A Developmentally Regulated, Neuron-specific Splice Variant of the Variable Subunit Bβ Targets Protein Phosphatase 2A to Mitochondria and Modulates Apoptosis
2003; Elsevier BV; Volume: 278; Issue: 27 Linguagem: Inglês
10.1074/jbc.m302832200
ISSN1083-351X
AutoresRuben K. Dagda, Julie A. Zaucha, Brian E. Wadzinski, Stefan Strack,
Tópico(s)Bone Metabolism and Diseases
ResumoHeterotrimeric protein phosphatase 2A (PP2A) is a major Ser/Thr phosphatase composed of catalytic, structural, and regulatory subunits. Here, we characterize Bβ2, a novel splice variant of the neuronal Bβ regulatory subunit with a unique N-terminal tail. Bβ2 is expressed predominantly in forebrain areas, and PP2A holoenzymes containing Bβ2 are about 10-fold less abundant than those containing the Bβ1 (previously Bβ) isoform. Bβ2 mRNA is dramatically induced postnatally and in response to neuronal differentiation of a hippocampal progenitor cell line. The divergent N terminus of Bβ2 does not affect phosphatase activity but encodes a subcellular targeting signal. Bβ2, but not Bβ1 or an N-terminal truncation mutant, colocalizes with mitochondria in neuronal PC12 cells. Moreover, the Bβ2 N-terminal tail is sufficient to target green fluorescent protein to this organelle. Inducible or transient expression of Bβ2, but neither Bβ1, Bγ, nor a Bβ2 mutant defective in holoenzyme formation, accelerates apoptosis in response to growth factor deprivation. Thus, alternative splicing of a mitochondrial localization signal generates a PP2A holoenzyme involved in neuronal survival signaling. Heterotrimeric protein phosphatase 2A (PP2A) is a major Ser/Thr phosphatase composed of catalytic, structural, and regulatory subunits. Here, we characterize Bβ2, a novel splice variant of the neuronal Bβ regulatory subunit with a unique N-terminal tail. Bβ2 is expressed predominantly in forebrain areas, and PP2A holoenzymes containing Bβ2 are about 10-fold less abundant than those containing the Bβ1 (previously Bβ) isoform. Bβ2 mRNA is dramatically induced postnatally and in response to neuronal differentiation of a hippocampal progenitor cell line. The divergent N terminus of Bβ2 does not affect phosphatase activity but encodes a subcellular targeting signal. Bβ2, but not Bβ1 or an N-terminal truncation mutant, colocalizes with mitochondria in neuronal PC12 cells. Moreover, the Bβ2 N-terminal tail is sufficient to target green fluorescent protein to this organelle. Inducible or transient expression of Bβ2, but neither Bβ1, Bγ, nor a Bβ2 mutant defective in holoenzyme formation, accelerates apoptosis in response to growth factor deprivation. Thus, alternative splicing of a mitochondrial localization signal generates a PP2A holoenzyme involved in neuronal survival signaling. Reversible phosphorylation is a key post-translational regulatory mechanism in all eukaryotic cells. The phosphorylation state of any given protein is determined by the balance of protein kinase and phosphatase activities acting on it. Although it has long been appreciated that kinases assemble into complex signaling networks, our understanding of protein phosphatase regulation is comparatively limited. Protein phosphatase 2A (PP2A) 1The abbreviations used are: PP2A, protein phosphatase 2A; EGFP, enhanced green fluorescent protein; EST, expressed sequence tag; GFP, green fluorescent protein; RT, reverse transcription; UTR, untranslated region. is one of four major classes of serine/threonine phosphatases (for a recent review, see Ref. 1Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Google Scholar). PP2A accounts for up to 1% of total protein in certain cell types, and together with PP1 it contributes greater than 90% of cellular Ser/Thr phosphatase activity (2Cohen P. Methods Enzymol. 