Protein Phosphatase 2A Is Regulated by Protein Kinase Cα (PKCα)-dependent Phosphorylation of Its Targeting Subunit B56α at Ser41*

Background: PP2A activity and intracellular targeting are regulated by post-translational modifications of B56 phosphoprotein subunits. Results: PP2A is inhibited by a PKCα-dependent phosphorylation of B56α at Ser41 leading to downstream functional effects. Conclusion: This inhibition may represent an important signaling pathway regulated by stimuli that activate PKCα. Significance: Our data focus B56α on a dynamic role in the interplay between protein kinases and PP2A. Protein phosphatase 2A (PP2A) is a family of multifunctional serine/threonine phosphatases consisting of a catalytic C, a structural A, and a regulatory B subunit. The substrate and therefore the functional specificity of PP2A are determined by the assembly of the enzyme complex with the appropriate regulatory B subunit families, namely B55, B56, PR72, or PR93/PR110. It has been suggested that additional levels of regulating PP2A function may result from the phosphorylation of B56 isoforms. In this study, we identified a novel phosphorylation site at Ser41 of B56α. This phosphoamino acid residue was efficiently phosphorylated in vitro by PKCα. We detected a 7-fold higher phosphorylation of B56α in failing human hearts compared with nonfailing hearts. Purified PP2A dimeric holoenzyme (subunits C and A) was able to dephosphorylate PKCα-phosphorylated B56α. The potency of B56α for PP2A inhibition was markedly increased by PKCα phosphorylation. PP2A activity was also reduced in HEK293 cells transfected with a B56α mutant, where serine 41 was replaced by aspartic acid, which mimics phosphorylation. More evidence for a functional role of PKCα-dependent phosphorylation of B56α was derived from Fluo-4 fluorescence measurements in phenylephrine-stimulated Flp293 cells. The endoplasmic reticulum Ca2+ release was increased by 23% by expression of the pseudophosphorylated form compared with wild-type B56α. Taken together, our results suggest that PKCα can modify PP2A activity by phosphorylation of B56α at Ser41. This interplay between PKCα and PP2A represents a new mechanism to regulate important cellular functions like cellular Ca2+ homeostasis.

Protein phosphatase 2A (PP2A) is a family of multifunctional serine/threonine phosphatases consisting of a catalytic C, a structural A, and a regulatory B subunit. The substrate and therefore the functional specificity of PP2A are determined by the assembly of the enzyme complex with the appropriate regulatory B subunit families, namely B55, B56, PR72, or PR93/ PR110. It has been suggested that additional levels of regulating PP2A function may result from the phosphorylation of B56 isoforms. In this study, we identified a novel phosphorylation site at Ser 41 of B56␣. This phosphoamino acid residue was efficiently phosphorylated in vitro by PKC␣. We detected a 7-fold higher phosphorylation of B56␣ in failing human hearts compared with nonfailing hearts. Purified PP2A dimeric holoenzyme (subunits C and A) was able to dephosphorylate PKC␣-phosphorylated B56␣. The potency of B56␣ for PP2A inhibition was markedly increased by PKC␣ phosphorylation. PP2A activity was also reduced in HEK293 cells transfected with a B56␣ mutant, where serine 41 was replaced by aspartic acid, which mimics phosphorylation. More evidence for a functional role of PKC␣-dependent phosphorylation of B56␣ was derived from Fluo-4 fluorescence measurements in phenylephrine-stimulated Flp293 cells. The endoplasmic reticulum Ca 2؉ release was increased by 23% by expression of the pseudophosphorylated form compared with wild-type B56␣. Taken together, our results suggest that PKC␣ can modify PP2A activity by phosphorylation of B56␣ at Ser 41 . This interplay between PKC␣ and PP2A represents a new mechanism to regulate important cellular functions like cellular Ca 2؉ homeostasis.
Protein phosphatase 2A (PP2A) 2 is one of the major classes of serine/threonine protein phosphatases affecting the phosphorylation status of many phosphoproteins in different cell types. It represents nearly half of the total cellular serine/threonine phosphatase activity and has been linked to the regulation of cellular signaling and (patho)physiology. To its multiple functions belong the regulation of the cell cycle, apoptosis, signal transduction, DNA replication, and myocardial contractility (for reviews see Refs. [1][2][3]. During the last decade, PP2A emerged as an important regulator of oncogenesis and Alzheimer disease (4,5). The backbone of this functional importance is formed by the broad diversity of PP2A subunit combinations. The PP2A core enzyme is composed of a heterodimer, including a scaffolding A and a catalytic C subunit (PP2A CA ). The association of regulatory B subunits with the core dimer confers substrate specificity and intracellular targeting of the heterotrimeric PP2A holoenzymes. Up to now, four distinct families of regulatory B subunits have been described, B55 (B), B56 (BЈ), PR72 (BЉ), and PR93/PR110 (Bٞ). The B56 subunits are the most diverse, consisting of ␣ (PPP2R5A), ␤ (PPP2R5B), ␥ (PPP2R5C), ␦ (PPP2R5D), and ⑀ (PPP2R5E) isoforms, which are differentially expressed in many tissues and cell types (6,7).
B56 subunits are crucial to regulate the subcellular targeting of the PP2A heterodimeric core enzyme to specific substrates and subcellular domains (6,8). The targeting is mediated by specific adaptor proteins allowing a differential regulation of PP2A activity. For example, ankyrin-B co-localizes with B56␣ in cardiomyocytes leading to tethering of PP2A CA to cellular ion pumps and channels like the sodium/calcium exchanger, Na/K-ATPase, and inositol 1,4,5-trisphosphate receptor (9). Besides this critical role in PP2A targeting, B56 subunits can also act as receptors of second messengers as shown for the lipid ceramide (10). Finally, PP2A activity and subcellular localization are regulated by post-translational modifications of B56 subunits. It has been demonstrated that most of the B56 family members are phosphoproteins (6). Several protein kinases (e.g. PKA and PKR) have been reported to phosphorylate B56 subunits (11,12). In detail, the phosphorylation of B56␦ at Ser 566 by PKA increases the PP2A activity that catalyzes dephosphorylation of DARPP-32, thereby coordinating the efficacy of dopaminergic neurotransmission in striatal neurons (12). Moreover, PKA-dependent phosphorylation of B56␦, which is anchored to PDE4D3 by muscle A kinase-anchoring protein, promotes the dephosphorylation of this cAMP-specific phosphodiesterase (13). This inhibits PDE4D3 activity and thereby mediates a cAMP-induced positive feedback mechanism after activation of adenylyl cyclase and B56␦ phosphorylation.
Previous work has shown the phosphorylation of PP2A by PKC␣ at one of its regulatory B subunits (14). These authors detected a 55-kDa band that became phosphorylated in the presence of PKC␣ but were not able to identify the isoform of this B subunit. The classical PKC isotypes (e.g. PKC␣) display a physiological requirement for Ca 2ϩ and diacylglycerol (15). The cPKC family members are known to play an important (patho-)physiological role in regulating cellular functions, including proliferation, differentiation, apoptosis, oncogenesis, and myocardial/vascular smooth muscle contraction (16), indicating that cPKC isotypes and PP2A are acting on the same signaling pathways and molecular targets. Indeed, the activation of PKC␣ by the phorbol ester PMA was followed by the occurrence of a membrane-associated PP2A heterotrimeric complex resulting in the dephosphorylation and desensitization of the kinase (17). Thus, the aim of this study was the identification and characterization of the missing link between PKC␣ and PP2A as several studies raised the possibility that B56␣ might mediate the kinase-phosphatase interaction. Here, we report that PKC␣ inhibits PP2A via phosphorylation of B56␣ at Ser 41 , leading to an altered ER Ca 2ϩ release.

