Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 13 de 13
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Stem Cell Res ; 40: 101563, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31494448

RESUMO

Development of neural tube has been extensively modeled in vitro using human pluripotent stem cells (hPSCs) that are able to form radially organized cellular structures called neural rosettes. While a great amount of research has been done using neural rosettes, studies have only inadequately addressed how rosettes are formed and what the molecular mechanisms and pathways involved in their formation are. Here we address this question by detailed analysis of the expression of pluripotency and differentiation-associated proteins during the early onset of differentiation of hPSCs towards neural rosettes. Additionally, we show that the BMP signaling is likely contributing to the formation of the complex cluster of neural rosettes and its inhibition leads to the altered expression of PAX6, SOX2 and SOX1 proteins and the rosette morphology. Finally, we provide evidence that the mechanism of neural rosettes formation in vitro is reminiscent of the process of secondary neurulation rather than that of primary neurulation in vivo. Since secondary neurulation is a largely unexplored process, its understanding will ultimately assist the development of methods to prevent caudal neural tube defects in humans.

2.
Nat Commun ; 10(1): 1804, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-31000703

RESUMO

Dishevelled (DVL) is the key component of the Wnt signaling pathway. Currently, DVL conformational dynamics under native conditions is unknown. To overcome this limitation, we develop the Fluorescein Arsenical Hairpin Binder- (FlAsH-) based FRET in vivo approach to study DVL conformation in living cells. Using this single-cell FRET approach, we demonstrate that (i) Wnt ligands induce open DVL conformation, (ii) DVL variants that are predominantly open, show more even subcellular localization and more efficient membrane recruitment by Frizzled (FZD) and (iii) Casein kinase 1 ɛ (CK1ɛ) has a key regulatory function in DVL conformational dynamics. In silico modeling and in vitro biophysical methods explain how CK1ɛ-specific phosphorylation events control DVL conformations via modulation of the PDZ domain and its interaction with DVL C-terminus. In summary, our study describes an experimental tool for DVL conformational sampling in living cells and elucidates the essential regulatory role of CK1ɛ in DVL conformational dynamics.


Assuntos
Caseína Quinase Iépsilon/metabolismo , Proteínas Desgrenhadas/metabolismo , Domínios PDZ/fisiologia , Via de Sinalização Wnt/fisiologia , Animais , Técnicas Biossensoriais , Caseína Quinase Iépsilon/genética , Proteínas Desgrenhadas/genética , Ensaios Enzimáticos/métodos , Transferência Ressonante de Energia de Fluorescência , Receptores Frizzled/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Microscopia de Fluorescência/métodos , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Oócitos , Fosforilação/fisiologia , Análise de Célula Única/métodos , Xenopus laevis
3.
J Biol Chem ; 293(48): 18477-18493, 2018 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-30309985

RESUMO

Frizzleds (FZDs) are receptors for secreted lipoglycoproteins of the Wingless/Int-1 (WNT) family, initiating an important signal transduction network in multicellular organisms. FZDs are G protein-coupled receptors (GPCRs), which are well known to be regulated by phosphorylation, leading to specific downstream signaling or receptor desensitization. The role and underlying mechanisms of FZD phosphorylation remain largely unexplored. Here, we investigated the phosphorylation of human FZD6 Using MS analysis and a phospho-state- and -site-specific antibody, we found that Ser-648, located in the FZD6 C terminus, is efficiently phosphorylated by casein kinase 1 ϵ (CK1ϵ) and that this phosphorylation requires the scaffolding protein Dishevelled (DVL). In an overexpression system, DVL1, -2, and -3 promoted CK1ϵ-mediated FZD6 phosphorylation on Ser-648. This DVL activity required an intact DEP domain and FZD-mediated recruitment of this domain to the cell membrane. Substitution of the CK1ϵ-targeted phosphomotif reduced FZD6 surface expression, suggesting that Ser-648 phosphorylation controls membrane trafficking of FZD6 Phospho-Ser-648 FZD6 immunoreactivity in human fallopian tube epithelium was predominantly apical, associated with cilia in a subset of epithelial cells, compared with the total FZD6 protein expression, suggesting that FZD6 phosphorylation contributes to asymmetric localization of receptor function within the cell and to epithelial polarity. Given the key role of FZD6 in planar cell polarity, our results raise the possibility that asymmetric phosphorylation of FZD6 rather than asymmetric protein distribution accounts for polarized receptor signaling.

