Subcellular Localization of Multiple PREP2 Isoforms Is Regulated by Actin, Tubulin, and Nuclear Export
2004; Elsevier BV; Volume: 279; Issue: 47 Linguagem: Inglês
10.1074/jbc.m406046200
ISSN1083-351X
AutoresKlaus Haller, Isabel Rambaldi, Eugene Daniels, Mark Featherstone,
Tópico(s)Cancer-related gene regulation
ResumoThe PREP, MEIS, and PBX families are mammalian members of the TALE (three amino acid loop extension) class of homeodomain-containing transcription factors. These factors have been implicated in cooperative DNA binding with the HOX class of homeoproteins, but PREP and MEIS interact with PBX in apparently non-HOX-dependent cooperative DNA binding as well. PREP, MEIS, and PBX have all been reported to reside in the cytoplasm in one or more tissues of the developing vertebrate embryo. In the case of PBX, cytoplasmic localization is due to the modulation of nuclear localization signals, nuclear export sequences, and interaction with a cytoplasmic anchoring factor, non-muscle myosin heavy chain II B. Here we report that murine PREP2 exists in multiple isoforms distinguished by interaction with affinity-purified antibodies raised to N- and C-terminal epitopes and by nuclear versus cytoplasmic localization. Alternative splicing gives rise to some of these PREP2 isoforms, including a 25-kDa variant lacking the C-terminal half of the protein and homeodomain and having the potential to act as dominant-negative. We further show that cytoplasmic localization is due to the concerted action of nuclear export, as evidenced by sensitivity to leptomycin B, and cytoplasmic retention by the actin and microtubule cytoskeletons. Cytoplasmic PREP2 colocalizes with both the actin and microtubule cytoskeletons and coimmunoprecipitates with actin and tubulin. Importantly, disruption of either cytoskeletal system redirects cytoplasmic PREP2 to the nucleus. We suggest that transcriptional regulation by PREP2 is modulated through the subcellular distribution of multiple isoforms and by interaction with two distinct cytoskeletal systems. The PREP, MEIS, and PBX families are mammalian members of the TALE (three amino acid loop extension) class of homeodomain-containing transcription factors. These factors have been implicated in cooperative DNA binding with the HOX class of homeoproteins, but PREP and MEIS interact with PBX in apparently non-HOX-dependent cooperative DNA binding as well. PREP, MEIS, and PBX have all been reported to reside in the cytoplasm in one or more tissues of the developing vertebrate embryo. In the case of PBX, cytoplasmic localization is due to the modulation of nuclear localization signals, nuclear export sequences, and interaction with a cytoplasmic anchoring factor, non-muscle myosin heavy chain II B. Here we report that murine PREP2 exists in multiple isoforms distinguished by interaction with affinity-purified antibodies raised to N- and C-terminal epitopes and by nuclear versus cytoplasmic localization. Alternative splicing gives rise to some of these PREP2 isoforms, including a 25-kDa variant lacking the C-terminal half of the protein and homeodomain and having the potential to act as dominant-negative. We further show that cytoplasmic localization is due to the concerted action of nuclear export, as evidenced by sensitivity to leptomycin B, and cytoplasmic retention by the actin and microtubule cytoskeletons. Cytoplasmic PREP2 colocalizes with both the actin and microtubule cytoskeletons and coimmunoprecipitates with actin and tubulin. Importantly, disruption of either cytoskeletal system redirects cytoplasmic PREP2 to the nucleus. We suggest that transcriptional regulation by PREP2 is modulated through the subcellular distribution of multiple isoforms and by interaction with two distinct