Characterization of the nuclear import pathways of galectin-3.
Galectin-3 (Gal-3), a pleiotropic beta-galactoside-binding protein, was shown to be involved in several nuclear-dependent functions, including up-regulation of transcriptional factors, RNA processing, and cell cycle regulation. Gal-3 compartmentalization in the nucleus versus the cytoplasm affects, in part, the malignant phenotype of various cancers. However, to date, the mechanism by which Gal-3 translocates into the nucleus remains debatable. Thus, we have constructed and expressed a variety of fusion proteins containing deletion mutants of Gal-3 fused with monomers, dimers, and trimers of enhanced green fluorescent protein and searched for the Gal-3 sequence motifs essential for its nuclear localization in vivo. In addition, a digitonin-permeabilized, cell-free transport in vitro assay was used to directly examine the mechanism of Gal-3 nuclear import. Partial deletions of the COOH-terminal region (114-250) of the human Gal-3 significantly decreases its nuclear translocation, whereas a peptide (1-115) was transported to the nuclei. The in vitro nuclear import assay revealed that there are at least two independent nuclear pathways for shuttling Gal-3 into the nucleus: a passive diffusion and an active transport. This is the first article providing direct evidence for the nuclear import mechanisms of Gal-3 and suggests that Gal-3 nuclear translocation is governed by dual pathways, whereas the cytoplasmic/nuclear distribution may be regulated by multiple processes, including cytoplasmic anchorage, nuclear retention, and or nuclear export. These results may lead to the development of a therapeutic modality aiming at abrogating Gal-3 translocation into the nucleus and thus hampering its activity during cancer progression and metastasis.
Nakahara
Susumu
S
Tumor Progression and Metastasis Program, Karmanos Cancer Institute, Wayne State University, Detroit, Michigan 48201, USA.
Oka
Natsuo
N
Wang
Yi
Y
Hogan
Victor
V
Inohara
Hidenori
H
Raz
Avraham
A
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D001693:Biological Transport, Active; D001943:Breast Neoplasms / Q000378:metabolism; D019556:COS Cells; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability / Q000187:drug effects; D002467:Cell Nucleus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004058:Diffusion; D004072:Digitonin / Q000494:pharmacology; D037502:Galectin 3 / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection
D000952:Antigens, Polyomavirus Transforming; D037502:Galectin 3; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004072:Digitonin