Identification and Functional Analysis of Two Ca2+-binding EF-hand Motifs in the B"/PR72 Subunit of Protein Phosphatase 2A*

Veerle JanssensDagger §, Jan JordensDagger , Ilse StevensDagger ||, Christine Van HoofDagger §, Ellen MartensDagger **, Humbert De SmedtDagger Dagger , Yves Engelborghs, Etienne WaelkensDagger , and Jozef GorisDagger §§

From the Dagger  Division of Biochemistry, Faculty of Medicine, Katholieke Universiteit Leuven, Herestraat 49, B-3000 Leuven,  Laboratory of Biomolecular Dynamics, Faculty of Science, Katholieke Universiteit Leuven, Celestijnenlaan 200 D, B-3001 Leuven, and Dagger Dagger  Laboratory of Physiology, Faculty of Medicine, Katholieke Universiteit Leuven, Herestraat 49, B-3000 Leuven, Belgium

Received for publication, November 18, 2002, and in revised form, January 8, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Protein phosphatase 2A (PP2A) is a multifunctional serine/threonine phosphatase that is critical to many cellular processes including cell cycle regulation and signal transduction. PP2A is a heterotrimer containing a structural (A) and catalytic (C) subunit, associated with one variable regulatory or targeting B-type subunit, of which three families have been described to date (B/PR55, B'/PR61, and B"/PR72). We identified two functional and highly conserved Ca2+-binding EF-hand motifs in human B"/PR72 (denoted EF1 and EF2), demonstrating for the first time the ability of Ca2+ to interact directly with and regulate PP2A. EF1 and EF2 apparently bind Ca2+ with different affinities. Ca2+ induces a significant conformational change, which is dependent on the integrity of the motifs. We have further evaluated the effects of Ca2+ on subunit composition, subcellular targeting, catalytic activity, and function during the cell cycle of a PR72-containing PP2A trimer (PP2AT72) by site-directed mutagenesis of either or both motifs. The results suggest that integrity of EF2 is required for A/PR65 subunit interaction and proper nuclear targeting of PR72, whereas EF1 might mediate the effects of Ca2+ on PP2AT72 activity in vitro and is at least partially required for the ability of PR72 to alter cell cycle progression upon forced expression.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Protein phosphatase 2A (PP2A),1 an essential serine/threonine phosphatase present in all eukaryotic cells, is a multifunctional enzyme of fundamental importance in signal transduction that regulates a wide variety of cellular events, including cell cycle progression, development, transcription, translation, DNA replication, and viral transformation (reviewed in Refs. 1 and 2). This extraordinary functional diversity can be explained by the existence of several mechanisms by which PP2A activity, substrate specificity, and subcellular localization are regulated (reviewed in Ref. 1). One of the major ways to achieve this is by the interaction, through a scaffolding A/PR65 subunit, of the PP2A catalytic subunit (PP2AC) with one of several regulatory B-type subunits. Although it has been suggested that a significant part of PP2AC can occur as a dimer with the A/PR65 subunit (PP2AD) within cells (3), recent evidence argues that PP2A is an obligate trimer (PP2AT) in vivo (4, 5). Nevertheless, there are a number of well documented cases at variance with this central "dogma," such as the association of PP2AC with alpha 4 without any other subunits (6), the complex formation of PP2AC, the B' subunit and cyclin G2, without the A subunit (7), and the association of PP2AD with some viral tumor antigens (8).

PP2A regulatory subunits are encoded by four multigene families, referred to as B, B', B", and B'''. All B-type subunits, with exception of B''', share two motifs for A/PR65 subunit binding (9). Because B''' subunits lack these binding motifs, their status as real PP2A subunits needs further evaluation. Each B subunit is thought to confer a set of specific functional characteristics to the phosphatase. For example, the B subunits have been implicated in the regulation of cytoskeletal protein assembly (10, 11), and in Drosophila S2 cells they mediate actions of PP2A on the mitogen-activated protein kinase signaling pathway (4, 5). The B' subunits, on the other hand, mediate PP2A function in the Wnt signaling cascade (12-14) and are required for the protective function of PP2A against apoptotic cell death in S2 cells (4, 5).

The functions of the B" subunit family are probably the least well understood. Five different B" isoforms have been identified in mammals: human PR72 and PR130 (the founding members of the family) (15), mouse PR59 (16), human PR48 (17), and the recently identified human G5PR (18). Although B" homologues have been described in plants (19), Xenopus laevis oocytes (20), Drosophila melanogaster (4, 5) and Caenorhabditis elegans (protein CO6G1.5, GenBank accession number AAK32946), they are manifestly absent in yeast. PR72 and PR130 are splice variants generated from a single gene and share an identical C terminus. PR72 has a muscle-specific expression, whereas PR130 is ubiquitous (15). In an in vitro assay, PP2AT72 is the only PP2A trimer that specifically stimulates simian virus 40 large T-antigen-dependent origin unwinding, an essential step in the initiation of viral DNA replication (21). PR59 was identified as interaction partner of the retinoblastoma-like p107 protein. Its overexpression results in G1/S arrest of cell cycle progression and coincides with increased amounts of hypophosphorylated and active p107 (16). PR48 was identified as a Cdc6-interacting protein (17). Cdc6 is an ATPase required for the initiation of DNA replication that primarily acts by recruiting the minichromosome maintenance complex to origins of replication (22). Recent results indicate, however, that PR48 is a partial clone that would better be renamed as "PR70," fitting with its real molecular mass (20). G5PR was identified as a germinal center-associated nuclear protein (GANP)-associated molecule by yeast two-hybrid screening (18). GANP carries DNA primase and minichromosome maintenance 3-binding activity and is thought to be involved in the regulation of DNA replication in activated germinal center B cells (23, 24). Interestingly, G5PR also strongly interacts with the tetratricopeptide repeat domain of protein phosphatase 5, another eukaryotic serine/threonine phosphatase (18). In general, most of the functions of the B" subunits described so far argue for important roles of these proteins in the regulation of the G1/S transition of the cell cycle and the regulation of DNA replication during S phase.

Calcium is an important second messenger within cells. Highly regulated changes in the intracellular Ca2+ concentration control biological processes as diverse as muscle contraction, fertilization, cell proliferation and division, gene transcription, and apoptosis (reviewed in Ref. 25). Many of these effects are modulated by multiple classes of Ca2+-binding proteins, some of which can in turn regulate multiple downstream effectors. Among the major intracellular Ca2+ receptors are calmodulin (CaM) and S100 proteins. Both are members of a large class of so-called "EF-hand" Ca2+-binding proteins, which share a common Ca2+-binding helix-loop-helix motif, the conformation of which essentially determines biological function. Specific binding of Ca2+ to the loop alters the conformation of the motif, involving the rearrangement of both helices in three-dimensional space (reviewed in Ref. 26). In many Ca2+ sensor proteins this conformational change exposes a hydrophobic effector protein-binding surface, which in turn allows them to bind to and regulate an array of secondary effector proteins (27).

