RESUMO
The adenomatous polyposis coli (APC) membrane recruitment (Amer) family proteins Amer1/Wilms tumour gene on the X chromosome and Amer2 are binding partners of the APC tumour suppressor protein, and act as negative regulators in the Wnt signalling cascade. So far, nothing has been known about the third member of the family, Amer3. Here we show that Amer3 binds to the armadillo repeat domain of APC, similarly to Amer1 and Amer2. Amer3 also binds to the Wnt pathway regulator conductin/axin2. Furthermore, we identified Amer1 as binding partner of Amer3. Whereas Amer1 and Amer2 are linked to the plasma membrane by an N-terminal membrane localization domain, Amer3 lacks this domain. Amer3 localizes to the cytoplasm and nucleus of epithelial cells, and this is dependent on specific nuclear import and export sequences. Functionally, exogenous Amer3 enhances the expression of a ß-catenin/T-cell factor-dependent reporter gene, and knockdown of endogenous Amer3 reduces Wnt target gene expression in colorectal cancer cells. Thus, Amer3 acts as an activator of Wnt signalling, in contrast to Amer1 and Amer2, which are inhibitors, suggesting a nonredundant role of Amer proteins in the regulation of this pathway. Our data, together with those of previous studies, provide a comprehensive picture of similarities and differences within the Amer protein family.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína da Polipose Adenomatosa do Colo/metabolismo , Polipose Adenomatosa do Colo/metabolismo , Núcleo Celular/metabolismo , Neoplasias Colorretais/metabolismo , Citoplasma/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Regulação para Cima , Via de Sinalização Wnt , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteína da Polipose Adenomatosa do Colo/antagonistas & inibidores , Proteína da Polipose Adenomatosa do Colo/química , Proteína da Polipose Adenomatosa do Colo/genética , Proteínas do Domínio Armadillo/química , Proteínas do Domínio Armadillo/metabolismo , Proteína Axina/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Células HEK293 , Humanos , Proteínas Mutantes , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Especificidade de Órgãos , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Sinais Direcionadores de Proteínas , Transporte Proteico , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genéticaRESUMO
The germline transmission of a mutation in the adenomatous polyposis coli (APC) gene leads to cancer of the gastro-intestinal tract upon somatic inactivation of the remaining allele in familial adenomatous polyposis (FAP) patients. APC mutations result in truncated products that have primarily lost the ability to properly regulate the level of the transcription factor ß-catenin. However, colorectal cancer cells from FAP patients always retain a truncated APC product and the reasons for this strong selective pressure are not understood. We describe here the surprising property for the transcriptional repressor C-terminal binding protein (CtBP) to promote the oligomerization of truncated APC through binding to the 15 amino acid repeats of truncated APC. CtBP can bind to either first, third or fourth 15 amino acid repeats, but not to the second. CtBP-mediated oligomerization requires both dimerization domains of truncated APC as well as CtBP dimerization. The analysis of the position of the mutations along the APC sequence in adenomas from FAP patients reveals that the presence of the first 15 amino acid repeat is almost always selected in the resulting truncated APC product. This suggests that the sensitivity of truncated APC to oligomerization by CtBP constitutes an essential facet of tumour development.
Assuntos
Proteína da Polipose Adenomatosa do Colo/química , Proteína da Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/metabolismo , Oxirredutases do Álcool/metabolismo , Neoplasias Colorretais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mutação , Proteínas do Tecido Nervoso/metabolismo , Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , Linhagem Celular , Linhagem Celular Tumoral , Proteínas Correpressoras , Neoplasias Colorretais/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Dimerização , Humanos , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de ProteínaRESUMO
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.