4.
Cell Signal ; 38: 85-96, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28668722

RESUMO

Frizzleds (FZDs) are unconventional G protein-coupled receptors, which activate diverse intracellular signaling pathways via the phosphoprotein Disheveled (DVL) and heterotrimeric G proteins. The interaction interplay of FZDs with DVL and G proteins is complex, involves different regions of FZD and the potential dynamics are poorly understood. In the present study, we aimed to characterize the function of a highly conserved tyrosine (Y2502.39) in the intracellular loop 1 (IL1) of human FZD4. We have found Y2502.39 to be crucial for DVL2 interaction and DVL2 translocation to the plasma membrane. Mutant FZD4-Y2502.39F, impaired in DVL2 binding, was defective in both ß-catenin-dependent and ß-catenin-independent WNT signaling induced in Xenopus laevis embryos. The same mutant maintained interaction with the heterotrimeric G proteins Gα12 and Gα13 and was able to mediate WNT-induced G protein dissociation and G protein-dependent YAP/TAZ signaling. We conclude from modeling and dynamics simulation efforts that Y2502.39 is important for the structural integrity of the FZD-DVL, but not for the FZD-G protein interface and hypothesize that the interaction network of Y2502.39 and H3484.46 plays a role in specifying downstream signaling pathways induced by the receptor.


Assuntos
Sequência Conservada , Proteínas Desgrenhadas/química , Proteínas Desgrenhadas/metabolismo , Receptores Frizzled/química , Receptores Frizzled/metabolismo , Tirosina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Análise Mutacional de DNA , Embrião não Mamífero/metabolismo , Células HEK293 , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Simulação de Dinâmica Molecular , Neoplasias/metabolismo , Neoplasias/patologia , Polimerização , Ligação Proteica , Transdução de Sinais , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Via de Sinalização Wnt , Xenopus laevis/embriologia
5.
Mol Cell Biol ; 2017 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-28674183

RESUMO

Dishevelled (DVL) proteins are key mediators of Wnt/ß-catenin signaling pathway. All DVL proteins contain three conserved domains -- DIX, PDZ and DEP. There is a consensus in the field that DIX domain is critical for Wnt/ß-catenin signaling but contradictory evidence exists regarding the function of the DEP domain. It has been difficult until recently to test the importance of the DEP domain rigorously because of the interference with endogenous DVL, expressed in all Wnt-responsive cell lines. In this study, we took advantage of DVL KO (DVL1/DVL2/DVL3-triple knockout) cells, fully deficient in Wnt3a-induced signaling events, and performed series of rescue experiments. Using this complementation assays we analyze the role of individual DVL isoforms. Further domain mapping of DVL1 showed that DVL1 DEP domain, and especially its N-terminal region, is both required and sufficient for Wnt3a-induced phosphorylation of LRP6 and TopFlash reporter activation. On the contrary, multiple DEP domain mutants deficient in the planar cell polarity (PCP) pathway could fully rescue the Wnt3a response. This study provides conclusive evidence that DVL DEP domain is essential for Wnt/ß-catenin signaling in mammalian cells and establishes an experimental system suitable for further functional testing of DVL.

