©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of a Novel Protein Phosphatase 2A Regulatory Subunit Highly Expressed in Muscle (*)

(Received for publication, September 25, 1995; and in revised form, December 21, 1995)

Mohsen Ahmadian Tehrani (§) Marc C. Mumby Craig Kamibayashi (¶)

From the University of Texas Southwestern Medical Center, Department of Pharmacology, Dallas Texas 75235-9041

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Differential association of regulatory B subunits with a core heterodimer, composed of a catalytic (C) and a structural (A) subunit, is an important mechanism that regulates protein phosphatase 2A (PP2A). We have isolated and characterized three novel cDNAs related to the B` subunit of bovine cardiac PP2A. Two human (B`alpha1 and B`alpha2) and a mouse (B`alpha3) cDNA encode for alternatively spliced variants of the B` subunit. The deduced primary sequences of these clones contain 12 of 15 peptides derived from the purified bovine B` subunit. Differences between the deduced sequences of the B`alpha splice variants and the cardiac peptide sequences suggest the existence of multiple isoforms of the B` subunit. Comparison of the protein and nucleotide sequences of the cloned cDNAs show that all three forms of B`alpha diverge at a common splice site near the 3`-end of the coding regions. Northern blot and reverse transcription-polymerase chain reaction analyses revealed that the B`alpha transcripts (4.3-4.4 kb) are widely expressed and very abundant in heart and skeletal muscle. The expressed human and mouse B`alpha proteins readily associated with the PP2A core enzyme in both in vitro and in vivo complex formation assays. Immunofluorescence microscopy revealed that epitope-tagged B`alpha was localized in both the cytosol and nuclei of transiently transfected cells. The efficiency of binding of all three expressed proteins to a glutathione S-transferase-A subunit fusion protein was greatly enhanced by the addition of the C subunit. Expression of the B`alpha subunits in insect Sf9 cells resulted in formation of ACbulletB`alpha heterotrimers with the endogenous insect A and C subunits. These results show that the B` subunit, which is the predominant regulatory subunit in cardiac PP2A, is a novel protein whose sequence is unrelated to other PP2A regulatory subunits. The nuclear localization of expressed B`alpha suggests that some variants of the B` subunit are involved in the nuclear functions of PP2A.


INTRODUCTION

Protein phosphorylation is an essential mechanism regulating a wide variety of cellular processes. The coordinate activity of protein kinases and phosphatases is required for normal signal transduction. Protein phosphatase 2A (PP2A) (^1)is a serine/threonine phosphatase that has been implicated in the control of the cell cycle(1) , growth and proliferation(2) , DNA replication(3) , viral transformation(4) , and morphogenetic events(5) . The PP2A holoenzyme is a heterotrimer composed of a 38-kDa catalytic (C) subunit, a 63-kDa structural (A) subunit, and a third subunit termed B or phosphatase regulatory (PR) subunit(6, 7) . There are at least five distinct families of proteins that interact with and regulate the PP2A core enzyme. These include the B, B`, PR72 (B"), and families of regulatory subunits and the small and middle tumor antigens of DNA tumor viruses(8) .

The diversity of PP2A regulatory subunits suggests specific physiological roles for individual holoenzymes. Genetic studies have shown that disruption of the yeast homolog of the Balpha subunit, CDC55, results in defects in budding and cytokinesis(9) . Decreased expression of the Drosophila Balpha subunit causes defects in mitosis, duplication of wing imaginal discs and is lethal in the late larval/early pupal stage of development.(5, 10) . Biochemical studies have demonstrated that the B subunits and tumor antigens affect the substrate specificity and activity of PP2A(11, 12, 13) . Sensitivity to polyamines, polycations, and ceramide is also dependent on the type of B subunit associated with the AC core complex(13, 14, 15) . These data support an emerging hypothesis that differential association of B subunits regulates PP2A function in vivo.

Molecular cloning has revealed diversity within the PP2A regulatory subunit families. Multiple isoforms (alpha, beta, ) of the B subunit and a splice variant of Balpha have been isolated from mammalian sources (9, 16, 17) . These proteins are 81-87% identical and diverge primarily at the amino termini. Two cDNAs encoding 72- and 130-kDa forms of the PR72 (B") subunit were obtained from human heart and brain libraries(18) . PR130 has an extension at the amino terminus that is generated either by alternative splicing or the use of an alternative promoter site. In this report, we describe the isolation and expression of novel cDNAs encoding human (B`alpha1 and B`alpha2) and mouse (B`alpha3) members of the B` familiy of PP2A regulatory subunits. The recombinant subunits readily form heterotrimeric complexes with the AC core enzyme in vitro and in vivo. Transient expression of human B`alpha in mammalian cells revealed the presence of both cytoplasmic and nuclear populations of the regulatory subunits. The B`alpha subunits may be important for the localization/translocation of PP2A into the nucleus.