EXPERIMENTAL PROCEDURES
Materials-[␥-32 P]ATP was obtained from Hartmann Analytic GmbH. Sf21 insect cells were supplied by Invitrogen. HEK293 cells were obtained from ATCC-LGC Standards. PMA was used to activate PKC (Sigma). All other chemicals were of reagent grade. A polyclonal antibody for phospho-Ser 41 B56␣ was generated in rabbit and affinity-purified by use of a peptide pair of 12 amino acids, comprising residues 37-48 of human B56␣ (Perbio Science). The phospho-specific peptide was synthesized with a phosphoserine residue at position 41 of B56␣.
Human Ventricular Tissue-Left ventricular (LV) tissue was received from patients undergoing heart transplantation due to end-stage heart failure resulting from ischemic (ICM) or dilated cardiomyopathy (DCM) and from nonfailing (NF) hearts that could not be transplanted due to medical reasons or blood group incompatibility (18 Cloning of Expression Vectors-cDNA from human left ventricle (BioChain Institute Inc.) was amplified using Pfu DNA polymerase (Promega) and B56␣ primers as follows. The forward primer included 1 bp of the 5Ј-UTR, and the reverse primer extended to bp 31 of the 3Ј-UTR downstream of the translational stop codon. The amplified cDNA fragment was inserted into SalI of the pJET1 cloning vector (Thermo Fisher Scientific). This construct was used as template for subsequent cloning experiments. PCR was utilized for generation of a B56␣ fragment containing a 5Ј-NdeI and a 3Ј-engineered PstI restriction enzyme site. The amplified fragment was subcloned inframe into the corresponding cloning sites of pAcHLTA (BD Biosciences). This vector contains a His 6 tag nucleotide sequence upstream of the multiple cloning site. Alternatively, a TaqDNA polymerase-amplified PCR product, using primers that generate a His 6 tag nucleotide sequence upstream of the 5Ј end of B56␣, was directly inserted into the pCR2.1-TOPO (Invitrogen). The His 6 -B56␣ cDNA was then excised from the pCR2.1-TOPO vector with BamHI/NotI and subcloned into corresponding coning sites of pVL1393 (BD Biosciences). In parallel, we also constructed a pCR2.1-TOPO vector that contained B56␣ without the His 6 tag nucleotide sequence as well as the stop codon. After digestion with XhoI/BamHI, the resulting B56␣ fragment was subcloned into the multiple cloning site of the pAcGFP1-N1 expression vector (Clontech). Thus, B56␣ was fused in-frame to the N terminus of AcGFP1 (green fluorescent protein from Aequorea coerulescens). Finally, the originally constructed B56␣ PCR fragment was inserted into the SalI site of the pIRES2-DsRed-Express vector (Clontech). This vector allows the control of an efficient transfection and expression of the gene of interest by detection of red fluorescence.
Mutagenesis of B56␣-Mutagenesis of wild-type B56␣ was performed according to the protocol of Braman et al. (19). This strategy allows the mutation of B56␣ at single residues by direct use of the harboring cloning vector. In detail, 200 ng of DNA of either pAcGFP1-B56␣, pIRES2-DsRed-Express-B56␣, or pAcHLTA-B56␣ were amplified by DNA polymerase with proofreading activity (Expand Long Template PCR System, Roche Applied Science) and 27-mer complementary primer pairs, including mismatched bases (S41A sense primer, 5Ј-cgctcccagggcgcgtcgcagtttcgc-3Ј; S41D sense primer, 5Ј-cgctcccagggcgactcgcagtttcgc-3Ј). The sample was digested by DpnI for 2 h at 37°C. After preparative agarose gel electrophoresis, the product bands were excised, and nucleic acids were extracted and purified with the Invisorb Spin DNA extraction kit (Invitek). Escherichia coli DH10B were transformed and plated on selective medium (pAcHLTA, ampicillin; pAcGFP1 and pIRES2-DsRed-Express, kanamycin). Colonies grown overnight were inoculated to liquid LB culture (including antibiotic agent), and plasmid DNA was extracted. The insertion of the mutation was verified by sequencing of the B56␣ coding region.
Cell Culture, Transfection, and Fluorescence Microscopy-Sf21 insect cells were cultured at 27°C in Grace's medium containing 10% FBS (v/v), 50 g/ml gentamicin, and 2.5 g/ml amphotericin B. Sf21 insect cells were co-transfected with a transfer plasmid (pAcHLTA or pVL1393) carrying cDNA of wild-type or mutated B56␣ and linearized wild-type baculovirus cDNA using the BaculoGold TM kit (BD Biosciences). Recombinant baculoviruses were enriched by the plaque purification method (20) and used for infection of insect cells for recombinant expression of wild-type and mutated B56␣. For this purpose, 9 ϫ 10 6 cells were seeded into monolayer culture and then infected with recombinant baculoviruses encoding wild-type B56␣ or B56␣ mutants. 72 h after infection, the cells were centrifuged, and the corresponding pellets were stored at Ϫ20°C.
HEK293 cells or Flp293 cells (derived from HEK293 cells that stably express the human ␣ 1A -adrenoreceptor (21)) were cultured (37°C and 5% CO 2 ) in Dulbecco's modified Eagle's medium (Invitrogen) with 10% FBS (v/v). Cells were transfected with expression vectors pAcGFP1-N1 or pIRES2-DsRed-Express (100 -200 ng) carrying wild-type or mutated B56␣ using Lipofectamine 2000 (Invitrogen) following the manufacturer's protocol for transient transfection of adherent cells. 72 h after transfection, the degree of expression of the B56␣-GFP fusion protein was tested by detection of the fluorescence signal using confocal microscopy (Zeiss LSM 710), and cells were washed in phosphate-buffered saline and harvested. Expression of PP2A subunits in cell pellets was also controlled by SDS-gel electrophoresis and immunoblotting. For stimulation experiments, cells were incubated with 10 M phenylephrine (PE, agonist on ␣-adrenoceptors), 1 M propranolol (blocks ␤-adrenoceptors), and 100 nM PMA (dissolved in 0.0001% dimethyl sulfoxide, DMSO) for the final 4 h before lysis.
For detection of the IP 3 -related fluorescence, Flp293 cells were seeded on round 18-mm diameter glass slides in 12-well plates (Nunc) and transfected using TurboFect TM in vitro transfection reagent (Fermentas) according to the manufacturer's protocol. Cells were transfected with 1 g of pIRES2-DsRed-Express-B56␣ (wild-type or mutants S41A/S41D). 48 h post-transfection, cells were incubated with 4 M of the calcium indicator Fluo-4 AM (Invitrogen) in Ca 2ϩ -free Tyrode's solution (140 mM NaCl, 4 mM KCl, 1 mM MgCl 2 , 5 mM HEPES, 10 mM glucose, pH 7.4) for 10 min in the incubation chamber. Then the dye was washed out using Ca 2ϩ -free Tyrode's solution. Detection of Fluo-4 fluorescence was recorded using a Zeiss LSM 710 confocal fluorescence microscope and ZEN software (excitation at 488 nm and Fluo-4 emission at 493-552 nm). Cells transfected with wild-type or mutant B56␣ pIRES-dsRed-Express constructs were identified by detection of dsRed fluorescence (excitation at 543 nm and dsRed emission at 552-747 nm). A possible spectral cross-talk between Fluo-4 and dsRed emissions was excluded by linear unmixing of the signals. 60 s after the start of the experiment, 100 M PE in 2 mM Ca 2ϩ Tyrode's solution was directly applied into the incubation chamber resulting in a final concentration of 1 mM Ca 2ϩ and 50 M PE.