1991; 201: 389-398Google Scholar, 3Lin X.H. Walter J. Scheidtmann K. Ohst K. Newport J. Walter G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14693-14698Google Scholar, 4Ruediger R. Van Wart Hood J.E. Mumby M. Walter G. Mol. Cell. Biol. 1991; 11: 4282-4285Google Scholar). PP2A enzymatic activity is conferred by a ∼36-kDa catalytic, or C subunit, which is highly conserved in evolution. Free C subunit is not known to exist in cells; rather it forms complexes with a variety of other proteins. The PP2A core dimer is composed of the C subunit and the scaffolding A (or PR65) subunit, and several other complexes containing one, but not the other subunit have also been described (5Murata K. Wu J. Brautigan D.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10624-10629Google Scholar, 6Lubert E.J. Hong Y. Sarge K.D. J. Biol. Chem. 2001; 276: 38582-38587Google Scholar, 7Bennin D.A. Arachchige Don A.S. Brake T. McKenzie J.L. Rosenbaum H. Ortiz L. DePaoli-Roach A.A. Horne M.C. J. Biol. Chem. 2002; 277: 27449-27467Google Scholar). The predominant form of PP2A, however, is the trimeric holoenzyme consisting of the core dimer complexed to a third variable regulatory subunit. In mammals, regulatory subunits are encoded by four gene families denoted B (or PR55), B′ (PR61, B56), B″ (PR48, PR59, PR72/130), and B‴ (striatin, SG2NA). Proposed functions of these subunits include regulation of catalytic activity, substrate specificity, and subcellular localization of PP2A. The PP2A B subunit family has four members (Bα–δ). The five B′ subunit genes (B′α–ϵ) encode phosphoproteins with diverse functions including regulation of wnt/β-catenin signaling (8Seeling J.M. Miller J.R. Gil R. Moon R.T. White R. Virshup D.M. Science. 1999; 283: 2089-2091Google Scholar, 9Yamamoto H. Hinoi T. Michiue T. Fukui A. Usui H. Janssens V. Van Hoof C. Goris J. Asashima M. Kikuchi A. J. Biol. Chem. 2001; 276: 26875-26882Google Scholar). The B″ family consists of four polypeptides that arise from three genes (PR72/130, PR48, PR59). B″ subunits are nuclear proteins that bind calcium and have been implicated in the regulation of the G1/S cell cycle transition (10Voorhoeve P.M. Hijmans E.M. Bernards R. Oncogene. 1999; 18: 515-524Google Scholar, 11Yan Z. Fedorov S.A. Mumby M.C. Williams R.S. Mol. Cell. Biol. 2000; 20: 1021-1029Google Scholar, 12Janssens V. Jordens J. Stevens I. Van Hoof C. Martens E. De Smedt H. Engelborghs Y. Waelkens E. Goris J. J. Biol. Chem. 2003; 278: 10697-10706Google Scholar). Recent RNA interference studies in Drosophila cells have demonstrated that B family subunits regulate mitogen-activated protein kinase signaling, whereas B′ family subunits protect cells from apoptosis (13Silverstein A.M. Barrow C.A. Davis A.J. Mumby M.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4221-4226Google Scholar, 14Li X. Scuderi A. Letsou A. Virshup D.M. Mol. Cell. Biol. 2002; 22: 3674-3684Google Scholar). Even though they were the first PP2A regulatory subunits to be identified, few functions of the mammalian B family have been uncovered to date. Structurally, B family subunits resemble β subunits of heteromeric G proteins in that they contain seven WD repeat motifs predicted to fold into a β-propeller (15Strack S. Ruediger R. Walter G. Dagda R.K. Barwacz C.A. Cribbs J.T. J. Biol. Chem. 2002; 277: 20750-20755Google Scholar). Bα, the most abundant B family member, is expressed in a variety of cell types and mediates dephosphorylation of the cytoskeletal proteins tau and vimentin by PP2A (16Sontag E. Nunbhakdi-Craig V. Bloom G.S. Mumby M.C. J. Cell Biol. 1995; 128: 1131-1144Google Scholar, 17Sontag E. Nunbhakdi-Craig V. Lee G. Bloom G.S. Mumby M.C. Neuron. 