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Originally published In Press as doi:10.1074/jbc.M406046200 on August 30, 2004

J. Biol. Chem., Vol. 279, Issue 47, 49384-49394, November 19, 2004
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Subcellular Localization of Multiple PREP2 Isoforms Is Regulated by Actin, Tubulin, and Nuclear Export*

Klaus Haller{ddagger}§, Isabel Rambaldi§, Eugene Daniels||, and Mark Featherstone, A Chercheur-National of the Fonds de la Recherche en Santé du Québec{ddagger}§**{ddagger}{ddagger}§§

From the {ddagger}McGill Cancer Centre and §Department of Biochemistry, Departments of **Medicine, {ddagger}{ddagger} Oncology, and ||Anatomy and Cell Biology, McGill University, Montreal, Quebec H3G 1Y6, Canada

Received for publication, June 1, 2004 , and in revised form, August 18, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Patterning of the embryonic antero-posterior axis comes under the control of HOX and TALE1 class homeoproteins (1). 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 (2). 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 (2). The ability of PBC proteins to interact with both HOX and MEIS/PREP partners results in DNA-bound heterotrimeric complexes that have been implicated in gene control in vitro and in vivo (3–8). However, the instances of HOX-independent gene control by TALE heterodimers have also been documented (9, 10), consistent with the presence of these proteins in tissues devoid of Hox gene expression.

In addition to the homeodomain, two conserved N-terminal domains, PBC-A and PBC-B, have been defined in PBC family proteins (11). These regions are implicated in numerous processes including nuclear export, transcriptional repression, MEIS/PREP interaction, and binding to non-muscle myosin heavy chain II B (2). 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 (9) with HR2 implicated in binding to PBC family proteins (12).

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 (13, 14). This distribution is critical for normal development, because forced nuclear accumulation of PBC proteins correlates with limb proximalization (13, 15, 16). Subcellular localization is controlled by protein kinase A (17) and by interaction with MEIS family proteins (14). MEIS binding masks NES while exposing NLS within the PBC N terminus (18–20). Reciprocally, MEIS is dependent on PBX for nuclear localization (21–24).

Recently, it has been shown in fly embryos and mammalian cells that PBX is retained in the cytoplasm by interaction with non-muscle myosin heavy chain II B (22), raising the possibility that subcellular localization of TALE class proteins could be influenced by signaling pathways impinging on the cytoskeleton.

The PREP (also known as PKNOX) (25) family has two members in mice and humans (9, 25–29). In complexes with PBX, PREP1 regulates the transcription of HOX-dependent (Hoxb2) and HOX-independent (urokinase plasminogen activator, glucagon) target genes (5, 9, 10). Both PREP1 and PREP2 have been shown to accumulate in the cytoplasm of cultured and embryonic cells (19, 28, 29). 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 (29). 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 PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Primary Antibodies—The PREP2 antibodies were raised as previously described (29). The N-terminal PREP2 antibody, PREP2NAB, is targeted against the region encoded by exon 2 (29), 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-{beta}-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 (30). 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 (18). 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 (18). 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 ReadyMixTM for quantitative PCR (S1816, Sigma) in a Roche LightCycler as described previously (31). 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 x 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 (29). 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.



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  		FIG. 5.
An alternative splice product encodes a PREP2 isoform of 25 kDa. A, partial sequence of a murine Prep2 cDNA (GenBankTM accession number AK084178 [GenBank] ) plus conceptually translated C terminus. Inverted triangles denote exon-exon and exon-intron junctions as indicated. Intron 4, which is spliced out of the cDNA encoding the originally reported PREP2 isoform, is retained in this variant. The probe used for Northern analysis (below) is underlined. B, Northern analysis of poly(A)+ RNA from undifferentiated murine P19 cells or adult mouse liver. For P19 cell poly(A)+ RNA, blots were hybridized with one of three probes: 5', the same probe from the 5' end of the full-length Prep2 cDNA as used previously (29); int, a 153-bp probe corresponding to the unique portion of the alternative splice variant (underlined in A) within intron 4; 3', a 156-bp probe from the 3' end of the originally reported Prep2 cDNA. For liver, a commercial blot of poly(A)+ RNA from adult tissues was hybridized with the "int" probe. Black arrowheads denote the position of hybridizing bands. C, the presumptive 25-kDa PREP2 isoform was tagged with HA, expressed in transiently transfected NIH3T3 cells, and detected by immunofluorescence with an anti-HA antibody. Localization was predominantly cytoplasmic.

 
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 4x 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. (32). 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 (29).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
A 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 (29). Although this result was consistent with other reports (19, 28), 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.