Luciferase Assay-Cultured HEK293 cells (see above) were maintained on 24-well plates (Corning Glass) and transfected using TurboFect TM in vitro transfection reagent (Fermentas) according to manufacturer's protocol. Cells were co-transfected with 200 ng of pAcGFP1-B56␣ (wild type or mutants), 200 ng of pAcGFP1-N1 as a negative control, 500 ng of the Photinus pyralis luciferase Icer promoter construct pP2Luc (22), and 50 ng of the control Renilla reniformis luciferase construct hRluc/TK (Promega) per well. 24 h post-transfection, cells were lysed and assayed using the Dual-Luciferase Reporter Assay System (Promega) as described in the manufacturer's protocol on a Mithras LB 940 microplate analyzer (Berthold Technologies). All luciferase results were normalized to the Renilla luciferase activity as internal control.
Affinity Purification of Tagged B56␣-Sf21 insect cell pellets were resuspended in a buffer (50 mM Tris-HCl, 150 mM NaCl, 10 mM imidazole, pH 8.0) containing lysozyme, benzonase, and a proteinase inhibitor mixture (Roche Applied Science). Fragmentation of cells was achieved by sonification on ice. Soluble and insoluble fractions were separated by centrifugation at 20,000 ϫ g for 30 min (4°C). The purification of His 6 -tagged wild-type or mutated B56␣ was performed by use of Ni-NTA affinity column chromatography according to the manufacturer's instructions (Qiagen). The purification procedure was controlled by loading small samples of all fractions on 8% polyacrylamide gels that were stained with Coomassie dye. All elution fractions were pooled, desalted, and concentrated by Amicon TM ultracentrifugal filter units (Millipore). The recombinant proteins were found to be ϳ95% pure by use of this method.
Phosphorylation of B56␣ by PKC␣-Recombinant wild-type or mutated B56␣ was phosphorylated by PKC␣ in a Ca 2ϩ -dependent manner. The standard reaction mixture contained 25 mM Tris-HCl, pH 7.5, 5 mM MgCl 2 , B56␣ (0.5-2 g per reaction), 5 mM NaF, 100 M ATP, [␥-32 P]ATP, 0.5 mM CaCl 2 , 100 g/ml phosphatidylserine, and 20 g/ml diolein. The reaction was initiated by addition of 0.2 l of recombinant PKC␣ (Sigma), incubated up to 60 min at 30°C, and terminated by addition of ice-cold EDTA (200 mM, pH 8.0). For determination of PKC activity, an aliquot was spotted on P81 phosphocellulose papers (Whatman), which were washed four times with 75 mM phosphoric acid. Filters were dried, and radioactivity was determined in a liquid scintillation counter. For visualization of PKC␣-dependent phosphorylation of wild-type and mutated B56␣, an aliquot of the reaction was added to 2.5% SDS buffer for the following separation of solubilized protein on 8% polyacrylamide gels. Gels were Coomassie-stained after electrophoresis, dried on a gel dryer (Bio-Rad), exposed for 24 h, and then analyzed by use of a STORM 860 (GE Healthcare). Moreover, aliquots of PKC␣-phosphorylated B56␣ reactions, where NaF was omitted from the mixture, were dephosphorylated subsequently by either recombinant catalytic subunit of PP1 (Sigma) or purified heterodimeric PP2A CA (Millipore), consisting of the catalytic C and scaffolding subunit A. Where indicated, dephosphorylation was performed in the absence or presence of 500 M inhibitor-2 of PP1 or 3 nM okadaic acid (inhibiting PP2A without affecting PP1 activity). Samples were analyzed by subjection to SDS-PAGE (see above) or detection of protein phosphatase activity.
Mass Spectrometry Analysis-Mass spectrometric (MS) analysis was performed using nanoAcquity UPLC (column BEH130 C 18 inner diameter 100 m, length 100 mm, pore size 130 Å, particle size 1.7 m, 30 min water/acetonitrile gradient) coupled to Q-Tof Premier (Waters Corp.). For best sensitivity, the protein content of one individual gel band (after tryptic in-gel digestion) was used for the detection of a putative phosphorylation site by tandem MS on the selected m/z value.
Protein Phosphatase Assay-Protein phosphatase activity was assayed using [ 32 P]phosphorylase a as substrate as described previously (23). Protein phosphatase activity was determined on purified heterodimeric PP2A CA in the absence or presence of PKC␣-(non)phosphorylated recombinant wild-type or mutated B56␣. In addition, protein phosphatase activity was also measured in HEK293 cells transfected with cDNA of wild-type or mutated B56␣. Briefly, cell pellets were sonicated at 4°C six times for 10 s in buffer containing 4 mM EDTA, pH 7.4, and 15 mM 2-mercaptoethanol. Lysed cells were centrifuged at 14,000 ϫ g for 20 min at 4°C, and the supernatants were used for determination of phosphorylase phosphatase activity. The dephosphorylation reactions were initiated by adding [ 32 P]phosphorylase a to a final concentration of 0.5 mg/ml (40,000 cpm/nmol) and carried out at 30°C for 20 min, except that time-dependent measurements were performed up to 40 min. Where appropriate, 3 nM okadaic acid was added before the initiation of the reaction. No more than 18% of the substrate was utilized in the assay to ensure linearity of the reaction.
Pulldown Assay-The amount of [ 32 P]phosphorylase a binding to B56␣ was determined by incubation of nonphosphorylated or PKC␣-phosphorylated His 6 -tagged wild-type B56␣ (4 g each) with purified heterodimeric PP2A CA under conditions described above. After addition of [ 32 P]phosphorylase a, the reaction mixture was incubated for 40 min at 30°C and then added to 25 l of Ni-NTA-agarose beads. Further incubation was performed for 2 h at 4°C on a rocking platform. After centrifugation at 14,000 ϫ g for 2 min, beads were washed three times with PBS, pH 7.4. Finally, bound His 6 -tagged B56␣ was eluted with 400 mM imidazole. SDS-solubilized eluted proteins were subjected to 10% polyacrylamide gels. The upper part of the gel was stained with Coomassie dye, dried, and then exposed for 24 h for further analysis of B56␣-bound 32 P-labeled phosphorylase a. The expression level of utilized B56␣ was tested by SDS-gel electrophoreses and subsequent immunoblotting.
Immunoprecipitation-Mouse cardiomyocytes were isolated as described previously (24). After incubation with 100 M phenylephrine, 1 M propranolol, and 100 nM PMA for 30 min at 37°C, cells were shock-frozen in liquid nitrogen. Cardiomyocytes were resuspended in 4°C cold PBS supplemented with 0.5% Triton X-100 and a protease inhibitor/phospho-STOP mixture (Roche Applied Science). Cell lysis was achieved by sonification six times for 10 s on ice. Thereafter, the homogenate was held on ice for an additional 30 min. Soluble and insoluble fractions were separated by centrifugation (14,000 ϫ g, 1 h, 4°C). For preclearing, the supernatant (0.5-1 g/l) was incubated with a mixture of protein A/G beads (Millipore) and 2 g of normal rabbit IgG (Millipore) for 1 h at 4°C. After centrifugation at 14,000 ϫ g for 5 min, the supernatant was incubated overnight with a polyclonal antibody against B56␣ (Bethyl, 2 g/mg supernatant protein) at 4°C on a rocking platform. Protein A/G beads were added for an incubation period of 2 h at 4°C. After centrifugation, beads were washed consecutively (three times) with PBS. The SDS-solubilized supernatant above the pelleted beads was processed for immunoblotting and anal-ysis of the phosphorylation status using the ProQ Diamond phosphoprotein stain (18).
