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Originally published In Press as doi:10.1074/jbc.M608069200 on October 20, 2006

J. Biol. Chem., Vol. 281, Issue 51, 39649-39659, December 22, 2006
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Importin-mediated Nuclear Translocation of Galectin-3*

Susumu Nakahara{ddagger}§, Victor Hogan{ddagger}, Hidenori Inohara§, and Avraham Raz{ddagger}1

From the {ddagger}Tumor Progression and Metastasis Program, Karmanos Cancer Institute, Wayne State University, Detroit, Michigan 48201 and the §Department of Otolaryngology and Sensory Organ Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan

Received for publication, August 22, 2006 , and in revised form, October 13, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Galectin-3 (Gal-3), a member of a beta-galactoside-binding protein family, is involved in RNA processing and cell cycle regulation through activation of transcription factors when translocated to the nucleus. We have previously shown that Gal-3 can import into the nucleus through at least two pathways; via passive diffusion and/or active transport (Nakahara, S., Oka, N., Wang, Y., Hogan, V., Inohara, H, and Raz, A. (2006) Cancer Res. 66, 9995-10006). Here, we investigated the process mediated by the active nuclear transport of Gal-3 and have identified a nuclear localization signal (NLS)-like motif in its protein sequence, 223HRVKKL228, that resembles p53 and c-Myc NLSs (378SRHKKL383, 322AKRVKL327), respectively. Moreover, trimers of enhanced green fluorescence protein (3xGFP) fused with this NLS-like sequence, which is too large to passively diffuse through the nuclear pores, accumulated in the cell nuclei. To gain insights into this newly identified nuclear import mechanism, the interaction between Gal-3 and importins (importins {alpha} and beta) that carry the NLS harboring nuclear proteins into the nucleus, was investigated. Pull-down assays and bimolecular fluorescence complementation (BiFC) analysis revealed that wild-type Gal-3, but not mutant Gal-3 (R224A), binds to importin-{alpha}. Down-regulation of importin-beta by RNA interference (RNAi) efficiently abrogates its nuclear accumulation. Furthermore, we provide evidence that impaired nuclear translocation of mutant Gal-3 protein (R224A) results in accelerated degradation compared with the wild-type protein. Thus, these results suggest that Gal-3 is translocated to the nucleus, in part, via the importin-{alpha}/beta route and that Arg224 amino acid residue of human Gal-3 is essential for its active nuclear translocation and its molecular stability.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Galectin-3 (Gal-3),2 a member of an evolutionary conserved family of beta-galactoside-binding proteins, is ubiquitously expressed and involved in diverse biological functions (1-3). This protein can be found not only in the cytoplasm, but also in the nucleus and even in the extracellular spaces (4, 5). It shuttles between the cytoplasm and the nucleus, where it undergoes post-translational modification, i.e. phosphorylation of a serine residue by the protein kinase; casein kinase 1 (CK1) that signals its export to the cytoplasm and serves as a molecular switch for the sugar binding ability (6-9). The nuclear localization of Gal-3 is probably associated with normal cell proliferation because it is a required factor in the splicing of pre-mRNA (10). In cancer, Gal-3 plays a significant role in tumor progression (11-16). It exerts a growth-promoting effect and cell cycle regulation through induction of cyclin D1 and c-Myc in the nuclei of human breast epithelial cells (11-13). In particular, up-regulation of cyclin D1 expression was induced through enhancement or stabilization of the nuclear protein-DNA complex formation at the cAMP-responsive element of the cyclin D1 promoter (14). In the nuclei of papillary thyroid cancer cells, Gal-3 interacts with the thyroid-specific TTF-1 and up-regulates its transcriptional activity, contributing to proliferation (15). In prostate cancer cells, nuclear Gal-3 suppressed malignancy whereas cytoplasmic Gal-3 promoted tumorigenicity through a mechanism yet to be determined (16). In vivo, the cytoplasmic versus nuclear expression of Gal-3 is associated with tumor invasion and metastasis (17-22). Loss of nuclear Gal-3 expression was reported in colon and prostate carcinomas (17, 18). Similarly, the levels of nuclear Gal-3 were markedly decreased during the progression from normal to cancerous states in tongue carcinomas (19). In lung carcinoma, the nuclear Gal-3 is a predictive factor of recurrence and/or worse clinical outcome (20, 21). In patients with esophageal squamous cell carcinoma, elevated expression of Gal-3 in the nuclei was suggested to be an important pathological parameter related to histological differentiation and vascular invasion (22).

Analysis of in vitro nuclear import assay using digitonin-permeabilized cells revealed that there are at least two pathways for the nuclear import of Gal-3, passive diffusion and active transport dependent on its N-terminal region (23). However, the possible role of the carbohydrate recognition domain (CRD) in Gal-3 nuclear translocation process was difficult to determine using this system because it bound to the cell surface glycoproteins and/or glycolipids through its beta-galactoside binding site, resulting in inhibition of transport (23). Thus, the complete mechanism of Gal-3 nuclear import is not fully understood and a different approach avoiding the use of digitonin-permeabilized cells is required to study and establish the critical role of CRD for Gal-3 nuclear import.

Here, we have identified an NLS-like sequence, 223HRVKKL228, in the C-terminal region of the human Gal-3 that is similar to p53 and c-Myc NLSs (24-26), and studied its function(s). NLS-containing proteins, like p53, are transported into the nucleus by the importin (karyopherin) {alpha}/beta complex (26-28). Importin-{alpha} is the receptor subunit that recognizes the NLS, a cluster of basic amino acid residues that complex with importin-beta for passage through the nuclear pore (29, 30). The presence of several isoforms of importin-{alpha} proteins in mammals recognizing variants of the classic NLS was reported (31), suggesting that the nuclear import process might be different for each protein depending in part on specifically regulated biological functions. This study also shows that deletion of the C-terminal region of Gal-3 protein without the NLS-like motif (1-222) results in complete impairment of nuclear accumulation, whereas a different C-terminal deletion protein that includes this motif (1-229) can be accumulated in the nucleus. In addition, substitution of Arg to Ala at position 224 (R224A) of the human Gal-3 efficiently abolished nuclear localization. To conclude, the studies shown here revealed that Gal-3 directly binds to importin-{alpha} proteins and complexes with the importin-{alpha}/beta complex in vivo. Moreover, the failure to translocate from the cytoplasm to the nucleus resulted in a rapid degradation of Gal-3.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Transfection—COS-7, HeLa and HT1080 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). These cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 5% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) and antibiotics (50 international units/ml penicillin and 50 µg/ml streptomycin) (Cellgro, Herndon, VA) under 5% CO2 at 37 °C. Transfection into COS-7 or HT1080 cells was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. For siRNA transfection to down-regulate the expression level of human importin-beta1, karyopherin beta1 siRNA (h) (sc-35736, Santa Cruz Biotechnology, Santa Cruz, CA) was purchased and used. Selection was performed via the addition of 1000 µg/ml G418 (Cellgro) to establish stable transfectants. Cell culture with addition of the reagents was performed as follows. In the case of leptomycin B treatment, at 24 h after transfections, COS-7 cells were treated with 30 ng/ml of leptomycin B (Sigma) for 4 h at 37 °C. In the case of cycloheximide (CHX) treatment, at 24 h after transfection COS-7 cells were treated with 100 ng/ml of CHX (Sigma) for the time indicated.