Although calcineurin (also known as protein phosphatase 2B) and protein phosphatase 7 are the only serine/threonine phosphatases known so far to be regulated directly by Ca2+ (28, 29), several reports have suggested a role for PP2A in Ca2+-dependent signaling. PP2A has been shown to associate with the CaM-dependent kinase IV (30) and with the CaM-binding proteins striatin and S/G2 nuclear autoantigen (also known as the B''' or PR93/PR110 subunits) (31). Moreover, PP2A has been implicated in the regulation of Ca2+-activated potassium channels by glucocorticoids (32, 33), in the regulation of L-type Ca2+ channels (34, 35), the cardiac ryanodine receptor RyR2 (36), and the inositol 1,4,5-trisphosphate receptor signaling complex (37). Inversely, calcium ions have also been shown to regulate PP2A activity in G-protein-coupled receptor signaling (38) or PP2A (re)localization in the establishment of cell-cell contacts between epithelial cells (39) and during mast cell secretion (40). However, the underlying mechanisms remain elusive.

In this report we identify two Ca2+-binding EF-hand motifs in human B"/PR72, demonstrating for the first time, the ability of calcium ions to directly interact with and regulate PP2A. By a mutational analysis of both domains, we evaluate the effects of Ca2+ on PP2AT72 subunit composition, subcellular targeting, catalytic activity, and function during the cell cycle. The results obtained suggest that both motifs serve different functions.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- 45CaCl2 (2.5 mCi/ml, 110.8 µg of Ca2+/ml), [35S]methionine, and protein G-Sepharose beads were obtained from Amersham Biosciences. Restriction enzymes and DNA-modifying enzymes were purchased from Fermentas. Calcium ionophore A23187, propidium iodide, and Hoechst 33342 were from Sigma. BAPTA-AM was supplied by Alexis Biochemicals; nocodazole was from Applichem. The bacterial expression vector for the His-tagged domain I of calpastatin was a generous gift of Dr. J. Elce (Cancer Research Laboratories, Department of Biochemistry, Queen's University, Kingston, Ontario, Canada). The protein was expressed and purified on nickel-agarose beads (Affiland) following standard procedures. Anti-EGFP antibodies were a kind gift of Dr. M. Beullens (Division of Biochemistry, Faculty of Medicine, Katholieke Universiteit Leuven, Belgium). Anti-PP2AC and anti-PR65 monoclonal antibodies were generously supplied by Dr. S. Dilworth (Department of Metabolic Medicine, Imperial College Faculty of Medicine, Hammersmith Hospital, London, UK). Pwo proofreading polymerase (used in all PCRs) was purchased from Roche Molecular Biochemicals.

Site-directed Mutagenesis of EF-hands 1 and 2-- Two point mutations were introduced in each EF-hand using a PCR-based method. For the introduction of a single point mutation into EF1, two separate PCRs were performed with PR72 cDNA as template: the first with 5'-ATATCATATGATGATCAAGGAAACATCTC-3' (start primer) and 5'-GTATCGAGACAGAGCGGCCTGGC-3' (reverse mutated primer) and the second with 5'-GCCAGGCCGCTCTGTCTCGATAC-3' (forward mutated primer) and 5'-TATAGGATCCCTATTCTTCATCCACTGATTG-3' (stop primer). The combined reaction products of these two PCRs were then used as template for a second amplification round with the start and stop primers only. The resulting PCR product was cloned into the EcoRV-digested pBluescript vector (Stratagene) and sequenced to confirm the introduction of the mutation. The second point mutation within EF1 was introduced very similarly with the start primer, 5'-GGTCGTGATCAGTAGCTAGTTCCCAG-3' (reverse mutated primer), 5'-CTGGGAACTAGCTACTGATCACGACC-3' (forward mutated primer), and the stop primer using the single EF1 point mutant as template. Together, the PR72 EF1 sequence 290DTDHDLYISQADL302 was changed into 290ATDHDLYISQAAL302. For mutation of EF2, the first mutation round was performed with the start primer, 5'-CATAGAAGTACTCCAATGCATACATGGAGAG-3' (reverse mutated primer), 5'-CTCTCCATGTATGCATTGGAGTACTTCTATG-3' (forward mutated primer) and the stop primer using PR72 as template. The second point mutation was generated very similarly with the start primer, 5'-GTCTCCATCCACAGCCATGCAGCGG-3' (reverse mutated primer), 5'-CCGCTGCATGGCTGTGGATGGAGAC-3' (forward mutated primer) and the stop primer using the single EF2 point mutant as template. In this way the wild-type EF2 sequence 364DVDGDGVLSMYEL376 was eventually changed into 364AVDGDGVLSMYAL376. The double mutation of EF1 and EF2 was generated via four consecutive mutation rounds using the same primers and the same PCR-based method.

Expression and Purification of Recombinant PR72 Polypeptides-- Wild-type PR72, N- and/or C-terminal truncations of wild-type PR72, and the single or double EF hand mutants thereof were cloned into pET15b, pET3C (Novagen), pGEX-4T2 or pGEX-4T3 (Amersham Biosciences) following standard molecular biology procedures. The resulting plasmids were transformed into BL21-pLys(RP) Escherichia coli bacteria for protein expression. Inductions were performed with 1 mM isopropyl-1-thio-beta -D-galactopyranoside for 2 h 30 min at 30 °C. Bacterial pellets were lysed by sonication in lysis buffer (50 mM Tris·HCl, pH 8.0, 2 mM EDTA, 1 mM dithiothreitol) supplemented with 2 mg/ml lysozyme, 10 µg/ml leupeptin, 1 mM benzamidine, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride. Inclusion bodies were purified (41) and solubilized in 7 M guanidinium hydrochloride. After dialysis against buffer B (200 mM Tris·HCl, pH 8.2, 500 mM NaCl), the majority of the proteins remained soluble.