Immunoblot Analysis-Protein expression analysis was performed on the following probes: 1) homogenates of wild-type and mutated B56␣ expressed in Sf21 insect (as His 6 -tagged proteins) or HEK293 cells (as GFP fusion proteins); 2) purified wild-type and mutated recombinant B56␣ (by Ni-NTA affinity column chromatography) and heterodimeric PP2A CA (Millipore); 3) immunoprecipitated mouse B56␣; 4) mouse heart lysates; and 5) ventricular homogenates of nonfailing and failing human hearts. To this end, Sf21 and HEK cell pellets were resuspended in a buffer containing 30 mM histidine, pH 7.4, 250 mM sucrose, and a protease inhibitor mixture (Roche Applied Science). Lysed homogenates were mixed with equal volumes of reducing 5% SDS sample buffer and boiled for 5 min before loading. Purified or immunoprecipitated B56␣ as well as PP2A CA were also solubilized in SDS sample buffer for further processing by gel electrophoresis. In addition, 50 mg of mouse heart tissue were homogenized at 4°C for 1 min in 0.5 ml of a medium containing 5% SDS and 10 mM NaHCO 3 using a Polytron PT-10 (Kinematica). Homogenates were centrifuged at 14,000 ϫ g for 20 min, and supernatant lysates were subjected to SDS-gel electrophoresis. Finally, human heart homogenates were prepared from frozen LV tissue as described previously (18). For immunoblot analysis of all proteins, 100 -200 g of individual samples were electrophoretically separated on 8 or 10% SDS-polyacrylamide gels (25,26). After transfer of proteins to nitrocellulose, the blots were incubated with different antibodies raised against the following proteins: human B56␣ of PP2A (Bethyl, amino acids 25-75), human B56␣ of PP2A (Acris, amino acids 417-446), human phosphoserine 41 B56␣ (custom-made), human B56␤ of PP2A (Thermo Scientific, amino acids 434 -497), human B56␥ of PP2A (Bethyl, amino acids 490 -524), human B56␦ of PP2A (Bethyl, amino acids 1-50), mouse A␣ of PP2A (Santa Cruz Biotechnology), human C␣ of PP2A (PTG), canine calsequestrin (24), rabbit GAPDH (Ambion), and Tetra-His epitopes (Qiagen). The amounts of bound antibodies were detected by use of secondary antibodies (ECL rabbit/goat IgG, HRP-linked whole antibody, GE Healthcare). Signals were visualized and quantified with the ECL Plus detection system (ECL Plus, GE Healthcare) and the STORM blot imaging system, respectively.
Quantitative RT-PCR-Total RNA was extracted from transfected HEK293 cells with the use of TRIzol (Invitrogen). Total RNA (1 g) was randomly reversely transcribed to cDNA using Transcriptor First Strand cDNA synthesis kit (Roche Applied Science). The real time RT-PCR was carried out using a Light-Cycler 480 System (Roche Applied Science), and detection was performed by the use of carboxyfluorescein-labeled universal ProbeLibrary probes (UPL, Roche Applied Science). Primers (Invitrogen) were designed using ProbeFinder Assay Design Center (Roche Applied Science). The PCRs were set up in a 96-well plate in a volume of 20 l. The reaction components were 1 l of undiluted cDNA, 10 l of LightCycler 480 Probes Master, 4 l of H 2 O, and 0.8 l for each primer and 0.4 l for the UPL probe (all 10 M). Reactions were incubated at 95°C for 10 min followed by 45 cycles at 95°C for 10 s, 60°C for 30 s, and 72°C for 1 s. Relative levels of particular cDNAs were deter-mined with the help of LightCycler 480 software with appropriate calibration curves obtained with different amounts of control cDNAs. Crossing points were determined by using the second derivative method. Relative quantification was performed by calculating relative expression ratios using the ⌬⌬C T method and the relative expression software tool (REST© Version 2.013; see Ref. 27,28). Random statistical analysis was performed with 10,000 iterations, and hypoxanthine-guanine phosphoribosyltransferase (Hprt) was used as a reference gene.
Statistical Analysis-Statistical differences between groups were calculated by analysis of variance or Kruskal-Wallis oneway analysis of variance on ranks followed by Bonferroni's or Dunn's post hoc tests, respectively. p Ͻ 0.05 was considered significant. Statistical analysis of real time PCR data were performed using REST© software (REST© Version 2.0.13; see Refs. 27,28). Random statistical analysis was performed with 10,000 iterations.

Identification of Ser 41 as a Novel PKC␣ Phosphorylation Site-
To study whether the regulatory subunit B is phosphorylated by PKC in vivo, isolated cardiomyocytes were treated with phenylephrine, propranolol, and PMA to achieve a maximum activation of PKC␣ (representing the main cardiac isoform). B56␣ was enriched in supernatants of lysed cells by immunoprecipitation, eluted from protein A/G beads, and separated by SDS-gel electrophoresis. The prominent phosphorylated mobility form detected by ProQ Diamond phosphoprotein stainings at a molecular mass of 56 kDa corresponds to mouse B56␣ (Fig. 1A). This was the first indication of a PKC-dependent phosphorylation of B56␣. In the next step, we tested whether B56␣ is also phosphorylated in vitro. For this purpose, we generated recombinant B56␣ by an insect cell expression system. The N-terminal His 6 -tagged B56␣ was purified by Ni-NTA affinity column chromatography. An example of a Coomassie-stained gel of the purification procedure is given for wild-type B56␣ (Fig. 1B). After elution, a single band at the expected height of the fusion protein was present in the desalted and concentrated fraction. This fraction was used for further expression analysis by immunoblotting using specific antibodies directed against the N-terminal region of B56␣ or the His 6 tag (Fig. 1C).
The purified recombinant B56␣ was subjected to in vitro phosphorylation by PKC␣ in the presence of [␥-32 P]ATP. The phosphorylation of B56␣ by PKC␣ was time-dependent ( Fig.  2A) reaching its maximum after 30 min. The maximal phosphorylation stoichiometry obtained after 30 min of incubation with PKC␣ was ϳ2.5 mol of phosphate incorporated per mol of B56␣ either in the absence or presence of purified heterodimeric PP2A CA . The 32 P-labeled wild-type B56␣ was detected by SDS-PAGE in an aliquot of the 60-min fraction ( Fig. 2A). To identify the site of 32 P incorporation into B56␣, we subjected the PKC␣-phosphorylated protein to MS analysis specifically targeting peptides of interest. For peptide 38 SQGSSQFR 45 , phosphorylation was indicated at its fourth amino acid, Ser 41 , by the gas phase fragmentation experiment (m/z 488.7 in Fig.  2B). This amino acid residue was predicted as a potential PKC␣ phosphorylation site by GPS 2.1, a group-based pre-diction system for kinase-specific phosphorylation sites (Fig.  2C) (29). Of note, this residue as well as adjacent amino acids are conserved between mammalian species, birds, fish, and amphibians (Fig. 2C).
The newly identified PKC␣ phosphorylation site was confirmed by mutagenesis of the Ser 41 residue to alanine (S41A). After a 30-min incubation period, the incorporation of 32 P into the B56␣ mutant S41A was reduced by 80% compared with the wild-type form (Fig. 3A). The remaining 32 P signal may result from unspecific phosphorylation of non-PKC sites. Moreover, we cannot exclude the presence of additional PKC␣ phosphorylation sites, which so far has possibly eluded detection due to the low abundance of phosphorylated peptides in the protein digest. Nevertheless, this has no impact on the inhibitory potency of B56␣ on PP2A. The PKC␣-dependent phosphorylation of B56␣ was also confirmed by an antibody specific for FIGURE 1. Phosphorylation of cardiomyocytes by PKC␣ and expression of recombinant B56␣. A, isolated mouse cardiomyocytes were lysed, homogenized, and then centrifuged resulting in a soluble (input) and insoluble (pellet) fraction. The soluble supernatant was incubated with a specific polyclonal antibody to precipitate mouse B56␣ (eluate). Phosphorylation signals of separated proteins were visualized by a ProQ Diamond stain. Total protein loading was examined by use of a SYPRO Ruby stain. An aliquot of all fractions was subjected to immunoblotting (IB) using a specific antibody directed against B56␣. PM, peppermint marker; LMW, low molecular weight marker. B, shown is a Coomassie dye of a Ni-NTA affinity chromatography purification for the supernatant fraction of insect cell cultures expressing His 6 -tagged human wild-type B56␣. Concentrated (Conc) elution fractions were used for the following detection step. C, expression of wild-type (WT) B56␣ was controlled by gel electrophoresis and subsequent immunoblotting. Blots were probed with antibodies specific for the N terminus (amino acids 25-75) of human B56␣ (upper panel) or tetra-His (lower panel) as described under "Experimental Procedures." Homogenates of insect cells infected with wild-type baculovirus were used as a control.