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  		FIG. 1.
Affinity-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.

 



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  		FIG. 2.
PREP2 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 (29) 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.

 
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 (29). 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 (29), 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.



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  		FIG. 3.
Multiple PREP2 isoforms are partitioned between the nucleus and cytoplasm and are sensitive to RNA interference. A, Western analysis of PREP2 isoforms in nuclear (N) and cytoplasmic (C) fractions of NIH3T3 cells. Lysates were run in parallel and probed with PREP2CAB or PREP2NAB (left panel). Cytoplasmic and nuclear fractions were also probed with antibodies against cytoplasmic (tubulin) and nuclear (TATA-binding protein, TBP) markers, whereas actin was used as a loading control (right panel). B, reduction in the level of Prep2 transcript-specific RT-PCR products from small interference-oligonucleotide-transfected NIH3T3 cells versus mock (upper panel) in comparison to gapdh control (lower panel). C, quantification of relative RT-PCR product abundance before (black bar) and after (gray bar) interference. D, Western analysis of NIH3T3 whole cell extracts detected with PREP2NAB and PREP2CAB before (lanes 1 and 3) and after (lanes 2 and 4) RNA interference. Actin was used as loading control.

 
To confirm the PREP2 identity of these isoforms, we performed siRNA analysis. A robust knockdown at the mRNA level could be detected (Fig. 3, B and C) and was accompanied by reduced protein levels for all PREP2 species (Fig. 3D). These results strongly support the PREP2 identity of all of the isoforms detected by PREP2NAB and PREP2CAB.

Alternative Splicing Gives Rise to a 25-kDa PREP2 Isoform Lacking the Homeodomain—We and others have identified cDNAs encoding the same PREP2 isoform in mouse and human. To determine whether the product of this open reading frame is proteolytically processed to place the N- and C-terminal epitopes on different peptides, we expressed PREP2 simultaneously tagged at its N terminus with HA and at its C terminus with GFP (Fig. 4, A–C). Importantly, both tags were found predominantly in the nucleus, arguing against proteolytic cleavage. Furthermore, expression and immunoblotting of a similar construct bearing a C-terminal Myc tag revealed no evidence of cleavage products (Fig. 4C). When HA tags were individually placed at either PREP2 terminus, again no cleavage products were observed (data not shown). These data suggest that PREP2 isoforms distinguished by their N and C termini are not derived by proteolytic cleavage of the product of the known open reading frame.



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  		FIG. 4.
PREP2 subcellular localization is regulated on multiple levels. A, schematic representation of PREP2 and PBX1A constructs fused to HA (red oval), GFP (green oval), or GAL-DBD (red rectangle). B, D, and E, fluorescence microscopy of COS-7 cells transfected with the indicated constructs. B, Double labeling of PREP2 protein at both termini reveals predominantly nuclear localization of both labels. C, substitution of the C-terminal GFP tag with a Myc tag followed by transfection into NIH3T3 cells and immunoblotting for both labels revealed no apparent cleavage products (lanes 3 and 4). The band indicated by the arrow (lane 4) was nonspecific, because it could also be detected in untransfected cells. Endogenous PREP2 detected by NAB and CAB is shown in lanes 1 and 2. D, coexpression of HA-tagged PBX1A (red; middle panel) directs PREP2-(1–267)-GFP (construct #2, green) to the nucleus (left panel; compare with Fig. 5I). Colocalization appears as yellow (right panel). E, endogenous NAB-reactive PREP2 is redirected to the nucleus (green) upon overexpression of HA-tagged PBX1A, which localizes to the nucleus (red), and the overlap is shown in yellow (right panel). N, nuclear; C, cytoplasmic.

 
PBX family members are the only known binding partners of PREP proteins (5, 9, 10, 29). Thus, if the immunoreactivity detected by PREP2NAB is indeed PREP2, it should be redistributed to the nucleus by overexpression of PBX. As previously shown with PREP1 (19), PBX directed strong nuclear localization of the PREP2 N terminus (Fig. 4D, compare with Fig. 6I). Importantly, PBX had a similar effect on endogenous PREP2 detected with PREP2NAB (Fig. 4E). These results further confirm the specificity of PREP2NAB.



