LZTS2 is a novel beta-catenin-interacting protein and regulates the nuclear export of beta-catenin - PubMed (original) (raw)

LZTS2 is a novel beta-catenin-interacting protein and regulates the nuclear export of beta-catenin

Gregory Thyssen et al. Mol Cell Biol. 2006 Dec.

Abstract

Beta-catenin plays multiple roles in cell-cell adhesion and Wnt signal transduction. Through the Wnt signal, the cellular level of beta-catenin is constitutively regulated by the multicomponent destruction complex containing glycogen synthase kinase 3beta, axin, and adenomatous polyposis coli. Here, we present multiple lines of evidence to demonstrate that LZTS2 (lucine zipper tumor suppressor 2) interacts with beta-catenin, represses the transactivation of beta-catenin, and affects the subcellular localization of beta-catenin. The LZTS2 gene is located at 10q24.3, which is frequently lost in a variety of human tumors. A functional nuclear export signal (NES) was identified in the C terminus of the protein (amino acids 631 to 641). Appending this motif to green fluorescent protein (GFP) induced nuclear exclusion of the GFP fusion protein. However, introducing point mutations in either one or two leucine residues of this NES sequence abolished the nuclear exclusion of the LZTS2 protein. The nuclear export of LZTS2 can be blocked by leptomycin B (LMB), an inhibitor of the CRM1/exportin-alpha pathway. Intriguingly, beta-catenin colocalizes with LZTS2 in the cytoplasm of cells in the absence of LMB but in the nuclei of cells in the presence of LMB. Increasing the LZTS2 protein in cells reduces the level of nuclear beta-catenin in SW480 cells. Taken together, these data demonstrate that LZTS2 is a beta-catenin-interacting protein that can modulate beta-catenin signaling and localization.

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Figures

FIG. 1.

FIG. 1.

Alignment of LZTS proteins. (A) Alignment of the three LZTS protein sequences, including LZTS1, also called FEZ1 (AAD23840), LZTS2, also called LAPSER1/KIAA1813 (AAH58938), and LZTS3, also called ProSapip1 (AAH38860). Identical and similar residues are highlighted in black and gray, respectively. Asterisks denote leucine or similar hydrophobic residues with spacing consistent with leucine zipper domains. The NES sequence characterized in the text is underlined. (B) Alignment of similar NES sites in orthologs of hLZTS2 (AAH58938) from Xenopus tropicalis (AAH75457), Mus musculus (NP_663478), Canis familiaris (XP_543975), and Pan troglodytes (XP_521656).

FIG. 2.

FIG. 2.

LZTS2 interacts with β-catenin in cells. (A) A schematic representation of the yeast two-hybrid assay for mapping the interaction between the β-catenin and LZTS2 proteins. (B) The cDNA fragments containing different portions of human LZTS2 were fused to a GAL4 transactivation domain, and the armadillo repeats of β-catenin were fused to the GAL4 DNA binding domain in pGBT9 vector. Numbers correspond to amino acid residues. Both of the plasmids were cotransformed into PJ69-4A cells as labeled in the figure and plated on SD-Ade-Leu-Trp plates or SD-Leu-Trp plates to monitor transformation efficiency. Three independent colonies were inoculated from each transformation for subsequent liquid β-Gal assays. The data for the liquid β-Gal assays are reported in relative units normalized by cell number (optical density at 600 nm [OD 600]). (C) The indicated fragments of β-catenin were examined by yeast two-hybrid assay for interaction with the C-terminal fragment of hLZTS2 (aa 447 to 669). (D) Equal amounts of GST-β-catenin fusion proteins were immobilized on a glutathione-Sepharose matrix. The binding of [35S]methionine-labeled LZTS2 to GST-β-catenin fusion proteins was analyzed by SDS-PAGE and visualized by autoradiography. (E and F) Whole-cell lysates of CV-1 cells transfected with full-length β-catenin and FLAG-tagged LZTS2 were immunoprecipitated with normal mouse IgG or a β-catenin (E) or FLAG (F) monoclonal antibody. The immunoprecipitates were analyzed by Western blotting with different antibodies as indicated. (G) Whole-cell lysates of SW480 cells were immunoprecipitated with the homemade LZTS2 antibody and normal rabbit IgG and analyzed by Western blotting.

FIG. 3.

FIG. 3.

LZTS2 represses β-catenin-mediated transcription. (A) One hundred nanograms of pGL3-OT (OT) or pGL3-OF (OF), 25 ng of pcDNA3-β-gal, 5 ng of TCF1 expression vector, 20 ng of β-catenin, and various amounts of pcDNA3-FLAG-hLZTS2 as indicated were transfected into LNCaP cells. Cells were cultured for 24 h in the regular media and luciferase, and β-Gal activities were measured as indicated above. Similar experiments were repeated with PC3 cells (B) and DU145 cells (C). (D) Either 100 ng of pGL3-OT (OT) or pGL3-OF (OF), 25 ng of pcDNA3-β-gal, and other shRNA or expression constructs as indicated in the figure were transfected into the human colon cancer cell line SW480. Luciferase and β-Gal activities were measured in panel C.

FIG. 4.

FIG. 4.

LZTS2 localizes in the cytoplasm and contains a leptomycin B-regulated NES motif. (A) The pcDNA3-FLAG-hLZTS2 expression vector was transfected into CV-1 cells. The ectopically expressed hLZTS2 was detected with FLAG monoclonal antibody and revealed by rhodamine-conjugated secondary antibody (red). The nuclei were counterstained with DAPI (blue). (B) Endogenous hLZTS2 was detected with a rabbit antibody in PC3 cells. (C) CV-1 cells were transfected with the pcDNA3-FLAG-hLZTS2 expression vector and then incubated in medium either with or without 60 ng/ml LMB. The ectopically expressed hLZTS2 was detected as in panel A. (D) PC3 cells were cultured in medium either with or without 60 ng/ml LMB for 24 h and then stained with the antibody against endogenous hLZTS2 (red). (E) The GFP fusion proteins containing the putative NES sequences of hLZTS2 and an HIV-Rev classical NES were generated in the pEGFP-C1 vector. Numbers correspond to amino acid residues. (F) The expression vectors containing the above GFP fusion proteins were transfected into CV-1 and PC3 cells. After 24 h of transfection, live cells were observed with an inverted fluorescence microscope. (G) A schematic representation of the NES sequence in hLZTS2. Either single or double mutations within the site were introduced. (H) The expression vectors of pcDNA3-FLAG-hLZTS2 containing either single or double point mutations were transfected into CV-1 cells. The cells were treated with LMB as indicated in panel C. The localization of the LZTS2 proteins was detected as in panel A.

FIG. 5.

FIG. 5.

LZTS2 affects the cellular localization of β-catenin. (A) pcDNA3-HA-β-catenin was transfected into CV-1 cells and incubated with or without 60 ng/ml LMB. The subcellular localization of the β-catenin (β-cat) was monitored by hemagglutinin monoclonal antibody and fluorescein isothiocyanate-conjugated secondary antibody (green). (B) Both pcDNA3-HA-β-catenin and pcDNA3-FLAG-hLZTS2 were transfected into CV-1 cells. Cells were then cultured with or without 60 ng/ml LMB for 24 h. The ectopically expressed proteins were detected with FLAG or hemagglutinin antibody and revealed with rhodamine or fluorescein isothiocyanate-conjugated secondary antibody, respectively. (C) As described above, the mutant of LZTS2 was used in the experiment to examine the colocalization with β-catenin. (D) PC3 cells were cultured in medium either with or without 60 ng/ml LMB. Endogenous β-catenin and LZTS2 proteins were detected by the specific antibodies against each protein and revealed with appropriate secondary antibodies. (E and F) Percentages of cells in which the localization of the β-catenin and LZST2 proteins in the nuclei (N), cytoplasm (C), or both (N and C) were assessed in CV-1 cells as described in the above experiments. (G) SW480 cells were transfected with either the pBS/U6-LZTS shRNA or pBS/U6 vector as a negative control and then fixed and stained for endogenous LZTS2 and β-catenin after 48 h. The rabbit anti-LZTS2 or mouse anti-β-catenin antibody was developed with the Alexa-Fluor goat anti-rabbit 594 (Invitrogen) (red) or Alexa-Fluor donkey anti-mouse 647 (pink) antibody, respectively. DAPI was used for nuclear visualization.

FIG. 6.

FIG. 6.

LZTS2 regulates the cellular level of β-catenin. The FLAG-tagged expression vectors containing wild-type APC (A) or LZTS2 (B) or the LZTS2 NES mutant (C) were transfected into SW480 cells. Cells were stained with FLAG and β-catenin antibodies for the ectopically expressed proteins and endogenous β-catenin, as indicated by arrows. (D) Percentages of positively stained β-catenin cells were measured from the above experiments. The intensity and localization of β-catenin staining in the nuclei (N), cytoplasm (C), or both (N and C) were analyzed in cells transfected with different expression constructs as indicated. (E) SW480 cells were infected with the pLentiviral vectors for either wild-type or mutated LZTS2. The nuclear extracts and whole-cell lysates were prepared from the above cells and used for Western blotting.

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