6.
Front Cell Dev Biol ; 5: 47, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28523267

RESUMO

Mammalian limb development is driven by the integrative input from several signaling pathways; a failure to receive or a misinterpretation of these signals results in skeletal defects. The brachydactylies, a group of overlapping inherited human hand malformation syndromes, are mainly caused by mutations in BMP signaling pathway components. Two closely related forms, Brachydactyly type B2 (BDB2) and BDB1 are caused by mutations in the BMP antagonist Noggin (NOG) and the atypical receptor tyrosine kinase ROR2 that acts as a receptor in the non-canonical Wnt pathway. Genetic analysis of Nog and Ror2 functional interaction via crossing Noggin and Ror2 mutant mice revealed a widening of skeletal elements in compound but not in any of the single mutants, thus indicating genetic interaction. Since ROR2 is a non-canonical Wnt co-receptor specific for Wnt-5a we speculated that this phenotype might be a result of deregulated Wnt-5a signaling activation, which is known to be essential for limb skeletal elements growth and patterning. We show that Noggin potentiates activation of the Wnt-5a-Ror2-Disheveled (Dvl) pathway in mouse embryonic fibroblast (MEF) cells in a Ror2-dependent fashion. Rat chondrosarcoma chondrocytes (RCS), however, are not able to respond to Noggin in this fashion unless growth arrest is induced by FGF2. In summary, our data demonstrate genetic interaction between Noggin and Ror2 and show that Noggin can sensitize cells to Wnt-5a/Ror2-mediated non-canonical Wnt signaling, a feature that in cartilage may depend on the presence of active FGF signaling. These findings indicate an unappreciated function of Noggin that will help to understand BMP and Wnt/PCP signaling pathway interactions.

7.
Proc Natl Acad Sci U S A ; 113(33): 9304-9, 2016 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-27486244

RESUMO

Dishevelled (DVL) is a key scaffolding protein and a branching point in Wnt signaling pathways. Here, we present conclusive evidence that DVL regulates the centrosomal cycle. We demonstrate that DVL dishevelled and axin (DIX) domain, but not DIX domain-mediated multimerization, is essential for DVL's centrosomal localization. DVL accumulates during the cell cycle and associates with NIMA-related kinase 2 (NEK2), which is able to phosphorylate DVL at a multitude of residues, as detected by a set of novel phospho-specific antibodies. This creates interfaces for efficient binding to CDK5 regulatory subunit-associated protein 2 (CDK5RAP2) and centrosomal Nek2-associated protein 1 (C-NAP1), two proteins of the centrosomal linker. Displacement of DVL from the centrosome and its release into the cytoplasm on NEK2 phosphorylation is coupled to the removal of linker proteins, an event necessary for centrosomal separation and proper formation of the mitotic spindle. Lack of DVL prevents NEK2-controlled dissolution of loose centrosomal linker and subsequent centrosomal separation. Increased DVL levels, in contrast, sequester centrosomal NEK2 and mimic monopolar spindle defects induced by a dominant negative version of this kinase. Our study thus uncovers molecular crosstalk between centrosome and Wnt signaling.


Assuntos
Autoantígenos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrossomo/metabolismo , Proteínas Desgrenhadas/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Quinases Relacionadas a NIMA/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Células HEK293 , Células HeLa , Humanos , Fosforilação , Via de Sinalização Wnt
8.
J Biol Chem ; 289(34): 23520-33, 2014 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-24993822

RESUMO

Dishevelled-3 (Dvl3), a key component of the Wnt signaling pathways, acts downstream of Frizzled (Fzd) receptors and gets heavily phosphorylated in response to pathway activation by Wnt ligands. Casein kinase 1ϵ (CK1ϵ) was identified as the major kinase responsible for Wnt-induced Dvl3 phosphorylation. Currently it is not clear which Dvl residues are phosphorylated and what is the consequence of individual phosphorylation events. In the present study we employed mass spectrometry to analyze in a comprehensive way the phosphorylation of human Dvl3 induced by CK1ϵ. Our analysis revealed >50 phosphorylation sites on Dvl3; only a minority of these sites was found dynamically induced after co-expression of CK1ϵ, and surprisingly, phosphorylation of one cluster of modified residues was down-regulated. Dynamically phosphorylated sites were analyzed functionally. Mutations within PDZ domain (S280A and S311A) reduced the ability of Dvl3 to activate TCF/LEF (T-cell factor/lymphoid enhancer factor)-driven transcription and induce secondary axis in Xenopus embryos. In contrast, mutations of clustered Ser/Thr in the Dvl3 C terminus prevented ability of CK1ϵ to induce electrophoretic mobility shift of Dvl3 and its even subcellular localization. Surprisingly, mobility shift and subcellular localization changes induced by Fzd5, a Wnt receptor, were in all these mutants indistinguishable from wild type Dvl3. In summary, our data on the molecular level (i) support previous the assumption that CK1ϵ acts via phosphorylation of distinct residues as the activator as well as the shut-off signal of Wnt/ß-catenin signaling and (ii) suggest that CK1ϵ acts on Dvl via different mechanism than Fzd5.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Caseína Quinase Iépsilon/metabolismo , Receptores Frizzled/metabolismo , Fosfoproteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Animais , Cromatografia Líquida , Proteínas Desgrenhadas , Ensaio de Desvio de Mobilidade Eletroforética , Células HEK293 , Humanos , Dados de Sequência Molecular , Fosfoproteínas/química , Fosforilação , Dobramento de Proteína , Frações Subcelulares/metabolismo , Espectrometria de Massas em Tandem , Transcrição Genética , Proteínas de Xenopus , Xenopus laevis
9.
Sci Signal ; 7(317): ra26, 2014 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-24643799