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  		FIG. 6.
Cytoplasmic forms of PREP2 are sensitive to the nuclear export inhibitor LMB. NIH3T3 cells were treated with LMB for 4 h at 0 (A), 5 (B), 25 (C), and 100 nM (D) and scored for nuclear accumulation of PREP2 using anti-PREP2NAB. E–H, DAPI staining (blue) reveals nuclei. I, the C-terminal deletion mutant PREP2-(1–267)-GFP (construct #2) localizes almost exclusively to the cytoplasm but relocates to the nucleus upon 150 nM LMB treatment (J). K, the PREP2 N terminus (amino acids 1–131) directs GAL-DBD to the cytoplasm as revealed by rhodamine (red)-conjugated immunoreactivity. Endogenous PREP2CAB reactivity (green) is nuclear, as expected.

 
To assess whether alternative splicing might give rise to different PREP2 isoforms differentially detected by our antibodies, we searched the publicly available databases. This scan yielded an alternatively spliced murine cDNA (GenBankTM accession number AK084178 [GenBank] ). As a result of the retention of intron 4, this transcript encodes a truncated peptide bearing a novel C terminus (Fig. 5A). This presumptive PREP2 isoform would be recognized by PREP2NAB but not by PREP2CAB and has a predicted molecular mass of 25 kDa corresponding to the smallest isoform detected by PREP2NAB (Fig. 3A).

To determine whether the alternatively spliced transcript encoding a 25-kDa PREP2 isoform is normally present in the cell, we designed primers specific to this form for use in RT-PCR. A specific band of the predicted size was detected from RNA of murine P19 embryonal carcinoma cells. To assess whether this message was present in mature processed mRNA, we performed Northern analysis on the poly(A)+ fraction of RNA from murine P19 embryonal carcinoma cells. We used three independent probes: a 5' probe previously used to detect Prep2 transcripts in adult and embryonic tissues (29); a 153-bp probe corresponding precisely to the intronic sequences uniquely present in the alternatively spliced cDNA; and a 3' probe downstream of the homeobox. As shown in Fig. 5B, the 5' probe detects two bands of 4.5 and 1.8 kb, comparable in size to the two transcripts noted previously (29). The probe, which lies within intron 4 and which is specific to the alternatively spliced cDNA, detects a 1.4-kb band in poly(A)+ mRNA of P19 cells. A similar band is likewise detected in the poly(A)+ fraction of adult liver (Fig. 5B) but not in several other tissues known to express Prep2 (data not shown). These results confirm the biological relevance of this alternatively spliced cDNA and strongly suggest that it is responsible for the 25-kDa PREP2 peptide detected with PREP2NAB. Interestingly, the 3' probe, located downstream of the homeobox, detected further RNAs of 4.7, 3.0, 2.6, and 1.4 kb, demonstrating multiple Prep2 transcripts that may well account for the additional peptide isoforms we detect in Western analysis.

To determine the subcellular localization of the 25-kDa PREP2 isoform encoded by the alternatively spliced transcript, we cloned the cDNA by RT-PCR and fused it to sequences encoding an HA tag. When expressed in 3T3 cells and detected by an anti-HA antibody, the 25-kDa isoform was found to localize predominantly to the cytoplasm (Fig. 5C), consistent with the largely cytoplasmic reactivity of PREP2NAB.

The PREP2 N Terminus Directs Cytoplasmic Localization via Multiple Mechanisms—Cytoplasmic localization directed by the PREP2 N terminus could be mediated by non-exclusive activities including nuclear export and cytoplasmic anchoring. Prep2 products are cytoplasmic in insect S2 cells (19). To test for a role for active nuclear export, we expressed PREP2 protein in S2 cells followed by treatment with the CRM-1 inhibitor LMB. S2 cells showed clear nuclear accumulation of PREP2NAB reactivity upon a 25 nM treatment for 4 h (data not shown). We then scored LMB-treated NIH3T3 cells for nuclear localization of endogenous PREP2NAB reactivity. Punctate nuclear staining appeared at concentrations of 5–25 nM LMB after 4 h of treatment (Fig. 6, B and C), whereas strong nuclear staining was observed at a concentration of 100 nM over the same time (Fig. 6D). To test whether the PREP2 N terminus can direct cytoplasmic localization, we fused residues 1–267 to GFP (Fig. 4A) and assessed subcellular distribution. Whereas unfused GFP is found throughout the cell, the addition of the PREP2 N terminus results in predominantly cytoplasmic localization (Fig. 6I). Although PREP2-(1–267)-GFP did not relocate to the nucleus upon 25 nM LMB treatment for 4 h, nuclear accumulation was detected after increasing the concentration to 100 nM (Fig. 6J). To extend these observations, we fused PREP2 residues 1–131, encompassing the PREP2NAB epitope, conserved regions HR1, and part of HR2, to the GAL4 DBD. The GAL4 DBD harbors two NLSs (33) and is normally found in the nucleus. However, fusion to the PREP2 N terminus results in almost total restriction of GAL4 immunoreactivity to the cytoplasm (Fig. 6K), supporting nuclear export and/or cytoplasmic retention roles for this region.