Plasmid Construction—The constructs for wild type (WT/GFP), C-terminal-deleted Gal-3 (1-222/GFP, 1-229/GFP), SV40NLS (NLS+x2GFP) fused with GFP, and 2x EGFP (2x GFP) for mammalian expression were described previously (23). For construction of C-terminal His-tagged wild type Gal-3 (Gal-3/C(WT)), the PCR product using the primers (a 5' primer, the same as above and a 3' primer, (5'-CGGAATTCTATCATGGTATATGAAGC-3') was subcloned into pcDNA3.1/Myc-His A (Invitrogen). C-terminal His-tagged deletion mutant Gal-3/C-(1-222) was made by subcloning the fragment cut from 1-222/GFP with EcoRI and ApaI into pcDNA3.1/Myc-His B (Invitrogen). For construction of N-terminal His-tagged wild-type Gal-3 (Gal-3/N(WT)) and deletion mutant (Gal-3/N-(63-250)), the fragment cut from pGEX/Gal-3 and cleaved Gal-3 (32) with EcoRI was subcloned into pcDNA3.1/His A (Invitrogen), respectively. For generation of a single amino acid mutant of each Gal-3 plasmid, a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used according to the manufacturer's protocol with specific primers as follows: Arg224 to Ala (R224A); 5' primer, 5'-GCTCACTTGTTGCAGTACAATCATGCGGTTAAAAAACTCAATG-3', 3' primer, 5'-CATTGAGTTTTTTAACCGCATGATTGTACTGCAACAAGTGAGC-3'; Lys226 to Ala (K226A), 5'-CTTGTTGCAGTACAATCATCGGGTTGCAAAACTCAATGAAATCAG-3', 3' primer, 5'-CTGATTTCATTGAGTTTTGCAACCCGATGATTGTACTGCAACAAG-3'; Lys227 to Ala (K227A), 5' primer, 5'-CTTGTTGCAGTACAATCATCGGGTTAAAGCACTCAATGAAATCAG-3', 3' primer, 5'-CTGATTTCATGAGTGCTTTAACCCGATGATTGTACTGCAACAAG-3', respectively). To create the expression vectors including Gal-3 NLS-like sequence (222NHRVKKLNE230) and its mutated sequence (R224A) fused with 3x EGFP (3x GFP/RVKKL, 3x GFP/AVKKL), two sets of synthesized oligonucleotides (for 3x GFP/RVKKL; 5'-CAATCATCGGGTTAAAAAACTCAATGAGGGCC-3' and 5'-CTCATTGAGTTTTTTAACCCGATGATTGGTAC-3', and for 3x GFP/AVKKL; 5'-CAATCATGCGGTTAAAAAACTCAATGAGGGCC-3' and 5'-CTCATTGAGTTTTTTAACCGCATGATTGGTAC-3') were annealed and subcloned into 3x GFP vector described previously (23). The DNA sequence of all mutants was confirmed at Wayne State University DNA Sequencing Core. The Escherichia coli expression vectors; pGEX-2T-PTAC58(importin-{alpha}1), pGEX-2T-hQip1(importin-{alpha}3), pGEX-5X-3-NPI-1(importin-{alpha}5), and pGEX-2T-HA-PTAC97(importin-beta) were kindly provided by Dr. Yoshihiro Yoneda (Osaka University Graduate School of Medicine, Osaka, Japan). For recombinant Gal-3 expression, pGEX-6P-2/Gal-3 was used as described previously (32) and pGEX-6P-2/Gal-3(R224A) was made and used as described above. For recombinant GFP-fused Gal-3(WT, R224A), the fragment cut with EcoRI and NotI from WT/GFP and R224A//GFP was subcloned into pGEX-6P-2 and expressed, respectively. For BiFC assays, cDNAs encoding importin-{alpha}1, -beta, Gal-3(WT), and Gal-3(R224A) were cut with appropriate restriction enzymes from the above vectors and subcloned into pE-YC or pe-YN vector (Fig. 5B, a gift from Dr. Yoel Kloog, Tel-Aviv University, Tel-Aviv, Israel), which expresses the C-terminal (YC) or N-terminal (YN) halves of YFP (33, 34).

GFP Expression and Immunofluorescence Microscopy—Cells on Lab-Tek II chamber slide (8-well) (Nalge Nunc International, Naperville, IL) were fixed with precooled (-30 °C) methanol for 30 min at 4 °C or 4% paraformaldehyde for 20 min at 25 °C. In the case of GFP expression, cells were washed with PBS and counterstained with 5 µg/ml Hoechst 33258 (Molecular Probes, Eugene, OR), then mounted with Crystal/Mount reagent (Biomeda, Foster City, CA). In the other cases, cells were permeabilized with 0.5% Triton X-100/PBS for 5 min after fixation. They were blocked with 10% goat serum in PBS for 30 min at room temperature and then incubated with anti-Myc antibody (Invitrogen) (1:400 dilution in PBS) for Gal-3/C detection, anti-Xpress antibody (Invitrogen) (1:400 dilution in PBS) for Gal-3/N detection at room temperature for 1 h, respectively. After washes with PBS, cells were incubated for 1 h with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG (Zymed Laboratories Inc., San Francisco, CA) for Myc and Xpress diluted 1:100. After washes with PBS, slides were then mounted with a drop of mounting reagent. Fluorescent signal was recorded with a Zeiss Laser Scanning Microscope 310 or an Olympus BX40 microscope.