RESUMO

Wnt signaling plays a central role in development, adult tissue homeostasis, and cancer. Several steps in the canonical Wnt/ß-catenin signaling cascade are regulated by ubiquitylation, a protein modification that influences the stability, subcellular localization, or interactions of target proteins. To identify regulators of the Wnt/ß-catenin pathway, we performed an RNA interference screen in Caenorhabditis elegans and identified the HECT domain-containing ubiquitin ligase EEL-1 as an inhibitor of Wnt signaling. In human embryonic kidney 293T cells, knockdown of the EEL-1 homolog Huwe1 enhanced the activity of a Wnt reporter in cells stimulated with Wnt3a or in cells that overexpressed casein kinase 1 (CK1) or a constitutively active mutant of the Wnt co-receptor low-density lipoprotein receptor-related protein 6 (LRP6). However, knockdown of Huwe1 had no effect on reporter gene expression in cells expressing constitutively active ß-catenin, suggesting that Huwe1 inhibited Wnt signaling upstream of ß-catenin and downstream of CK1 and LRP6. Huwe1 bound to and ubiquitylated the cytoplasmic Wnt pathway component Dishevelled (Dvl) in a Wnt3a- and CK1ε-dependent manner. Mass spectrometric analysis showed that Huwe1 promoted K63-linked, but not K48-linked, polyubiquitination of Dvl. Instead of targeting Dvl for degradation, ubiquitylation of the DIX domain of Dvl by Huwe1 inhibited Dvl multimerization, which is necessary for its function. Our findings indicate that Huwe1 is part of an evolutionarily conserved negative feedback loop in the Wnt/ß-catenin pathway.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fosfoproteínas/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , Via de Sinalização Wnt , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Desgrenhadas , Células HEK293 , Humanos , Espectrometria de Massas , Interferência de RNA , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , beta Catenina/metabolismo
10.
Toxicol Sci ; 122(2): 349-60, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21602191

RESUMO

ß-catenin is a key integrator of cadherin-mediated cell-cell adhesion and transcriptional regulation through the Wnt/ß-catenin pathway, which plays an important role in liver biology. Using a model of contact-inhibited liver progenitor cells, we examined the interactions of Wnt/ß-catenin signaling with the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, which mediates the toxicity of dioxin-like compounds, including their effects on development and hepatocarcinogenesis. We found that AhR and Wnt/ß-catenin cooperated in the induction of AhR transcriptional targets, such as Cyp1a1 and Cyp1b1. However, simultaneously, the activation of AhR led to a decrease of dephosphorylated active ß-catenin pool, as well as to hypophosphorylation of Dishevelled, participating in regulation of Wnt signaling. A sustained AhR activation by its model ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), led to a downregulation of a number of Wnt/ß-catenin pathway target genes. TCDD also induced a switch in cytokeratin expression, where downregulation of cytokeratins 14 and 19 was accompanied with an increased cytokeratin 8 expression. Together with a downregulation of additional markers associated with stem-like phenotype, this indicated that the AhR activation interfered with differentiation of liver progenitors. The downregulation of ß-catenin was also related to a reduced cell adhesion, disruption of E-cadherin-mediated cell-cell junctions and an increased G1-S transition in liver progenitor cell line. In conclusion, although ß-catenin augmented the expression of selected AhR target genes, the persistent AhR activation may lead to downregulation of Wnt/ß-catenin signaling, thus altering differentiation and/or proliferative status of liver progenitor cells.