PREP2 Interacts with the Actin and Microtubule Cytoskeletons—Three-dimensional reconstruction of GAL4-PREP2-(1–131) confocal immunofluorescent images revealed a cytoplasmic staining pattern highly reminiscent of cytoskeletal structures (Fig. 6K). This suggested that interaction with one or more components of the cytoskeleton may retain PREP2 in the cytoplasm, as has been noted for the action of non-muscle myosin heavy chain II B on PBX (22). To test this proposal, we performed colocalization studies in synchronized NIH3T3 cells using PREP2NAB and an anti-tubulin antibody. NIH3T3 cells showed a clear overlap of PREP2 with microtubules at all of the stages of mitosis (Fig. 7, A and B). To further verify these observations, we transfected GAL4-PREP2-(1–131) (Fig. 4, construct 3) into COS-7 cells and assayed for colocalization with tubulin (Fig. 7C). Again, we observed strong colocalization of the portion representing the PREP2 N terminus and tubulin. As expected, PREP2CAB-reactive signal did not colocalize with tubulin (Fig. 7F).



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  		FIG. 7.
PREP2 colocalizes with the microtubule and actin cytoskeletons. A, NIH3T3 cells were synchronized at G2/M, released for 40 min, and examined in M phase. PREP2 (green) and DAPI (blue) labeling was scored at different mitotic stages as indicated. B, double labeling using PREP2NAB (green) and anti-tubulin-specific antibody (red) reveals colocalization (yellow) at the mitotic spindle aster. The monochromatic image is also shown (lower right). C, colocalization of GAL-tagged PREP2-(1–131) (green) with microtubules (red) in transfected COS-7 cells is shown in the overlay (yellow). D, PREP2NAB (green) and rhodamine-conjugated phalloidin (red) were used to detect PREP2 and F-actin in untransfected COS-7 cells. Overlay demonstrates that the endogenous PREP2 N terminus colocalizes with actin (yellow and arrows). E, GAL-tagged PREP2-(1–131) (green) colocalizes with F-actin (red) in the periphery of COS-7 cells as shown in the overlay (lower panel, yellow). F, endogenous nuclear PREP2 recognized by PREP2CAB does not colocalize with tubulin. Images C and F are three-dimensional reconstructions from multiple confocal "z" planes.

 
To verify the association of PREP2 with tubulin, we performed coimmunoprecipitation assays on untransfected NIH3T3 cell extracts. PREP2NAB coprecipitated {beta}-tubulin (55 kDa) from untreated cells but not from cells treated with the microtubule-stabilizing agent, taxol (Fig. 8A, upper panel). Interaction with PREP2CAB was similar but much weaker (Fig. 8A, lower panel). Thus, PREP2 isoforms bearing the N-terminal epitope associate with microtubules in a specific manner sensitive to tubulin-interacting drugs.



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  		FIG. 8.
PREP2 is associated with F- and G-actin. A, coimmunoprecipitation assay of NIH3T3 cell extracts using PREP2NAB (top panel) versus PREP2CAB (bottom panel). AB, antibody. Proteins were separated by 10% SDS-PAGE and immunoblotted simultaneously for actin (42 kDa, black arrowhead) and {beta}-tubulin (55 kDa, gray arrowhead) with specific antibodies. Lane 1, input; lane 2, beads only; lane 3, anti-PREP2NAB, lane 4, anti-PREP2NAB on taxol-treated cells. Note the absence of the tubulin signal (lane 4) when cells were treated with the microtubule-stabilizing drug, taxol. IP, immunoprecipitation; IB, immunoblotting. B, fractionation of cellular extracts from NIH3T3 cells into 100,000-g supernatant (S) and pellet (P) fractions reveals association of PREP2 with F-actin. Cells were treated with the following drugs: lanes 1 and 3, 10 µM F-actin-depolymerizing drug cytochalasin D added to lysis buffer; lanes 2 and 4, untreated. Equal amounts were separated by 10% SDS-PAGE, and proteins were detected by Western blotting. Upper panel, PREP2 full-length and 25-kDa isoforms sediment with the F-actin-containing pellet (lanes 4). Lower panel, note the shift in G- to F-actin ratios following drug treatment (lanes 1 versus 3). WB, Western blotting. C, NIH3T3 cells were either transfected with the FLAG-tagged actin mutant (G13R) or assayed for endogenous actin. Cells were lysed in actin stabilization buffer with and without the F-actin stabilization drug phalloidin and fractionated as in B. WT, wild type. Equal volumes of the fractions were separated by 10% SDS-PAGE and actin detected by Western blotting. Under physiological conditions, the distribution of F-versus G-actin was approximately equal (top left panel), whereas phalloidin shifted the balance toward F-actin (top right panel). G13R does not contribute to the F-actin pool (bottom left panel). Even under F-actin-stabilizing conditions, the signal in the supernatant (G-actin) remains stronger (bottom right panel, compare with upper right panel). D, immunoprecipitation assays show the association of PREP2 with G-actin. FLAG-tagged actin G13R-expressing cell extracts were precipitated with PREP2NAB (upper panel) or PREP2CAB (lower panel), blotted, and probed with anti-FLAG antibody. Lane 1, input; lane 2, immunoprecipitation; lane 3, control. E, precipitation of FLAG-tagged actin G13R-expressing cell extracts with M2-anti-FLAG-agarose beads and detection of PREP2 confirming the association of PREP2 and G-actin. Cyto D, cytochalasin D.