45Ca2+ Overlay Assay-- Ca2+ binding to the recombinant (fusion) proteins was measured after transfer to Immobilon P membranes (42). Briefly, membranes were washed overnight at room temperature in nominally Ca2+-free incubation medium containing 10 mM imidazole, pH 7.0, 60 mM KCl, and 1 mM MgCl2. Binding was carried out in the same medium supplemented with 1.5 µCi/ml 45CaCl2 for 10 min at room temperature. Membranes were washed during 2 min in 50% ice-cold ethanol/water mixture and air-dried. Bands were visualized by autoradiography.

Fluorescence Spectroscopy-- Protein concentrations of purified recombinant proteins, comprising amino acids (aa) 262-449 of PR72, were evaluated by absorbance measurement at 280 nm and by the BCA quantification method (Pierce). Equal amounts of PR72 aa262-449 or PR72 EF(1 + 2)mut aa262-449 were diluted in buffer B and excited at 295 nm. The fluorescence spectra between 300 and 410 nm were recorded in a Photon Technology International spectrofluorometer in the absence or the presence of different amounts of CaCl2 buffered by 1 mM EGTA. Free Ca2+ concentration was calculated with the CaBuf program (available at ftp.cc.kuleuven.ac.be/pub/droogmans/cabuf.zip).

GST-Pull Downs-- [35S]Methionine-labeled proteins were obtained from pBluescript vectors containing the coding regions of PR72, PR72 EF1mut, PR72 EF2mut, or PR72 EF(1 + 2)mut, using the TNT-coupled rabbit reticulocyte lysate system (Promega) with the appropriate RNA polymerase (T3 or T7). The GST-PR65alpha subunit of PP2A (PR65-GST) and the free GST protein were produced in E. coli BL21-pLys cells following standard procedures, and purified on glutathione-Sepharose beads (Amersham Biosciences) according to the manufacturer's instructions. The GST pull-down binding reactions contained 20 µl of [35S]methionine-labeled proteins, 1 µg of GST or PR65-GST, 20 µl of prewashed glutathione-Sepharose beads, and NENT100 buffer (20 mM Tris·HCl, pH 7.4, 1 mM EDTA, 0.1% Nonidet P-40, 25% glycerol, 100 mM NaCl) to a final volume of 500 µl. If appropriate, either 2 mM EGTA or 4 mM CaCl2 was added to the reaction mix. Incubation was done for 4 h at 4 °C on a rotating wheel. The beads were washed five times with 1 ml of NENT300 (NENT with 300 mM NaCl) containing 2 mg/ml bovine serum albumin and either 2 mM EGTA or 4 mM CaCl2. Bound proteins were eluted by addition of 20 µl of SDS sample buffer and boiling. The eluted proteins were analyzed by SDS-PAGE and imaged using an Amersham Biosciences PhosphorImager.

Cell Culture-- Monkey COS7, human U2OS, and rat L6 cells were supplied by the American Type Culture Collection (Manassas, VA). COS7 and U2OS cells were cultured in Dulbecco's modified Eagle's medium containing 1 g/liter glucose (BioWhittaker) supplemented with 2 mM L-glutamine (Invitrogen), 100 units/ml penicillin (Invitrogen), 100 mg/ml streptomycin (Invitrogen), and 10% fetal calf serum (Biochrom KG). Rat L6 myoblasts were grown in Dulbecco's modified Eagle's medium containing 5 g/liter glucose (BioWhittaker) supplemented with 2 mM L-glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin, and 10% fetal calf serum. Nuclear and cytoplasmic extracts of subconfluent L6 cells were prepared as described (43).

Two-hybrid Systems-- To generate VP-16 fusion proteins for use in mammalian double-hybrid experiments, PCR fragments of PR72, PR72 EF1mut, PR72 EF2mut, PR72 EF(1 + 2)mut, A/PR65alpha , B/PR55alpha , and B'/PR61gamma 1 were cloned into the EcoRV restriction site of the pSNATCH-II plasmid, encoding residues 413-498 of the VP16 activating region (44). For the expression of PP2A regulatory subunits fused to residues 1-147 of the yeast GAL4 transcription factor, PCR-generated fragments encoding PR72, PR72 EF1mut, PR72 EF2mut, PR72 EF(1 + 2)mut1, A/PR65alpha , B/PR55alpha , and B'/PR61gamma were inserted in the SmaI restriction site of the pAB-Gal4 plasmid (45). The luciferase reporter plasmid pUAS-TATA-luc contains 5 GAL4-binding sites in front of a minimal promoter (TATA box). pSNATCH-II, pAB-Gal4, and pUAS-TATA-luc were a kind gift of Dr. F. Claessens (Division of Biochemistry, Faculty of Medicine, Katholieke Universiteit Leuven, Leuven, Belgium). All transfections for the mammalian double-hybrid experiments were performed in COS7 cells in 24-well culture plates (Nunc) with FuGENE 6 Transfection Reagent (Roche Molecular Biochemicals). 400 ng of pUAS-TATA-luc, 400 ng of pAB-Gal4, and 40 ng of the pSNATCH-II plasmid per well was used, along with 40 ng of a beta -galactosidase reporter plasmid (pEF1-beta Gal) to correct for different transfection efficiencies. In the latter plasmid beta -galactosidase expression is driven by the human translation elongation factor 1 promoter. Transfected cells were lysed in 100 µl of passive lysis buffer (Promega), and luciferase and beta -galactosidase activities were measured in 10 µl of the extracts using the assay systems from Promega and Tropix, respectively.

For yeast two-hybrid experiments, GAL4 binding and activation domain fusion proteins were generated by cloning PCR fragments of full-length PR72 (wild-type or mutated in either or both EF-hands) and of several PR72 deletions in the pGBT9 and pGAD424 vectors (Clontech), respectively. The PR72 deletion fragments used encompassed aa1-99, aa1-219, aa1-358, aa123-473, aa353-529, and aa219-529. All transformations were performed in the PJ69-4A Saccharomyces cerevisiae strain (46) harboring adenine, beta -galactosidase, and histidine reporter plasmids under the control of GAL4 upstream-activating sequences. For the evaluation of positive interactions, only the expression of the adenine and beta -galactosidase reporters were assayed.