1996; 17: 1201-1207Google Scholar, 18Turowski P. Myles T. Hemmings B.A. Fernandez A. Lamb N.J. Mol. Biol. Cell. 1999; 10: 1997-2015Google Scholar). The recently identified Bδ subunit is most similar to Bα and is also expressed in multiple tissues (19Strack S. Chang D. Zaucha J.A. Colbran R.J. Wadzinski B.E. FEBS Lett. 1999; 460: 462-466Google Scholar). Bβ and Bγ genes, on the other hand, give rise to neuron-specific members of the B family of PP2A subunits with distinct temporal and spatial expression patterns in brain (20Strack S. Zaucha J.A. Ebner F.F. Colbran R.J. Wadzinski B.E. J. Comp. Neurol. 1998; 392: 515-527Google Scholar). Forced expression of Bγ, but not other PP2A regulatory subunits, promotes neuronal differentiation of PC12 cells, an effect that appears to be mediated by activation of the mitogen-activated protein kinase cascade at the level or upstream of the Ser/Thr kinase B-Raf (21Strack S. J. Biol. Chem. 2002; 277: 41525-41532Google Scholar). An important role of Bβ in neuronal survival was suggested by the discovery that the neurodegenerative disorder spinocerebellar ataxia type 12 is caused by a trinucleotide repeat expansion in the promoter region of the human Bβ gene (PPP2R2B) (22Holmes S.E. O'Hearn E.E. McInnis M.G. Gorelick-Feldman D.A. Kleiderlein J.J. Callahan C. Kwak N.G. Ingersoll-Ashworth R.G. Sherr M. Sumner A.J. Sharp A.H. Ananth U. Seltzer W.K. Boss M.A. Vieria-Saecker A.M. Epplen J.T. Riess O. Ross C.A. Margolis R.L. Nat. Genet. 1999; 23: 391-392Google Scholar). Thus, dysregulated Bβ gene expression may be detrimental to neurons, ultimately leading to the massive cerebral and cerebellar atrophy seen in spinocerebellar ataxia type 12 patients. In this report, we characterize a novel splice product of the Bβ gene which is induced upon neuronal differentiation. The unique N-terminal extension of Bβ2 is shown to target the protein to mitochondria, where Bβ2 accelerates neuronal cell death after survival factor deprivation. Isolation of the Bβ2 cDNA—Duplicate filters containing 1 × 106 plaque-forming units from a rat brain cDNA library in the λZap II vector (Stratagene) were screened with a random primed, [α-32P]dCTP-labeled probe corresponding to the full-length mouse Bβ1 cDNA (a gift from Dr. Nat Heintz, Rockefeller University). After four rounds of screening, a partial cDNA containing the 5′-UTR and the N-terminal third of the Bβ2 coding sequence was isolated. The full-length coding sequence for Bβ2 was obtained by reverse transcription-PCR (RT-PCR) from total rat brain RNA with primers complementary to the unique 5′-coding sequence and common 3′-UTR (5′-coding sequence/forward primer: 5′-AAA TGC TTC TCT CGT TAC CT-3′; 3′-UTR/reverse primer: 5′-GGT TTG ACT AGT ATT CAG TAT GTG-3′). The Bβ2 cDNA sequence was submitted to GenBank and is available under accession number AY251277. Generation of FLAG- and Green Fluorescent Protein (GFP)-tagged Bβ Constructs and Site-directed Mutagenesis—Primers complementary to the N terminus of Bβ1 and Bβ2 and to the beginning of the common region (TEAD) fitted with a HindIII cloning site in conjunction with two nested reverse primers including sequence complementary to the Bβ C-terminal end, the FLAG epitope tag, and a SalI cloning site were used to PCR amplify Bβ1, Bβ2, and BβΔN, respectively. PCR fragments were ligated into pcDNA5/TO or pEGFP-N1 to generate fusion proteins with C-terminal FLAG and FLAG-GFP sequences, respectively. Bβ11–32-GFP and Bβ21–35-GFP were constructed by excising C-terminal sequences from the full-length Bβ1/2-pEGFP-N1 plasmids by EcoRI/XmaI digestion, filling in the overhangs with Klenow polymerase, and religating the plasmids. The Bβ2 RR168EE mutant was constructed by full plasmid synthesis using Pfu Ultra polymerase according to instructions for the QuikChange mutagenesis kit (Stratagene). All constructs were fully sequenced at the University of Iowa DNA Facility. Antibodies—A peptide derived from the N terminus of Bβ2 (CFSRYLPYIFRPPNT) was coupled to keyhole limpet hemocyanin via the sulfhydryl group of the N-terminal cysteine, and polyclonal antibodies were generated in rabbits and affinity purified by standard techniques (23Harlow E. Lane D.P. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 53-138Google Scholar). Bβ1 and pan-B subunit antibodies have been described previously (20Strack S. Zaucha J.A. Ebner F.F. Colbran R.J. Wadzinski B.E. J. Comp. Neurol. 1998; 392: 515-527Google Scholar). Monoclonal antibodies to the PP2A A subunit were a kind gift from Gernot Walter (University of California San Diego), and PP2A C subunit antibodies were purchased from Transduction Laboratories. The adenine nucleotide translocase antibody was provided by Harmut Wohlrab (Boston Biomedical Research Institute). Ribonuclease Protection Analyses—The Bβ2 cDNA library clone was subcloned into pBluescript KS+ and in vitro transcribed using T7 polymerase. Total RNA was isolated from selected rat organs and brain regions using TriZol reagent according to the manufacturer's instruction (Molecular Research Center). Ribonuclease protection was carried out as described previously (20Strack S. Zaucha J.A. Ebner F.F. Colbran R.J. Wadzinski B.E. J. Comp. Neurol. 1998; 392: 515-527Google Scholar, 24Zaucha J. Westphal R. Wadzinski B. Methods Mol. Biol. 1998; 93: 279-291Google Scholar). Competitive RT-PCR—Total RNA (0.5–1.0 μg) was reverse transcribed and PCR amplified in the same 25-μl reaction with reagents from the Access RT-PCR kit (Promega, Madison, WI) and the following primers (0.5 μm each): common reverse, 5′-GAC ATC AAG CCA GCC AAC ATG GAG G-3′; Bβ1 forward, 5′-TGC CCC CCT CTC CTG TGA GAC-3′; Bβ2 forward, 5′-ACC ATC CTC TCT TCC AGC TGC C-3′. Aliquots of PCRs were separated on 1% agarose gels and ethidium bromide-stained bands were quantified by image analysis using NIH Image software. The ratio of the 749-bp Bβ1 and 619-bp Bβ2 PCR products was found to be independent of the number of PCR cycles; 35 cycles were routinely used. Cell Culture—COS-M6 and PC6-3 cells were cultured and transfected as described previously (15Strack S. Ruediger R. Walter G. Dagda R.K. Barwacz C.A. Cribbs J.T. J. Biol. Chem. 2002; 277: 20750-20755Google Scholar, 21Strack S. J. Biol. Chem. 2002; 277: 41525-41532Google Scholar). The adult hippocampal progenitor cell line HC2S2 was generously provided by Fred Gage (Salk Institute) and cultured in the presence of 20 ng/ml fibroblast growth factor-2 on laminin- and polyornithine-coated plates according to published protocols (25Hoshimaru M. Ray J. Sah D.W. Gage F.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1518-1523Google Scholar, 26Berger F. Gage F.H. Vijayaraghavan S. J. Neurosci. 1998; 18: 6871-6881Google Scholar). Immunoprecipitation and Phosphatase Activity Assays—COS-M6 cells were transfected in six-well plates using LipofectAMINE 2000 (BD Biosciences), and C-terminally FLAG-tagged Bβ subunits were immunoprecipitated with FLAG antibody-agarose conjugates (Sigma) as described (15Strack S. Ruediger R. Walter G. Dagda R.K. Barwacz C.A. Cribbs J.T. J. Biol. Chem. 2002; 277: 20750-20755Google Scholar), except that the immunoprecipitation buffer lacked protein phosphatase inhibitors. Aliquots of immunoprecipitates were solubilized in SDS sample buffer for immunoblot analyses. For PP2A activity assays, immunoprecipitates were stored at –20 °C in 50% glycerol, 10% ethylene glycol, 20 mm Tris, pH 7.5, 5 mm dithiothreitol, 2 mm EDTA, and 0.1% Triton X-100. 