 
We then asked whether PREP2 might also associate with the actin cytoskeleton. Fluorescent detection of F-actin with rhodamine-conjugated phalloidin revealed colocalization with the PREP2NAB epitope (Fig. 7D). Furthermore, overlapping signals could be detected in the cell periphery of GAL4-PREP2-(1–131)-transfected COS-7 cells (Fig. 7E). In addition, PREP2NAB revealed a strong interaction between PREP2 and actin (42 kDa) in coimmunoprecipitation experiments (Fig. 8A). Again, the signal was much weaker with PREP2CAB.

Based on this result, we expected PREP2 to copurify with F-actin in a F/G-actin fractionation assay. Under physiological conditions, the F/G-actin ratio in NIH 3T3 cells is roughly equal. To shift the F/G-actin ratio, we used cytochalasin D, an F-actin-disrupting agent. PREP2 was detected in the F-actin-containing pellet by PREP2NAB (Fig. 8B) and was reduced by treatment with cytochalasin D (compare lanes 3 and 4). Together, these results support an interaction between PREP2 and F-actin but do not exclude association with G-actin.

To test whether PREP2 also interacts with G-actin, we made use of a non-polymerizing {beta}-actin mutant (G13R) that does not contribute to the cellular F-actin pool (Fig. 8C) (32). Extracts of NIH3T3 cells expressing FLAG-tagged actin G13R were precipitated with PREP2NAB and PREP2CAB and Western blotted against anti-FLAG antibody (Fig. 8D). Although coprecipitation was observed for both PREP2 immunoreactive species, a much stronger interaction with the actin mutant was revealed with PREP2NAB (Fig. 8D, lane 2). These results were confirmed by the reverse experiment in which immunoprecipitation was performed with anti-FLAG beads followed by detection with the anti-PREP2 antibodies (Fig. 8E and data not shown).

The embryonic limb bud displays tight regulation of TALE class homeoproteins at a number of levels. We have previously shown that cytoplasmically restricted PBX is colocalized with assemblies bearing non-muscle myosin heavy chain II B in the distal mouse limb bud. Moreover, we have shown that PREP2 species detected with PREP2NAB are also found in the cytoplasm of limb bud cells (29). Therefore, we examined the distribution of actin, tubulin, and PREP2NAB-reactive species in this structure. PREP2 isoforms recognized by PREP2NAB strongly colocalize with the microtubule (Fig. 9, A–D) and actin (Fig. 9, E–H) cytoskeletons in this embryonic tissue. Taken together, these findings indicate that the N-terminally distinct isoforms of PREP2 are associated with microtubules, tubulin, and polymerized actin (F-actin) as well as G-actin.



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  		FIG. 9.
PREP2 colocalizes with actin and tubulin in the mouse embryo. Double staining with PREP2NAB (A) and anti-tubulin-specific antibodies (B) of frontal limb-bud sections of 10.5-dpc embryos demonstrates colocalization in the distal anterior region (C). Higher magnification reveals a clear overlap (examples given by arrows, D). Double labeling with anti-PREP2NAB (E) and anti-actin (F) reveals colocalization in the apical ectodermal ridge (AER) of transverse limbbud sections (G). H, higher magnification of the area boxed in G.

 
Disruption of the Actin or Microtubule Cytoskeletons Provokes PREP2 Relocalization—The actin and microtubule cytoskeletons control gene expression by influencing the nucleocytoplasmic transport of transcriptional regulators (22, 34–37). Therefore, we asked whether perturbation of these structures might trigger a change in PREP2 subcellular localization. Indeed, a 2-h treatment with 10 µg/ml nocodazole, a drug that disrupts microtubules by binding to {beta}-tubulin and preventing the formation of interchain-disulfide bridges, relocated the N-terminal epitope of PREP2 to the nucleus of COS-7 cells (Fig. 10, A–D). Under these conditions, the PREP2 N-terminal epitope was also observed in nucleoli (data not shown), contrary to the nuclear staining pattern observed with PREP2CAB. Similarly, cytochalasin D, a drug that disrupts actin microfilaments, provoked a dose-dependent relocalization of PREP2NAB reactivity. A 2-h exposure to 10 µM cytochalasin D redistributed PREP2 within the cytoplasm (Fig. 10, E–G), whereas 1 h at twice this concentration relocated the protein to the nucleus (Fig. 10, H–J). It has been reported that actinomycin D can block nuclear export (38). To confirm that relocalization of PREP2 to the nucleus was due to the loss of actin cytoskeleton integrity, we also treated cells with swinholide A, an F-actin-severing drug that does not affect nuclear export (38). A 2-h exposure to 50 nM swinholide A also induced efficient nuclear accumulation of endogenous PREP2NAB-reactive species as well as an ectopically expressed fusion of PREP2 residues 1–267 linked to GFP (data not shown). Together, these data demonstrate that both the actin and microtubule cytoskeletons coregulate the nuclear availability of PREP2 through interaction with distinct PREP2 isoforms.



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  		FIG. 10.
Disrupting the actin or microtubule cytoskeleton relocates PREP2. COS-7 cells were treated with the microtubule-destabilizing drug, nocodazole (33 µM or 10 µg/ml for 2 h) and scored for localization of PREP2 using PREP2NAB (green) and anti-{beta}-tubulin (red). Nuclear relocalization of PREP2NAB reactivity was clearly visible following treatment with nocodazole (A). The microtubule cytoskeleton was depolymerized and diffusely distributed (B), whereas tubulin remained in newly formed plasma membrane processes (arrows) that overlapped with residual cytoplasmic PREP2NAB reactivity (C). An intact microtubule cytoskeleton is shown in untreated cells (D). Treatment with the F-actin-disrupting drug cytochalasin D (10 µM for 2 h) led to a redistribution of PREP2 (E). Doubling the drug concentration (1 h) relocated the majority of PREP2 (H) and actin (I) immunoreactivity to the nucleus. Actin was visualized with an anti-actin antibody (F and I). G, overlap of E and F. J, nuclei in H and I stained with DAPI.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
A fundamental means to regulate transcription is through the control of nuclear access. In addition to the expected role of PBX in controlling PREP2 subcellular localization, here we show that CRM-1-dependent nuclear export and association with the actin and microtubule cytoskeletons also impinge on this process. Throughout this study, we used two antibodies raised against N-and C-terminal epitopes of PREP2. We have validated the specificity of our antibodies in several ways including the use of Western blotting of in vitro and in vivo translated proteins, immunoprecipitation, preadsorption, and preimmune serum. Perhaps most tellingly for the specificity of PREP2NAB, the material reactive with this antibody is coprecipitated with PBX in immunoprecipitation assays (data not shown), is strongly translocated to the nucleus upon overexpression of PBX1A, a known PREP2 partner (Fig. 4E), and is decreased following siRNA knockdown of Prep2 message (Fig. 3D).

A key finding from immunofluorescence studies is that PREP2NAB reactivity is largely restricted to the cytoplasm, whereas that of PREP2CAB is nuclear. Additionally, up to five distinct PREP2 isoforms are distributed between cytoplasmic and nuclear fractions in Western blots. These findings raise two major questions. First, how are multiple PREP2 isoforms produced? Second, what are the mechanisms governing the subcellular distribution of PREP2 isoforms?

Processes that could generate PREP2 isoforms of differing mobility in SDS-PAGE include alternative start codon usage, alternative splicing, and post-translational modification, whereas we have shown that proteolytic cleavage is less likely. Although the start codon at position 2 of the published Prep2 sequence appears optimal, there are other AUGs both upstream and downstream. The use of these starts could add or remove 2.5 or 1.8 kDa, respectively, and may well account for some of the isoforms we detect. Importantly, Northern analyses using additional probes located in intronic and 3' sequences have detected multiple Prep2 transcripts in mature poly(A)+ RNA in addition to those noted previously. Clearly, these additional products have the potential to encode the different PREP2 isoforms detected with our antibodies. Following the observation of alternatively spliced intron-4-containing Prep2 cDNAs in the public data base, we used a specific probe to confirm that intron 4 is indeed facultative and present in a 1.4-kb transcript in poly(A)+ RNA of adult liver and P19 cells. One such intron-4-containing transcript would in fact encode a 25-kDa PREP2 isoform recognized by PREP2NAB but not PREP2CAB, just as we observe for the 25-kDa isoform in our Western analyses. Together, these observations strongly suggest that at least some PREP2 isoforms and specifically the 25-kDa variant are the result of alternative splicing.