EGFP Experiments-- PCR fragments of PR72, PR72 EF1mut, PR72 EF2mut, and PR72 EF(1 + 2)mut were cloned into the SmaI restriction site of pEGFP-C1 (Clontech). 1 µg of each plasmid was transfected into COS7 cells grown on a glass coverslip in a 6-cm dish (Nunc) using FuGENE 6 transfection reagent. 24-36 h after transfection cells were washed in phosphate-buffered saline and fixed in ice-cold methanol for 20 min. Following the addition of 3 µM Hoechst 33342 for 10 min to stain the nuclei, cells were briefly rinsed in water, mounted in mounting medium (Sigma), and examined by a fluorescence microscope (Diaplan, Leitz, Germany) equipped with a digital camera (DC200, Leica Microsystems). For anti-EGFP immunoprecipitations, transfected cells were lysed in Tris-buffered saline supplemented with 0.1% Nonidet P-40. Before addition of the anti-EGFP antibody, lysates were precleared with 10 µl of protein G-Sepharose. Immunoprecipitates were washed four times in lysis buffer and once with 0.1 mM LiCl before addition of SDS sample buffer, boiling, SDS-PAGE, and Western blotting. Blots were developed with a mixture of anti-PP2AC and anti-PR65 monoclonal antibodies using the enhanced chemiluminescence detection system (Amersham Biosciences).

Measurement of PP2A Activity-- Protein phosphatase assays were performed with 32P-labeled phosphorylase a as the substrate in the absence of protamine stimulation, essentially as described (15). PP2AT72, purified from rabbit skeletal muscle (15), was preincubated with calpastatin (an inhibitor of the m-calpain protease) and different amounts of CaCl2 for 10 min at 30 °C. Subsequently, different amounts of EGTA were added together with the substrate. This mixture was further incubated for 10 min at 30 °C. The reactions were stopped by trichloroacetic acid precipitation, and the amount of free 32P-labeled phosphate was counted. Similar experiments were performed by including the Ca2+ and/or EGTA concentrations directly in the phosphatase assay. Because this approach basically resulted in the same data, this indicated that Ca2+/EGTA effects are "instantaneous," and prior incubation of Ca2+ and/or EGTA with the enzyme is not required.

FACS Analysis-- Asynchronously growing U2OS cells were transfected with pEGFP-C1, PR72-EGFP, PR72 EF1mut-EGFP, PR72 EF2mut-EGFP, or PR72 EF(1 + 2)mut-EGFP in four 6-cm dishes per plasmid. 24 h after transfection, cells were trypsinized, seeded into 10-cm dishes, and allowed to grow for another 24 h before FACS analysis. If nocodazole (1 µg/ml) was used, it was added at this point for another 16 h before FACS analysis. If BAPTA-AM (10 µM) was used, it was added 6 h before the addition of nocodazole. Cells were fixed for 5 min in 4% paraformaldehyde at room temperature to preserve the EGFP signal. After washing, the cell pellet was incubated in 0.5 ml of phosphate-buffered saline containing 100 µg/ml propidium iodide and 0.1% RNase for at least 1 h at room temperature. The samples were analyzed with a Beckman Instruments Coulter Epics XL flow cytometer (Analis) on FL1 (for EGFP) and FL3 (for propidium iodide) using standard procedures and the System IITM software (Analis) for quantification.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Two Conserved EF-hands within PR72 Bind Ca2+ with Different Affinities-- Analysis of the primary amino acid structure of the B" family members revealed the presence of two well conserved EF-hand domains (termed EF1 and EF2) (Fig. 1A). These motifs are well known Ca2+-binding domains and are present, very often in tandem, in many Ca2+-binding proteins (with calmodulin being the most renown example) (reviewed in Ref. 26). Given their high degree of conservation, within the family as well as throughout evolution, and given their (almost) perfect match with the known consensus sequence (Fig. 1A), it was not unlikely to predict that the B" members are genuine Ca2+-binding proteins. To check this, we used the human B"/PR72 protein as a "model" for the other family members, and we performed all further experiments with this particular B" isoform.


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Fig. 1.   A, alignment of putative EF-hand motifs of some eukaryotic B" subunits. Amino acids matching with the EF consensus sequence are in boldface. Positions 1, 3, and 12 are the most conserved. In most cases there is a glycine at position 6. Amino acids shown in brackets are optional; amino acids in braces are excluded; x is any amino acid. In PR72, EF1 composes aa290-302 and EF2 aa364-376. B, 45Ca2+ overlay assay on recombinant PR72-derived polypeptides. Several recombinant PR72-derived proteins were subjected to Western blotting and incubated with 45CaCl2 in nominally Ca2+-free medium (see "Experimental Procedures"). Bands are visualized by autoradiography. M, protein marker; lane 1, GST; lane 2, GST-PR72 aa238-420; lane 3, GST-PR72 aa238-420 EF1mut; lane 4, GST-PR72 aa238-420 EF2mut; lane 5, GST-PR72 aa238-420 EF(1 + 2)mut; lane 6, GST-PR72 aa238-358; lane 7, GST-PR72 aa238-358 EF1mut; lane 8, GST-PR72 aa353-410; lane 9, GST-PR72 aa353-410 EF2mut; lane 10, PR72-Nhis; lane 11, PR72 aa1-452; lane 12, PR72 aa123-473; lane 13, PR72-Nhis EF(1 + 2)mut; lane 14, PR72 aa1-452 EF(1 + 2)mut; lane 15, PR72 aa123-473 EF(1 + 2)mut. C, Coomassie Brilliant Blue staining of recombinant PR72-derived polypeptides. SDS-PAGE loading control of 45Ca2+ overlay assay in B.

To test whether PR72 could bind Ca2+ in vitro, a 45Ca2+ overlay assay was performed on several recombinant PR72-derived proteins, expressed and purified from E. coli bacteria (see "Experimental Procedures") (Fig. 1B). The results show that full-length PR72, as well as GST fusion proteins of PR72 aa238-358 (comprising EF1), of PR72 aa353-410 (comprising EF2), and of PR72 aa238-420 (comprising both EF1 and EF2), but not GST alone bind Ca2+. Moreover, GST-PR72 aa353-410 (comprising only EF2) seemed to bind significantly more Ca2+ than GST-PR72 aa238-358 (comprising only EF1), although approximately equal amounts of both proteins were loaded (Fig. 1, B and C, compare lane 6 and lane 8). This could suggest that the affinity of Ca2+ for EF2 is much higher than for EF1.

To ensure that Ca2+ binding to full-length PR72 or the fragments thereof indeed occurred via the proposed EF-hands, several mutations known to destroy Ca2+ binding to these motifs were introduced either in the full-length protein or in the fragments thereof (see "Experimental Procedures"). Briefly, in each motif the aspartate at position 1 plus the aspartate or glutamate at position 12 were changed into alanines. Similarly, these mutated proteins were expressed and purified from bacteria and subjected to 45Ca2+ overlay (Fig. 1B). The data show that mutation of EF1 abolishes Ca2+ binding to GST-PR72 aa238-358 (comprising only EF1) (lane 7), and similarly, mutation of EF2 abolishes Ca2+ binding to GST-PR72 aa353-410 (comprising only EF2) (lane 9). However, single mutation of EF1 within GST-PR72 aa238-420 (comprising both EF1 and EF2) has only minor effects on Ca2+ binding to this protein, whereas mutation of EF2 almost completely destroys Ca2+ binding (Fig. 1B, lane 3 and lane 4). This clearly demonstrates that both EF motifs bind calcium ions with different affinities.

