototypical serine/arginine-rich (SR) protein, ASF/SF2, was identified as a binding partner. Dephosphorylation of SR alternative splicing factors results in their inactivation and their transport between nucleus and cytoplasm.52-54 Interestingly, ASF/SF2 is a key splicing regulator linked to hypertrophic cardiac growth and heart failure.30,55 Thus, the conclusions of proteomics results were tested in B56γ1 overexpression studies which reveal for the first time that PP2A/B56γ1 can regulate the phosphorylation state of ASF/ SF2 in intact cardiac myocytes. It is important to exploit mass spectrometric methods to identify the particular multiple phosphorylation sites that must exist on this protein. However, we could identify only two tryptic peptides of this lowabundance protein, neither of which contains Ser or Thr residues. Future studies will be directed toward obtaining more protein for such an analysis. Finally, in the future, it will be important to identify the mRNA splicing reactions that are regulated by this dephosphorylation event. An unexpected finding in the proteomic analysis was the identification of SERCA2a as an associated partner for B56γ1. In addition to SERCA2a, we also identified two more Ca-ATPase splice variants, cardiac slow twitch2 and SERCA3a (Table 1). SERCA2a is likely the only positive identification among the three Ca-ATPases, since all three are homologous by greater than 77%, including the MASCOT-matched sequences. Another identified protein, sarcalumenin, is likely part of this PP2A-

Local Signaling of Protein Phosphatase PP2A Gamma 1

SERCA2a complex.56 These proteomic-based conclusions are in accord with the new results here that adenovirally overexpressed B56γ1 localizes to Z-line structures in cardiac myocytes. It is important to note that a nearly identical cardiomyocyte Z-line distribution for SERC2a has been previously reported to be nearly identical to that of HA-B56γ1 reported here.28 One should interpret with caution localization based on virally driven expressed protein. However, we have previously shown that adenovirus based HA-B56γ1 expression results in only a 2-fold increase in B56γ1 in cardiac cells and that endogenous and HA-tagged B56γ1 both target to nuclear speckles.19 Clearly. the best approach would be to co-label both endogenous SERCA2a and B56γ1 to determine if they colocalize in cardiac myocytes. The lack of specific anti-B56γ1 precluded such analyses at this time. A priority for this study was to further validate these proteomic results with in situ phosphorylation studies. While SERCA2a itself is not known to be phosphorylated, the activity of this Ca2+ pump is controlled by phosphorylation-dephosphorylation cycles of Ser16 and Thr17 in the associated regulatory protein, phospholamban (PLB).33-36 An important finding here was that B56γ1 overexpression resulted in selective decreases in Ser16 phosphorylation of PLB in intact cardiac myocytes. While increases in PP2A activity and the loss in phosphorylation of Ser16 have been associated with reduced SERCA2a activity in heart failure,57 this is the first evidence for the role of PP2A targeting and B subunit function in SERCA2a regulation. The identification of several Ca2+ handling proteins, including SERCA2a, PLB, sarcalumenin, and calmodulin (Table 1), is intriguing, since calcium is the crucial signaling ion linking excitation to contraction in cardiac myocytes.1,58,59 These findings motivated a set of experiments that examined the effects of B56γ1 on contractility. Although more calcium signaling experiments need to be done, the marked decrease in contractility in B56γ1 overexpressing cells is consistent with the Ca2+ handling targets revealed in the proteomics analysis. In summary, this study demonstrates the utility of an affinity purification-based proteomic approach combined with the functional validation to identify physiologically relevant interacting complexes for the phosphatase PP2A targeting protein, B56γ1. While the details of each protein interaction remain to be defined, these results provide a global view of the diverse interactions of a single B subunit that target PP2A to multiple strategic sites within the cardiac cell. Abbreviations: PP2A, protein phosphatase 2A; HA, hemagglutinin; PBS, phosphate-buffered saline; IPTG, isopropyl-β-Dthiogalactopyranoside; MS, mass spectrometry; LC, liquid chromatography; SR, sarcoplasmic reticulum; EC, excitationcontraction; PLB, phospholamban; DHPR, dihydropyridine receptor; GST, glutathione S-transferase.

Acknowledgment. We thank Shirley Gaa and Marcus Palmer for expert technical assistance. We gratefully acknowledge the Applied Biosystems Mass Spectrometry Facility at The Johns Hopkins School of Medicine. We also thank Dr. Zhu Weizhong at NIH for providing PLB antibodies. This work was supported by the National Institutes of Health (AG14637; HL070709 to T.B.R.). References (1) Bers, D. M.; Guo, T. Calcium signaling in cardiac ventricular myocytes. Ann. N. Y. Acad. Sci. 2005, 1047, 86-98.

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PR060619L