RESUMO
ß-Arrestin is a scaffold protein that regulates signal transduction by seven transmembrane-spanning receptors. Among other functions it is also critically required for Wnt/ß-catenin signal transduction. In the present study we provide for the first time a mechanistic basis for the ß-arrestin function in Wnt/ß-catenin signaling. We demonstrate that ß-arrestin is required for efficient Wnt3a-induced Lrp6 phosphorylation, a key event in downstream signaling. ß-Arrestin regulates Lrp6 phosphorylation via a novel interaction with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-binding protein Amer1/WTX/Fam123b. Amer1 has been shown very recently to bridge Wnt-induced and Dishevelled-associated PtdIns(4,5)P2 production to the phosphorylation of Lrp6. Using fluorescence recovery after photobleaching we show here that ß-arrestin is required for the Wnt3a-induced Amer1 membrane dynamics and downstream signaling. Finally, we show that ß-arrestin interacts with PtdIns kinases PI4KIIα and PIP5KIß. Importantly, cells lacking ß-arrestin showed higher steady-state levels of the relevant PtdInsP and were unable to increase levels of these PtdInsP in response to Wnt3a. In summary, our data show that ß-arrestins regulate Wnt3a-induced Lrp6 phosphorylation by the regulation of the membrane dynamics of Amer1. We propose that ß-arrestins via their scaffolding function facilitate Amer1 interaction with PtdIns(4,5)P2, which is produced locally upon Wnt3a stimulation by ß-arrestin- and Dishevelled-associated kinases.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Arrestinas/metabolismo , Membrana Celular/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteína Wnt3A/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Arrestinas/genética , Western Blotting , Células Cultivadas , Proteínas Desgrenhadas , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Células HEK293 , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Camundongos , Camundongos Knockout , Microscopia Confocal , Antígenos de Histocompatibilidade Menor , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ligação Proteica , Interferência de RNA , Proteínas Supressoras de Tumor/genética , Proteína Wnt3A/genética , beta-ArrestinasRESUMO
Phosphorylation of the Wnt receptor low-density lipoprotein receptor-related protein 6 (LRP6) by glycogen synthase kinase 3ß (GSK3ß) and casein kinase 1γ (CK1γ) is a key step in Wnt/ß-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 CK1γ, recruits Axin and GSK3ß 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/ß-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.
Assuntos
Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteínas de Membrana/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas Wnt/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Western Blotting , Membrana Celular/metabolismo , Células Cultivadas , Imunofluorescência , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Rim/citologia , Rim/metabolismo , Proteínas Relacionadas a Receptor de LDL/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Proteínas de Membrana/genética , Fosforilação , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Proteínas Supressoras de Tumor , Proteínas Wnt/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Wnt/ß-catenin signaling is negatively controlled by the adenomatous polyposis coli (APC) tumor suppressor, which induces proteasomal degradation of ß-catenin as part of the ß-catenin destruction complex. Amer2 (APC membrane recruitment 2; FAM123A) is a direct interaction partner of APC, related to the tumor suppressor Amer1/WTX, but its function in Wnt signaling is not known. Here, we show that Amer2 recruits APC to the plasma membrane by binding to phosphatidylinositol 4,5-bisphosphate lipids via lysine-rich motifs and that APC links ß-catenin and the destruction complex components axin and conductin to Amer2. Knockdown of Amer2 increased Wnt target gene expression and reporter activity in cell lines, and overexpression reduced reporter activity, which required membrane association of Amer2. In Xenopus embryos, Amer2 is expressed mainly in the dorsal neuroectoderm and neural tissues. Down-regulation of Amer2 by specific morpholino oligonucleotides altered neuroectodermal patterning, which could be rescued by expression of a dominant-negative mutant of Lef1 that interferes with ß-catenin-dependent transcription. Our data characterize Amer2 for the first time as a negative regulator of Wnt signaling both in cell lines and in vivo and define Amer proteins as a novel family of Wnt pathway regulators.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células HEK293 , Humanos , Placa Neural/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Wnt/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis , beta Catenina/genéticaRESUMO
Amer1/WTX binds to the tumor suppressor adenomatous polyposis coli and acts as an inhibitor of Wnt signaling by inducing ß-catenin degradation. We show here that Amer1 directly interacts with the armadillo repeats of ß-catenin via a domain consisting of repeated arginine-glutamic acid-alanine (REA) motifs, and that Amer1 assembles the ß-catenin destruction complex at the plasma membrane by recruiting ß-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 ß-catenin destruction complex and promoting stabilization of Axin at the plasma membrane.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Membrana Celular/metabolismo , Transdução de Sinais/fisiologia , Proteínas Supressoras de Tumor/metabolismo , Proteínas Wnt/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Proteína Axina , Membrana Celular/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Camundongos , Proibitinas , Estrutura Terciária de Proteína , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Relação Estrutura-Atividade , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética , Proteínas Wnt/genética , Proteínas de Xenopus , Xenopus laevis , beta Catenina/genética , beta Catenina/metabolismoRESUMO
The X-linked WTX/AMER1 protein constitutes an important component of the ß-catenin destruction complex that can both enhance and suppress canonical ß-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 ß-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.
Assuntos
Alelos , Densidade Óssea/genética , Mutação , Osteosclerose , Crânio , Proteínas Supressoras de Tumor , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Mutantes , Osteosclerose/genética , Osteosclerose/metabolismo , Osteosclerose/patologia , Crânio/metabolismo , Crânio/patologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
APC is a multifunctional tumor suppressor protein that negatively controls Wnt signaling, but also regulates cell adhesion and migration by interacting with the plasma membrane and the microtubule cytoskeleton. Although the molecular basis for the microtubule association of APC is well understood, molecular mechanisms that underlie its plasma membrane localization have remained elusive. We show here that APC is recruited to the plasma membrane by binding to APC membrane recruitment 1 (AMER1), a novel membrane-associated protein that interacts with the ARM repeat domain of APC. The N-terminus of AMER1 contains two distinct phosphatidylinositol(4,5)-bisphosphate [PtdIns(4,5)P(2)]-binding domains, which mediate its localization to the plasma membrane. Overexpression of AMER1 increases APC levels and redirects APC from microtubule ends to the plasma membrane of epithelial cells. Conversely, siRNA-mediated knockdown of AMER1 reduces the overall levels of APC, promotes its association with microtubule ends in cellular protrusions and disturbs intercellular junctions. These data indicate that AMER1 controls the subcellular distribution of APC between membrane- and microtubule-associated pools, and might thereby regulate APC-dependent cellular morphogenesis, cell migration and cell-cell adhesion.