SummaryAMER1 regulates the distribution of the tumor suppressor APC between microtubules and the plasma membrane

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...

Phosphorylation of the Wnt receptor low-density lipoprotein receptor-related protein 6 (LRP6) by glycogen synthase kinase 3b (GSK3b) and casein kinase 1c (CK1c) is a key step in Wnt/b-catenin signalling, which requires Wnt-induced formation of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ). Here, we show that adenomatous polyposis coli membrane recruitment 1 (Amer1) (also called WTX), a membrane associated PtdIns(4,5)P 2 -binding protein, is essential for the activation of Wnt signalling at the LRP6 receptor level. Knockdown of Amer1 reduces Wnt-induced LRP6 phosphorylation, Axin translocation to the plasma membrane and formation of LRP6 signalosomes. Overexpression of Amer1 promotes LRP6 phosphorylation, which requires interaction of Amer1 with PtdIns(4,5)P 2 . Amer1 translocates to the plasma membrane in a PtdIns(4,5)P 2 -dependent manner after Wnt treatment and is required for LRP6 phosphorylation stimulated by application of PtdIns(4,5)P 2 . Amer1 binds CK1c, recruits Axin and GSK3b to the plasma membrane and promotes complex formation between Axin and LRP6. Fusion of Amer1 to the cytoplasmic domain of LRP6 induces LRP6 phosphorylation and stimulates robust Wnt/b-catenin signalling. We propose a mechanism for Wnt receptor activation by which generation of PtdIns (4,5)P 2 leads to recruitment of Amer1 to the plasma membrane, which acts as a scaffold protein to stimulate phosphorylation of LRP6.

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The X-linked WTX/AMER1 protein constitutes an important component of the β-catenin destruction complex that can both enhance and suppress canonical β-catenin signaling. Somatic mutations in WTX/AMER1 have been found in a proportion of the pediatric kidney cancer Wilms' tumor. By contrast, germline mutations cause the severe sclerosing bone dysplasia osteopathia striata congenita with cranial sclerosis (OSCS), a condition usually associated with fetal or perinatal lethality in male patients. Here we address the developmental and molecular function of WTX by generating two novel mouse alleles. We show that in addition to the previously reported skeletal abnormalities, loss of Wtx causes severe midline fusion defects including cleft palate and ectopic synostosis at the base of the skull. By contrast, deletion of the C-terminal part of the protein results in only mild developmental abnormalities permitting survival beyond birth. Adult analysis, however, revealed skeletal defects including changed skull morphology and an increased whole-body bone density, resembling a subgroup of male patients carrying a milder, survivable phenotype. Molecular analysis in vitro showed that while β-catenin fails to co-immunoprecipitate with the truncated protein, partial recruitment appears to be achieved in an indirect manner using AXIN/AXIN2 as a molecular bridge. Taken together our analysis provides a novel model for WTX-caused bone diseases and explains on the molecular level how truncation mutations in this gene may retain some of WTX-protein functions. © 2018 American Society for Bone and Mineral Research.