Assuntos
Fígado/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Caderinas/genética , Adesão Celular , Diferenciação Celular , Linhagem Celular , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1B1 , Regulação para Baixo/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Dibenzodioxinas Policloradas/toxicidade , Ratos , Ratos Endogâmicos F344 , Receptores de Hidrocarboneto Arílico/genética , Proteínas Wnt/genética , Via de Sinalização Wnt , beta Catenina/genética
11.
J Biol Chem ; 286(12): 10396-410, 2011 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-21285348

RESUMO

Dishevelled (Dvl) is a key component in the Wnt/ß-catenin signaling pathway. Dvl can multimerize to form dynamic protein aggregates, which are required for the activation of downstream signaling. Upon pathway activation by Wnts, Dvl becomes phosphorylated to yield phosphorylated and shifted (PS) Dvl. Both activation of Dvl in Wnt/ß-catenin signaling and Wnt-induced PS-Dvl formation are dependent on casein kinase 1 (CK1) δ/ε activity. However, the overexpression of CK1 was shown to dissolve Dvl aggregates, and endogenous PS-Dvl forms irrespective of whether or not the activating Wnt triggers the Wnt/ß-catenin pathway. Using a combination of gain-of-function, loss-of-function, and domain mapping approaches, we attempted to solve this discrepancy regarding the role of CK1ε in Dvl biology. We analyzed mutual interaction of CK1δ/ε and two other Dvl kinases, CK2 and PAR1, in the Wnt/ß-catenin pathway. We show that CK2 acts as a constitutive kinase whose activity is required for the further action of CK1ε. Furthermore, we demonstrate that the two consequences of CK1ε phosphorylation are separated both spatially and functionally; first, CK1ε-mediated induction of TCF/LEF-driven transcription (associated with dynamic recruitment of Axin1) is mediated via a PDZ-proline-rich region of Dvl. Second, CK1ε-mediated formation of PS-Dvl is mediated by the Dvl3 C terminus. Furthermore, we demonstrate with several methods that PS-Dvl has decreased ability to polymerize with other Dvls and could, thus, act as the inactive signaling intermediate. We propose a multistep and multikinase model for Dvl activation in the Wnt/ß-catenin pathway that uncovers a built-in de-activation mechanism that is triggered by activating phosphorylation of Dvl by CK1δ/ε.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fosfoproteínas/metabolismo , Transdução de Sinais/fisiologia , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Caseína Quinase Idelta/genética , Caseína Quinase Idelta/metabolismo , Caseína Quinase Iépsilon/genética , Caseína Quinase Iépsilon/metabolismo , Proteínas Desgrenhadas , Células HEK293 , Humanos , Camundongos , Mapeamento de Peptídeos , Fosfoproteínas/genética , Fosforilação/fisiologia , Receptor PAR-1/genética , Receptor PAR-1/metabolismo , Proteínas Wnt/genética , beta Catenina/genética
12.
Breast Cancer Res ; 12(3): R30, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20507565