Ca2+ Binding Induces Conformational Changes in PR72-- It is well known that Ca2+ binding can result into drastic conformational changes within EF-hand proteins, changing the relative position of helix E and helix F from a closed to a more open configuration (reviewed in Refs. 26 and 47). This property of Ca2+ forms the basis for its regulatory capacity, because conformational changes within proteins often affect their biological activities.

To assess whether Ca2+ binding to PR72 could result into conformational changes, measurements of the intrinsic tryptophan fluorescence of wild-type and mutated PR72 recombinant proteins were conducted in the presence of different amounts of calcium ions. Such fluorescence spectra are dependent on the micro- and/or macro-environment of the emitting Trp residues within the protein. Optimal results were obtained with a rather short PR72 fragment comprising both EF-hand motifs (aa262-449). This polypeptide contains only three Trp residues, two of which are in the very near vicinity of each EF-hand (one is found three aa N-terminally from EF1 and one 5 aa N-terminally from EF2). Using this fragment obviously diminishes the risk that (small) changes in fluorescence of only a few Trp residues in the near vicinity of the EF motifs may become undetectable because of the intrinsic fluorescence of a lot of other Trp residues of which the physicochemical environment is not changed by Ca2+ binding. The data show that addition of Ca2+ to the PR72 aa262-449 apoprotein, in any of the concentrations used, leads to a significant increase in fluorescence intensity (Fig. 2A), whereas this is not the case for the PR72 aa262-449 protein in which both EF-hands are mutated (Fig. 2B). This is indicative for a conformational change induced by binding of calcium ions to the EF hands. Moreover, the spectra of PR72 aa262-449 EF(1 + 2)mut and PR72 aa262-449 in the absence of Ca2+ are very similar (overlay Fig. 2C), suggesting that the introduction of the four point-mutations per se does not affect the overall protein conformation of the wild-type apoprotein.


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Fig. 2.   Intrinsic Trp fluorescence spectrum of recombinant PR72 aa262-449 and PR72 aa262-449 EF(1 + 2)mut in the presence of varying Ca2+ concentrations. Equal amounts of purified PR72 aa262-449 (A) and PR72 aa262-449 EF(1 + 2)mut (B) were excited at 295 nm. The fluorescence spectrum between 300 and 410 nm was recorded in the presence of different amounts of CaCl2 buffered by 1 mM EGTA in buffer B (pH 8.2, see "Experimental Procedures"). The calculated free Ca2+ concentrations are indicated. An overlay of the fluorescence spectra of recombinant PR72 aa262-449 and PR72 aa262-449 EF(1 + 2)mut in the absence of Ca2+ (presence of 1 mM EGTA) is shown in C.

Effects of Ca2+ on the Interaction of PR72 with the A/PR65 Subunit-- The A/PR65 subunit binding domain of PR72 was determined by analysis of the interaction of several deletion mutants of PR72 with A/PR65alpha in yeast two-hybrid assays. This yielded a rough estimation of the interaction domain. The smallest PR72 fragment tested was still able to interact with the A subunit, composed of amino acids 219-473 (results not shown). This domain contains about 80% of the proposed A Subunit Binding Domain 1 (ASBD1, aa197-302) and the complete ASBD2 (aa342-399) (9). Remarkably, also both Ca2+-binding motifs are located within these ASBDs, suggesting that calcium ions might affect the interaction of PR72 with the A/PR65 subunit (and consequently, with the core enzyme).

To test this hypothesis, we performed mammalian two-hybrid assays in COS7 cells. This system has the advantage that interactions can be easily quantified and/or evaluated in the presence of extracellular stimuli, such as calcium ionophore or BAPTA-AM treatment. In the absence of any stimulus, the A/PR65 subunit interaction is observed with wild-type PR72 and PR72 EF1mut but not with PR72 EF2mut or PR72 EF(1 + 2)mut (Fig. 3A). Identical results were obtained in the yeast two-hybrid system (results not shown), suggesting that the integrity of EF2 and therefore its Ca2+-binding capacity are vital for binding to A/PR65. Furthermore, the comparison of A subunit binding to B"/PR72 with that to B/PR55alpha and B'/PR61gamma 1 in the mammalian two-hybrid system reveals that B"/PR72 relatively shows the strongest interaction with A/PR65, whereas the interaction with B/PR55alpha is the weakest (Fig. 3A). Treatment of the cells with 5-10 µM BAPTA-AM, a cell-permeable Ca2+ chelator, resulted in a slight (albeit nonspecific) decrease of the observed PR72-PR65 interaction, because a parallel decrease was also observed for the PR55-PR65 and the PR61-PR65 interactions upon BAPTA-AM addition (data not shown). Similarly, treatment of the cells with 2-10 µM A23187, a calcium ionophore, led to inconclusive results, because in this case an overall inhibition of the transcriptional response of the reporter genes (luciferase as well as beta -galactosidase) was observed (data not shown).


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Fig. 3.   A, mammalian two-hybrid results in COS7 cells. PR72, the PR72 EF mutants, PR55alpha , and PR61gamma 1 were fused to the DNA binding domain of GAL4 (pAB-GAL4-derived plasmids). PR65alpha was fused to the transactivating region of VP16 (pSNATCH-II-derived plasmids). The indicated combinations of both expression plasmids were transfected into COS7 cells together with the luciferase reporter plasmid pUAS-TATA-luc and the beta -galactosidase vector pEF1-beta Gal as internal control. The indicated luciferase values represent the mean of at least three independent experiments and are normalized against the measured beta -galactosidase values, and eventually against the mean of the luciferase values measured for the PR72-PR65 interaction, which was set to 100. B, GST pull-down assay with A/PR65alpha -GST and 35S-labeled PR72 or the PR72 EF-mutants. 35S-Labeled proteins were produced by in vitro coupled transcription-translation in reticulocyte lysates. Bacterial recombinant GST or GST-PR65alpha immobilized on glutathione-Sepharose beads were incubated with the labeled proteins, in the presence of 2 mM EGTA or 4 mM CaCl2. After stringent washings, the resin-bound proteins were eluted with SDS-loading buffer, analyzed by SDS-PAGE, and visualized by autoradiography.