cytoskeletal systems. Patterning of the embryonic antero-posterior axis comes under the control of HOX and TALE 1The abbreviations used are: TALE, three amino acid loop extension; HR, homology region; NLS, nuclear localization signal; NES, nuclear export sequence; PREP2NAB, N-terminal PREP2 antibody; PREP2CAB, C-terminal PREP2 antibody; DBD, DNA-binding domain; HA, hemagglutinin; TRITC, tetramethylrhodamine isothiocyanate; LMB, leptomycin B; GFP, green fluorescent protein; siRNA, small interfering RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT, reverse transcriptase; DAPI, 4′,6-diamidino-2-phenylindole; dpc, days post coitum.1The abbreviations used are: TALE, three amino acid loop extension; HR, homology region; NLS, nuclear localization signal; NES, nuclear export sequence; PREP2NAB, N-terminal PREP2 antibody; PREP2CAB, C-terminal PREP2 antibody; DBD, DNA-binding domain; HA, hemagglutinin; TRITC, tetramethylrhodamine isothiocyanate; LMB, leptomycin B; GFP, green fluorescent protein; siRNA, small interfering RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT, reverse transcriptase; DAPI, 4′,6-diamidino-2-phenylindole; dpc, days post coitum. class homeoproteins (1Krumlauf R. Cell. 1994; 78: 191-201Abstract Full Text PDF PubMed Scopus (1730) Google Scholar). Members of the TALE class are characterized by sequence relatedness and by a three amino acid loop extension between helices one and two of the homeodomain (2Featherstone M. Lufkin T. Murine Homeobox Gene Control of Embyronic Patterning and Organogenesis. Elsevier Science Publishers B.V., Amsterdam2003: 1-42Google Scholar). Families within the TALE group include PBC, MEIS, and PREP. Among the PBC family members, the fly extradenticle and vertebrate PBX proteins have been most intensively studied. PBC proteins bind DNA cooperatively with many but not all HOX proteins. Additionally, they form stable DNA-binding heterodimers with MEIS and PREP family members (2Featherstone M. Lufkin T. Murine Homeobox Gene Control of Embyronic Patterning and Organogenesis. Elsevier Science Publishers B.V., Amsterdam2003: 1-42Google Scholar). 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Murine Homeobox Gene Control of Embyronic Patterning and Organogenesis. Elsevier Science Publishers B.V., Amsterdam2003: 1-42Google Scholar). Vertebrate MEIS proteins and fly homothorax are members of the MEIS family, whereas PREP is represented in vertebrates but not in flies. The related MEIS and PREP families share N-terminal regions termed homology region 1 (HR1) and HR2 (9Berthelsen J. Zappavigna V. Mavilio F. Blasi F. EMBO J. 1998; 17: 1423-1433Crossref PubMed Scopus (149) Google Scholar) with HR2 implicated in binding to PBC family proteins (12Knoepfler P.S. Calvo K.R. Chen H. Antonarakis S.E. Kamps M.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14553-14558Crossref PubMed Scopus (167) Google Scholar).Many TALE class homeoproteins have been shown to reside in the cytoplasm at certain times or sites of embryonic development. For example, extradenticle and PBX are cytoplasmic in the distal limb primordia of insects and vertebrates, respectively (13Gonzalez-Crespo S. Abu-Shaar M. 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EMBO J. 2003; 22: 89-99Crossref PubMed Scopus (53) Google Scholar) and by interaction with MEIS family proteins (14Rieckhof G.E. Casares F. Ryoo H.D. Abu-Shaar M. Mann R.S. Cell. 1997; 91: 171-183Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). MEIS binding masks NES while exposing NLS within the PBC N terminus (18Saleh M. Huang H. Green N.C. Featherstone M.S. Exp. Cell Res. 2000; 260: 105-115Crossref PubMed Scopus (52) Google Scholar, 19Berthelsen J. Kilstrup-Nielsen C. Blasi F. Mavilio F. Zappavigna V. Genes Dev. 1999; 13: 946-953Crossref PubMed Scopus (198) Google Scholar, 20Abu-Shaar M. Ryoo H.D. Mann R.S. Genes Dev. 1999; 13: 935-945Crossref PubMed Scopus (192) Google Scholar). Reciprocally, MEIS is dependent on PBX for nuclear localization (21Choe S.K. Vlachakis N. Sagerstrom C.G. Development. 2002; 129: 585-595PubMed Google Scholar, 22Huang H. Paliouras M. Rambaldi I. Lasko P. Featherstone M. Mol. Cell. 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Genomics. 1997; 41: 193-200Crossref PubMed Scopus (40) Google Scholar) family has two members in mice and humans (9Berthelsen J. Zappavigna V. Mavilio F. Blasi F. EMBO J. 1998; 17: 1423-1433Crossref PubMed Scopus (149) Google Scholar, 25Chen H. Rossier C. Nakamura Y. Lynn A. Chakravarti A. Antonarakis S.E. Genomics. 1997; 41: 193-200Crossref PubMed Scopus (40) Google Scholar, 26Ferretti E. Schulz H. Talarico D. Blasi F. Berthelsen J. Mech. Dev. 1999; 83: 53-64Crossref PubMed Scopus (60) Google Scholar, 27Imoto I. Sonoda I. Yuki Y. Inazawa J. Biochem. Biophys. Res. Commun. 2001; 287: 270-276Crossref PubMed Scopus (28) Google Scholar, 28Fognani C. Kilstrup-Nielsen C. Berthelsen J. Ferretti E. Zappavigna V. Blasi F. Nucleic Acids Res. 2002; 30: 2043-2051Crossref PubMed Scopus (39) Google Scholar, 29Haller K. Rambaldi I. Kovacs E.N. Daniels E. Featherstone M. Dev. Dyn. 2002; 225: 358-364Crossref PubMed Scopus (20) Google Scholar). In complexes with PBX, PREP1 regulates the transcription of HOX-dependent (Hoxb2) and HOX-independent (urokinase plasminogen activator, glucagon) target genes (5Ferretti E. Marshall H. Pöpperl H. Maconochie M. Krumlauf R. Blasi F. Development. 2000; 127: 155-166Crossref PubMed Google Scholar, 9Berthelsen J. Zappavigna V. Mavilio F. Blasi F. EMBO J. 1998; 17: 1423-1433Crossref PubMed Scopus (149) Google Scholar, 10Herzig S. Fuzesi L. Knepel W. J. Biol. Chem. 2000; 275: 27989-27999Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Both PREP1 and PREP2 have been shown to accumulate in the cytoplasm of cultured and embryonic cells (19Berthelsen J. Kilstrup-Nielsen C. Blasi F. Mavilio F. Zappavigna V. Genes Dev. 1999; 13: 946-953Crossref PubMed Scopus (198) Google Scholar, 28Fognani C. Kilstrup-Nielsen C. Berthelsen J. Ferretti E. Zappavigna V. Blasi F. Nucleic Acids Res. 2002; 30: 2043-2051Crossref PubMed Scopus (39) Google Scholar, 29Haller K. Rambaldi I. Kovacs E.N. Daniels E. Featherstone M. Dev. Dyn. 2002; 225: 358-364Crossref PubMed Scopus (20) Google Scholar). When we used an affinity-purified antibody directed against an N-terminal epitope to assess PREP2 distribution in the mouse embryo, we noted widespread but essentially cytoplasmic expression (29Haller K. Rambaldi I. Kovacs E.N. Daniels E. Featherstone M. Dev. Dyn. 2002; 225: 358-364Crossref PubMed Scopus (20) Google Scholar). We report here that a second affinity-purified antibody directed against the PREP2 C terminus reveals nuclear staining and that between them the two antibodies recognize at least five distinct PREP2 isoforms. One of these isoforms has a molecular mass of 25 kDa and is the result of alternative splicing. We further show that cytoplasmic PREP2 is associated with the actin and microtubule cytoskeletons and that this cytoplasmic localization is dependent on CRM-1-mediated nuclear export and the integrity of cytoskeletal networks.EXPERIMENTAL PROCEDURESPrimary Antibodies—The PREP2 antibodies were raised as previously described (29Haller K. Rambaldi I. Kovacs E.N. Daniels E. Featherstone M. Dev. Dyn. 2002; 225: 358-364Crossref PubMed Scopus (20) Google Scholar). The N-terminal PREP2 antibody, PREP2NAB, is targeted against the region encoded by exon 2 (29Haller K. Rambaldi I. Kovacs E.N. Daniels E. Featherstone M. Dev. Dyn. 2002; 225: 358-364Crossref PubMed Scopus (20) Google Scholar), whereas the C-terminal PREP2 antibody (PREP2CAB) is targeted against residues 341–392. Cross-reactivity to PREP1 was removed from PREP2CAB by passing over a column bearing a fusion of glutathione S-transferase to murine PREP1 residues 322–380. The following antibodies were also used: anti-GAL4 DNA-binding domain (DBD) mouse monoclonal antibody clone RK5C1 raised against residues 94–147 (sc-510, Santa Cruz Biotechnology); mouse monoclonal anti-β-tubulin antibody clone tub 2.1 (T-4026, Sigma); mouse monoclonal anti-HA-11 antibody (MMS-101R, BabCO); mouse monoclonal anti-actin antibody, clone AC 40 (A470, Sigma); anti-PBX1 (P-20) antibody (sc-889, Santa Cruz Biotechnology); anti-TATA-binding protein antibody (SI 1) (sc 273, Santa Cruz Biotechnology); anti-FLAG-agarose beads (A2220, Sigma); and rhodamine-conjugated phalloidin (R-415, Molecular Probes).Secondary Antibodies—The secondary antibodies used were as follows: marina blue-conjugated anti-mouse IgG (M-10991, Molecular Probes); rhodamine (TRITC)-conjugated goat anti-mouse IgG (115-025-003, Jackson Laboratories); peroxidase-conjugated goat anti-mouse IgG (115-035-003, Jackson Laboratories); peroxidase-conjugated anti-rabbit IgG (A0545, Sigma); and fluorescein isothiocyanate-conjugated anti-rabbit IgG (F-7512, Sigma).Drugs—The drugs used were as follows: cytochalasin D (C8273, Sigma); swinholide A (350-088-C010, Alexis Biochemicals); phalloidin (P2141, Sigma); paclitaxel (T1912, Sigma); nocodazole (M1404, Sigma); leptomycin B (LMB) (L2913, Sigma) kindly provided by Dr. M. Yoshida; and taxol (T1912, Sigma).Constructs—The full-length Prep2 coding region was PCR-amplified with NheI site-containing primers, subcloned into the vector TOPO II (Invitrogen), and sequenced (pPrep2-Topo). The Prep2-containing NheI fragment was then subcloned into the vector pFRED 143, 5′ to the GFP-coding region (construct A). pFRED 143 contains a humanized version of a strong mutant of GFP (Kyoji Horie and George N. Pavlakis, National Cancer Institute-Advanced Bioscience Laboratories.) A DNA fragment encoding the N terminus of PREP2 (residues 1–267) was fused to the GFP-coding region in the same manner (construct 2). Construct A was partially digested with NheI/XbaI, and the resulting full-length Prep2-gfp fusion was introduced into XbaI-digested pRC/CMV plasmid resulting in construct 1. Gal-O45 is a mammalian expression vector for the GAL4 DBD (30Rambaldi I. Nagy Kovács E. Featherstone M.S. Nucleic Acids Res. 1994; 22: 376-382Crossref PubMed Scopus (31) Google Scholar). For construct 3, pPrep2-Topo was digested with EcoRI/BamHI subcloned into BamHI-digested and -blunted Gal-045 3′ to the GAL-DBD-coding region. The cloning of PBX1-HA has been reported previously (18Saleh M. Huang H. Green N.C. Featherstone M.S. Exp. Cell Res. 2000; 260: 105-115Crossref PubMed Scopus (52) Google Scholar). The expression vector for the G13R actin mutant was a generous gift from the Treisman laboratory. For constructing the plasmid HA-PREP2-Myc, pPrep2-Topo was digested with NheI and subcloned into pHAv (18Saleh M. Huang H. Green N.C. Featherstone M.S. Exp. Cell Res. 2000; 260: 105-115Crossref PubMed Scopus (52) Google Scholar). This construct was further cleaved with BamHI/ApaI and inserted into pcDNA4/Myc-His A (Invitrogen).Cell Culture and Transfection—COS-7, NIH3T3, and primary mouse embryonic fibroblasts were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen). Drosophila S2 cells were cultured in Schneider's Drosophila medium (Invitrogen) with 10% fetal bovine serum. For immunohistological analysis, cells were grown on 12-mm-diameter round coverslips. Transfection was performed using LipofectAMINE Plus (NIH3T3) or LipofectAMINE 2000 (COS-7) reagents (Invitrogen).Cytoplasmic and Nuclear Fractionation of Cell Extracts—NIH3T3 cells were scraped from confluent tissue culture dishes and washed twice in phosphate-buffered saline, and cytoplasmic extracts were obtained by resuspending in hypotonic buffer (10 mm Tris, pH 7.4, 10 mm NaCl, 3 mm MgCl, 0.5 mm dithiothreitol, 0.05% Nonidet P-40). Nuclei were collected by centrifugation, and nuclear proteins were extracted in radioimmune precipitation assay buffer (50 mm HEPES, pH 7.5, 150 mm NaCl, 0.1% SDS, 1% sodium deoxycholate, 1% Triton X-100). Complete EDTA-free protease inhibitor tablets (1873580, Roche Diagnostics) were added to each buffer. 40 μg of total protein were separated on a 10% SDS gel and analyzed by Western blotting.RNA Interference—A mixture of two double-stranded siRNA oligonucleotides (Qiagen) was transfected into NIH3T3 cells using LipofectAMINE Plus, and transfection efficiency was monitored by nonsilencing fluorescein-labeled control siRNA. RNA was extracted using TRIzol reagent followed by real-time PCR using SYBR Green Taq ReadyMix™ for quantitative PCR (S1816, Sigma) in a Roche LightCycler as described previously (31Rastegar M. Kobrossy L. Nagy Kovacs E. Rambaldi I. Featherstone M. Mol. Cell. Biol. 2004; 24: 8090-8103Crossref PubMed Scopus (56) Google Scholar). Quantification of the LightCycler data was performed using standard curves of serially diluted plasmid DNA of known concentration. Sequences for primers are as follows: GAPDH sense primer 5′-AACGACCCCTTCATTGAC-3′; GAPDH antisense primer 3′-TCCACGACATACTCAGCAC-5′; Prep2 sense primer 5′-CCAGGTCCTGAGGATC-3′ (16 bp); and Prep2 antisense primer (5′-CATGGAATTGGGGGAATTCTGCAGGAGGTC-3′ (30 bp). PCR products were run on a 1% agarose gel and stained with ethidium bromide. The sequences of siRNA oligonucleotides (one strand only) were as follows: (a) 5′-CCCAGAUCCUGCUCCCAAA-3′; (b) 5′-UGUCUGGAGUCUCCAAUAA-3′; and (c) 5′-CCAAGAUGCACAGUGAUAA-3′. Mixtures of siRNAs a and c or b and c gave comparable results.Northern Analysis—Total RNA was isolated from P19 embryonal carcinoma cells using TRIzol reagent. Poly(A)+ RNA was then purified using the GenElute mRNA kit (Sigma). 3 μg of poly(A)+ RNA per well were run on a 1.2% formaldehyde-agarose gel and transferred to a Nytran plus membrane (Schleicher & Schuell). Blots were sliced to give one well of fractionated mRNA per membrane strip and prehybridized and hybridized in Express hybridization solution and washed according to the manufacturer's instructions. 2 × 106 cpm/ml of 32P-labeled and denatured Prep2 probe were used in the hybridizations. Blots were exposed to x-ray film with screens either overnight (375-nucleotide 5′ probe) or for 7 days (156-nucleotide 3′ probe and 153-nucleotide intron 4 (int) probe). An adult mouse tissue poly(A)+ blot purchased from Clontech was described previously (29Haller K. Rambaldi I. Kovacs E.N. Daniels E. Featherstone M. Dev. Dyn. 2002; 225: 358-364Crossref PubMed Scopus (20) Google Scholar). The region used for probe marked int is shown in Fig. 5A, whereas the 3′ probe spanned the sequence coding for PREP2 amino acids 340–391.Coimmunoprecipitation—NIH3T3 cells grown to confluency in 10-cm tissue culture dishes were harvested and washed in phosphate-buffered saline. Cells were lysed in buffer, pH 7.9, containing 50 mm NaCl, 1 mm EDTA, 0.5% Triton X-100, and 20 mm HEPES. Cell debris was removed by a - min spin in a microcentrifuge, and the lysate was precleared with 100 μl of protein A-agarose (16-156, Upstate Biotechnology). The immunoprecipitation was performed for 1 h at 4 °C using 1.5–2 mg of whole cell extract and 1 μg of antibody/mg protein. Protein A-agarose beads were applied for 2 h in a volume of 100 μl at 4 °C. The complexes were washed twice with cold phosphate-buffered saline and eluted with a 1:1 ratio of water and 4× SDS sample buffer. The proteins were separated on an 8% SDS gel and analyzed by Western blotting with the appropriate antibodies. Taxol was used at a concentration of 2 μm overnight.Actin Fractionation—The protocol for actin fractionation was adapted from Posern et al. (32Posern G. Sotiropoulos A. Treisman R. Mol. Biol. Cell. 2002; 13: 4167-4178Crossref PubMed Scopus (190) Google Scholar). Phalloidin was added for 10 min to the lysis buffer by using 10 μm drug. Cyochalasin D was applied at a concentration of 10 μm for 1 h to cells in culture.Immunofluorescence Staining—Cells were grown on coverslips and fixed in methanol for 15 min at -20 °C, rinsed in phosphate-buffered saline, and permeabilized with 0.1% Triton X-100 for 2 min. Primary antibodies were applied for 2 h, whereas secondary antibodies were applied for 1 h. Cells were mounted with GelTol water-based mounting medium from Fisher (catalog number 28-607-87). Pictures were taken with a Zeiss Axiovert 100 M confocal microscope and a Nikon digital camera DXM 1200 attached to a Nikon ECLIPSE E800 microscope. The protocol for immunohistological analysis of embryonic sections as well as the procedures for fixation and sectioning has been described previously (29Haller K. Rambaldi I. Kovacs E.N. Daniels E. Featherstone M. Dev. Dyn. 2002; 225: 358-364Crossref PubMed Scopus (20) Google Scholar).RESULTSA C-terminal Antibody Detects PREP2 in the Nucleus—We have previously reported that an affinity-purified antibody (PREP2NAB) directed against the murine PREP2 N terminus (amino acids 18–64) gives largely cytoplasmic reactivity in numerous tissues of 8.5–12.5-dpc mouse embryos (29Haller K. Rambaldi I. Kovacs E.N. Daniels E. Featherstone M. Dev. Dyn. 2002; 225: 358-364Crossref PubMed Scopus (20) Google Scholar). Although this result was consistent with other reports (19Berthelsen J. Kilstrup-Nielsen C. Blasi F. Mavilio F. Zappavigna V. Genes Dev. 1999; 13: 946-953Crossref PubMed Scopus (198) Google Scholar, 28Fognani C. Kilstrup-Nielsen C. Berthelsen J. Ferretti E. Zappavigna V. Blasi F. Nucleic Acids Res. 2002; 30: 2043-2051Crossref PubMed Scopus (39) Google Scholar), it was surprising for a protein with a transcriptional function. Therefore, we raised and tested a second affinity-purified antibody (PREP2CAB) directed against the murine PREP2 C terminus (amino acids 341–392). In Western analysis, both PREP2NAB and PREP2CAB were highly specific for PREP2, failing to recognize the closely related PREP1, two additional members of the TALE class of homeoproteins, and numerous peptides within rabbit reticulocyte lysates (Fig. 1). A signal was not detected with these antibodies in untransfected Drosophila S2 cells or when preimmune sera were used instead of primary antibodies (data not shown). Moreover, preabsorption of the anti-sera with PREP2 peptides abolished reactivity in Western and immunofluorescence studies (Fig. 2, K and L) (data not shown). Thus, PREP2NAB and PREP2CAB are specific for PREP2.Fig. 1Affinity-purified anti-PREP2 antibodies specifically recognize PREP2 but not other PREP2-related proteins of the TALE class. Top panel, 35S-labeled products produced by in vitro translation. Bottom panels, in vitro translated products from parallel reactions were probed with affinity-purified anti-PREP2 antibodies. Mock, unprogrammed reticulocyte lysate.View Large Image Figure ViewerDownload (PPT)Fig. 2PREP2 isoforms localize to different subcellular compartments. A and C–F, immunohistological analysis of 10.5-dpc mouse embryonic sections with PREP2CAB. The tissue distribution of PREP2CAB reactivity was as previously reported for PREP2NAB (29Haller K. Rambaldi I. Kovacs E.N. Daniels E. Featherstone M. Dev. Dyn. 2002; 225: 358-364Crossref PubMed Scopus (20) Google Scholar) but was in contrast predominantly nuclear. This is shown in the nucleus (arrow) of choroid plexus epithelium (A), in the precartilage mesenchyme (C), by the cartilage in the intervertebral discs (D), in the dermal mesenchyme in the hind brain area (E), and in the mesenchyme between the spinal ganglia (SG) (F). Note the absence of nuclear staining with PREP2NAB in choroid plexus epithelium (B). G–L, immunofluorescent detection of PREP2 in NIH3T3 cells. G, PREP2NAB reactivity is predominantly cytoplasmic (93% cells). H, nuclear reactivity of PREP2CAB (95% cells). I and J, DAPI-stained nuclei corresponding to G and H. K and L, preabsorption with glutathione S-transferase-PREP2 fusion protein abolishes immunoreactivity of both PREP2NAB and PREP2CAB.View Large Image Figure ViewerDownload (PPT)To assess the embryonic pattern of PREP2CAB reactivity, we performed immunohistological analysis on embryonic sections of 10.5-dpc mouse embryos (Fig. 2, A–F). The tissue distribution of PREP2 protein was consistent with our previous observations (29Haller K. Rambaldi I. Kovacs E.N. Daniels E. Featherstone M. Dev. Dyn. 2002; 225: 358-364Crossref PubMed Scopus (20) Google Scholar). However, in striking contrast to results obtained with PREP2NAB, we now observed predominantly nuclear staining. Tissues with marked nuclear signals included the epithelium of the choroid plexus (Fig. 2A), precartilagenous mesenchyme (Fig. 2C), cartilage of the intervertebral discs (Fig. 2D), dermal mesenchyme in the hindbrain area (Fig. 2E), and mesenchyme between the spinal ganglia (Fig. 2F). As noted previously (29Haller K. Rambaldi I. Kovacs E.N. Daniels E. Featherstone M. Dev. Dyn. 2002; 225: 358-364Crossref PubMed Scopus (20) Google Scholar), PREP2NAB gave cytoplasmic signals in these same tissues (Fig. 2B). These data suggested that PREP2 isoforms differing at their N and C termini localize to different subcellular compartments.To confirm and extend these results, we used PREP2NAB and PREP2CAB to examine PREP2 distribution in cells in culture (Fig. 2, G–L). In NIH3T3 cells, the PREP2NAB signal was consistently cytoplasmic but with faint and reproducible reactivity in the nucleus (Fig. 2G), whereas that of PREP2CAB was strongly nuclear (Fig. 2H). These observations were confirmed in a number of cell lines including COS-7, transfected S2, HEK293, and primary mouse embryonic fibroblasts (data not shown). Preincubation with fusions of glutathione S-transferase to the PREP2 N or C termini abolished reactivity (Fig. 2, K and L). Thus, PREP2 isoforms distinguished by N- and C-terminal-specific antibodies localize to distinct subcellular compartments in both embryonic and cultured cells.Distinct Isoforms of PREP2 Localize to Different Subcellular Compartments—To understand the basis of this differential localization, we used Western blot analysis to examine PREP2 in cytoplasmic and nuclear fractions of cultured cells. Confirming the results observed in immunofluorescence studies, PREP2NAB- and PREP2CAB-reactive species were distributed in a mutually exclusive fashion between the nuclear and cytoplasmic compartments of untransfected NIH3T3 (Fig. 3A) and COS-7 (data not shown). Strikingly, five PREP2 isoforms were detected, none of which was recognized by both antibodies and none of which was found in both subcellular compartments. Consistent with data from immunofluorescence, the majority of PREP2NAB-reactive material was found as high molecular mass species in the cytoplasmic fraction but also as a less abundant 25-kDa nuclear isoform.Fig. 3Multiple PREP2 isoforms are partitioned between the nucleus and cytoplasm and are sensitive to RNA interference. A, Western
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