Structural and Functional Characterization of the Wnt Inhibitor APC Membrane Recruitment 1 (Amer1)

Abstract

Amer1/WTX binds to the tumor suppressor adenomatous polyposis coli and acts as an inhibitor of Wnt signaling by inducing ␤-catenin degradation. We show here that Amer1 directly interacts with the armadillo repeats of ␤-catenin via a domain consisting of repeated arginine-glutamic acid-alanine (REA) motifs, and that Amer1 assembles the ␤-catenin destruction complex at the plasma membrane by recruiting ␤-catenin, adenomatous polyposis coli, and Axin/Conductin. Deletion or specific mutations of the membrane binding domain of Amer1 abolish its membrane localization and abrogate negative control of Wnt signaling, which can be restored by artificial targeting of Amer1 to the plasma membrane. In line, a natural splice variant of Amer1 lacking the plasma membrane localization domain is deficient for Wnt inhibition. Knockdown of Amer1 leads to the activation of Wnt target genes, preferentially in dense compared with sparse cell cultures, suggesting that Amer1 function is regulated by cell contacts. Amer1 stabilizes Axin and counteracts Wnt-induced degradation of Axin, which requires membrane localization of Amer1. The data suggest that Amer1 exerts its negative regulatory role in Wnt signaling by acting as a scaffold protein for the ␤-catenin destruction complex and promoting stabilization of Axin at the plasma membrane.The canonical Wnt/␤-catenin signaling pathway is a key pathway in embryonic development and disease. Aberrant activation of Wnt signaling leads to the development of tumors (1, 2). The binding of Wnt ligands to the transmembrane receptors Frizzled (Fz) 2 and low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6) leads to the stabilization of cytoplasmic ␤-catenin, which enters the nucleus and activates target gene expression by interacting with DNA-binding proteins of the T-cell factor/lymphoid enhancer factor (TCF/lymphoid enhancer factor) family (3, 4). In the absence of Wnts, ␤-catenin is degraded in the proteasome after its ubiquitination by the E3 ligase ␤-transducin repeat-containing protein (␤-TrCP), which recognizes ␤-catenin phosphorylated at specific N-terminal residues. ␤-catenin phosphorylation is accomplished by the coordinated action of CK1 and glycogen synthase kinase 3 (GSK3) and takes place in a multiprotein complex assembled by the scaffold proteins adenomatous polyposis coli (APC) and Axin or its homologue Axin2/Conductin (4, 5). Because Axin binds to ␤-catenin, GSK, CK1, and APC, and APC has multiple binding sites for ␤-catenin, it is generally believed that one function of this ␤-catenin destruction complex is to bring ␤-catenin into close vicinity to the kinases, thereby allowing efficient phosphorylation (6).It is generally thought that ␤-catenin phosphorylation and degradation is a constitutive process, keeping the amount of ␤-catenin in the cytoplasm and nucleus low in the absence of Wnts. Wnt binding to the receptor complex leads to the recruitment of Axin to the plasma membrane and phosphorylation of LRP5/6 at cytoplasmic PPPSPXS motifs (7-10). Phosphorylated PPPSPXS m...

Amer2 Protein Is a Novel Negative Regulator of Wnt/β-Catenin Signaling Involved in Neuroectodermal Patterning

“…Cell Culture and Transfections-Cells were cultured and transfected as reported (5). HEK293T-pBAR/Renilla and RKOpBAR/Renilla cells (4) were cultured in medium supplemented with 2 g/ml puromycin (Sigma).…”

“…The Amer1, conductin, LGR5 (leucine-rich repeat-containing G-protein-coupled receptor 5), and GAPDH primers were described previously (5). The xAmer2 probe was generated from the cDNA clone IRAKp961P02150Q in pCMV-Sport6.…”

“…RT-PCR-RNA isolation, cDNA synthesis, and PCR was performed as reported (5). For Xenopus RT-PCR, RNA was extracted from five embryos using the High Pure RNA isolation kit from Roche Applied Science.…”

“…Reporter Assays-T-cell factor-␤-catenin-dependent reporter assays were performed in HEK293T or RKO cells stably expressing a ␤-catenin-responsive firefly luciferase reporter (pBAR) along with Renilla luciferase as an internal control (4) using 300 ng of Amer2 plasmids and 200 ng of monomeric red fluorescent protein-dominant-active LRP6 or 150 ng of mYFP-␤-catenin S33Y as reported (5). Where indicated, Wnt3A-conditioned medium was added for 4 -6 h. For knockdown of Amer2, cells were transfected with 40 pmol of siRNA.…”

“…Amer2 (APC membrane recruitment 2; FAM123A) was previously shown by our group to interact with APC via two domains that bind the Armadillo repeats of APC (3). These domains are conserved in the related proteins Amer1/WTX (Wilms tumor gene on the X-chromosome; FAM123B), which acts as a negative regulator of Wnt signaling (4,5), and Amer3 (FAM123C), which has not been characterized so far (6). Amer1 is mutated in Wilms tumors and in the inherited disease osteopathia striata congenita with cranial sclerosis (7,8).…”