Additionally, post-translational modification or processing could change the apparent molecular mass while masking or removing the N- or C-terminal epitopes. For example, phosphorylation can alter protein conformation and mobility in SDS-PAGE. Moreover access to a given epitope could be directly or indirectly blocked. Lastly, proteolytic cleavage could generate isoforms of differing molecular masses that contain only one or the other (or neither) of the epitopes studied here. For example, the 25-kDa N-terminal and 47-kDa C-terminal peptides detected in our study could arise by cleavage of the 62-kDa "full-length" protein. However, our experiments with N- and C-terminally tagged PREP2 suggest that this is not the case.

PREP2NAB detects a 25-kDa PREP2 isoform in nuclear extracts despite the fact that immunofluorescent signals detected with this antibody are predominantly cytoplasmic. Because the extraction protocol (buffers and temperatures) used to obtain cytoplasmic and nuclear fractions is known to disrupt the cytoskeleton and given our demonstration that cytoskeletal disruption triggers nuclear translocation, the procedure may provoke rapid relocalization of normally cytoplasmic 25-kDa PREP2 to the nucleus. Moreover, protein-protein interactions masking N- and C-terminal epitopes may be relatively stable in immunofluorescence but not in denaturing SDS-PAGE, thereby minimizing nuclear immunofluorescent detection. Lastly, the staining detected with PREP2NAB is not exclusively cytoplasmic (Figs. 2G and 9B). Given that immunofluorescent signal strength does not necessarily correlate with that obtained by Western analysis, it may be that the weak nuclear reactivity of PREP2NAB in immunofluorescence is indeed due to the 25-kDa isoform. Nonetheless, the expression of a tagged 25-kDa product shows marked cytoplasmic accumulation, suggesting that the endogenous 25-kDa isoform does contribute to PREP2NAB immunoreactivity outside the nucleus.

Our observation of a 25-kDa PREP2 isoform residing largely in the cytoplasm but capable of accumulating in the nucleus suggests a role in transcriptional regulation. Whereas the 25-kDa PREP2 isoform lacks the homeodomain, it retains all of HM1 and HM2. This region of the MEIS/PREP family is known to be sufficient for interaction with PBX. Thus, the N terminus of MEIS family members binds to PBX family members in vitro and alters PBX function in vivo (39, 40). Moreover, the deletion of the PREP1 homeodomain actually augments association with PBX (8). Thus, the 25-kDa PREP2 isoform could reasonably function as a dominant-negative capable of preventing full-length forms of PREP and MEIS family members from interacting with PBX family peptides. Given the importance of DNA binding by MEIS/PREP family members for the function of HOX-PBX-MEIS/PREP trimers (3) or PBX-MEIS/PREP dimers (5), significant expression of the 25-kDa PREP2 isoform could significantly modulate target gene regulation. This does not exclude possible novel functions conferred on the 25-kDa peptide by the unique 50 residues at its C terminus. However, such a function may not be evolutionarily conserved because a similar splicing event in human PREP2 (in which intron 4 is retained in the mature transcript) would encode a C terminus only poorly conserved with its murine counterpart.

The masking and unmasking of NESs and NLSs through intermolecular and intramolecular interactions have been shown to play a crucial role in the localization of PBX and extradenticle (18–20). In this study, we provide evidence that the N-terminal portion of PREP2 harbors either a NES or a region that associates with NES-containing protein(s). In the presence of the nuclear export inhibitor LMB, PREP2 accumulates in the nucleus of diverse cell types including NIH3T3, COS-7, and S2. This is also true for the C-terminally truncated version PREP2-(1–267)-GFP, although higher LMB concentrations were necessary to achieve nuclear staining. This may have been due to the absence of two putative NLSs in the C-terminal portion of PREP2. One of these NLSs was proposed to lie between residues 348 and 354 (PKAKKIK) (27), and we tentatively assigned the other NLS to residues 274–280 (KKSKNKR). Substitution of the two presumptive NLSs in the PREP2 C terminus with the GAL4 DBD resulted in cytoplasmic staining only. Therefore, PREP2 N-terminal signals directing cytoplasmic localization dominate over NLSs in GAL-DBD and in larger PREP2 isoforms.