33P-Labeled substrates (see below) were diluted to 0.2–0.5 mg/ml (2,000–10,000 cpm/μl) in 2 mg/ml bovine serum albumin, 50 mm Tris, pH 7.5, 2 mm EDTA, 2 mm EGTA, 2 mm dithiothreitol, 1 mm benzamidine, 1 mg/ml leupeptin. Phosphatase reactions were started by the addition of 5 μl of PP2A immunoprecipitates to 20 μl of diluted substrate, incubated for 30 min at 30 °C with intermittent agitation on an Eppendorf shaking incubator, and terminated by the addition of trichloroacetic acid to a final concentration of 20%. After centrifugation at 22,000 × g, acid-soluble 33P was quantified by liquid scintillation counting. Less than 20% substrate dephosphorylation occurred under our assay conditions, and activities were inhibited completely by 2.5 nm okadaic acid, a specific inhibitor of PP2A at this concentration. Preparation of Phosphatase Substrates—Partially dephosphorylated casein or bovine brain myelin basic protein (5 mg/ml, Sigma) was phosphorylated by protein kinase A catalytic subunit (0.25 units/μl, Sigma) for 2–16 h at 30 °C in buffer containing 1 mm ATP, 100 μCi of [γ-33P]ATP, 50 mm Tris, pH 7.5, 10 mm MgCl2, 2mm dithiothreitol, 1 mm EGTA, and 0.01% Triton X-100. Phosphorylation reactions were stopped by the addition of 20% trichloroacetic acid, followed by centrifugation at 22,000 × g and successive washing of the pellet in 10% trichloroacetic acid, 70% ethanol, and 100% acetone. After the last wash, 33P-labeled substrates were dissolved in 50 mm Tris, pH 7.5, and stored aliquoted at –80 °C. Confocal Imaging of GFP Fusion Proteins—PC6-3 cells were seeded on collagen-coated, chambered no. 1 cover glasses (20-mm2 chamber, Nalge Nunc) and transfected with 1 μg of GFP fusion protein plasmids using LipofectAMINE 2000. 24–48 h post-transfection, cells were imaged live on a Zeiss LSM 510 laser scanning confocal microscope at the Central Microscopy Facility of the University of Iowa. In some experiments, MitoTracker Red CMXRos (Molecular Probes) was added to 100 nm to stain mitochondria. Subcellular Fractionation—PC6-3 cells were plated at 2 * 106 cells/dish in 60-mm dishes and transfected 1 day later with 5 μg/dish GFP fusion protein plasmids using LipofectAMINE 2000. Three days later, cells were dislodged by scraping in 0.5 ml of mitochondria isolation buffer (0.25 m sucrose, 20 mm HEPES, pH 7.4, 1 mm EGTA, 0.5 mm EDTA, 1 mm dithiothreitol, 1 mm benzamidine, 5 μg/ml leupeptin, 0.5 mm phenylmethylsulfonyl fluoride. After addition of 2 mm MgCl2, cells were disrupted by nitrogen cavitation (20 min, 1,000 p.s.i). Unbroken cells and nuclei were removed by two successive centrifugations at 800 × g for 5 min. The postnuclear supernatant was supplemented with 5mm EDTA and further fractionated by centrifugation at 22,000 × g for 15 min. The supernatant was designated the soluble protein fraction; the pellet was washed once in mitochondria isolation buffer and designated the crude membrane fraction containing mitochondria. Generation of Tetracycline-inducible PC6-3 Cell Lines—Tetracycline-inducible (T-Rex system, BD Biosciences) PC6-3 cell lines stably expressing FLAG epitope-tagged B family PP2A regulatory subunits were generated as described previously (21Strack S. J. Biol. Chem. 2002; 277: 41525-41532Google Scholar). Between 40 and 60 blasticidine- and hygromycin-resistant clones were expanded and tested for inducible expression by immunoblotting for the FLAG epitope tag. In positive clones, maximum protein expression was achieved after 24 h treatment with 1 μg/ml doxycycline. Cell Death Assays—Tetracycline-inducible PC6-3 cell lines were seeded at 10,000 cells/well in collagen-coated 96-well plates and grown for 72 h in regular growth medium (10% horse serum, 5% fetal bovine serum in RPMI 1640) in the presence of vehicle (0.1% ethanol) or 1 μg/ml doxycycline. After two washes, serum-free RPMI 1640 ± doxycycline was added, and cell density was assayed by MTS tetrazolium reduction to formazan according to the manufacturer's instructions (CellTiter 96® AQueous nonradioactive cell proliferation assay, Promega). Formazan production was quantified after 3 h by absorbance measurement at 490 nm using a 96-well plate reader. The MTS assay was repeated after 24 h in serum-free medium, and cell survival was expressed as the ratio of the two measurements. Previous apoptosis studies with PC6-3 cells have documented excellent correlation between cell counts and metabolic activity as assayed by tetrazolium salt reduction (27Pittman R.N. Wang S. DiBenedetto A.J. Mills J.C. J. Neurosci. 