To confirm the former data in vitro, GST pull-down assays were performed with GST-PR65alpha and in vitro translated and radioactively labeled PR72 protein or the EF-hand mutants thereof, either as such (without any special treatment), in the presence of a Ca2+ chelator (2 mM EGTA) or in the presence of 4 mM CaCl2. Irrespective of the binding conditions, a specific interaction of GST-PR65alpha was observed with PR72 and PR72 EF1mut but not with PR72 EF2mut or PR72 EF(1 + 2)mut (Fig. 3B). Moreover, in the presence of extra calcium ions, the interaction of PR72 and PR72 EF1mut with GST-PR65alpha was slightly increased compared with the binding in the presence of a calcium chelator (Fig. 3B). Taking the limitations of the use of chelators into account, the data suggest that Ca2+ binding to EF2, a high affinity Ca2+-binding site, is a prerequisite for interaction of PR72 with the A subunit.

Effects of Ca2+ on Phosphatase Activity of PP2AT72 in Vitro-- In order to test the effect of CaCl2 on phosphatase activity, different (buffered) concentrations of Ca2+ were tested on purified PP2AT72 in an in vitro assay with phosphorylase a as the substrate. Calpastatin was added in order to block any residual m-calpain activity, known to be present in some PP2AT72 preparations (15). It should be noted that the standard purification of PP2AT72 from rabbit skeletal muscle is performed in buffers containing 1 mM EGTA all through the procedure (15). We therefore presume that EF1 (the low affinity binding site) may be (partially) in a Ca2+-free state, whereas EF2 (the high affinity binding site) is likely still loaded with Ca2+, because, according to our data, this is necessary for the interaction with PP2AD. Only high concentrations of Ca2+ seem to have an inhibitory effect on the phosphorylase phosphatase activity of PP2AT72 in vitro (Fig. 4A). These inhibitory effects are therefore probably mediated by the low affinity binding site EF1. In the absence of any added Ca2+, EGTA is without effect or only slightly stimulatory (Fig. 4B). This slight stimulation could correlate with some dissociation of PR72 from the trimer because it is known that PP2AT72 has a lower specific activity than PP2AD with phosphorylase a as the substrate (48). However, because the effect is so small, these data seem to confirm that EGTA, even at very high concentrations, is hardly able to affect the interaction of PR72 with the core enzyme. In the presence of both Ca2+ and EGTA, complex titration curves are observed (Fig. 4C); lower Ca2+ concentrations stimulate the activity maximally 2-fold, depending on the EGTA concentration, whereas inhibition by the higher Ca2+ concentrations seems to be more pronounced at higher EGTA concentrations. To investigate the individual contribution of EF1 and EF2 on the inhibitory as well as on the stimulatory effects of Ca2+ in the in vitro phosphatase assay, we tried to reconstitute PP2AT72 from purified PP2AD and the bacterially expressed and purified PR72, PR72 EF1mut, PR72 EF2mut, and PR72 EF(1 + 2)mut proteins, but these reconstitution experiments failed.


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Fig. 4.   Effects of calcium ions on phosphorylase a phosphatase activity of PP2AT72 in vitro. PP2AT72 purified from rabbit skeletal muscle was appropriately diluted and assayed in the presence of calpastatin and different amounts of Ca2+ (A) or EGTA (B) for 10 min at 30 °C with 32P-labeled phosphorylase a as the substrate. C, the enzyme was preincubated in the presence of calpastatin and different amounts of calcium for 10 min at 30 °C. Different amounts of EGTA were added together with 32P-labeled phosphorylase a, and the incubation was continued for another 10 min at 30 °C before trichloroacetic acid precipitation and measurement of the free 32P-labeled phosphate. The final EGTA and Ca2+ concentrations in the assay mixture are indicated.

Effects of Ca2+ on the Subcellular Localization of PR72-- The localization of PR72 and the putative Ca2+ effects on this phenomenon were initially evaluated by expression of EGFP fusion proteins of wild-type PR72 and of the PR72 EF-hand mutants in COS7 cells. The data show that wild-type PR72 and PR72 EF1mut are predominantly nuclear, whereas PR72 EF2mut and PR72 EF(1 + 2)mut are clearly excluded from the nucleus (Fig. 5A). Moreover, immunoprecipitations with anti-EGFP antibodies revealed co-immunoprecipitation of PP2AC and A/PR65 with PR72 and PR72 EF1mut but not with PR72 EF2mut or PR72 EF(1 + 2)mut (Fig. 5B), nicely confirming our former observations. Treatment of the transfected cells with 5-10 µM BAPTA-AM or 2-10 µM A23187, however, failed to affect the distribution of EGFP-PR72 or any of the EGFP-PR72 mutants (data not shown). The presence of PR72 in the nucleus was further confirmed by cell fractionation of L6 cells (rat myoblasts), where the majority of endogenous PR72 is present in the nuclear fraction (Fig. 5C).


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Fig. 5.   A, localization of wild-type and mutant EGFP-PR72 fusion proteins in COS7 cells. COS7 cells were transfected with pEGFP-C1-derived plasmids encoding EGFP fusions of PR72 or the PR72 EF mutants. After fixation of the cells and nuclear staining with Hoechst 33342, EGFP expression was examined with the fluorescence microscope. B, co-immunoprecipitation of PP2AC and A/PR65 with PR72-derived EGFP fusion proteins. Lysates of COS7 cells transfected with the upper EGFP fusion constructs were immunoprecipitated (IP) with a polyclonal anti-EGFP antibody. Whole cell lysates and the immunoprecipitations were then subjected to Western blotting and blots were developed with a mixture of anti-PP2AC and anti-PR65 monoclonal antibodies. C, Western blot of PR72 in nuclear and cytoplasmic extracts of L6 cells. Nuclear and cytoplasmic extracts of rat myoblast L6 cells, expressing PR72 were subjected to Western blotting. Blots were developed with a polyclonal anti-PR72 antibody raised against a C-terminal peptide of PR72 (15).

These remarkable results suggest that the integrity of EF2 is not only required for binding to the core enzyme but also for proper subcellular (nuclear) targeting of PR72. Apparently, PR72 cannot enter the nucleus unless incorporated within a trimeric PP2A complex. Moreover, these data suggest that withdrawal of B"/PR72 from the trimer does not necessarily lead to its degradation within living cells, which is in contrast with some observations done for the B/PR55 subunit (49). To ensure that the EGFP tag did not influence our observations, we also transiently overexpressed wild-type PR72, PR72 EF1mut, PR72 EF2mut, and PR72 EF(1 + 2)mut in human U2OS cells from the pCEP4 vector, and in all cases the wild-type or mutant PR72 proteins were expressed (results not shown). This confirms that PR72 mutants unable to bind to the A/PR65 subunit are not rapidly degraded within cells.