MATERIALS AND METHODS

Molecular Cloning of the Human B`-Subunit

PP2A (ACbulletB`) was purified from bovine cardiac tissue, and partial amino acid sequence of the B` subunit determined as described previously(12) . The peptides derived from the bovine cardiac B` subunit were used to search for similar sequences in the GenBank data base using the BLAST network service of the National Center for Biotechnology Information. This search revealed significant homologies between the peptides and a random cDNA cloned from human KG1 cells (accession number D26445). The sequence of this cDNA was used for the design of oligonucleotide primers. An 854-bp fragment was amplified by polymerase chain reaction (PCR) as described previously (13) from a human umbilical vein epithelial cell (HUVEC) cDNA library (kindy provided by Drs. J. Battey and Mark Akeson, NIDCD) using a sense primer 5`-CTGAGTGTCTACCATCCCCAG-3` (nucleotides 775-795) and an antisense primer 5`-TGGAATTCGTTTGAACTTCTAATCT-3` (nucleotides 1605-1629) in which several bases were mutated (underlined) to generate an EcoRI restriction site. The thermal profile (94 °C, 1 min; 50 °C, 1 min; 72 °C, 1 min) was carried out for 30 cycles. The PCR fragment was subcloned into pCRII (Invitrogen) and the sequence was verified. Random priming of this fragment was used to generate a radiolabeled probe (10^9 dpm/µg) to screen the HUVEC ZAP cDNA library. Prehybridization and hybridization of filters containing 10^6 independent recombinants was performed at 65 °C in 5 times SSC, 5 times Denhardt's solution, 0.1% SDS, and 100 µg/ml herring sperm DNA. The filters were washed 4 times (5 min) in 2 times SSC, 0.1% SDS at room temperature, followed by a 20-min wash in 0.4 times SSC, 0.1% SDS at 65 °C, and subjected to autoradiography at -20 °C. Five positive clones (HB`-2, 3, 5, 6, 7) were obtained after two rounds of plaque purification. Recovery of plasmids from the HUVEC cDNA library was carried out according to the manufacturer's protocol (Stratagene). The cDNAs were sequenced on both strands by the dideoxynucleotide chain termination method(19) .

Yeast Two-hybrid Screening

The A subunit of PP2A (20) was inserted into the SmaI/SalI site of pAS1-CYH2 to generate a fusion with the GAL4 DNA binding domain. Yeast transformed with this plasmid were used to screen a mouse T-lymphocyte cDNA library, constructed as fusions with the GAL4 activation domain (10^6 transformants), using the two-hybrid assay(21) . Positive colonies were selected on Trp, Leu, and His medium in the presence of 50 mM 3-aminotriazole and screened for beta-galactosidase activity by the colony lift method(22) . The activation domain pACT-fusion plasmids were extracted from beta-galactosidase negative colonies (25) after growth on Leu, cycloheximide (100 µg/ml) medium to remove the pAS1-A subunit plasmid. Sequences of the fusion plasmids were determined and used in BLAST searches of the nucleic acid and protein data bases.

GAL4 DNA binding domain fusions of human B`alpha, p53, and the A subunit were tested for binding specificity with GAL4 activation domain fusions of the A and C subunits of PP2A. The A and C subunits of PP2A were inserted into the EcoRI/XhoI and SmaI/SalI sites of pACTII, respectively. NcoI/BamHI fragments from the pRcCMV-B`alpha plasmids (see below) were subcloned into the appropriate sites in the GAL4 DNA binding domain vector, pAS1-CYH2. Expression of beta-galactosidase activity in the two-hybrid assay was determined as described above(22) .

Northern Analysis and Reverse Transcription PCR

A 1.5-kb EcoRI fragment (nucleotides 2501-4064 of B`alpha1 clone HB`-7) was used to probe a mRNA blot of human tissues (Clontech). Prehybridization was carried out in 50% formamide, 10 times Denhardt's solution, 5 times SSPE, 2% SDS, and 100 µg/ml herring sperm DNA at 42 °C. Hybridization was carried out in the same solution with the randomly primed, radiolabeled (1.5 times 10 dpm/µg) EcoRI fragment for 16 h at 42 °C. The filter was washed twice (20 min) in 2 times SSC, 0.05% SDS at room temperature followed by a 20-min wash in 0.2 times SSC, 0.1% SDS at 50 °C. The blot was subjected to autoradiography for 6 h at -80 °C with an intensifying screen.

Poly(A) RNA was isolated from mouse tissues, and first strand cDNA was synthesized as described previously(23) . The mouse cDNA was used in a PCR with oligonucleotide primers corresponding to nucleotides 837-857 (sense 5`-CTGAGTGTCTACCATCCCCAG-3`) and 1413-1436 (antisense 5`-CTCGGACTTGCGGCGTGCAAGAGG-3`) of B`alpha2. The thermal profile (94 °C, 1 min; 55 °C, 1 min; 72 °C, 1 min) was carried out for 30 cycles.

Construction of B`alpha Expression Plasmids

To facilitate the insertion of the HUVEC B`alpha cDNAs into various expression vectors, a short fragment (nucleotides 77-158) of clone HB`-7 (B`alpha1) was amplified by PCR and subcloned into pCRII. The antisense primer was 5`-GAACATCTCGAATATGGAGAAGGA-3`, while several bases of the sense primer (5`-GGGGATCCAACCATGGTGGTGGATGCG-3`) were mutated (underlined) to generate BamHI and NcoI restriction sites. Nucleotides 130-1662 of HB`-7 (B`alpha1) was excised and inserted into the PflMI/ApaI site of pCRII. Since clone HB`-5 (B`alpha2) lacked a portion of the 5`-coding sequence, an AvrII/ApaI fragment from this cDNA was ligated into the same site of pCRII-B`alpha1 to generate full-length pCRII-B`alpha2. HindIII and HindIII/ApaI fragments from pCRII-B`alpha1, containing the complete open reading frame, were subcloned into the mammalian expression vector, pRcCMV (Invitrogen). A HpaI/ApaI fragment was removed from pRcCMV-B`alpha1 and replaced with the corresponding fragment from pCRII-B`alpha2 to create pRcCMV-B`alpha2. Donor plasmids for insect cell expression were constructed by inserting BamHI/NheI fragments from pRcCMV-B`alpha1 and pRcCMV-B`alpha2 into the BamHI/SpeI site of pFASTBAC (Life Technologies, Inc.). SpeI/NheI fragments of pRcCMV-B`alpha1 and pRcCMV-B`alpha2 were also inserted into pMamNeo (Clontech). BamHI/SalI fragments from the pMamNeo constructs were then subcloned into the BglII/SalI site of pFLAG-CMV2 (Eastman Kodak Co.) to generate plasmids for expression of epitope-tagged B`alpha. The cDNA for the human Balpha subunit of PP2A (13) was subcloned into the BamHI site of pFLAG-CMV2.

In Vitro Translation of B`alpha and GST-A Binding Assays

A PCR product was amplified from the mouse T-lymphocyte pACT-B`alpha3 plasmid using a sense primer 5`-TAATACGACTCACTATAGGGAGACCACATGGATGATGTATATAACTATCATTTC-3`, containing a T7 promoter site and an in-frame initiator methionine and using an antisense primer against the pACT vector (5`-CTACCAGAATTCGGCATGCCGGTAGAGGTGTGGTCA-3`). In vitro translation products were synthesized with the mouse B`alpha3 PCR fragment, pRcCMV-B`alpha1, and pRcCMV-B`alpha2 as templates using the TnT T7 coupled reticulocyte lysate system (Promega). GST and GST-A subunit fusion proteins were prepared from Escherichia coli and used in binding assays with the [S]methionine-labeled B`alpha proteins(2) . In some cases, purified bovine cardiac C subunit (2.5 µg) was preincubated for 30 min (4 °C) with GST (16 µg) and the GST-A subunit fusion protein (16 µg) prior to addition of labeled B`alpha translation products. Bound proteins were eluted with 10 mM glutathione, resolved by SDS-PAGE, followed by fluorography.

Association of Expressed B`alpha with the A and C Subunits of PP2A in Sf9 Cells

Recombinant baculoviruses encoding the B`alpha subunits were generated using the BAC to BAC expression system according to the manufacturer's protocol (Life Technologies, Inc.). Insect cells (4 times 10^7) were infected with recombinant B`alpha baculoviruses(20) , harvested 48 h postinfection, and washed twice with phosphate-buffered saline (PBS). Subsequent operations were performed at 4 °C. The cells were resuspended in 3 ml of HS buffer (10 mM imidazole, pH 6.5, 1 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 10% glycerol) and homogenized in a Duall homogenizer. The homogenate was centrifuged at 12,000 times g for 15 min, and the supernatant was loaded onto a heparin-Sepharose column (5 ml) equilibrated in HS buffer at a flow rate of 1 ml/min. The column was washed with the same buffer until the A returned to base line. The column was eluted with 20 ml each of 0.1, 0.3, and 0.5 M NaCl in HS buffer, and 1-ml fractions were collected. An aliquot of the fractions was analyzed by immunoblotting with B`alpha specific antiserum. An aliquot (0.25 ml) of fractions 40 (0.3 M NaCl elution) and 60 (0.5 M NaCl elution), which contained B`alpha, was adjusted to 0.5 ml with HS buffer and applied to a Superdex 200 10/30 gel filtration column in HS buffer containing 0.5 M NaCl. A second aliquot of fraction 60 was incubated with the purified AC form of bovine cardiac PP2A (20 µg) for 30 min prior to chromatography. Chromatography was carried out at a flow rate of 0.25 ml/min, and 0.5-ml fractions were collected. The fractions were analyzed by immunoblotting with antibodies against the C (24) and B`alpha subunits.

Expression of B`alpha in Mammalian Cells

Mouse NIH 3T3 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% calf serum. Cells were transfected with pFLAG-CMV2 constructs using the liposome method(2) . The cells were harvested 48 h post-transfection and washed twice with PBS. Cells were Dounce homogenized, and nuclei were isolated by centrifugation (800 times g, 10 min) through a 1 mM imidazole, pH 7.4, 0.25 M sucrose cushion. The supernatant was recentrifuged at 12,000 times g (10 min), and the resulting supernatant was the cytosolic fraction. The nuclei were washed once, and nuclear extracts were prepared according to Krainer et al.(25) . Cytosolic and nuclear proteins were resolved by SDS-PAGE and analyzed for the expression of B`alpha1, B`alpha2, and Balpha by immunoblotting.