Western Blot Analysis—Total cell lysates or cytoplasmic and nuclear proteins were prepared as described previously (9, 23). Concentration of extracted protein was measured by means of a Bio-Rad protein assay reagent. Adjusted proteins were separated by SDS-PAGE (8-12.5%) and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 0.1% casein in PBS with 0.1% Tween 20 (PBS-T) and then incubated with primary antibodies for 1 h at room temperature. After washes for 5 min with PBS-T, it was incubated with the secondary fluorescence-labeled antibodies for 1 h at room temperature and finally washed three times with PBS-T. The fluorescence-labeled bands were detected with the Odyssey system (LI-COR Biosciences, Lincoln, NE). The density of the band was also determined with the Odyssey software. Primary and secondary antibodies were used as follows; TIB166 antibody (ATCC) at 1:250 dilution, IRDye 800-conjugated anti-GFP antibody (Rockland, Gilbertsville, PA) at 1:2500, His6 antibody (BD Biosciences) at 1:5000, beta-tubulin antibody (Sigma) at 1:2000, histone H1 antibody (Calbiochem, San Diego, CA) at 1:100, beta-actin antibody (Sigma) at 1:5000, GST antibody (Amersham Biosciences) at 1:500, importin-beta1 (H-300) (Santa Cruz Biotechnology) at 1:100, Alexa Fluor 680-conjugated antimouse, anti-rat, anti-rabbit, anti-goat antibody (Molecular Probes) at 1:5000, IRDye 800 conjugated anti-mouse, anti-rat, anti-rabbit antibody (Rockland) at 1:5000.

In Vitro Binding Assay—Recombinant GST-fused importin proteins (importin-{alpha}1, {alpha}3, {alpha}5, beta1) were expressed and purified as described previously (35). Recombinant Gal-3 proteins (wild type, R224A mutant) were expressed and purified as described previously (23, 32). Lysates from bacteria expressing GST (20 µg) or GST-importins (each 15 µg) were incubated with GSH-Sepharose 4B (Amersham Biosciences) and resuspended as 1:1 slurry with transport buffer (23). The beads were mixed with 1 µg of Gal-3 and dissolved in final 150 µl of transport buffer. After incubation for 1.5 h at room temperature, the beads were washed five times with transport buffer and subject to SDS-PAGE followed by Western blot analysis.

BiFC Analysis—The plasmids for BiFC chimeras [YC/{alpha}1, YN/beta, YC/WT, YN/WT, YC/224, and YN/224] were transiently co-transfected with the indicated pair into COS-7 cells by means of Lipofectamine 2000 (Invitrogen). 24-36 h after transfection, COS-7 cells were plated for 15 h at 30 °C on coverslips to induce maturation of the YFP fluorophore (33, 34). Cells were fixed in 2% paraformaldehyde, and counterstained with Hoechst 33258 to clearly define nuclear areas. Fluorescence images were acquired with an Olympus BX40 microscope and processed with imaging software (MCID).

Lactose Precipitation—Lactose-conjugated agarose was used to check lactose binding capacity of wild-type Gal-3 and its mutants (10). Briefly, whole cell extracts (200 µg) from each transfected cell line were mixed with 15 µl of 1:1 lactose-conjugated agarose slurry (Sigma) followed by agitation at 4 °C for 1.5 h. At the same time, 10% of whole cell extracts was saved as an input. After three washes with 0.5% Triton X-100 in PBS, samples were subject to SDS-PAGE followed by Western blot analysis.

In Vitro Degradation Assay—Recombinant GFP-fused Gal-3 and mutant Gal-3 (R224A) proteins were expressed and purified as described above. Each GFP-fused Gal-3 (25 ng) was mixed with 10 µg of whole cell lysates (protease inhibitor-free) from COS-7 cells and dissolved in final 15 µl of PBS. After incubation at 37 °C for indicated time, samples were subject to SDS-PAGE followed by Western blot analysis.

RNA Isolation and Reverse Transcription (RT)-PCR—Total RNAs from transfected COS-7 cells were isolated with TRIzol reagent (Invitrogen) according to the manufacturer's instructions. RT-PCR analysis was performed using AccessQuick RT-PCR system (Promega, Madison, WI) with 1 µg of RNA from each cell as a template. For detection of GFP mRNA expression, 40 cycles (95 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min) were performed with the following primers: 5' primer, 5'-CGGGATCCATCGCCACCATGGT-3' and 3' primer, 5'-CGGGATCCCTTGTACAGCTCGTC-3'. For detection of beta-actin mRNA as an internal control, 25 cycles (95 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min) were performed with the following primers: 5' primer, 5'-TGACGGGGTCACCCACACTGTGCCCAT-3' and 3' primer, 5'-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3'. After RT-PCR, samples were electrophoresed on 1.0% agarose gel containing SYBR Gold nucleic acid gel stain reagent (Molecular Probes), and the intensity of each band was detected with a Kodak Digital Science Image System.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Subcellular Localization of NLS-like Sequence Deleted or Point-mutated Gal-3 Proteins—To address the possibility that the 223HRVKKL228 sequence of Gal-3 is functionally homologous with p53 and c-Myc NLSs (Refs. 24 and 25, Fig. 1A) and is involved in Gal-3 nuclear import, we initially constructed several GFP-fused Gal-3 expression vectors, transfected them into cells, and analyzed subcellular localization of those products with a fluorescence microscope. We constructed fusion proteins whereby wild-type Gal-3 fused with GFP at its C terminus (WT/GFP) and its point mutants in the target sequence by sitedirected mutagenesis where we have substituted Arg224, Lys226, and Lys227 with Ala (R224A, K226A, K227A), respectively, and in addition constructed two C-terminal-deleted mutants proteins with or without the above sequence (1-229/GFP and 1-222/GFP), respectively (Fig. 1B). To confirm the correct expression of the deletion and point mutants of EGFP-fused Gal-3 as well as 2x EGFP (2x GFP) proteins, Western blotting of the transfected COS-7 cell lysates was performed using antibodies to GFP and Gal-3 (TIB166) (Fig. 1C). All plasmids expressed proteins of the expected molecular weights in Western blotting of either GFP (Fig. 1C, green), and/or Gal-3 (Fig. 1C, red) indicated the correct expression of Gal-3 polypeptides (Fig. 1C, yellow). Thereafter, each plasmid was transfected into COS-7 cells and cultured for 24 h at 37 °C. The cells were then fixed and visualized under a fluorescence microscope. The representative microscopic fields are depicted in Fig. 1D. EGFP expression of 1-222/GFP, which does not include the above NLS-like Gal-3 sequence, was outside the nucleus (Fig. 1D, panels a and a'). However, 1-229/GFP Gal-3-protein, which contains that sequence, was found to be localized in the nuclear compartment (Fig. 1D, panels b and b'), implying that the consecutive amino acid residues (223-229) of Gal-3 partially control its cellular distribution. Moreover, high expression of R224A mutant protein was located predominantly outside the nucleus (Fig. 1D, panels d and d'), whereas the GFP expression patterns of K226A and K227A protein mutants were diffusely spread throughout the cytoplasm (Fig. 1D, panels e, e',f, and f'). Whereas, the expression of wild-type Gal-3 protein was found to be randomly distributed in both the nucleus and cytoplasm (Fig. 1D, panels c and c'). The fluorescence in the cells transfected with 2x GFP showed the predominant cytoplasmic localization of EGFP (Fig. 1D, panels g and g') as described previously (23). However, we observed that the expression pattern of each protein had variety and diversity, therefore, to evaluate and determine the cellular distribution of each protein, we categorized their patterns into three major groups (mostly localized in the nucleus, N > C; equally localized in the cell, n = C; mostly spotted around the nucleus (spot C) and counted over 300 cells that expressed GFP in each plasmid-transfected cells. As shown in Fig. 1E, the nuclear localization ratio of 1-229/GFP was ~10%, whereas the ratio of 1-222/GFP was ~0%. In addition, the spotted pattern ratio of 1-222/GFP highly increased compared with R224A/GFP (Fig. 1D, 1-222, R224A, shadowed bar, p < 0.0001). There were no significant differences of subcellular distribution among WT/GFP, K226A/GFP, and K227A/Gal-3 proteins (Fig. 1E, WT, K226A, K227A, p > 0.05).