Effects of Ca2+ on the Ability of PR72 to Induce Cell Cycle Arrest-- Similar to B"/PR48 (17) and B"/PR59 (16), we noticed that forced expression of B"/PR72 in U2OS cells leads to a G1/S phase arrest. This arrest became apparent by comparing the propidium iodide-stained DNA profiles of PR72-EGFP-transfected and pEGFP-C1-transfected cells by flow cytometry (results not shown). This effect was more pronounced after prior blockage of the dividing cells in G2/M by the addition of nocodazole, a spindle de-polymerizing agent (Fig. 6A). Interestingly, if the same experiment was repeated in PR72 EF1mut-EGFP, PR72 EF2mut-EGFP, and PR72 EF(1 + 2)mut-EGFP expressing cells, the EF1 and EF(1 + 2) mutants partially lost the ability to induce the G1/S arrest, whereas the EF2 mutant even generated a more pronounced G1/S arrest (Fig. 6A). These results suggest that the ability of PR72 to induce a G1/S cell cycle arrest is at least partially dependent on the integrity of EF1. The administration of 10 µM BAPTA-AM induced a slight increase in the amount of cells in the G1 phase in EGFP-, PR72 EF1mut-EGFP-, and PR72 EF(1 + 2)mut-EGFP-transfected cells, whereas the opposite is true for the PR72-EGFP- and PR72 EF2mut-EGFP-transfected cells. These results confirm that Ca2+ binding to the low affinity EF1-binding site is (partially) necessary to generate the growth arrest, probably via a Ca2+-dependent interaction with a substrate or another binding partner. The more pronounced G1/S arrest in cells where PR72 EF2mut is overexpressed suggests that the mechanism by which PR72 induces the G1/S arrest likely occurs via competition of monomeric PR72 with a B"-containing PP2A holoenzyme for binding to this substrate or binding partner. The EF2 mutant indeed lacks the interaction with the core enzyme and therefore would have a more pronounced dominant negative effect than the wild-type protein.


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Fig. 6.   Forced expression of PR72 alters cell cycle progression: flow cytometry profiles of DNA content of EGFP, PR72-EGFP, PR72 EF1mut-EGFP, PR72 EF2mut-EGFP, and PR72 EF(1 + 2)mut-EGFP expressing U2OS cells in the presence of nocodazole, without (A) or with (B) the prior addition of BAPTA-AM. 24 h after transfection with the diverse expression plasmids, asynchronously growing U2OS cells were seeded from 6- to 10-cm dishes to induce growth. 24 h later cells were supplied with 1 µg/ml nocodazole for another 16 h before FACS analysis. If appropriate, 10 µM BAPTA-AM was added 6 h before nocodazole administration. The profiles of DNA content (propidium iodide fluorescence, FL3 channel) were selectively measured in EGFP-positive cells (FL1 channel). Estimates of the percentage of cells in G1, S, and G2/M stages of the cell cycle are included.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

This is the first report of a direct regulatory effect of calcium ions on protein phosphatase 2A. In the present study, we demonstrate that the B"/PR72 regulatory subunit of PP2A is a "classical" Ca2+-binding protein of the EF-hand type. PR72 contains two well conserved EF-hand motifs, which apparently exhibit different affinities for Ca2+ in an overlay assay. Because it is known that this type of assay merely detects high affinity binding sites (42), EF1 (aa290-302) could be catalogued as a low affinity binding site because of its poor Ca2+ binding, whereas EF2 (aa364-376) is clearly a high affinity binding site. Although both amino acid sequences of EF1 and EF2 conform to the overall EF-hand consensus, the difference in affinity could be explained by the presence of a glycine residue at position 6 within EF2, whereas EF1 has a leucine at this position. According to some reports (47), it is important that a relatively small amino acid is present at this particular position in between both helices E and F in order for calcium ions to bind effectively. Moreover, we have shown that Ca2+ binding to PR72 results in a significant conformational change, which is dependent on the integrity of the EF-hands. This structural change could be visualized by an increase in the intrinsic tryptophan fluorescence of the PR72 aa262-449 apoprotein upon the addition of Ca2+.

We further investigated the functionality of both EF-hands by a site-directed mutagenesis approach. We mutated in each EF-hand the glutamate or aspartate residues at positions 1 and 12, which are both involved in the Ca2+ coordination (47), into alanines. These mutations did not only abolish Ca2+ binding to these motifs, but also changed the overall protein structure relatively little. This is an important observation, making it highly unlikely that specific defects of the EF mutants (as observed in further experiments) can be explained by conformational changes, which are sometimes inherent to the introduction of the mutations themselves.

By having established by a yeast two-hybrid approach that the minimal A subunit interacting domain of PR72 comprises both EF-hands, we were prompted to investigate whether these motifs are directly involved in A subunit binding. By a combination of both yeast and mammalian double-hybrid experiments, in vitro GST-PR65 pull-down assays, and anti-EGFP co-immunoprecipitations, we have shown that mutation of Asp-290 and Asp-301 within EF2 destroys binding of PR72 to the A subunit. Given that these mutations also destroy Ca2+ binding to EF2, this could suggest that Ca2+ binding to EF2 and the resulting conformational change are required for A subunit binding. Alternatively, Asp-290 and Asp-301 may be structurally important amino acids that are directly involved in protein-protein contacts with the A subunit. However, the identical intrinsic fluorescence spectra of PR72 aa262-449 and PR72 aa262-449 EF(1 + 2)mut argue against this explanation. Because EF2 is a high affinity Ca2+-binding site in vitro, it would not be unlikely that it constitutively binds calcium ions in vivo, where the normal intracellular Ca2+ concentration is a few hundred nanomolars. An accurate determination of the affinity constant of EF2 for Ca2+ binding would make this more clear. The fact, however, that calcium chelators, even at high concentrations, are hardly capable of affecting the PR72-PR65 interaction in vitro as well as in vivo suggests that either the affinity of EF2 for Ca2+ is very high or that Ca2+ is inaccessible for these chelators because it is embedded within the PP2AT72 or PR72 protein structure.

