Abstract
Beta-catenin is a key component of the Wnt signaling pathway that functions as a transcriptional co-activator of Wnt target genes. Upon UV-induced DNA damage, beta-catenin is recruited for polyubiquitination and subsequent proteasomal degradation by a unique, p53-induced SCF-like complex (SCF(TBL1)), comprised of Siah-1, Siah-1-interacting protein (SIP), Skp1, transducin beta-like 1 (TBL1), and adenomatous polyposis coli (APC). Given the complexity of the various factors involved and the novelty of ubiquitination of the non-phosphorylated beta-catenin substrate, we have investigated Siah-1-mediated ubiquitination of beta-catenin in vitro and in cells. Overexpression and purification protocols were developed for each of the SCF(TBL1) proteins, enabling a systematic analysis of beta-catenin ubiquitination using an in vitro ubiquitination assay. This study revealed that Siah-1 alone was able to polyubiquitinate beta-catenin. In addition, TBL1 was shown to play a role in protecting beta-catenin from Siah-1 ubiquitination in vitro and from Siah-1-targeted proteasomal degradation in cells. Siah-1 and TBL1 were found to bind to the same armadillo repeat domain of beta-catenin, suggesting that polyubiquitination of beta-catenin is regulated by competition between Siah-1 and TBL1 during Wnt signaling.
Highlights
-Catenin is a ubiquitous transcriptional activator in the canonical Wnt signaling pathway involved in cellular processes ranging from embryogenesis, cell proliferation, cell fate, and survival to adult stem cell differentiation and oncogenesis [1, 2]
The Siah-1 RING E2-binding domain is linked to a substrate binding domain that directly recruits, and mediates polyubiquitination of many substrates including Deleted in Colorectal Cancer (DCC), nuclear co-repressor (NCoR), c-Myb, and synphilin-1 [12,13,14,15]
-catenin polyubiquitination is observed even in the absence of TBL1, Skp1 or Siah-1-interacting protein (SIP) (Fig. 1B, lanes 6 – 8). These results show that the polyubiquitination of -catenin by SCF(TBL1) can be reconstituted in vitro, and only Siah-1 is required for -catenin polyubiquitination
Summary
Bacterial Protein Expression and Purification—Full-length Siah-1 (residues 1–282) was expressed as a His6-maltose-binding protein (MBP) fusion protein. The SBD of Siah-1 (residues 90 –282) was subcloned in a pET28a vector (Novagen) with an N-terminal His tag containing a thrombin cleavage site. SIP and Skp were expressed as His fusion constructs from pET28a vectors as previously described [18]. Fulllength murine -catenin (residues 1–781) and four truncation mutant constructs (Nt: residues 1–133), (Ntϩarm: residues 1– 671), (arm: residues 134 – 671) and (armϩCt: residues 1–781) were expressed as glutathione S-transferase (GST) fusion proteins from pGEX vectors as previously described [19]. -catenin constructs were affinity purified on glutathione-Sepharose 4B (Amersham Biosciences) followed by cleavage of the GST fusion tag, Source Q, and size exclusion chromatography, as previously described [19]. Ubiquitination reactions analyzed by Western blotting using C-terminal -catenin primary antibody (Cell Signaling) and visualized with goat anti-rabbit-horseradish peroxidase by SuperSignal West Pico Chemiluminescent. All antibodies used in the Western blot are commercially available: -catenin (BD), TBL1 (Abcam), TBLR1 (Bethyl Laboratories), and ␣-tubulin (Santa Cruz Biotechnology)
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