Figure 1
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  		FIGURE 1.
Subcellular localization of EGFP fused with wild-type and mutated Gal-3. A, sequence alignment of Gal-3, p53, and c-Myc proteins. Bold characters indicate basic amino acid residues. B, schema of the deletion and point mutants of Gal-3 fused with EGFP (Gal-3/GFP). Gray-shadowed characters indicate the point-mutated amino acid residues in the NLS-like motif. C, Western blotting of GFP (green) and Gal-3 (red) was simultaneously performed using transiently transfected COS-7 cells. Identical bands of GFP and Gal-3 colored yellow. Molecular standards (Mr) are shown in the left side.(Lane 1, 1-222/GFP; lane 2, 1-229/GFP; lane 3, WT/GFP; lane 4, R224A/GFP; lane 5, K226A/GFP; lane 6, K227A/GFP; lane 7, x2GFP). D, subcellular localization of EGFP-fused wild type and mutant Gal-3 in COS-7 cells. COS-7 cells were transiently transfected with the plasmids depicted in A and observed 24 h after transfection by fluorescence microscopy. A typical image (x400) was selected in each transfected cell. Panels a-g, fluorescent signals of the cells expressing EGFP-labeled proteins; panels a'-g', nuclear DNA stained with Hoechst 33258 of the cells in the same fields as in panels a-g, respectively. E, relative proportion of cells with different subcellular localizations of EGFP-fused Gal-3. EGFP expression analyses of more than 300 cells are summarized. Bars represent mean ± S.D. of three independent experiments. Percentages of cells with EGFP concentrated in nuclei (nucleus (N) > cytoplasm (C), black bar), throughout the entire cell (n = C, white bar), and spotted in the cytoplasm (spot C, gray bar) are shown.

 
Because GFP fusion proteins might change the original biological function of Gal-3 because of its large molecular mass, and because it was reported that the region fused with tags could affect the localization pattern of several proteins (37), we constructed both N- and C-terminal His-tagged Gal-3 expression vectors. In addition to the wild type, one C-terminal-deleted Gal-3-(1-222) and three point mutant proteins (described above), we constructed a mammalian expression vector for the deleted N-terminal region Gal-3 (Gal-3/N-(63-250)) (Fig. 2A). To determine how these exogenous His-tagged Gal-3 products distribute in the cell, we first performed immunofluorescence analysis after transfection of each plasmid into COS-7 cells cultured for 24 h, and visualized them under the microscope. The representative immunostaining data using anti-Myc antibody of C-terminal His-tagged Gal-3 (Gal-3/C) are depicted in Fig. 2B. As shown in Fig. 2B, wild-type Gal-3 (Gal-3/C(WT)) was evenly distributed throughout the cell, slightly different from the pattern of GFP-fused wild-type Gal-3 distribution (Fig. 1D, panels c and c'), probably because of the difference between direct observation of GFP expression and indirect immunofluorescent methodology. The staining patterns of R224A mutant and C-terminal-deleted Gal-3-(1-222) proteins were similar to the results of GFP-fused protein expression, respectively (Fig. 2B, 224, 1-222). The staining patterns of both K226A and K227A mutants were almost similar to that of wild type (data not shown). We confirmed there was no staining with control normal mouse IgG (Fig. 2B, CTRL). The representative immunostaining patterns using anti-Xpress antibody of N-terminal His-tagged Gal-3 proteins (Gal-3/N) were shown in Fig. 2C. The patterns of wild-type Gal-3 and three point mutants (R224A, K226A, K227A) were almost similar to those of C-terminal His-tagged Gal-3 (Fig. 2C, WT, 224, the data of 226 and 227 are not shown), indicating that any tags of the terminal regions do not alter the subcellular localization of Gal-3. As a new insight, N-terminal-deleted Gal-3 (Gal-3/N-(63-250)) could also exist in the nucleus (Fig. 2C, 63-250). We also confirmed there was no staining with normal mouse IgG (Fig. 2C, CTRL). Next, the observed distributions of each Gal-3 protein were confirmed by Western blot analysis using anti-His antibodies after subcellular fractionation of each plasmid transfected COS-7 cell. The results of the Western blot analyses in subcellular localization strongly support the lack of nuclear localization of Gal-3/C(R224A), Gal-3/C-(1-222), and Gal-3/N(R224A) proteins, respectively, whereas the other mutant proteins including wild-type Gal-3 were localized in the nucleus (Fig. 2, D and E, upper panel). To show the successful subcellular fractionation, we reprobed the same membranes with anti-beta-tubulin antibodies for cytoplasmic fraction and anti-histone H1 antibodies for nuclear fraction (Fig. 2, D and E, middle and lower panel). In addition, we performed these experiments using additional cell lines (BT-549, human breast carcinoma and HEK 293, human fetal kidney cell), and the results were found to be similar (data not shown). Taken together, Arg224 residue in the NLS-like motif of Gal-3 is essential for nuclear localization of Gal-3.