We showed intimate association between PREP2NAB-reactive material and the actin and microtubule cytoskeleton by both immunofluorescent colocalization and coimmunoprecipitation. Additionally, we demonstrated cytochalasin D-sensitive cosedimentation of PREP2 with F-actin. Overall, our data show the association between PREP2 with F- and G-actin and with microtubules/tubulin. A functional role for these associations is implied by our observation that drug-induced perturbation of the actin or microtubule cytoskeletons results in nuclear accumulation of PREP2NAB-reactive species.

How might association with cytoskeletal elements control PREP2 distribution in the cell? First, direct association with G-actin or tubulin in the nucleus could facilitate exposure of one or more NESs in the PREP2 N terminus. This would be consistent with our demonstration that PREP2 interacts with G-actin and with the fact that G-actin can be found in the nucleus (41). Alternatively, PREP2 may not encode an NES but could be exported from the nucleus because of its association with G-actin, which does have NES (41). Second and non-exclusively, direct binding to F-actin or microtubules could anchor PREP2 in the cytoplasm. We note that the action of nocodazole and cytochalasin D does not allow easy discrimination between these possibilities, because their effect may not be attributed to depolymerization of cytoskeletal elements but to the masking of interaction sites on G-actin or free tubulin (37). Nonetheless, interaction with the filamentous forms of actin and tubulin strongly supports a cytoplasmic anchoring function.

Association of PREP2 with cytoskeletal structures could also be indirect via one of the many peptides that contact these polymers. Some proteins, such as bullous pemphigoid antigen, have binding sites for both F-actin and microtubules (42). Linkage to such a protein could explain our observation that disruption of either cytoskeletal system is sufficient to direct the majority of PREP2 to the nucleus. This implies a mechanism involving cross-talk between the two cytoskeletal systems, which could be provided by a cross-linking protein, and is consistent with extensive cooperation between actin and microtubules in numerous cellular processes (43).

A number of studies have established the importance of cytoskeletal interactions in regulating nuclear access to transcription factors. Thus, Myc-interacting zinc finger 1, a regulator of the low density lipoprotein receptor gene, is sequestered in the cytoplasm by association to microtubules but is directed to the nucleus upon treatment with microtubule-depolymerizing agents (36). In a variation on this theme, binding to tubulin inhibits TGF-{beta}-mediated phosphorylation and perhaps nuclear translocation of Smad2, Smad3, and Smad4 (34). On the other hand, actin filaments retain the Nrf2 (NF-E2-related factor 2) in the cytoplasm until filament disruption provoked by phosphatidylinositol 3-kinase signaling sends a Nrf2-actin complex to the nucleus (44). Conversely, association of the MAL coactivator with G-actin prevents nuclear translocation in a manner reversible by Rho-induced F-actin formation (37). In the case of the glucocorticoid receptor, association with microtubules, including the mitotic spindle, may allow direct control of cell growth by glucocorticoids (35). These examples highlight the importance of the cytoskeleton in relaying cell-signaling cues to a variety of transcription factors and raise the possibility that multiple signaling pathways may impinge on interactions with the cytoskeleton to control PREP2 function. With regard to the ubiquitous embryonic expression profile of PREP2 protein (29), we speculate that PREP2 may be preferentially regulated at the protein level. This could have the advantage of making certain isoforms, perhaps with distinct regulatory potential, rapidly functional, both because the protein is already present and because it could be directed quickly to the nucleus by the cytoskeletal network.

In summary, our results, along with reports of cytoplasmically localized PBX, MEIS, and HOX family members (24, 45–47), suggest that the control of subcellular distribution is broadly employed to regulate homeoprotein function.


   FOOTNOTES
 
* This work was supported by Operating Grant 36409 from the Canadian Institutes of Health Research (CIHR). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Supported by a McGill Graduate Studies Fellowship. Back

§§ To whom correspondence should be addressed: McGill Cancer Center, McGill University, McIntyre Medical Science Bldg., Rm. 714, 3655 Promenade Sir W. Osler, Montreal, Quebec H3G 1Y6, Canada. Tel.: 514-398-8937; Fax: 514-398-6769; E-mail: mark.featherstone{at}mcgill.ca.

1 The 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. Back


   ACKNOWLEDGMENTS
 
We thank R. Treisman and G. Posern for the G13R actin mutant, K. Yoshida for LMB, U. Stochaj, W. Mushynski, Feng Gu, and A. Nepveu for helpful discussions.



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 ABSTRACT
 INTRODUCTION
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 DISCUSSION
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