the phosphorylated Ser 41 site of human B56␣. The B56␣ phosphoserine 41 antibody recognized recombinant wild-type B56␣ phosphorylated with PKC␣ (Fig. 3B). The nonphosphorylated wild-type B56␣ was also detected by this antibody. However, this was to a much lesser extent, suggesting a nonexclusive specificity for phosphoserine 41 of B56␣. Antibodies against the C-terminal region (Fig. 3B) or N-terminal region (data not shown) of B56␣ detected a comparable loading of nonphosphorylated and PKC␣-phosphorylated protein samples. The phospho-specific antibody was also studied on homogenates of human LV tissue of nonfailing and failing hearts (Fig. 3C). We detected a higher phosphorylation level of B56␣ at Ser 41 in failing hearts suffering from ischemic or dilated cardiomyopathy compared with nonfailing hearts (Fig. 3D). In contrast, the expression of total B56␣ was reduced by 61% in DCM hearts (Fig. 3E). Thus, the ratio of phosphorylated to total B56␣ was increased by 220 and 600% in ICM and DCM hearts, respectively, compared with NF hearts (Fig. 3F). The expression of GAPDH, which served as a control, was unchanged between all groups (data not shown).  ). B, purified recombinant wild-type B56␣ (2 g) was phosphorylated by PKC␣ and subjected to mass spectrometry. MS/MS spectrum exhibits phosphorylation of Ser 41 . The phosphorylated sample was separated on a gel, then excised from the gel, and subjected to tryptic digestion. The peptide mixture was interrogated for phosphorylated species using targeted peptide sequencing on selected masses. The base peak chromatogram (BPI) shows the low relative concentration of phosphorylated peptide SQGSSQFR. The m/z (x axis) and relative intensity (y axis) of the signals are shown. C, part of the B56␣ amino acid sequence deduced from the cDNA sequence of different species is reported in single-letter code. Identical residues are highlighted in black, and variant residues from the human sequence are highlighted in gray. The arrow indicates Ser 41 , and the asterisks denote phosphorylatable serine residues.

Phosphorylation of B56␣ by PKC␣ Is Reversed by PP2A-To
investigate whether the phosphorylation of B56␣ can be reversed, PKC␣-phosphorylated wild-type B56␣ was incubated with different protein phosphatases. Dephosphorylation of B56␣ was reversed by PP2A but not PP1, another major serine/ threonine protein phosphatase (Fig. 4A). The specificity of a PP2A-dependent dephosphorylation was further confirmed by preincubation with 3 nM okadaic acid, which totally blocks the enzyme activity. In addition, the inhibition of PP1 by one of its potent endogenous inhibitors, inhibitor 2, had no influence on the phosphorylation level of wild-type B56␣ (Fig. 4A). PP2A subunits of the dephosphorylation reaction were detected by immunoblotting using specific antibodies (Fig. 4B). The ratio of all PP2a subunits, namely the purified heterodimeric PP2A CA and the recombinant wild-type B56␣, is denoted. Mouse heart homogenate was loaded as a control demonstrating that the tagged recombinant B56␣ fusion protein runs higher on SDS gels than endogenous native B56␣ (Fig. 4B).
PKC␣-dependent Phosphorylation of B56␣ Augments Its Inhibition on PP2A-To test whether B56␣ can influence the PP2A activity per se, we incubated different amounts of recombinant wild-type B56␣ with purified heterodimeric PP2A CA . B56␣ inhibited PP2A activity on phosphorylase a in a concentration-dependent manner (Fig. 5A). (1 g each) were exposed to PKC␣ in the presence of [␥-32 P]ATP. The reaction was terminated by addition of SDS sample buffer, and solubilized proteins were separated on 8% polyacrylamide gels. Gels were Coomassie-stained after electrophoresis and then treated as described under "Experimental Procedures." Shown are an autoradiography of radiolabeled phosphoproteins (upper panel) and the quantification of 32 P incorporated into B56␣-WT/S41A (lower panel). *, p Ͻ 0.05 versus WT. B, recombinant wild-type B56␣ (1 g) was phosphorylated in vitro by PKC␣. For controls, PKC␣, Ca 2ϩ , diacylglycerol, and phosphatidylserine were omitted from the reaction mixture. After SDS-PAGE, the separated proteins were transferred to nitrocellulose membranes. The blot was consecutively probed with a phospho-specific antibody raised against Ser 41 of B56␣ (upper panel) and with an antibody raised against the C-terminal region of B56␣, detecting the total level of the recombinant protein (lower panel). C, expression of PKC␣-phosphorylated (pSer 41 ) and native B56␣ were measured in failing human hearts. Representative immunoblots, including GAPDH as a loading control, are given. B56␣ phosphorylated at Ser 41 (D) and total B56␣ (E) were identified in homogenates of LV tissue received from patients suffering on ICM or DCM and from NF hearts with specific antibodies. F, ratio of phospho-Ser 41 to total B56␣ was determined. *, p Ͻ 0.05 versus NF; ϩ, p Ͻ 0.05 versus ICM. , respectively. Samples were analyzed by subjection to SDS-PAGE and Coomassie staining after electrophoresis. Dried gels with 32 P-labeled B56␣ were exposed, and the autoradiographies are shown. B, detection of PP2A subunits was performed by subjection of the dephosphorylation reaction (including both purified wild-type B56␣ and heterodimeric PP2A CA ) and mouse heart lysates as a control to 8% SDS-PAGE. Immunoblotting was performed to detect the scaffolding (A␣), the catalytic (C␣), or regulatory subunit (B56␣). Shown is the ratio of PP2A subunits under normal reaction conditions (1ϫ) and higher loading (25ϫ). rec., recombinant B56␣; nat., native (endogenous) B56␣.
Wild-type B56␣ displayed high inhibitory activity on PP2A CA with an IC 50 of 2 nM. PP2A activity was completely inhibited by wild-type B56␣ at concentrations of ϳ100 nM. In the next step, we studied whether phosphorylation by PKC␣ can modulate the inhibitory effect of B56␣ on PP2A activity. The PKC␣-phosphorylated form shifted the dose-response curve to the left resulting in an IC 50 of 0.5 nM (Fig. 5A). To evaluate the mechanism(s) of PP2A inhibition, we measured the interaction between [ 32 P]phosphorylase a and B56␣ in the presence of purified PP2A CA . The binding of 32 P-labeled phosphorylase a was lower in PKC␣-phosphorylated B56␣ compared with nonphosphorylated B56␣ (Fig. 5B). The assay was performed under conditions where the difference in PP2A inhibition was maximal between nonphosphorylated and phosphorylated B56␣. The B56␣ mutant S41D reduced PP2A activity on phosphorylase a to a comparable left shift as seen under application of PKC␣-phosphorylated wild-type B56␣ (Fig. 5C). Therefore, S41D mimics phosphorylated wild-type B56␣. S41D exhibited a high inhibitory activity on PP2A CA with an IC 50 of 0.5 nM, which is the same potency as measured for phosphorylated wild-type B56␣. These data suggest that the activity of the heterodimer PP2A CA depends directly on the PKC␣-mediated phosphorylation of B56␣. The mutant S41A, lacking the PKC␣ phosphorylation site, had an inhibitory potency similar to that of wild-type B56␣, which under the same condition was ϳ1.5 nM (Fig. 5D). When Ser 41 of B56␣ was mutated to alanine, the inhibitory effect of PKC␣ phosphorylation on PP2A activity was abolished (Fig. 5D), indicating that this phosphorylation site is crucial for regulation of PP2A by PKC␣-dependent phosphorylation of B56␣. In addition, recombinant (non)phosphorylated wild-type B56␣ had no effect on PP1 activity (data not shown).