Figure 2
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  		FIGURE 2.
Subcellular localization of His-tagged Gal-3. A, schema of Gal-3 fused with Myc-His in its C-terminal region (I, Gal-3/C) and with His-Xpress in its N-terminal region (II, Gal-3/N). B, immunofluorescent staining (x630) with anti-Myc antibody was performed to detect Gal-3/C expression after transfection to COS-7 cells. No staining was observed in vector only transfected cells (CTRL). C, immunofluorescent staining (x630) with anti-Xpress antibody was performed to detect Gal-3/N after transfection. Parental cells were used as a control stain (CTRL). D, subcellular localization of various Gal-3/C was investigated after transfection into COS-7 cells. Cytoplasmic protein (Cy, each 30 µg) and nuclear protein (N, each 15 µg) were subjected to SDS-PAGE (12.5%) followed by Western blotting with anti-His for detection of Gal-3/C. The same membrane was reprobed with anti-beta-tubulin, anti-histone H1 for detection of cytoplasmic and nuclear fraction, respectively. As a control, vector only was expressed and analyzed (CTRL). An arrow indicates nonspecific band detected in nuclear fraction of the cell. E, subcellular localization of various Gal-3/N was investigated after transfection into COS-7 cells. Cytoplasmic protein (Cy, each 30 µg) and nuclear protein (N, each 20 µg) were subjected to SDS-PAGE (12.5%) followed by Western blotting with anti-His for detection of Gal-3/N. As a control, vector only was expressed and analyzed (CTRL). An arrow indicates nonspecific band that is mainly detected in the nuclear fraction of the cell.

 
Because the possible role of a sugar binding activity for Gal-3 nuclear import was questioned (23), we examined whether the nuclear translocation of mutated Gal-3 protein (R224A) lost its sugar binding capacity. Thus, we examined the sugar binding of each mutant Gal-3 protein by lactose-agarose precipitation (LP) assay using the endogenous expressed Gal-3 protein present in cell lysates of COS-7 cells transfected with His-tagged Gal-3 plasmids. The result indicated that Gal-3(R224A) also maintained its sugar binding activity similar to the wild-type Gal-3 proteins (Fig. 3A, upper panel). To validate the efficiency of this methodology, the same membranes were reprobed with anti-Gal-3 antibodies, positive control (Fig. 3A, middle panel) or anti-beta-actin antibodies as a negative control of lactose binding (Fig. 3A, lower panel).

Next, we examined whether Gal-3(R224A) protein was predominantly distributed outside the nucleus because of the lack of a nuclear retention. Because nuclear localization of Gal-3 can be attributed to a nuclear export signal (NES) located at the C-terminal region, 228LNEISKLGI236 in human Gal-3 (39), and the Arg224 residue is located near this NES, one might argue that the cytoplasmic localization of Gal-3(R224A) is caused by the lack of nuclear retentive capacity by enhancement of its nuclear export. Leptomycin B (LMB) is known to inhibit the nuclear export of Gal-3 through NES (23, 39); thus, we treated GFP-fused Gal-3-transfected cells with LMB and followed its effect on the localization of each Gal-3 protein. The representative data are depicted in Fig. 3B. LMB significantly inhibited the nuclear export of the WT/GFP protein (Fig. 3, A and B, WT, black bar, p = 0.01). Under the same experimental condition, the localization of R224A/GFP was not changed because of the LMB treatment (Fig. 3, B and C, 224, p > 0.05), suggesting that R224A/GFP protein cannot migrate into the nucleus, similarly, the localization of 1-222/GFP protein in which NES was deleted, was also not changed (Fig. 3, B and C, 1-222, p > 0.05). Taken together, these results suggested that Gal-3(R224A) protein lacks nuclear import activity independent of its beta-galactoside binding.


Figure 3
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  		FIGURE 3.
Characterization of nuclear translocation impaired Gal-3(R224A). A, lactose binding capacity of Gal-3(R224A). COS-7 cells transfected with each Gal-3/C expression vector were harvested and lactose precipitation (LP) was performed as described under "Experimental Procedures." Input (10% of whole cell extracts), and LP samples were subject to SDS-PAGE (10%) followed by Western blotting using anti-His antibody. The same membrane was reprobed with anti-Gal-3 antibody (TIB166) for positive control of LP and anti-beta-actin antibody for negative control of LP, respectively. B, effect of leptomycin B on subcellular localization of Gal-3(R224A). COS-7 cells transfected with various Gal-3/GFP were treated with 30 ng/ml of leptomycin B (LMB(+)) or only Me2SO (LMB(-)) for 4 h at 37°C. Typical images of each treatment are shown (x400). Panels a-f, fluorescent signals of the cells expressing EGFP-labeled proteins; panels a'-f', nuclear DNA stained with Hoechst 33258 of the cells in the same fields as in panels a-f, respectively. C, relative proportion of cells with different subcellular localizations of EGFP-fused Gal-3 after LMB treatment. EGFP expression analyses of more than 200 cells are summarized. Bars represent mean ± S.D. of two independent experiments. Percentages of cells with EGFP concentrated in nuclei (nucleus (N) > cytoplasm (C), black bar), throughout the entire cell (n = C, white bar), and spotted in the cytoplasm (spot C, gray bar) are shown.

 
NLS-like Sequence of Gal-3—Additional studies are required to evaluate the essential role of the NLS-like sequence (223HRVKKL228) as well as the Arg224 residue for Gal-3 nuclear import. Nuclear localization was observed for 1-229/GFP, K226A/GFP, and K227A/GFP, but not for 1-222/GFP and R224A/GFP (Fig. 1D), therefore, we focused on further characterizing the putative nuclear localization domain of amino acid residues 222NHRVKKLNE230 (aa 222-230) and the significance of its Arg residue. To confirm that aa 222-230 can mediate the nuclear import of Gal-3 and the Arg224 residue can play a critical role in that process, plasmids encoding aa 222-230 and its Arg residue mutated peptides fused with trimer of EGFP (3x EGFP), respectively, to avoid the passive diffusion into the nucleus were constructed (3x GFP/RVKKL, 3x GFP/AVKKL, Fig. 4A), and the subcellular localization of those fusion proteins as well as 3x GFP was examined 48 h after transfection in COS-7 (Fig. 4B) and BT549 (Fig. 4C) cells by fluorescence microscopy. As expected from the deletion mutant analysis, 3x GFP/RVKKL was predominantly directed into the nucleus (Fig. 4, B and C, panel a''). On the other hand, the replacement of Arg to Ala in that sequence (3x GFP/AVKKL) or trimer of EGFP (3x EGFP) abrogated the nuclear localization in both COS-7 and BT549 cells (Fig. 4, B and C, panels b'' and c''). Collectively, these results indicate that the 222NHRVKKLNE230 sequence of human Gal-3 constitutes a functional NLS, one in which Arg224 in particular plays a critical role.


Figure 4
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  		FIGURE 4.
Effect of an NLS-like motif of Gal-3 responsible for nuclear localization. A, schematic diagram of the trimer of EGFP (x3GFP) fusion proteins used to identify the effect of Gal-3 NLS-like motif. COS-7 cells (B) and BT549 cells (C) were transiently transfected with plasmids encoding x3GFP/RVKKL, x3GFP/AVKKL, and x3GFP for 48 h, followed by observation by fluorescence microscopy (x400). Panels a-c, fluorescent signals of the cells expressing EGFP-labeled proteins; panels a'-c', nuclear DNA stained with Hoechst 33258 of the cells in the same fields as in panels a-c, respectively; panels a''-c'', combined images of panels a-c and a'-c', respectively.

 
Gal-3 Importins Interaction—Because nuclear import of Gal-3 has a sequence motif similar to that of p53 and c-Myc NLSs (Fig. 1A) and this 222NHRVKKLNE230 sequence should be a functional NLS (Fig. 4), we examined whether Gal-3 could directly bind to importin-{alpha} proteins, e.g. binding partners of NLS-containing proteins. And because importin-{alpha} proteins have been categorized into three major groups according to the structure (31, 40), we first performed GST pull-down assay using three importin-{alpha} proteins ({alpha}1, {alpha}3, {alpha}5). As shown in Fig. 5A, wild type Gal-3 protein directly binds to all importin-{alpha} proteins, whereas failing to bind to importin-beta or GST alone (Fig. 5A, upper panel). On the other hand, mutant Gal-3 (R224A) protein could not efficiently bind to any of the importin-{alpha} proteins (Fig. 5A, middle panel), suggesting that the Arg224 residue is essential for Gal-3 binding to importin-{alpha} proteins and continuous basic amino acid residues of that region function as an NLS. The proper expression of each recombinant protein was confirmed with Western blotting of GST (Fig. 5A, lower panel).

To further investigate the interaction of Gal-3-importins in vivo, we tested the complex formation of these proteins using BiFC analysis. The BiFC approach is based on the formation of a fluorescent complex when two fragments of a fluorescent protein are brought together by an interaction between proteins fused to the fragments (33, 34). Complementary fragments of YFP (YN or YC) were fused to the N-terminal region of importin-beta, importin-{alpha}1, Gal-3(WT), and Gal-3(R224A), respectively (Fig. 5B). A selected pair of these plasmids was co-transfected into COS-7 cells, and we confirmed the expression of the two proteins by Western blotting using polyclonal anti-GFP antibody (Fig. 5C). Two distinct protein bands in each lane indicate the successful double protein expression in each transfection. We also performed Western blotting using anti-Gal-3 and antiimportin-beta antibodies and confirmed the identity of proteins bands (data not shown). Using the same transfection procedure, we monitored the transfected COS-7 cells by fluorescence microscopy. Fluorescence was detected within 24-48 h after transfection in cells expressing pairs of importin-beta + importin-{alpha}1, importin-{alpha}1 + Gal-3(WT or R224A), importin-beta + Gal-3(WT or R224A) proteins. BiFC was distributed throughout the cell (Fig. 5D). No fluorescence was detected in cells transfected with plasmids of importin-{alpha}1 + Gal-3(R224A) and importin-beta + Gal-3(R224A), respectively (Fig. 5D). Thus, formation of the bimolecular fluorescent complex required specific interaction between importins and Gal-3(WT) proteins. Of note, no fluorescence was observed in cells that expressed combination of importins and Gal-3(R224A), suggesting that these proteins do not form complexes in the cell.


Figure 5
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  		FIGURE 5.
Gal-3(WT), but not mutant Gal-3 (R224A), can interact with importin-{alpha}/beta complex. A, direct interactions between Gal-3 and various importin proteins were analyzed with GST pull-down assay. Recombinant wild-type Gal-3 or mutant Gal-3 (R224A) protein was incubated with GSH-Sepharose 4B loaded with GST or various GST-importins. Bound fractions were analyzed with Western blotting of anti-Gal-3 antibody (TIB166). The expression of recombinant proteins was confirmed with anti-GST antibody. Input, loading control; GST, GST-bound fraction; {alpha}1, GST-importin-{alpha}1-bound fraction; {alpha}3, GST-importin-{alpha}3-bound fraction; {alpha}5, GST-importin-{alpha}5-bound fraction; beta, GST-importin-beta1-bound fraction. B, schematic diagram of the fusion proteins for BiFC analysis. C, Western blotting of GFP was performed using COS-7 cell lysates concurrently transfected with YC- and YN-containing plasmids. Molecular standards (Mr) are shown in the left side. Lane 1, YN/beta+YC/{alpha}1; lane 2, YN/WT+YC/{alpha}1; lane 3, YN/beta+YC/WT; lane 4, YN/224+YC/{alpha}1; lane 5, YN/beta+YC/224. D, visualization of interactions among importins and Gal-3 using BiFC analysis. The proteins indicated to the left of each set of images were coexpressed in COS-7 cells, and the fluorescence emissions of the cells were imaged 48 h after transfection (x400). Panels a-e, fluorescent signals of the cells expressing YFP-labeled proteins; panels a'-e', nuclear DNA stained with Hoechst 33258 of the cells in the same fields as in panels a-e, respectively.

 
Importin-beta Contributes to the Nuclear Translocation of Gal-3—Next, we examined whether Gal-3 is carried by the importin-{alpha}/beta complex into the nucleus, and used the RNA interference (RNAi) approach to inhibit the expression of importin-beta to evaluate its effect on the cellular localization of Gal-3/GFP. The cellular localization of SV40NLS fused with 2x EGFP (NLS+2 x GFP, Ref. 23), which is a characterized carrier protein of the importin-{alpha}/beta complex, is used as a control. Each plasmid and a siRNA duplex targeted to human importin-beta were co-transfected to HT1080 cells. At 48 h after transfection, the cells were harvested, and the expression level of endogenous importin-beta was analyzed by Western blotting. As shown in Fig. 6A, this treatment specifically reduced the expression level of importin-beta protein as compared with beta-tubulin. The transfected cells with importin-beta siRNA and Gal-3/GFP showed distinct perinuclear localization with several punctuate cytoplasmic staining patterns (Fig. 6B, panels b and b', Fig. 6C), whereas the non-transfected cells with siRNA showed the entire cellular distribution of Gal-3/GFP (Fig. 6B, panels a and a'', Fig. 6C). Similarly, NLS+2 x GFP in the cell transfected with siRNA was predominantly localized in the cytoplasmic space (Fig. 6B, panels d and d'', Fig. 6C). These results demonstrate the functional and the specific interaction of importin-beta is required for Gal-3 nuclear translocation.

Outcome of the Lack of Nuclear Localization Ability of Deleted and Mutated Gal-3 Proteins—Because the mutant Gal-3(R224A) protein did not show typical cytoplasmic distribution, we hypothesized that it may be rapidly degraded. Thus, we have used CHX, a reagent that can inhibit de novo protein synthesis (41), to establish whether the rate at which Gal-3 protein level declines with this CHX treatment is a gauge of protein instability (42). As shown in Fig. 7A, we examined the stability of wild type of Gal-3 (Gal-3/C(WT)) and mutant Gal-3 (Gal-3/C(R224A)) at different time points after CHX addition to the transfected COS-7 cells. It appeared that Gal-3/C(R224A) protein, which cannot enter the nucleus, was rapidly degraded (Fig. 7A, 224), whereas wild-type Gal-3 was very stable over the6 h of the assay (Fig. 7A, WT). We also examined the stability of Gal-3/C-(1-222), and the results were similar to that of Gal-3/C(R224A) (data not shown). In addition, we also monitored the amount of Gal-3 proteins secreted into the conditioned medium after CHX treatment and found that Gal-3/C(WT), but not Gal-3/C(R224A), was secreted out of the cell (data not shown). Next, recombinant proteins were introduced to evaluate the stability of mutant Gal-3 (R224A) protein using an in vitro degradation assay. As shown in Fig. 7B, both recombinant Gal-3 proteins (Gal-3/GFP(WT) and Gal-3/GFP(R224A)) were degraded in ~6 h when incubated with COS-7 cell lysates, with no detectable difference in the degradation rate between them. These results suggest that lack of nuclear translocation of Gal-3 (1-222, R224A) protein results in its in vivo rapid degradation. Thus, we tested whether the protein expression level of Gal-3(R224A) would decrease after prolonged culture. Cell selection was performed after 3 weeks to establish the stable transfectants of Gal-3/GFP(WT) and Gal-3/GFP(R224A). Three different transfectants were established, e.g. one Gal-3/GFP(WT) transfectant (Fig. 7C, WT) and two Gal-3/GFP(R224A) transfectants (Fig. 7C, 224a, and b), and performed Western blotting and RT-PCR for detection of GFP. Gal-3/GFP(WT), but not Gal-3/GFP(R224A), could be detected at the protein level (Fig. 7C, upper panel). In contrast, Gal-3/GFP(R224A) could be clearly detected at the mRNA level as well as Gal-3/GFP(WT) (Fig. 7C, lower panel). We confirmed the control of no protein and no mRNA expressions of GFP in parental cells (Fig. 7C, CTRL) and detected beta-actin expression as an internal control in all cells (Fig. 7C, beta-actin). In addition, we observed these transfectants under the microscope and again the expression of Gal-3/GFP(R224A) protein was not detected (data not shown). Taken together, these results suggest that Arg224 mutation as well as C-terminal deletion affect the Gal-3 protein stability and expression.


Figure 6
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  		FIGURE 6.
Effect of importin-beta siRNA on Gal-3 nuclear accumulation. Total cell lysates (15 µg) were prepared from HT1080 cell 48 h after specific importin-beta siRNA transfection and subjected to Western blotting of importin-beta and beta-tubulin using the same membrane (A). Images of cells (B, x400) and relative proportion of cells (C) expressing Gal-3/GFP or NLS+x2GFP in control cultures or after importin-beta siRNA transfection. Panels a-d, fluorescent signals of the cells expressing EGFP-labeled proteins; panels a'-d', nuclear DNA stained with Hoechst 33258 of the cells in the same fields as in panels a-d, respectively; panels a''-d'', combined images of panels a-d and a'-d', respectively. ((-), control culture; (+), siRNA transfection)).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Here, we report that a consecutive basic amino acid residues in the C-terminal domain of the human Gal-3, i.e. 223HRVKKL228 that resembles a sequence of one of the p53 NLSs (p53 has three NLSs in its sequence, Refs. 24 and 26) or that of c-Myc NLS (25) (Fig. 1A), could function as its NLS. A single amino acid substitution of Arg224 residue in this motif results in the protein retention in the cytoplasm, whereas both Lys226 and Lys227 residues are not essential for Gal-3 nuclear import despite consecutive basic amino acids (Fig. 1, D and E). This agrees with reports showing the significance of an Arg residue, but not Lys, in the nuclear import of protein (43-45). In particular, a single mutation of Arg306 to Ala or even Lys in the major NLS of p53 abolished its nuclear import (42). Next, we examined the interaction between Gal-3 and importin-{alpha} proteins, key molecules essential for NLS-containing the protein nuclear import. Gal-3 (WT) could bind directly to importin-{alpha} proteins, whereas mutant Gal-3 (R224A) protein could not (Fig. 5A). BiFC analysis using Gal-3(WT) and Gal-3(R224A) proteins supported these findings (Fig. 5D). In addition, down-regulation of importin-beta by siRNA treatment abrogated nuclear localization of Gal-3, suggesting that Gal-3 could be imported to the nucleus via the importin-{alpha}/beta complex.