Another remarkable result was the abolishment of the proper nuclear localization of PR72 by mutation of EF2, suggesting that its incorporation within the trimer is required for its subcellular targeting. This could indicate that the trimer context is required for the interaction with a specific import protein or, alternatively, with a modifying enzyme that takes care of a specific modification necessary for nuclear import. It should be noticed that PR72 contains two putative nuclear localization signals within its primary structure (15), which may be functional nuclear targeting motifs as well. This issue awaits further clarification.

A role for EF1, the low affinity calcium-binding site, is suggested by the in vitro measurement of PP2AT72 phosphatase activity toward phosphorylase a, where only the addition of high Ca2+ concentrations resulted in inhibitory effects. Whether calcium ions also affect PP2A activity toward the as yet unknown physiological substrate(s) of PP2AT72 awaits further investigation. Unfortunately, the effects of Ca2+ on the phosphatase activity of a PP2AT72 enzyme with a mutated EF1 or EF2 motif could not be assessed, because we were unable to reconstitute such an enzyme in vitro with PP2AD and bacterially expressed and purified PR72 or its mutants. Nevertheless, EF1 is (due to its relatively low affinity for Ca2+) likely the site with the highest regulatory potential in vivo, because it might act as a "calcium sensor" that transiently binds Ca2+ upon local or temporal rises in the intracellular Ca2+ concentration. But whether Ca2+ binding to EF1 would result in activation or inhibition of PP2AT72 toward a particular in vivo substrate cannot be predicted from our in vitro experiments.

However, that EF1 may operate as a calcium sensor is supported by the cell cycle experiments, where it became clear that the ability of PR72 to induce a G1/S arrest in U2OS cells is at least partially dependent on the integrity and the Ca2+ recruiting ability of EF1. Note that in this case the addition of BAPTA-AM did have an effect on the properties of the wild-type protein. We propose that forced expression of monomeric PR72 may act as a dominant negative, influencing phosphatase activity indirectly. Because PR72 has a higher affinity for the A/PR65 subunit than PR55 or PR61 (Fig. 3A), it could be integrated in a trimer by expelling B/PR55 and B'/PR61 from their holoenzymes or by interacting with a pre-existing dimer. Dephosphorylation of specific substrates for PP2AT55, PP2AT61, or PP2AD would therefore be inhibited, whereas specific PP2AT72 substrates might be stimulated. On the other hand, "free" PR72 may compete with PP2AT72 for an interacting protein that would normally mediate a specific PP2AT72 effect. This would lead to inhibition of dephosphorylation of specific PP2AT72 substrates. Therefore, here again, it cannot be predicted whether overexpression of PR72 would lead to inhibition or stimulation of dephosphorylation of some PP2A substrates. However, PP2A activity is required for the firing of replication origins (50, 51), and this may be mediated by PR72 (21) or another B" family member. Binding of PR72 with a relevant substrate in this case likely occurs via the Ca2+-bound EF1 motif, and Ca2+ binding to EF1 would likely stimulate PP2A activity. Cdc6 is one of the candidates to be this binding partner, because it interacts with PR48 and with aa354-1150 of PR130 (a region that encompasses the common part of PR130 and PR72) in yeast two-hybrid assays (17). The more pronounced dominant negative effect of the PR72 EF2 mutant, which lacks the interaction with the core enzyme and the proper subcellular localization, additionally suggests that this binding partner is (initially) not necessarily present in the nucleus.

Compared with B'/PR61gamma 1 and especially with B/PR55alpha , B"/PR72 strongly interacts with the A/PR65 subunit in the mammalian two-hybrid system. This opens up the possibility that PR72 effectively competes with both PR61gamma 1 and PR55alpha for binding to PP2AD. Moreover, and in contrast with the reported data on the PR55 subunit (49), our data suggest that PR72 mutants failing to interact with the core enzyme are not highly unstable within cells. This suggests that in contrast with PP2AC, A/PR65 (4, 5), and B/PR55 (49), the cell lacks a control mechanism to avoid the presence of free B"/PR72, provided of course that such a population would exist. These findings therefore contribute to a better understanding of the dynamics of the various PP2A complexes in vivo.

Together, we have demonstrated a role for Ca2+ in PP2AT72 subunit assembly, nuclear targeting, catalytic activity, and PR72-mediated cell cycle regulation. Whether the other B"/PR72 family members also bind Ca2+ will have to be determined but is highly likely, given the conservation of the EF motifs within these proteins. This is obviously true for the PR130 subunit, a splice variant generated from the same gene as PR72 that shares both EF-hands. In the light of a recent report (52) demonstrating an interaction of PR130 with the ryanodine type 2 receptor, this is certainly an interesting observation. Moreover, given the muscle-specific expression of PR72 and the important role of calcium in muscle-specific processes, such as muscle contraction, our data open up a new avenue for further research at the interface between calcium signaling and protein phosphorylation/dephosphorylation.

    ACKNOWLEDGEMENTS

We appreciate the help and critical comments of Dr. I. Sienaert (former member of the Laboratory of Physiology, Faculty of Medicine, Katholieke Universiteit Leuven) and the expert technical assistance of Fabienne Withof and Roos Verbiest. We thank Dr. F. Claessens, Dr. M. Beullens, Dr. S. Dilworth, and Dr. J. Elce for their willingness to provide plasmids or antibodies.

    FOOTNOTES

* This work was supported in part by the Inter-University Poles of Attraction, the Geconcerteerde Onderzoeksacties van de Vlaamse Gemeenschap, and the Fonds voor Wetenschappelijk Onderzoek (F.W.O.) Vlaanderen.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Postdoctoral fellow of the F.W.O.-Vlaanderen.

|| Recipient of a postdoctoral fellowship from the Onderzoeksfonds Katholieke Universiteit Leuven.

** Predoctoral fellow supported by European Grant QLK3-CT-2000- 01038.

§§ To whom correspondence should be addressed: Afdeling Biochemie, Gasthuisberg O&N, Herestraat 49, B-3000 Leuven, Belgium. Tel.: 32-16-345-796; Fax: 32-16-345-995; E-mail: jozef.goris@med.kuleuven.ac.be.

Published, JBC Papers in Press, January 10, 2003, DOI 10.1074/jbc.M211717200

    ABBREVIATIONS

The abbreviations used are: PP2A, protein phosphatase 2A; aa, amino acid; ASBD, A subunit binding domain; CaM, calmodulin; GANP, germinal center-associated nuclear protein; PP2AC, PP2A catalytic subunit; PP2AD, dimeric form of PP2A; PP2AT, trimeric form of PP2A; PP2AT72, PR72-containing PP2A trimer; EGFP, enhanced green fluorescent protein; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid; GST, glutathione S-transferase; FACS, fluorescence-activated cell sorter.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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