Phosphorylation of B56␣ at Ser 41 Inhibits PP2A Activity in Vivo-The effects of PKC␣-dependent B56␣ phosphorylation at Ser 41 on type-2A phosphatase activity were further confirmed in vivo by measuring total protein phosphatase activity in HEK293 cells transfected with the expression vector pAcGFP1 carrying either wild-type or mutated B56␣ (S41A or S41D). The construction of the expression vector allows the visual control of the transfection efficiency by GFP fluorescence (Fig. 6A). By use of this vector, we achieved abundant expression of recombinant B56␣-GFP fusion proteins (Fig. 6B). The endogenous B56␣ expression in HEK293 cells was not influenced by transfection of the empty vector, and it was not detectable in cells exhibiting expression of recombinant B56␣. The expression of exogenous B56␣ (Table 1) and of endogenous scaffolding A␣ and catalytic C␣ subunits was not different between wild-type and mutated B56␣ (Fig. 6C). Moreover, exogenous B56␣ did not affect the protein expression of other B56 subunits (Fig. 6D and Table 1). The mRNA expression of single B56 subunits was only slightly reduced in transfected HEK293 cells expressing wild-type B56␣ or S41A (Table 1). Confocal microscopy revealed that recombinant B56␣ is located mainly in the cytosol of transfected HEK293 cells (Fig.  6E). To determine whether the depression in protein phosphatase activity associated with phosphorylation of B56␣ at Ser 41 corresponds to similar changes in an in vivo system, total phosphatase activity was measured in homogenates of transfected HEK293 cells (Fig. 6F). Stimulation of PKC by phenylephrine and PMA (30) resulted in a reduced total phosphatase activity in cells transfected with wild-type B56␣. This decrease was abolished in homogenates of HEK293 cells expressing S41A. When cells were transfected with pAcGFP1 carrying the B56␣ mutant, S41D, total phosphatase activity was already depressed under basal conditions to the same level as observed in HEK293 cells transfected with wild-type B56␣ after stimulation. Pharmacological stimulation of S41D-transfected HEK293 cells was not able to further augment the inhibition of total phosphatase activity (Fig. 6F).
Protein phosphatase activity in transfected HEK293 cell homogenates was also assayed in the presence of okadaic acid, which allows the discrimination between PP1 and PP2A activity. Purified PP2A CA was completely inhibited at a concentration of 3 nM okadaic acid, which selectively inhibits PP2A (Fig. 7A). Thus, the remaining protein phosphatase activity in HEK293 cells, transfected with the empty pAcGFP1 vector, represents PP1 activity. By use of 3 nM okadaic acid, we found decreased PP2A activity in wild-type B56␣-transfected HEK293 cells after stimulation of PKC with both phenylephrine and PMA (Fig.  7B). This effect on PP2A activity was abolished in cells expressing S41A. Consistently, expression of S41D, the pseudophosphorylated form of B56␣, was associated with a depressed PP2A FIGURE 5. PKC␣-phosphorylated B56␣ inhibits PP2A activity. A, increasing amounts of recombinant (non)phosphorylated wild-type (WT or WT-p) B56␣ were added to purified heterodimeric PP2A CA , and protein phosphatase activity was measured as described under "Experimental Procedures." Curves were fitted for analysis of inhibitory concentrations (IC 50 , dashed line). B, PP2A was incubated in the absence (input) and presence of nonphosphorylated or phosphorylated His 6 -tagged wild-type B56␣ (4 g each). After coupling of tagged B56␣ species to Ni-NTA-agarose, the amount of bound 32 Plabeled phosphorylase a (Phos a) was detected. The immunological detection of bead-coupled B56␣ was used as a control (Ctr.). C, PP2A activity was determined in response to recombinant wild-type or pseudophosphorylated (S41D) B56␣. The dashed line indicates half-maximal PP2A activity. D, recombinant (non)phosphorylated mutated (S41A or S41A-p) B56␣ was added to purified heterodimeric PP2A CA for measurement of protein phosphatase activity.
activity under basal conditions compared with wild-type B56␣ (Fig. 7B). Under conditions of maximal PKC stimulation, no additional effect on PP2A activity was measurable. Taken together, these findings indicate that phosphorylation of B56␣ at Ser 41 and resulting regulation of PP2A occurs not only in vitro but also in living cells.
Phosphorylation of B56␣ at Ser 41 Increases IP 3 -mediated ER Ca 2ϩ Release-Finally, the functional relevance of PKC␣-dependent phosphorylation of B56␣ was examined in regard to downstream cellular signaling. For this purpose, ␣ 1A -adrenoreceptor-expressing Flp293 cells were transfected with wild-type or mutated B56␣ expression constructs using the pIRES2-DsRed-Express vector that allows a discrimination between transfected (red fluorescence) and nontransfected control cells (Fig. 8A). The IP 3 -induced Fluo-4 fluorescence was detected in transfected and nontransfected cells under application of 50 M phenylephrine (Fig. 8B). Transfection of Flp293 cells with wildtype B56␣ resulted in a 22% lower IP 3 -mediated Ca 2ϩ release FIGURE 6. Expression of GFP-tagged B56␣ in HEK293 cells. A, shown is a schema of the construct for expression of the B56␣-GFP fusion protein in HEK293 cells. The cDNA of either wild-type or mutated (S41A or S41D) B56␣ was subcloned in-frame into the multiple cloning site of the pAcGFP1 expression vector. B-D, expression of PP2A subunits was tested by immunoblotting in HEK293 cells transfected with either pAcGFP1-N1 alone (AcGFP) or the expression vector carrying wild-type or mutated B56␣. Antibodies used were directed against the regulatory subunit B56␣ (B and C), the catalytic subunit C␣, the scaffolding subunit A␣ (C), and the regulatory B56␣ subunits ␤, ␥, and ␦ (D). E, fusion of wild-type or mutated B56␣ to GFP allowed the expression control in HEK293 cells by confocal microscopy. The left panel shows the morphology of native nontransfected HEK293 cells, and the middle and right panels exhibit fluorescence in cells transfected with pAcGFP1 and pAcGFP1-B56␣-WT, respectively. F, total protein phosphatase activity was measured under basal conditions and after stimulation with 10 M phenylephrine and 100 nM PMA in HEK293 cells transfected with wild-type or mutated (S41A or S41D) B56␣ cDNA. *, p Ͻ 0.05 versus basal; ϩ, p Ͻ 0.05 versus corresponding WT group.

TABLE 1 Quantification of B56 subunit mRNA and protein levels in transfected HEK cells
Calculation of relative expression ratios using Hprt mRNA as a reference gene and statistical randomization test analysis were performed by application of the relative expression software tool (REST 2009© Version 2.0.13 ).
*, p Ͻ 0.05 versus pAcGFP1-N1 control vector (Ctr.). measured as the F 1 /F 0 peak amplitude of individual Ca 2ϩ spikes (Fig. 8C). Lack of the PKC␣ phosphorylation site by mutation of Ser 41 to alanine resulted in an ER Ca 2ϩ release amplitude that was comparable with the wild-type form of B56␣. This is consistent with the similar basal inhibitory activity of both nonphosphorylated B56␣ isoforms (wild type and S41A) on PP2A CA (Fig. 5, A and D) and the comparable PP2A activity under basal (nonstimulated) conditions in HEK cells transfected with wild-type and mutated (S41A) B56␣ (Fig. 7B). We co-transfected Flp293 cells also with a pIRES2-DsRed-Express construct expressing the B56␣ mutant S41D, which imitates a constitutive phosphorylation by PKC␣ at this site. This mutation enhanced the ER Ca 2ϩ release amplitude by 23% compared with wild-type B56␣ resulting in similar Fluo-4 fluorescence F 1 /F 0 peak amplitudes in transfected and nontransfected cells (Fig. 8C). Ca 2ϩ decay kinetics were unchanged between all groups (Fig. 8D). In addition, application of 1 M isoprenaline was not sufficient to elicit Ca 2ϩ spikes in ␣ 1A -adrenoreceptorexpressing Flp293 cells (data not shown). Thus, our data suggest that PKC␣ can modulate PP2A activity and thereby IP 3 R activity in vivo by phosphorylation of B56␣.
B56␣ Phosphorylation Changes cAMP-response Element-mediated Transcriptional Activation-Furthermore, we investigated the relevance of the Ser 41 phosphosite for transcriptional gene regulation by expression of either wild-type or mutated B56␣ together with a luciferase reporter gene construct containing the inducible cAMP early repressor (Icer) promoter in HEK293 cells. Lack of the PKC␣ phosphorylation site by mutation of Ser 41 to alanine, S41A, resulted in a luciferase expression that was comparable with the wild-type form of B56␣ (data not shown). This is consistent with the similar inhibitory activity of both nonphosphorylated B56␣ isoforms (wild-type and S41A) on PP2A CA (Fig. 5, A and D). In contrast, transcriptional activity of the Icer promoter was decreased by 20 Ϯ 2% in S41D-transfected HEK293 cells (p Ͻ 0.05, n ϭ 20 from three independent transfections).

DISCUSSION
The findings of this study provide in vitro and in vivo evidence for the functional relevance of a novel phosphorylation site in B56␣ at Ser 41 . The regulatory function of B56␣ phosphorylation on PP2A activity was confirmed by several independent lines of evidence using a mutant B56␣ subunit (S41A) that cannot be phosphorylated by PKC␣ and by the generation of a specific antibody against Ser 41 -phosphorylated B56␣. Ser 41 phosphorylation was increased in LV tissue of failing human hearts. PP2A activity was inhibited by PKC␣-dependent phosphorylation at this B56␣ phosphosite. The decrease in PP2A activity was observed both in vitro on recombinant B56␣ and in cultured HEK293 cells by use of a B56␣ mutant (S41D) mimicking phosphorylated B56␣. The transient transfection of wild-type B56␣ decreased IP 3 -mediated ER Ca 2ϩ release in cultured Flp293 cells, although this decrease was not observed after transfection of the S41D mutant. Thus, the PKC␣-dependent phosphorylation of B56␣ regulates the ER Ca 2ϩ release.
Previous work has demonstrated that most of the B56 subunits are phosphoproteins, with phosphorylation on serine (6). This is in contrast to the regulation of the PP2A catalytic subunit C, occurring upon both serine/threonine and tyrosine phosphorylation. The phosphorylation of B56 subunits by different protein kinases can affect either the assembly of the PP2A heterotrimer or its enzymatic activity depending on the localization of the phosphosite. Letourneux et al. (31) found that ERK-phosphorylated B56␥1 exhibited a decreased binding to A and C subunits. This is in contrast to a previous report showing that the formation of the PP2A trimeric holoenzyme occurred independently of its phosphorylation state (6). ERKdependent phosphorylation of B56␥1 was associated with a lower PP2A activity on ERK and was localized to phospho-Ser 327 (31). The phosphorylation of B56␥3 by checkpoint kinase Chk2 resulted in an increase in PP2A activity against myelin basic protein as well as on autophosphorylated Chk2 (32). An increase of PP2A activity was also observed for phosphorylation of B56␣ by PKR (11) and of B56␦ by PKA or PKC at Ser 566 (12,33). In this study, we found a depressed PP2A activity after a PKC␣-dependent phosphorylation of B56␣ at Ser 41 . A lower PP2A activity was also observed after phosphorylation of recombinant wild-type B56␣ by PKA, whereas CaM kinase II-phosphorylated B56␣ enhanced PP2A activity (data not shown). This mechanism may explain, at least in part, the increased PP2A activity in CaM kinase II-overexpressing hearts of transgenic mice (34). Thus, besides the diversity of B56 subunits, the phosphorylation of B56 isoforms may contribute to the substrate specificity of PP2A trimeric holoenzymes. Moreover, the phosphorylation studies raise the question on how the phosphorylation of B56 isoforms at multiple phosphoacceptor sites may influence the catalytic activity of PP2A by structural changes.
The B56 subunits exhibit a superhelical structure comprising 18 ␣-helices, which are organized into eight HEAT-like repeats (35). There are a number of highly conserved amino acid residues to interact with the A and C subunits. The interaction to the A subunit is mediated by two binding domains located on the convex side of B56 subunit C-terminal HEAT-like repeats. The interface between both subunits is relatively weak but is enhanced by binding of the methylated C-terminal tail of the C subunit to form a stable heterotrimeric structure. The interaction with the C subunit is mediated by three interfaces, including the intra-repeat loop 2, HEAT-like repeats 4 -6, and intra-repeat loops of HEAT repeats 6 -8 of B56␥1 (35). However, there are no direct contacts between B56␥ and residues near the active site of the catalytic subunit C, suggesting that regulatory B56 subunits cannot directly modify the catalytic site or activity. Thus, the major role of B56 isoforms is rather to tether the PP2A holoenzyme complex to its substrates. This is caused by formation of the PP2A heterotrimer bringing the concave surface of B56 subunits to the active site pocket of the catalytic subunit C. The acidic concave may shape a surface that recruits different phosphoproteins through electrostatic interactions (36). Therefore, the phosphorylation of B56 subunits at different sites at the concave surface, giving an additional negative charge to the modified protein, may favor charge-charge repulsionorattractionbetweenB56isoformsandthephosphorylated substrates leading to a limited or improved access of PP2A substrates to the catalytic site, respectively (Fig. 9A). This hypothesis is supported by data from this study showing that phosphorylase a exhibited a lower binding to PKC␣-phosphorylated B56␣ compared with nonphosphorylated B56␣, which might explain the reduced PP2A activity when extra-charged B56␣ (ϭ phosphorylated by PKC␣) was applied (Figs. 5B and 9A). Thus, different protein kinases can compel B56␣-associated PP2A to a lower (by PKC␣) or higher (by CaM kinase II) activity depending on the phosphorylation at different specific phosphosites of the regulatory subunit. Alternatively, the assembly of the PP2A trimeric holoenzyme may be disturbed due to the charge repulsion between the PKC␣-phosphorylated B56␣ and a negatively charged surface on the A subunit binding area. The phosphorylation of B56␣ at Ser 41 occurs at the N-terminal tail of the protein, a region that is unlikely to be involved in the formation of the PP2A heterotrimer. However, it may affect transient interactions between the B56␣ core domain and the AC dimer because B56 isoforms, truncated at the N-termi-nal sequence, did not interact with A and C subunits in vivo (37)(38)(39).
The functional relevance of the newly identified phosphosite in B56a was examined by transfection of cultured Flp293 cells with a pseudophosphorylated B56␣ mutant (S41D), which resulted in a 23% increase in the F 1 /F 0 amplitude of Fluo-4 fluorescence compared with wild-type B56␣. This increase was paralleled by a reduced PP2A activity under basal conditions in Flp293 cell homogenates (Fig. 7B) suggesting that the IP 3 R function is modulated by the PKC␣-dependent phosphorylation of B56␣. PP2A as well as PP1 are components of the IP 3 R macromolecular signaling complex (40). It was demonstrated that PP1 facilitates the dephosphorylation of the PKA-phosphorylated IP 3 R, reversing the increase of the channel sensitivity to activation by IP 3 (41). The physiological significance of PP2A in regulating the IP 3 R function remained unclear because both basal and dopamine-induced PKA phosphorylation of the IP 3 R were not affected by 10 nM okadaic acid (41). However, our data rather suggest a more sophisticated model of IP 3 R regulation by the phospho-B56␣/PP2A heterotrimer than a simple FIGURE 9. Schematic models of regulating PP2A by PKC␣-dependent phosphorylation of B56␣. Schemata depict the summary of the results of this study. A, PKC␣-dependent phosphorylation of B56␣ (part of the PP2A holoenzyme) leads to an extra-charged phospho-Ser 41 and therefore a change of the substrate recognition surface of B56␣. Subsequently, PP2A substrates like phosphorylase a (Phos a) exhibit a lower binding to the enzyme. PP2A is able to reverse the PKC␣-dependent phosphorylation of B56␣. The depiction of the PP2A holoenzyme model was modified (53). B, Ca 2ϩ -dependent activation of PKC␣ by PE stimulation of ␣ 1 -adrenergic receptors and PMA is followed by phosphorylation of B56␣ at its specific phosphosite, Ser 41 . This may result in a restricted access of PP2A to the IP 3 R (dashed line) reflected by a reduced PP2A activity in living HEK293 cells. A higher phosphorylation level of the IP 3 R due to a lower PKC␣-forced membrane-associated PP2A activity may then cause an increased Ca 2ϩ release from the ER. DAG, diacylglycerol; PIP 2 , phosphatidylinositol 4,5-bisphosphate.
on-off of the catalytic activity of the PP2A heterodimer. This focuses on a potential role of protein kinases (e.g. PKC␣) in influencing PP2A activity. Normally, PKC␣ and PP2A CA heterodimers are co-localized in the cytosol of resting cells (17,42). Activation of PKC␣ by (auto)phosphorylation or PMA induces its translocation along with PP2A because of their physical association from the cytosol to the membrane (17,42,43). From these studies, it can be suggested that a PKC␣-dependent phosphorylation of B56␣, which is mimicked by the B56␣ mutant S41D, results in a lower membrane-associated PP2A activity. This is followed by a higher PKA-mediated phosphorylation and activation of the IP 3 R, i.e. an increased ER Ca 2ϩ release (Fig.  9B). The presence of PP2A also correlated with a PKC␣ phosphatase activity in membrane fractions suggesting the inhibition and desensitization of PKC␣ through dephosphorylation by PP2A (14). In addition, not only PKC␣ but also PKC␣-phosphorylated B56␣ is a target for PP2A leading to dephosphorylation of the regulatory subunit as shown in our studies. This suggests a multitude of autoregulatory mechanisms to restore normal PP2A activity as demonstrated for PKR-dependent phosphorylation of B56␣ (11). It is also conceivable that the increased ER Ca 2ϩ release in S41D-expressing Flp293 cells leads to activation of the CaM kinase II, which phosphorylates B56␣ (data not shown) and reverses the inhibition of the PP2A activity. Alternatively, the activation of PP2B by the released Ca 2ϩ may dephosphorylate DARPP-32, which relieves the inhibition of PP1 and closes the feedback loop by dephosphorylating the IP 3 R (41).
Previous findings showed that the PP2A is able to dephosphorylate the transcription factor cAMP-response elementbinding protein (CREB) at Ser 133 , which inhibits its transcriptional activity (44). A prominent target gene promoter of CREB is the inducible cAMP early repressor (Icer) (45). Accordingly, an inhibition of PP2A by the B56␣ mutant S41D should increase Icer promoter activity. However, a luciferase reporter gene assay in HEK293 cells revealed that expression of S41D is followed by decreased transcriptional activity of the Icer gene promoter compared with wild-type B56␣. Shanware et al. (46) reported that B56␥-associated PP2A inhibits CREB hyperphosphorylation at Ser 121 resulting in a decreased binding of CREB to the CREB-binding protein. The histone acetyltransferase activity of CREB-binding protein is able to enhance the ability of CREB to activate the transcription of its target genes (47). Hence, an inhibition of PP2A activity by the B56␣ mutant S41D, which is associated with a hyperphosphorylation of CREB at Ser 121 and a decreased interaction with CREB-binding protein, may explain the observed decreased Icer promoter activity. Although the exact mechanism underlying the inhibition of cAMP-response element-mediated gene transcription by S41D is not fully understood, the altered promoter activity suggests the possible relevance of the Ser 41 phosphosite of B56␣ for the regulation of gene transcription.
The higher phosphorylation level of B56␣ at Ser 41 in failing human hearts suggests an important role of the PKC␣-dependent phosphorylation of B56a not only under physiological conditions (e.g. modulation of IP 3 R function) but also in disease states. There is evidence that PKC␣ activity regulates cardiac contractility. The phosphorylation of PKC␣ substrates (e.g. inhibitor-1 of PP1, G-protein-coupled receptor kinase 2, cardiac troponin I) is thereby associated with a reduced sarcoplasmic reticulum Ca 2ϩ load, an uncoupling of ␤-adrenergic receptors, a lower myofilament Ca 2ϩ sensitivity, and a decreased contractility (48). Consistently, the increased PKC␣ expression and activity contributes to myofilament dysfunction in failing human hearts and in experimental congestive heart failure (49,50). Moreover, heart-directed overexpression or adenovirusmediated gene transfer of PKC␣ resulted in a reduced ventricular performance (51). Thus, it is conceivable that a depressed PP2A activity due to a higher PKC␣-dependent phosphorylation of B56␣ may contribute to the effects of increased PKC␣ on cardiac troponin I phosphorylation in hypertrophy and endstage heart failure. A model of protein phosphatase regulation was demonstrated for the PKC␣-dependent phosphorylation of PP1 inhibitor-1 leading to inhibition of sarcoplasmic reticulum Ca 2ϩ uptake and diminished contractile response (51). These authors did not detect changes in PP2A activity in homogenates of PKC␣-overexpressing hearts. However, this does not rule out the possibility that PP2A activity is altered in certain subcellular compartments relevant for cardiac contractility and/or progression of heart failure. The stimulation of PKC␣, which is physically associated with PP2A, initiated its translocation to membrane fractions in general (17,42,43). In cardiomyocytes, the activation of PKC␣ by phorbol esters was followed by the translocation of the enzyme to the T-tubular membrane network (51) where phosphosubstrates of PP2A (e.g. ryanodine receptor type 2, L-type Ca 2ϩ channel, phospholamban) are localized. Consistently, PKC␣ overexpression in REH cells was associated with a depressed PP2A activity in mitochondrial membrane fractions (52). The lower PP2A activity was not paralleled by a decreased expression of the catalytic subunit C␣ but a reduction of the B56␣ protein levels indicating the redistribution of PP2A subunits. Thus, taking into account the close physical association of PKC␣ and PP2A as well as the wide ranging effects of this protein kinase-phosphatase complex in the heart, the investigation of the exact mechanism of how PKC␣ regulates PP2A and vice versa under (patho)physiological conditions is of considerable interest in future studies.
In summary, here we present data showing for the first time that PP2A activity is regulated by a newly identified phosphosite on B56␣. The phosphorylation at Ser 41 by PKC␣ converts B56␣ into a more potent inhibitor of PP2A. The inhibition of PP2A activity was associated with an increase in the IP 3 -mediated ER Ca 2ϩ release and an altered cAMP-response element-mediated transcriptional activity in vivo. Moreover, the PKC␣-dependent Ser 41 phosphorylation of B56a may play an important role in the pathophysiology of human heart failure.