RESUMO

INTRODUCTION: Breast cancer is one of the most common types of cancer in women. One of the genes that were found mutated in breast cancer is casein kinase 1 epsilon (CK1epsilon). Because CK1epsilon is a crucial regulator of the Wnt signaling cascades, we determined how these CK1epsilon mutations interfere with the Wnt pathway and affect the behavior of epithelial breast cancer cell lines. METHODS: We performed in silico modeling of various mutations and analyzed the kinase activity of the CK1epsilon mutants both in vitro and in vivo. Furthermore, we used reporter and small GTPase assays to identify how mutation of CK1epsilon affects different branches of the Wnt signaling pathway. Based on these results, we employed cell adhesion and cell migration assays in MCF7 cells to demonstrate a crucial role for CK1epsilon in these processes. RESULTS: In silico modeling and in vivo data showed that autophosphorylation at Thr 44, a site adjacent to the breast cancer point mutations in the N-terminal lobe of human CK1epsilon, is involved in positive regulation of the CK1epsilon activity. Our data further demonstrate that, in mammalian cells, mutated forms of CK1epsilon failed to affect the intracellular localization and phosphorylation of Dvl2; we were able to demonstrate that CK1epsilon mutants were unable to enhance Dvl-induced TCF/LEF-mediated transcription, that CK1epsilon mutants acted as loss-of-function in the Wnt/beta-catenin pathway, and that CK1epsilon mutants activated the noncanonical Wnt/Rac-1 and NFAT pathways, similar to pharmacological inhibitors of CK1. In line with these findings, inhibition of CK1 promoted cell migration as well as decreased cell adhesion and E-cadherin expression in the breast cancer-derived cell line MCF7. CONCLUSIONS: In summary, these data suggest that the mutations of CK1epsilon found in breast cancer can suppress Wnt/beta-catenin as well as promote the Wnt/Rac-1/JNK and Wnt/NFAT pathways, thus contributing to breast cancer development via effects on cell adhesion and migration. In terms of molecular mechanism, our data indicate that the breast cancer point mutations in the N-terminal lobe of CK1epsilon, which are correlated with decreased phosphorylation activities of mutated forms of CK1epsilon both in vitro and in vivo, interfere with positive autophosphorylation at Thr 44.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Caseína Quinase Iépsilon/genética , Movimento Celular , MAP Quinase Quinase 4/metabolismo , Mutação/genética , Fatores de Transcrição NFATC/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Western Blotting , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patologia , Caseína Quinase Iépsilon/química , Caseína Quinase Iépsilon/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Imunoprecipitação , Fosforilação , Conformação Proteica , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
13.
FASEB J ; 24(7): 2417-26, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20215527

RESUMO

Dishevelled (Dvl) is a multifunctional effector of different Wnt cascades. Both canonical Wnt3a and noncanonical Wnt5a stimulate casein-kinase-1 (CK1) -mediated phosphorylation of Dvl, visualized as electrophoretic mobility shift [phosphorylated and shifted Dvl (ps-Dvl)]. However, the role of this phosphorylation remains obscure. Here we report the functional interaction of ps-Dvl with the receptor tyrosine kinase Ror2, which is an alternative Wnt receptor and is able to inhibit canonical Wnt signaling. We demonstrate interaction between Ror2 and ps-Dvl at the cell membrane after Wnt3a or Wnt5a stimulus dependent on CK1. Ps-Dvl interacts with the C-terminal proline-serine-threonine-rich domain of Ror2, which is required for efficient inhibition of canonical Wnt signaling. We further show that the Dvl C terminus, which seems to be exposed in ps-Dvl and efficiently binds Ror2, is an intrinsic negative regulator of the canonical Wnt pathway downstream of beta-catenin. The Dvl C terminus is necessary and sufficient to inhibit canonical Wnt/beta-catenin signaling, which is dependent on the presence of Ror2. Furthermore, both the Dvl C terminus and CK1epsilon can inhibit the Wnt5a/Ror2/ATF2 pathway in mammalian cells and Xenopus explant cultures. This suggests that phosphorylation of Dvl triggers negative feedback regulation for different branches of Wnt signaling in a Ror2-dependent manner.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Caseína Quinase I/metabolismo , Retroalimentação Fisiológica , Fosfoproteínas/metabolismo , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Transdução de Sinais , Proteínas Wnt/metabolismo , Animais , Células COS , Cercopithecus aethiops , Proteínas Desgrenhadas , Humanos , Camundongos , Fosforilação , Proteína Wnt-5a , Proteína Wnt3 , Proteína Wnt3A , Xenopus , Proteínas de Xenopus
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA