Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Jan 12;8(1):671.
doi: 10.1038/s41598-017-18421-8.

Beta-catenin Cleavage Enhances Transcriptional Activation

Affiliations
Free PMC article

Beta-catenin Cleavage Enhances Transcriptional Activation

Tatiana Goretsky et al. Sci Rep. .
Free PMC article

Abstract

Nuclear activation of Wnt/β-catenin signaling is required for cell proliferation in inflammation and cancer. Studies from our group indicate that β-catenin activation in colitis and colorectal cancer (CRC) correlates with increased nuclear levels of β-catenin phosphorylated at serine 552 (pβ-Cat552). Biochemical analysis of nuclear extracts from cancer biopsies revealed the existence of low molecular weight (LMW) pβ-Cat552, increased to the exclusion of full size (FS) forms of β-catenin. LMW β-catenin lacks both termini, leaving residues in the armadillo repeat intact. Further experiments showed that TCF4 predominantly binds LMW pβ-Cat552 in the nucleus of inflamed and cancerous cells. Nuclear chromatin bound localization of LMW pβ-Cat552 was blocked in cells by inhibition of proteasomal chymotrypsin-like activity but not by other protease inhibitors. K48 polyubiquitinated FS and LMW β-catenin were increased by treatment with bortezomib. Overexpressed in vitro double truncated β-catenin increased transcriptional activity, cell proliferation and growth of tumor xenografts compared to FS β-catenin. Serine 552-> alanin substitution abrogated K48 polyubiquitination, β-catenin nuclear translocation and tumor xenograft growth. These data suggest that a novel proteasome-dependent posttranslational modification of β-catenin enhances transcriptional activation. Discovery of this pathway may be helpful in the development of diagnostic and therapeutic tools in colitis and cancer.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Nuclear low-molecular weight (LMW) β-catenin predominates in colon cancer IEC. Protein isolates from biopsy-derived normal and colorectal cancer (CRC) IEC were probed with antibodies specific for distinct β-catenin epitopes. (A) Normal and CRC IEC fraction lysates (cytosolic (Cyto), membrane (Memb) and nuclear (Nucl) were probed sequentially for β-catenin antibodies directed to different epitopes as shown. Purity controls were achieved by probing for α-tubulin (cytosol), E-cadherin (membrane) and lamin B1 (nucleus). (B) Nuclear lysates from normal and CRC biopsies were probed for phospho-β-cateninSer552 (pβ-Cat552). WB for lamin B1 serves as loading control. Arrows indicate positions of LMW β-catenin. Full size membrane scans for WBs can be seen in Suppl. Fig. SS1.
Figure 2
Figure 2
β-catenin truncation is proteasome dependent. (A) Cytosolic and nuclear lysates from HT29 cells, treated with bortezomib (Borte) were immunoprecipitated for lysine 48 polyubiquitin chain (K48) and probed for N, C and pβ-Cat552 epitopes of β-catenin. (B) HT29 cells treated with bortezomib were fractionated to cytosolic (Cyto), membranous (Memb), soluble (Nucl) and chromatin-bound (Chrom) nuclear fractions. WBs were run sequentially for β-catenin antibodies specific for epitopes as indicated. Solid arrows depict decreases in LMW β-catenin with bortezomib. Open arrows depict increases in full length β-catenin after bortezomib treatment. The braces indicate pβ-Cat552 likely processed in the nuclear proteasome. α-tubulin is a loading and purity control for cytosolic fraction, histone H3 - for chromatin bound nuclear fraction. Full size membrane scans for WBs can be seen in Suppl. Fig. SS2.
Figure 3
Figure 3
Inhibitors of chymotrypsin-like activity of the proteasome diminish β-catenin transcriptional activity. (A) HT29 cells transfected with TCF/LEF reporter were treated with inhibitors as indicated. Luciferase activity is shown in cells incubated with protease inhibitors. Asterisks indicate statistically significant p values compared to control. p < 0.001. (B) HT29 cells were treated as in A and nuclear chromatin-bound fractions probed for pβ-Cat552. Fibrillarin served as a loading control. Full size membrane scans for WBs can be seen  in Suppl. Fig. SS3.
Figure 4
Figure 4
TCF4 binds proteasome-sensitive LMW-β-catenin. (A) IEC nuclear fractions from normal (N) and CRC biopsies were immunoprecipitated by anti-TCF4 and probed for pβ-Cat552, core region β-catenin and TCF4. (B) Nuclear fractions from CRC biopsies were used for IP with different β-catenin antibodies. Precipitated proteins were probed with anti-TCF4 antibody. Control WBs with N and C terminal, core region specific and pβ-Cat552 antibodies can be seen in Suppl. Fig. S6D. (C) Normal, CRC and CRC treated with MG132 IEC nuclear fraction were immunoprecipitated by anti-TCF4 and probed for pβ-Cat552. Input and loading control WBs for these samples can be seen on Suppl. Fig. S6E. (D) Chromatin-bound nuclear fractions of HT29 cell, untreated and treated with bortezomib (input and loading controls in Fig. 2B), were immunoprecipitated by anti-TCF4 and probed for anti-pβ-Cat552 and core region β-catenin antibodies. Full size membrane scans for WBs can be seen at Suppl. Fig. SS4.
Figure 5
Figure 5
TNF, LiCl and carcinogenic transformation increase abundance of chromatin-bound LMW-β-catenin and TCF4 binding. (A) NCM460 cells were treated with LiCl and TNF and fractionated as in Fig. 2B. WBs were probed sequentially with anti-pβ-Cat552 and anti-core region β-catenin antibodies. WBs for α-tubulin, e-cadherin, laminB1 and histone H3 represent loading controls in sub-cellular fractions. (B) Chromatin-bound fractions from (A) were immunoprecipitated by anti-TCF4 and probed for pβ-Cat552 and core region specific antibodies. (C) Chromatin-bound fractions from NCM460 and indicated colon cancer cell lines were immunoprecipitated by anti-TCF4 and probed for pβ-Cat552. Input control WB was also probed for core region β-catenin and fibrillarin. Full size membrane scans for WBs can be seen in Suppl. Fig. SS5.
Figure 6
Figure 6
Overexpressed double truncated β-catenin increases transcriptional activity. (A) NCM460 cells were infected with FS, ∆∆ and ∆N142 β-catenin constructs tagged with His (N terminus) and Flag (C terminus). WBs were probed sequentially with anti-Flag and anti-His antibodies. Fibrillarin served as loading and purity controls for chromatin bound fractions. (B) Total cell lysates of NCM460 cells overexpressing FS β-catenin were precipitated with anti-Flag antibody or (Co2+) sepharose (His tag specific). Proteins were resolved on SDS PAGE and WBs probed sequentially with N and C termini specific anti-β-catenin antibodies. (C) Schematic representation of β-catenin constructs used in (A and B). (D) Chromatin bound fractions from NCM460 cells are shown: vector control, FS, ∆∆ and ∆N89 β-catenin overexpressing cell lines immunoprecipitated with anti-TCF4 and probed with pβ-Cat552 and core region antibodies. Fibrillarin serves as a loading control for input. (E) FS, ∆∆ and ∆N89 β-catenin overexpressing NCM460 cell lines were co-transfected with TCF/LEF reporter plasmid and β-catenin-induced luciferase activities measured. *p = 0.002, **p = 0.001, ***p = 0.003. Flag WB of total cell lysates used in (D and E) (to evaluate levels of overexpressed β-catenin) can be seen on Suppl. Fig. S8C. Full size membrane scans for WBs can be seen  in Suppl. Fig. SS6.
Figure 7
Figure 7
β-catenin serine 552 phosphorylation enhances translocation to chromatin-bound fraction and increases xenograft tumor growth. (A) RKO cells were transfected with FS, ∆∆, FS 552A and ∆∆ 552A β-catenin and fractionated. WBs were probed with anti-Flag antibody. Fibrillarin served as loading and purity controls for chromatin-bound fractions. (B) RKO cell lines overexpressing wild type  and mutated β-catenin were co-transfected with TCF/LEF reporter plasmid and luciferase assay performed. *p = 0.002, **p = 0.001, ***p = 0.005. (C) RKO cells were treated with 20nM epoxomicin and cytosolic lysates precipitated with K48 specific antibody. WBs were developed with Flag tag antibody. Actin served as a loading control. (D) Xenograft mice were injected with RKO cells transfected with control and overexpressing β-catenin constructs. The graph represents volumes of developed tumors. *p = 0.016; **p = 0.04; ***p = 0.003. n = 7. Full size membrane scans for WBs can be seen in Suppl. Fig. SS7. (E) Proposed mechanism of β-catenin transcriptional activation.

Similar articles

See all similar articles

Cited by 3 articles

References

    1. Gottardi CJ, Gumbiner BM. Distinct molecular forms of beta-catenin are targeted to adhesive or transcriptional complexes. J Cell Biol. 2004;167:339–349. doi: 10.1083/jcb.200402153. - DOI - PMC - PubMed
    1. Chien AJ, Conrad WH, Moon RT. A Wnt survival guide: from flies to human disease. J Invest Dermatol. 2009;129:1614–1627. doi: 10.1038/jid.2008.445. - DOI - PMC - PubMed
    1. Logan CY, Nusse R. The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol. 2004;20:781–810. doi: 10.1146/annurev.cellbio.20.010403.113126. - DOI - PubMed
    1. Clevers H, Nusse R. Wnt/beta-catenin signaling and disease. Cell. 2012;149:1192–1205. doi: 10.1016/j.cell.2012.05.012. - DOI - PubMed
    1. Bienz M, Clevers H. Linking colorectal cancer to Wnt signaling. Cell. 2000;103:311–320. doi: 10.1016/S0092-8674(00)00122-7. - DOI - PubMed

Publication types

MeSH terms

Feedback