Immunofluoresence Microscopy

NIH3T3 cells transfected with pFLAG-B`alpha1, pFLAG-B`alpha2, or pFLAG-Balpha were trypsinsized after 24 h and grown on glass coverslips. The cells were fixed 48 h post-transfection by incubation in absolute methanol for 5 min at -20 °C and washed twice with PBS. Nonspecific sites were blocked by incubation in PBS containing 5% goat serum (1 h). Subsequent antibody incubations (1 h) and washes were carried out in PBS containing 1% goat serum. The cells were incubated with monclonal anti-FLAG 5 antibody (Kodak) at a concentration of 10 µg/ml followed by a Cy3-conjugated affinity-purified goat anti-mouse antibody (Jackson ImmunoResearch Labs) at a dilution of 1:400. The coverslips were mounted with Fluoromount-G (Fisher) and examined with an Olympus AX70 microscope (600times magnification).

Production of B`alpha Specific Antisera

A synthetic peptide (CPQAQKDPKKDR) corresponding to amino acids 431-441 of B`alpha2 and residues 471-480 of B`alpha1 was coupled to keyhole limpet hemocyanin. The amino-terminal cysteine was added to facilitate the coupling. Peptide conjugation and polyclonal antibody production were carried out as described previously(13) .

Protein Analyses

Protein concentrations were determined by the Bradford method (26) using bovine serum albumin as standard. SDS-PAGE was carried out in 0.75-mm-thick slab gels containing 7.5 or 10% acrylamide concentration(27) . Immunoblot analyses were performed using Fab(2) fragments of horseradish peroxidase-conjugated donkey anti-rabbit (1:10,000) or sheep anti-mouse (1:10,000) and the enhanced chemiluminescence system (Amersham Corp.) to detect bound antibody(13) .


RESULTS

Molecular Cloning of cDNAs Encoding B`alpha

The major form of regulatory subunit in purified bovine cardiac PP2A is related to the B` subunit originally identified in a form of rabbit muscle PP2A termed PP2A(0)(17) . Partial amino acid sequence of the bovine cardiac PP2A B` subunit was obtained from tryptic and cyanogen bromide peptides. 15 peptides were sequenced, and a total of 177 residues were determined. A search of the protein data bases with these peptides indicated a high degree of similarity with the translated sequence of a randomly cloned human cDNA from myeloid KG1 cells (accession number D26445). The KG1 cDNA was 3.7 kb and the longest open reading frame encoded for a protein of 475 amino acids. The deduced sequence of the KG1 cDNA contained 12 of the 15 peptides obtained from bovine cardiac B`. The similarity strongly suggested that the human cDNA was related to the bovine B` subunit. A Saccharomyces cerevisiae cDNA, RTS1 (accession number U06630) encoding a suppressor of a defect in the ROX3 transcription factor was also found to contain some of the bovine B` peptides. Although the similarity is much lower, it appears that this clone is a yeast homolog of the mammalian B` subunit (Fig. 1).


Figure 1: Alignment of the deduced amino acid sequences of human B`alpha1, B`alpha2, mouse B`alpha3, and residues 127-561 of yeast RTS1. Peptides derived from purified bovine cardiac B` are shown below the aligned sequences. The asterisks represent identities, and the dashed lines indicate gaps. The bipartite nuclear localization signal in B`alpha1 is underlined. The nucleotide sequences of human B`alpha1 (accession number U37352), and mouse B`alpha3 (accession number U37353) have been deposited in the GenBank data base. The accession number for human B`alpha2 (KG1 ORFY) is D26445 and for yeast RTS1 is U06630.



Oligonucleotide primers were designed from the human sequence in the data base, and a 854-bp PCR product was amplified from a HUVEC ZAP cDNA library. The PCR product was sequenced and found to be identical to nucleotides 775-1629 of the KG1 cDNA. Five positive clones were isolated from the HUVEC cDNA library with the 854-bp probe using stringent hybridization and wash conditions. The largest cDNA (HB`-7) was 4064 bp long and nearly identical in sequence to the KG1 cDNA. Two base changes were present in the 3`-untranslated region of the HUVEC cDNA; nucleotide 2082 was G instead of A, and nucleotide 3847 was A instead of G. The HUVEC cDNA extends the 5`- and 3`-untranslated regions of the KG1 sequence by 62 and 182 nucleotides, respectively. The sequence around the putative initiator codon, AGCAGGATGGTGG, conforms to the consensus motif for translation initiation in vertebrates(28) . Four potential polyadenylation sites (AATAAA) are present at nucleotides 2480, 3677, 3851, and 4027. There is also an insertion of 117 nucleotides in the HB`-7 cDNA that is absent in the KG1 cDNA. The insertion occurs after nucleotide 1385 and encodes for an additional 39 amino acids (433-472). We have designated this cDNA B`alpha1. The deduced amino acid sequence encodes for a protein of 514 residues with a predicted molecular weight of 59,995 and an isoelectric point of 6.1 (Fig. 1).