1993; 13: 3669-3680Google Scholar). For nuclear morphology assays, native PC6-3 cells or tetracycline-inducible cells were seeded at 200,000 cells/well in 20-mm2 chambered cover glasses. Native PC6-3 cells were transiently transfected with 1 μg/chamber GFP fusion protein plasmids using LipofectAMINE 2000 and cultured for 48 h, whereas inducible cells were treated with vehicle or doxycycline for 72 h prior to serum deprivation. After 24 h under serum-free conditions, cultures were fixed in 3.7% paraformaldehyde in phosphate-buffered saline, incubated with the blue fluorescent nuclear stain Hoechst 33342 at 1 μg/ml for 5 min and mounted on slides. Random microscopic fields (6–12 fields/culture, 50–200 cells/field) were captured on an epifluorescence microscope, and images were coded and analyzed blind to the experimental condition. Cells with condensed, irregular, or fragmented nuclei were scored as apoptotic. Identification of a Novel PP2A Regulatory Subunit—A rat brain cDNA library was screened with a PP2A/Bβ cDNA probe to identify novel isoforms of this brain-specific PP2A regulatory subunit. A clone was isolated with an insert of 431 bp, of which the first 223 bases are novel, and the last 208 bp are identical to the coding sequence for amino acid residues 23–91 of rat Bβ (28Hatano Y. Shima H. Haneji T. Miura A.B. Sugimura T. Nagao M. FEBS Lett. 1993; 324: 71-75Google Scholar). Conceptual translation of this partial cDNA predicts an isoform of Bβ with a novel 5′-UTR and N-terminal extension of 24 amino acids. The full-length cDNA for Bβ2 was isolated by RT-PCR from rat brain RNA using primers flanking the coding sequence (GenBank accession number AY251277). Data base searches with the unique rat Bβ2 sequence identified several human and mouse ESTs with high degrees of sequence conservation at the nucleotide level and 100% amino acid identity in the coding region. The murine Bβ2 ortholog was recently described and named Bβ.2 (29Schmidt K. Kins S. Schild A. Nitsch R. Hemmings B. Gotz J. Eur. J. Neurosci. 2002; 16: 2039-2048Google Scholar). EST data base searches also identified a Bβ2 ortholog from rainbow trout (accession number CA376753) which has three conservative substitutions in the N-terminal tail. No Bβ2-related sequences were found in other EST or genome data bases, suggesting that Bβ2 has evolved in the vertebrate subphylum. The chromosomal organization of exons encoding human Bβ1 and Bβ2 was determined by computer-aided alignment of the Bβ cDNAs with human and murine genome data bases and is shown in Fig. 1A. The gene structure of the human and murine Bβ genes is highly conserved as has been noted previously (29Schmidt K. Kins S. Schild A. Nitsch R. Hemmings B. Gotz J. Eur. J. Neurosci. 2002; 16: 2039-2048Google Scholar). The alternate N termini of Bβ1 and Bβ2 are encoded by exons separated by ∼150 kb. Because the transcription start site for the human Bβ1 mRNA is less than 600 nucleotides upstream of the initiation codon (30Mayer R.E. Hendrix P. Cron P. Matthies R. Stone S.R. Goris J. Merlevede W. Hofsteenge J. Hemmings B.A. Biochemistry. 1991; 30: 3589-3597Google Scholar), the Bβ2 transcript appears to be generated by use of an alternate promoter upstream of exon 1.2 and splicing of exon 1.2 to the first common exon (Fig. 1A). Of note, human EST BC031790 predicts an alternate Bβ mRNA in which exon 1.2 is fused out of frame to exon 1.1 and the rest of the coding sequence. The existence of this apparently incompletely spliced EST supports the notion that Bβ2 is generated by cis splicing of a huge pre-mRNA (∼500 kb) spanning the Bβ locus. Based on prior structure modeling and site-directed mutagenesis of the related Bγ subunit (15Strack S. Ruediger R. Walter G. Dagda R.K. Barwacz C.A. Cribbs J.T. J. Biol. Chem. 2002; 277: 20750-20755Google Scholar), the variant Bβ2 N terminus is predicted to extend from a β-propeller core structure encoded by common exons 2–9 (Fig. 1B). Characterization of Bβ2 Expression—To demonstrate that Bβ2 is expressed at the protein level, we generated polyclonal antibodies by immunizing rabbits with a peptide from the unique N-terminal tail of Bβ2. The resulting antibody reacted specifically with heterologously expressed Bβ2 and displayed no cross-reactivity with Bβ1 or other PP2A regulatory subunits (Fig. 2, and not shown). Although Bβ1 could be detected in total brain lysates (Ref. 20Strack S. Zaucha J.A. Ebner F.F. Colbran R.J. Wadzinski B.E. J. Comp. Neurol. 1998; 392: 515-527Google Scholar and Fig. 2), antibody detection of a protein with the size predicted for Bβ2 (52,000) necessitated enrichment of PP2A holoenzymes by microcystin-Sepharose affinity purification (20Strack S. Zaucha J.A. Ebner F.F. Colbran R.J. Wadzinski B.E. J. Comp. Neurol. 1998; 392: 515-527Google Scholar). COS cell lysates expressing FLAG epitope-tagged Bβ splice variants were used as standards and immunoblotted with Bβ isoform-specific and FLAG-directed antibodies to compare detection strengths of Bβ1 and Bβ2 antibodies. Thus normalizing for antibody affinities and titers, the relative abundance of Bβ1- and Bβ2-containing PP2A holoenzymes in rat brain was estimated to be ∼10:1. The low abundance of Bβ2 precluded an analysis of its spatial and temporal expression pattern at the protein level. Therefore, we performed ribonuclease protection assays with probes corresponding to the divergent domains to map the expression of Bβ isoforms in rat brain regions. Bβ1 and Bβ2 transcripts were detected at comparable levels in all forebrain structures, except in olfactory bulb, where relatively more Bβ1 was expressed (Fig. 3 and Ref. 20Strack S. Zaucha J.A. Ebner F.F. Colbran R.J. Wadzinski B.E. J. Comp. Neurol. 1998; 392: 515-527Google Scholar). The cerebellum contained low levels of both Bβ splice forms. We reported previously that B family regulatory subunits exhibit distinct developmental expression profiles in brain (20Strack S. Zaucha J.A. Ebner F.F. Colbran R.J. Wadzinski B.E. J. Comp. Neurol. 1998; 392: 515-527Google Scholar). The neuronal Bγ isoform is induced during postnatal brain development, whereas Bα and Bβ1 show constant expression and a slight postnatal decline in expression, respectively. Ribonuclease protection assays with a Bβ2-specific probe revealed that this isoform has an expression pattern similar to Bγ, with near undetectable expression at birth rising to adult levels by postnatal day 14 (Fig. 4A). Thus, alternative promoter use and splicing of the Bβ gene appear to be regulated developmentally. We developed a competitive RT-PCR protocol to assay changes in relative abundance of Bβ1 and Bβ2 transcripts rapidly. In this assay, reverse transcription of mRNA is carried out using a reverse primer that anneals to the common region of Bβ1 and Bβ2 followed by PCR amplification with the common primer and two competing forward primers complementary to Bβ1- and Bβ2-specific sequences (Fig. 4B). This technique was tested by analyzing changes in Bβ gene expression during rat brain maturation (Fig. 4C). Although this assay clearly showed postnatal up-regulation of the Bβ2 transcript, the 70% drop in Bβ1 message levels detected by ribonuclease protection could not be demonstrated by RT-PCR for reasons that are unclear at present. Because Bβ1 protein levels show an ∼2-fold decline during postnatal development (20Strack S. Zaucha J.A. Ebner F.F. Colbran R.J. Wadzinski B.E. J. Comp. Neurol. 1998; 392: 515-527Google Scholar), the ribonuclease protection data are likely a better indicator of the absolute abundance of the Bβ1 mRNA. Our data indicate that Bβ2 mRNA is found specifically in mature brain, but do not rule out a non-neuronal (e.g. glial) origin of expression. To address this issue, multiple cell lines of neuronal (PC6-3, PC12, B104, SHSY5Y, Neuro2A), glial (C6, Ng108), and other (COS, HEK293, NIH3T3, MCF7) origin were analyzed for Bβ isoform expression by competitive RT-PCR. Although all neuronal cell lines tested expressed Bβ1, none had detectable levels of Bβ2 (data not shown). To examine alternative splicing of the Bβ locus in a cell line that more closely resembles primary forebrain neurons, we turned to HC2S2 cells, a neuronal progenitor cell line derived from rat hippocampus (25Hoshimaru M. Ray J. Sah D.W. Gage F.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1518-1523Google Scholar). HC2S2 cells are conditionally immortalized by a tetracycline-repressible v-myc oncogene and differentiate into phenotypic neurons upon addition of tetracycline or doxycycline to the medium (Fig. 4D). Competitive RT-PCR with Bβ1 and Bβ2 primers was performed on HC2S2 cultures treated for up to 4 days with doxycycline. Bβ2 mRNA was already detectable in dividing HC2S2 cells, and levels increased further relative to Bβ1 as cells differentiated into neurons (Fig. 4E). These data strongly indicate a neuronal locus of Bβ2 expression. The time course of Bβ isoform expression in differentiating HC2S2 cells closely parallels that seen in postnatal maturation of the brain (compare Fig. 4, C and E). With the caveats inherent to a comparison between neuronal differentiation in vitro and brain development in the intact organism, these data suggest that the HC2S2 cell line may be an appropriate model system for Bβ gene regulation studies. The Bβ2 N Terminus Does Not Affect Holoenzyme Formation or Catalytic Activity—Previous structure-function studies indicated that the variable N terminus of Bγ is dispensable for binding to the A and C subunits (15Strack S. Ruediger R. Walter G. Dagda R.K. Barwacz C.A. Cribbs J.T. J. Biol. Chem. 2002; 277: 20750-20755Google Scholar). A role of the Bγ N terminus in directing the PP2A holoenzyme to specific substrates in the mitogen-activated protein kinase pathway was suggested by analyses of chimeras between this neuronal specific regulatory protein and the widely expressed Bα subunit (21Strack S. J. Biol. Chem. 2002; 277: 41525-41532Google Scholar). To investigate whether the differentially spliced N termini of Bβ1 and Bβ2 play a role in formation or catalytic activity of the PP2A holoenzyme, the two isoforms were FLAG epitope tagged at the C terminus and transiently expressed in COS-M6 cells. A deletion mutant lacking the divergent N terminus, BβΔN, was also constructed and analyzed in parallel. The ectopically expressed proteins were immuno-isolated with anti-FLAG resin and analyzed for association with endogenous A and C subunits by immunoblotting. Bβ1, Bβ2, and BβΔN could be expressed to similar levels and associated with equivalent amounts of A and C subunits (Fig. 5A). Aliquots of the immunoprecipitates were then assayed for dephosphorylation of two model substrates, myelin basic protein and casein phosphorylated in vitro by protein kinase A (Fig. 5B). Myelin basic protein was a better substrate than the more acidic casei
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