Dataset Information

Deconstructing the ßcatenin destruction complex: mechanistic roles for the tumor suppressor APC in regulating Wnt signaling.

ABSTRACT: Negatively regulating signaling by targeting key effectors for ubiquitination/destruction is essential for development and oncogenesis. The tumor suppressor adenomatous polyposis coli (APC), an essential negative regulator of Wnt signaling, provides a paradigm. APC mutations occur in most colon cancers. Acting in the "destruction complex" with Axin, glycogen synthase kinase 3, and casein kinase, APC targets ßcatenin (ßcat) for phosphorylation and recognition by an E3 ubiquitin-ligase. Despite 20 years of work, the internal workings of the destruction complex and APC's role remain largely mysterious. We use both Drosophila and colon cancer cells to test hypotheses for APC's mechanism of action. Our data are inconsistent with current models suggesting that high-affinity ßcat-binding sites on APC play key roles. Instead, they suggest that multiple ßcat-binding sites act additively to fine-tune signaling via cytoplasmic retention. We identify essential roles for two putative binding sites for new partners--20-amino-acid repeat 2 and conserved sequence B--in destruction complex action. Finally, we demonstrate that APC interacts with Axin by two different modes and provide evidence that conserved sequence B helps ensure release of APC from Axin, with disassembly critical in regulating ßcat levels. Using these data, we suggest a new model for destruction complex action in development, which also provides new insights into functions of truncated APC proteins in cancer.

SUBMITTER: Roberts DM

PROVIDER: S-EPMC3103401 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Deconstructing the ßcatenin destruction complex: mechanistic roles for the tumor suppressor APC in regulating Wnt signaling.

Roberts David M DM Pronobis Mira I MI Poulton John S JS Waldmann Jon D JD Stephenson Elise M EM Hanna Shahnaz S Peifer Mark M

Molecular biology of the cell 20110406 11

Negatively regulating signaling by targeting key effectors for ubiquitination/destruction is essential for development and oncogenesis. The tumor suppressor adenomatous polyposis coli (APC), an essential negative regulator of Wnt signaling, provides a paradigm. APC mutations occur in most colon cancers. Acting in the "destruction complex" with Axin, glycogen synthase kinase 3, and casein kinase, APC targets ßcatenin (ßcat) for phosphorylation and recognition by an E3 ubiquitin-ligase. Despite 20 ...[more]

PMID: 21471006

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Project description:Mutation of the tumor suppressor adenomatous polyposis coli (APC) is considered an initiating step in the genesis of the vast majority of colorectal cancers. APC inhibits the Wnt-signaling pathway by targeting the proto-oncogene ?-catenin for destruction by cytoplasmic proteasomes. In the presence of a Wnt signal, or in the absence of functional APC, ?-catenin can serve as a transcription cofactor for genes required for cell proliferation such as cyclin-D1 and c-Myc. In cultured cells, APC shuttles between the nucleus and the cytoplasm, with nuclear APC implicated in the inhibition of Wnt target gene expression. Adopting a genetic approach to evaluate the functions of nuclear APC in the context of a whole organism, we generated a mouse model with mutations that inactivate the nuclear localization signals (NLSs) of Apc (Apc(mNLS)). Apc(mNLS/mNLS) mice are viable and fractionation of mouse embryonic fibroblasts (MEFs) isolated from these mice revealed a significant reduction in nuclear Apc as compared with Apc(+/+) MEFs. The levels of Apc and ?-catenin protein were not significantly altered in small intestinal epithelia from Apc(mNLS/mNLS) mice. Compared with Apc(+/+) mice, Apc(mNLS/mNLS) mice showed increased proliferation in epithelial cells from the jejunum, ileum and colon. These same tissues from Apc(mNLS/mNLS) mice showed more mRNA from three genes upregulated in response to canonical Wnt signal, c-Myc, axin-2 and cyclin-D1, and less mRNA from Hath-1, which is downregulated in response to Wnt. These observations suggest a role for nuclear Apc in the inhibition of canonical Wnt signaling and the control of epithelial proliferation in intestinal tissue. Furthermore, we found Apc(Min/+) mice, which harbor a mutation that truncates Apc, to have an increased polyp size and multiplicity if they also carry the Apc(mNLS) allele. Taken together, this analysis of the novel Apc(mNLS) mouse model supports a role for nuclear Apc in the control of Wnt target genes, intestinal epithelial cell proliferation and polyp formation.

Project description:In colorectal cancer cells, APC, a tumor suppressor protein, is commonly expressed in truncated form. Truncation of APC is believed to disrupt degradation of ?-catenin, which is regulated by a multiprotein complex called the destruction complex. The destruction complex comprises APC, Axin, ?-catenin, serine/threonine kinases, and other proteins. The kinases CK1? and GSK -3?, which are recruited by Axin, mediate phosphorylation of ?-catenin, which initiates its ubiquitination and proteosomal degradation. The mechanism of regulation of ?-catenin degradation by the destruction complex and the role of truncation of APC in colorectal cancer are not entirely understood. Through formulation and analysis of a rule-based computational model, we investigated the regulation of ?-catenin phosphorylation and degradation by APC and the effect of APC truncation on function of the destruction complex. The model integrates available mechanistic knowledge about site-specific interactions and phosphorylation of destruction complex components and is consistent with an array of published data. We find that the phosphorylated truncated form of APC can outcompete Axin for binding to ?-catenin, provided that Axin is limiting, and thereby sequester ?-catenin away from Axin and the Axin-recruited kinases CK1? and GSK -3?. Full-length APC also competes with Axin for binding to ?-catenin; however, full-length APC is able, through its SAMP repeats, which bind Axin and which are missing in truncated oncogenic forms of APC, to bring ?-catenin into indirect association with Axin and Axin-recruited kinases. Because our model indicates that the positive effects of truncated APC on ?-catenin levels depend on phosphorylation of APC, at the first 20-amino acid repeat, and because phosphorylation of this site is mediated by CK1?, we suggest that CK1? is a potential target for therapeutic intervention in colorectal cancer. Specific inhibition of CK1? is predicted to limit binding of ?-catenin to truncated APC and thereby to reverse the effect of APC truncation.

Dataset Information

Deconstructing the ßcatenin destruction complex: mechanistic roles for the tumor suppressor APC in regulating Wnt signaling.

Publications

Deconstructing the ßcatenin destruction complex: mechanistic roles for the tumor suppressor APC in regulating Wnt signaling.

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