The remaining cDNAs were found to be partial clones, three of which corresponded to B`alpha1. The fourth cDNA (HB`-5) lacked 617 nucleotides of the 5` sequence, terminated at nucleotide 3138 of B`alpha1, and did not contain the 117-nucleotide insertion. This cDNA corresponded to the KG1 sequence in the data base, and we have designated it B`alpha2. The deduced amino acid sequence encodes for a protein of 475 residues with a predicted molecular weight of 55,958 and an isoelectric point of 6.5 (Fig. 1).

An additional B`alpha cDNA was isolated in an independent screen of a mouse T-lymphocyte library for proteins that interact with the A subunit of PP2A using the yeast two-hybrid assay. One of the cDNAs that interacted with the GAL4 DNA binding domain-A subunit fusion protein was a mouse homolog of human B`alpha. This cDNA (B`alpha3) was 1.35 kb, lacked a portion of the 5` sequence, and contained 37 nucleotides of the 3`-untranslated sequence. The partial open reading frame encodes for a protein of 435 residues, with a predicted molecular weight of 51,158. An alignment of the human, mouse, and a portion of the yeast RTS1 primary sequences is shown in Fig. 1. There are three conserved substitutions in the mouse sequence compared with human B`alpha1 (88% identity) and B`alpha2 (92% identity). The mouse sequence terminates 10 residues after the B`alpha1 insertion begins.

Based on the nucleotide sequence identity, the two human forms are likely to arise by alternative splicing of a single gene. A putative splice acceptor site boundary, CCCAGG (nucleotides 764-769 in B`alpha2, 1319-1324 in the KG1 cDNA, and 1379-1384 in B`alpha1), is present at the point of divergence between the B`alpha1 and B`alpha2 sequences. Consistent with these observations is the conservation of the same splice site junction in the mouse B`alpha3 cDNA (nucleotides 1231-1236). The presence of the 39-amino acid insertion in B`alpha1 produces a bipartite nuclear targeting signal (29) that is absent in both B`alpha2 and B`alpha3. The yeast homolog of B`alpha diverges from its mammalian counterparts at the amino and carboxyl termini, which are not shown in Fig. 1. The overall identity between the yeast protein and B`alpha1, B`alpha2, and mouse B`alpha3 is 39, 40, and 43%, respectively. However, if the divergent termini are omitted and the comparison done is on residues 127-561 of the yeast protein, the identity increases to 68%.

Expression and Distribution of B`alpha mRNA

The levels of transcripts encoding human B`alpha were analyzed in poly(A) RNA isolated from human tissues (Fig. 2A). A single major transcript of 4.4 kb was detected in all tissues examined. The B`alpha mRNA was very abundant in heart, skeletal muscle, and brain. Lower levels were present in the pancreas, kidney, lung, and placenta. Liver had a very low level of the B`alpha mRNA that was detectable with longer exposure times (data not shown).


Figure 2: Expression and tissue distribution of B`alpha mRNA. Panel A, Poly(A) RNA (2 µg) from the indicated human tissues was hybridized with a B`alpha specific probe as described under ``Materials and Methods.'' Panel B, first strand cDNA was prepared from poly(A) RNA isolated from mouse tissues and used in a PCR with B`alpha specific primers as described under under ``Materials and Methods.'' An aliquot of each reaction was resolved on a 1% agarose gel and stained with ethidium bromide (0.5 µg/ml). The migration of molecular size standards (kb) are indicated to the left of each panel.



The probe used in the Northern analysis does not distinguish between the alternatively spliced forms of human B`alpha. Therefore, expression of the alternatively spliced forms of B`alpha mRNA was examined in mouse tissues by reverse transcription PCR (Fig. 2B). Two major bands were amplified from each tissue that had mobilities identical to the B`alpha1 (717 bp) and B`alpha2 (600 bp) controls. Similar amounts of cDNA were present in each PCR reaction, since the ubiquitously expressed mRNA for cyclophilin was amplified to similar levels with all of the cDNAs (data not shown). Approximately the same ratios of B`alpha1 and B`alpha2 were observed in mouse tissues except liver and especially brain, where B`alpha2 was more prevalent. In contrast to the human Northern blot, mouse skeletal muscle contained significantly lower levels of the B`alpha transcripts. Interestingly, bands of 650 and 750 bp were also amplified with the heart, brain, and skeletal muscle cDNAs. Although we have not identified these products, they may be due to the presence of additional splice variants of B`alpha or other isoforms of the B` regulatory subunit.

Association of Recombinant B`alpha Subunits with PP2A

Several approaches were taken to show that the human and mouse B`alpha cDNAs encode proteins that interact with the PP2A core enzyme. Yeast strains containing GAL4 activation domain fusions (GAD) of the A and C subunits of PP2A were transformed with plasmids encoding fusions between the GAL4 DNA binding domain (GDB) and human B`alpha1, B`alpha2, p53, and the A subunit of PP2A (Table 1). Both of the GDB-fusions of human B`alpha interacted with the GAD-A subunit fusion and activated transcription of the HIS3 and lacZ genes in the two-hybrid assay. No hybrids were formed between either GDB-B`alpha fusions and activation domain fusions of the PP2A C subunit or p53. As a positive control, beta-galactosidase expression was activated by the GDB-A subunit in the strain containing the GAD-C fusion.



In vitro transcription/translation of B`alpha cDNAs in reticulocyte lysates directed the synthesis of [S]methionine-labeled human and mouse B`alpha proteins. The expressed proteins had apparent molecular masses of 60 kDa (Fig. 3A, lane 1, B`alpha1), 56-kDa (Fig. 3A, lane 2, B`alpha2), and 52-kDa (Fig. 3A, lane 3, B`alpha3), which were nearly identical to the predicted molecular weights of each splice variant. The interaction of the expressed proteins with the A subunit was assayed using a GST-A subunit fusion protein. A low level of binding to GST-A was observed with B`alpha1 (Fig. 3B, lane 1), B`alpha2 (Fig. 3C, lane 1), and B`alpha3 (Fig. 3D, lane 1). However, preincubation of the GST-A fusion protein with the C subunit of PP2A significantly enhanced the binding of all three forms of B`alpha (lane 2). The interaction of the B`alpha proteins with the A subunit was specific, as no binding was observed with GST alone (lane 3) or when GST was preincubated with the C subunit (lane 4). The minor bands present in the GST-A lanes may be due to proteolysis of B`alpha1 and B`alpha2 or initiation from an internal methionine.


Figure 3: Binding of B`alpha proteins to a GST-A subunit fusion protein. Panel A, SDS-PAGE of in vitro translation products of human B`alpha1 (lane 1), human B`alpha2 (lane 2), and mouse B`alpha3 (lane 3) cDNAs. A control sample containing no cDNA template was applied to lane 4. An aliquot of the translation mixture containing B`alpha1 (panel B), B`alpha2 (panel C), and B`alpha3 (panel D) were assayed for binding to a GST-A subunit fusion protein as described under under ``Materials and Methods.'' Lane 1, GST-A subunit; lane 2, GST-A subunit + purified bovine C subunit (2.5 µg); lane 3, GST fusion protein; lane 4, GST fusion protein + purified bovine C subunit (2.5 µg). The migration of molecular mass standards including bovine serum albumin (66-kDa), glutamate dehydrogenase (55-kDa), ovalbumin (44-kDa), and aldolase (40-kDa) are shown to the left of each panel.



Recombinant B`alpha proteins were also expressed in the baculovirus-insect cell system. Homogenates from infected cells were partially purified by heparin-Sepharose and size exclusion chromatography to determine if the expressed B`alpha proteins interacted with the endogenous AC core enzyme. The B`alpha proteins in infected Sf9 cell extracts were eluted from heparin-Sepharose with both 0.3 and 0.5 M NaCl (data not shown). When B`alpha1 in the 0.3 M NaCl fraction was applied to a gel filtration column, the peak of immunoreactivity co-eluted with the C subunit of PP2A (Fig. 4A) in fractions 24-26, which correspond to the elution position of purified cardiac ACbulletB` (M(r) = 156,000). The 0.5 M NaCl heparin-Sepharose fraction, which did not contain any detectable C subunit (data not shown), eluted in fractions 27-29, corresponding to the predicted molecular weight of monomeric B`alpha1 (Fig. 4C; left half of panel). Incubation of the 0.5 M NaCl heparin-Sepharose fraction with purified bovine cardiac AC dimer shifted the peak of B`alpha1 to fractions 24-26, corresponding to heterotrimeric PP2A (Fig. 4C; right half of panel). Similar results were obtained with recombinant B`alpha2 (Fig. 4, B and D). Formation of the ACbulletB`alpha complexes was not disrupted by chromatography in 0.5 M NaCl. These properties of the expressed B`alpha1 and B`alpha2 proteins are identical to those described previously for the B` subunit purified from cardiac tissue(20) . These data demonstrate that the cloned B`alpha proteins bind directly to the A subunit of PP2A and readily form heterotrimeric complexes with the AC core enzyme in vitro and in vivo.


Figure 4: In vivo and in vitro reconstitution of expressed B`alpha proteins with the AC core enzyme of PP2A. Homogenates of insect cells infected with baculoviruses encoding B`alpha1 (panels A and C) and B`alpha2 (panels B and D) were partially purified on a heparin-Sepharose column. Aliquots of the fraction eluted with 0.3 M NaCl (panels A and B) and 0.5 M NaCl (left half of panels C and D) were separately chromatographed on a Superdex 200 10/30 column. A second aliquot of the 0.5 M NaCl fractions (right half of panels C and D) were incubated with purified bovine AC (20 µg) for 30 min (4 °C) prior to chromatography. The fractions from the Superdex column were analyzed by immunoblotting for B`alpha1 (panels A and C), B`alpha2 (panels B and D), and the C subunit of PP2A (panels A and B). The homogenates were used as positive controls (+) for the expression of B`alpha1 and B`alpha2. The migration of molecular mass standards including bovine serum albumin (66-kDa), glutamate dehydrogenase (55-kDa), and aldolase (40-kDa) are shown to the left of each panel.



Localization of B`alpha in Mammalian Cells

Indirect immunofluorescence of NIH3T3 cells expressing FLAG-B`alpha revealed both cytoplasmic and nuclear populations of the recombinant proteins. Transient expression of both B`alpha1 (Fig. 5A) and B`alpha2 (Fig. 5C) led to diffuse cytoplasmic and pronounced nuclear staining. In contrast, transient expression of FLAG-Balpha (Fig. 5E) led only to cytoplasmic staining. The predominantly cytoplasmic localization of FLAG-Balpha is consistent with the distribution of endogenous Balpha in CV1 cells(30) . Similar amounts of FLAG-B`alpha and FLAG-Balpha were present in whole cell extracts as determined by immunoblotting with the anti-FLAG antibody (data not shown). The differential localization of FLAG-B`alpha1 and FLAG-B`alpha2 in the nucleus and cytoplasm and FLAG-Balpha in the cytoplasm was confirmed by immunoblots of cytosolic and nuclear extracts from the transfected cells (data not shown). Control experiments in which the primary or secondary antibody was omitted led to a loss of immunostaining. Preadsorbtion of the anti-FLAG antibody with the antigenic peptide also abolished the immunoreactivity (data not shown). Phase contrast images (Fig. 5, B, D, and F) showed that nontransfected cells lacked any significant immunofluorescence.


Figure 5: Localization of transiently expressed human B`alpha in NIH3T3 cells. NIH3T3 cells were transfected with pFLAG-CMV2-B`alpha1 (panels A and B), pFLAG-CMV2-B`alpha2 (panels C and D), or pFLAG-CMV2-Balpha (panels E and F). The cells were fixed and stained with an anti-FLAG antibody and analyzed by indirect immunofluorescence microscopy (panels A, C, and E) as described under ``Materials and Methods.'' Panels B, D, and F are phase contrast images of the same field of cells shown in panels A, C, and E.




DISCUSSION

We have isolated three novel cDNAs related to the bovine cardiac B` regulatory subunit of PP2A. All three forms appear to be generated from a single gene by alternative splicing. Transcripts of B`alpha are widely expressed in human and mouse tissues and are especially abundant in muscle. The high level of B`alpha transcripts detected in heart and skeletal muscle is consistent with the biochemical composition of PP2A purified from these tissues(17, 24) . These B` subunits have no apparent homology to the B or PR72/130 (B") regulatory subunits or other proteins that interact with and regulate PP2A, including viral tumor antigens and the phosphotyrosyl phosphatase activator protein(31) . Interaction of these proteins with PP2A is apparently not due to regions of conserved sequence but may involve domains of similar higher order structure.

Heterotrimeric complexes of PP2A were formed when recombinant human and mouse B`alpha proteins were reconstituted with the AC core enzyme in vivo and in vitro. All three forms of B`alpha interacted with a GST-A subunit fusion protein and with a GAL4 activation domain-A subunit fusion protein in the yeast two-hybrid assay. A role for the C subunit in stabilizing oligomeric complexes was originally suggested by reconstitution experiments with porcine cardiac PP2A(32) . This idea is supported by chemical cross-linking studies (13, 20) , and analysis of A subunit mutants(33, 34) . The increased binding of recombinant B`alpha proteins to GST-A in the presence of C is also consistent with a role in stabilizing the ACbulletB` heterotrimer. We do not know if the endogenous yeast A subunit participates in the two-hybrid interaction. However, no hybrids were formed between GAD-C and GDB-B`alpha, suggesting that B`alpha has very weak affinity for C and that the yeast A subunit homolog does not participate in hybrid formation. Another possibility is that the fusion with the DNA binding domain to the amino terminus of B`alpha hinders interaction with GAD-C.

The interaction assays suggested that the B`alpha subunits have different apparent affinities for PP2A. Of the three proteins, B`alpha1 displayed the strongest interaction in both the yeast two-hybrid and GST-A binding assays. It is not known whether the absence of 8 amino acids from the amino terminus of mouse B`alpha3 affects binding to PP2A. However, there is evidence that a portion of the amino terminus of the Balpha and Bbeta subunits is involved in binding to the AC core enzyme (13) . The fact that B`alpha1 and B`alpha2 have different apparent affinities for the AC complex suggests that regions near the carboxyl terminus are also important for subunit interactions.

The purified bovine cardiac B` subunit has a very high affinity (IC = 0.58 nM) for the AC form of PP2A(20) . Although cardiac B` and B`alpha2 have the same mobility in SDS-PAGE, functional data and other lines of evidence suggest that human B`alpha2 and bovine B` are not the same protein. Three of the peptides derived from the bovine cardiac protein were not found in the human or mouse B`alpha sequences. Although these peptides may have been generated from a contaminant(s), they were not similar to any other sequences in the data bases. Anti-peptide antibodies raised against the human B`alpha proteins cross-reacted with the bovine cardiac subunit; however, no cross-reactivity with the human proteins was observed with an antiserum (E005) raised against purified bovine cardiac B` (data not shown). Taken together, these data indicate that the human B`alpha and bovine cardiac B` subunits are closely related isoforms of a larger B` family. (^2)

PP2A has generally been regarded as a soluble cytoplasmic enzyme, but significant amounts of PP2A are also present in the nucleus(35, 36) . Partial purification of rat liver nuclear PP2A suggests that the nuclear enzyme is a heterotrimer; however, the type of B subunit in nuclear PP2A has not been identified(37) . A significant feature of all three B`alpha variants is the presence of a cluster of basic residues near the carboxyl terminus and a consensus bipartite nuclear localization signal in B`alpha1 (residues 462-478). The bipartite motif has been found in 60% of all known nuclear proteins and less than 4% of nonnuclear proteins(29) . Although the bipartite nuclear localization signal is absent in B`alpha2, localization of FLAG-B`alpha2 was similar to that of B`alpha1 in our transient assays. While some nuclear proteins contain large T antigen-type nuclear targeting signals or the bipartite motifs, there are many nuclear proteins that do not contain a concensus sequence for nuclear localization(29) . The only significant feature that has been recognized in these proteins is the presence of clusters of basic residues within the signaling domain. The basic residues present in the carboxyl termini of the B`alpha proteins may serve as a signal for nuclear import. Mutational analysis of this region will be required to determine the critical residues for nuclear localization.

Exclusion of transiently expressed FLAG-Balpha from the nucleus is consistent with previous immunofluorescence data showing that ACbulletBalpha is largely cytoplasmic and that a subpopulation is associated with the microtubule cytoskeletal network(30) . We have not quantitated the amount of FLAG-Balpha that is associated with endogenous AC in the transient assays. However, other studies have shown that expressed small t antigen interacts with endogneous AC in mammalian cells(2, 38) . In addition, we have shown here that expressed B`alpha subunits form heterotrimeric complexes with endogenous AC in Sf9 cells. Presumably, some FLAG-Balpha associates with AC and is bound to microtubules, preventing nuclear import. We have no evidence that ACbulletB`alpha binds to microtubules, and failure of the ACbulletB`alpha complex to interact with the cytoskeleton may allow nuclear uptake. Regardless of the mechanism, it is clear that expressed B`alpha subunits are highly localized within the nucleus, while the Balpha subunit is not. This result suggests that some members of the B` family may be present in nuclear PP2A and that B` may be important in nuclear functions of PP2A. PP2A has been implicated in the dephosphorylation of nuclear cAMP-response element binding protein(37, 38) . The ACbulletB`alpha heterotrimer may be involved in the control of cAMP-mediated changes in gene transcription. Yeast RTS1 is a suppressor of a mutation in the ROX3 transcription factor. The similarity of RTS1 and B`alpha also suggests a role for the mammalian homolog in regulating the activity of transcription factor(s).


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants GM49505 and HL31107 (to M. C. M.) and American Heart Association (Texas affiliate) Grant 93G-091 (to C. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U37352 [GenBank]and U37353[GenBank].

§
Supported by a grant from the G. Harold and Leila Mathers Foundation to Dr. John Minna.

To whom correspondence and reprint requests should be addressed: University of Texas Southwestern Medical Center, Dept. of Pharmacology, 5323 Harry Hines Blvd., Dallas, TX 75235-9041. Tel.: 214-648-7908; Fax: 214-648-8626; ckamib{at}mednet.swmed.edu.

(^1)
The abbreviations used are: PP2A, protein phosphatase 2A; PR, phosphatase regulatory; bp, base pair(s); PCR, polymerase chain reaction; HUVEC, human umbilical vein epithelial cell; kb, kilobase pair(s); PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; PBS, phosphate-buffered saline; GDB, GAL4 DNA binding domain; GAD, GAL4 activation domain.

(^2)
While this manuscript was under review, McCright and Virshup (39) reported the isolation of a new family of PP2A regulatory subunit (accession numbers L42373[GenBank]-L42375[GenBank]). Comparison of the sequences indicates that their cDNAs are members of the B` family of regulatory subunits. The B56 isoform that they report corresponds to our B`alpha cDNAs.


ACKNOWLEDGEMENTS

We thank Robert Estes for assisting in the purification of cardiac PP2A. We also thank Dr. Clive Slaughter and Carolyn Moomaw for sequencing of the cardiac B` subunit. The HUVEC cDNA library was provided by Drs. Jim Battey and Mark Akeson (NIDCD, National Institutes of Health, Rocksville, MD), and the GAL4 DNA binding domain-p53 fusion plasmid was provided by Dr. Stanely Fields (State University of New York, Stony Brook, NY). The pAS-CYH2 and pACTII plasmids and mouse lymphocyte cDNA library were kindly provided by by Dr. S. Elledge (Baylor University Medical Center). We also thank Drs. Hussein Naim and Timothy Quil for assistance with the microscopy.


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