Importin-mediated nuclear translocation of galectin-3.
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, (223)HRVKKL(228), that resembles p53 and c-Myc NLSs ((378)SRHKKL(383), (322)AKRVKL(327)), 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 Arg(224) amino acid residue of human Gal-3 is essential for its active nuclear translocation and its molecular stability.
Nakahara
Susumu
S
Tumor Progression and Metastasis Program, Karmanos Cancer Institute, Wayne State University, Detroit, Michigan 48201, USA.
Hogan
Victor
V
Inohara
Hidenori
H
Raz
Avraham
A
D021581:Active Transport, Cell Nucleus; D020816:Amino Acid Motifs; D000818:Animals; D001120:Arginine / Q000737:chemistry; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D037502:Galectin 3 / Q000737:chemistry / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D028884:Karyopherins / Q000737:chemistry / Q000502:physiology*; D009154:Mutation; D019913:Nuclear Localization Signals; D011487:Protein Conformation
D037502:Galectin 3; D028884:Karyopherins; D019913:Nuclear Localization Signals; D049452:Green Fluorescent Proteins; D001120:Arginine