Figure 7
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  		FIGURE 7.
Rapid degradation of Gal-3 that cannot enter the nucleus in a living cell. A, COS-7 cells transiently transfected with each Gal-3/C expression vector were treated with cycloheximide (CHX(+)) or only Me2SO ((-)) and harvested at the indicated time. Each fraction was subjected to Western blotting of anti-His and anti-beta-actin antibodies. (WT; Gal-3/C(WT)-transfected cells, 224; Gal-3/C (224)-transfected cells)). B, recombinant GFP-fused Gal-3 (WT, 224) was incubated with whole cell extracts (10 µg) from COS-7 cells. After incubation for indicated time, samples were subjected to Western blotting of GFP and beta-actin. (WT, EGFP-fused wild-type Gal-3; 224, EGFP-fused mutant Gal-3 (R224A)). C, proteins and RNAs were extracted from COS-7 cells stably transfected with wild-type and mutant Gal-3/GFP. Equal amount of proteins (20 µg) were subjected to Western blotting of anti-GFP and anti-beta-actin antibodies (I). RT-PCR was performed for detection of GFP and beta-actin mRNA expression, respectively, and each amplified cDNA was electrophoresed (II). (CTRL: parental COS-7 cells, WT; Gal-3/GFP(WT) transfected COS-7 cells, 224a, b; Gal-3/GFP (224) transfected COS-7 cells, and these two cell lines were independently established.)

 
However, a question arises regarding the biological significance of Gal-3 nuclear import by means of an importin-{alpha}/beta complex because we previously reported that Gal-3 migrated into the nucleus by two pathways; passive diffusion and active transport dependent on its N-terminal sequence (23). In this regard, it is important to point out that the localization patterns of 1-222/GFP, 1-229/GFP, and R224A/GFP were different in the transfected cells (Fig. 1E). The difference between 1-222/GFP and 1-229/GFP subcellular distribution might show the dependence of Gal-3 nuclear import on the importin-{alpha}/beta complex (Fig. 1). Regarding the subcellular distribution pattern, wild-type Gal-3 is predominantly localized in a cytoplasmic compartment despite the presence of functional importin-{alpha}/beta complex. It is probable that Gal-3 nuclear migration depends in part on the affinity to importin-{alpha} proteins, which is likely to be weak (Fig. 5A). To support this, the localization of trimer of EGFP fused with Gal-3 NLS-like sequence did not show the typical nuclear accumulation (Fig. 4) as shown when SV40NLS harboring protein was expressed (23). In BiFC analysis, the number of cells expressing maturated YFP, i.e. YN/beta + YC/{alpha}1 >> YN/WT + YC/{alpha}1 > YN/beta + YC/WT, may also suggest a low affinity of Gal-3 to importin-{alpha} proteins (data not shown). Moreover, Davidson et al. (46) implied that the static observations of the nuclear versus cytoplasmic localization of murine Gal-3 must reflect a balance among at least four key parameters, i.e. nuclear import, nuclear export, cytoplasmic anchorage, and binding and retention in the nucleus, so subcellular localization of Gal-3 should be regulated in a complicated manner in vivo.

Regarding nuclear export, there are also some similarities between Gal-3 and p53. Serine phosphorylation is the only known post-translational modification for Gal-3, and its phosphorylation seems to be related to changes in its subcellular distribution (4, 39, 47). In particular, phosphorylation of Gal-3 is required for its nuclear export in response to chemotherapeutic drugs (9). In the case of p53, two serine residues located within the N-terminal NES (two NESs have been found in p53) are phosphorylated following DNA damage (26, 48), resulting in blockage of p53 nuclear export leading to its nuclear retention. Of note, ubiquitination can also regulate the nuclear-cytoplasmic shuttling of p53 (26, 49). Ubiquitination of p53 protein in the nucleus signals its export from the nucleus followed by cytoplasmic degradation in the proteasome system. Here, we found that loss of nuclear migration ability results in a rapid cytoplasmic degradation of Gal-3 mutant proteins (1-222, R224A) (Fig. 7). One might think that this degradation is not caused by simple inhibition of nuclear import but induced by rapid protein misfolding and/or aggregation followed by trapping in certain cytoplasmic compartments. In this regard, we performed double staining with Mitotracker or Lysotracker (Molecular Probes) using Gal-3/GFP-transfected cells, but no mutant Gal-3 could be detected in either the mitochondria or lysosomes (data not shown). Also, we checked the rate of degradation by addition of lactacystin and chloroquine, a proteasome and lysosome inhibitor, respectively, but there were no significant differences with these treatments (data not shown). Taken together, the detailed mechanism of mutant Gal-3 rapid degradation is still unclear, but we believe that active nuclear import and serine phosphorylation of Gal-3 are necessary for stability expression of Gal-3 protein.

In summary, we hypothesized that, like p53, the nuclear transport of Gal-3 plays a role in the regulation of its turnover in a living cell. The Gal-3 protein could be imported into the nucleus by either of passive diffusion, N-terminal region-dependent active transport, or importin-{alpha}/beta complex-dependent transport. This Gal-3 nuclear localization is dependent on the Arg224 residue involved in the interaction with importin-{alpha}/beta proteins leading to nuclear import of Gal-3, a process likely to be essential for its protein stabilization.


   FOOTNOTES
 
* This work was supported by Grant R37CA46120-19 from the National Institutes of Health. 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

1 To whom correspondence should be addressed: Tumor Progression and Metastasis Program, Karmanos Cancer Institute, Wayne State University, Detroit, MI 48201. Tel.: 313-833-0960; Fax: 313-831-7518; E-mail: raza{at}karmanos.org.

2 The abbreviations used are: Gal-3, Galectin-3; WT, wild type; NLS, nuclear localization signal; EGFP, enhanced green fluorescent protein; YFP, yellow fluorescent protein; BiFC, bimolecular fluorescence complementation; PBS, phosphate-buffered saline; GST, glutathione S-transferase; NES, nuclear export signal; LMB, leptomycin B; CTRL, control; CHX, cycloheximide; aa, amino acid; RNAi, RNA interference assay. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Yoshihiro Yoneda, Toshihiro Sekimoto, Yoichi Miyamoto (Osaka University Graduate School of Medicine, Japan) for providing the E. coli expression vectors (pGEX-2T-PTAC58(importin-{alpha}1), pGEX-2T-hQip1(importin-{alpha}3), pGEX-5X-3-NPI-1(importin-{alpha}5), pGEX-2T-HA-PTAC97(importin-beta)), and also helpful advice. We also thank Vivian Powell for secretarial help.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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The stability of AID and its function in class-switching are critically sensitive to the identity of its nuclear-export sequence
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