RESUMO
Nephronophthisis-related ciliopathies (NPHP-RC) are degenerative recessive diseases that affect kidney, retina, and brain. Genetic defects in NPHP gene products that localize to cilia and centrosomes defined them as "ciliopathies." However, disease mechanisms remain poorly understood. Here, we identify by whole-exome resequencing, mutations of MRE11, ZNF423, and CEP164 as causing NPHP-RC. All three genes function within the DNA damage response (DDR) pathway. We demonstrate that, upon induced DNA damage, the NPHP-RC proteins ZNF423, CEP164, and NPHP10 colocalize to nuclear foci positive for TIP60, known to activate ATM at sites of DNA damage. We show that knockdown of CEP164 or ZNF423 causes sensitivity to DNA damaging agents and that cep164 knockdown in zebrafish results in dysregulated DDR and an NPHP-RC phenotype. Our findings link degenerative diseases of the kidney and retina, disorders of increasing prevalence, to mechanisms of DDR.
Assuntos
Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Exoma , Doenças Renais Císticas/genética , Proteínas dos Microtúbulos/metabolismo , Animais , Cílios/metabolismo , Técnicas de Silenciamento de Genes , Genes Recessivos , Humanos , Proteína Homóloga a MRE11 , Camundongos , Proteínas , Transdução de Sinais , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismoRESUMO
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.
Assuntos
Caseína Quinase I/metabolismo , Proteínas Desgrenhadas/fisiologia , Receptores Frizzled/metabolismo , Sequência de Aminoácidos , Anticorpos/imunologia , Membrana Celular/metabolismo , Proteínas Desgrenhadas/química , Epitélio/metabolismo , Tubas Uterinas/metabolismo , Feminino , Receptores Frizzled/química , Células HEK293 , Humanos , Espectrometria de Massas , Fosfoproteínas/imunologia , Fosforilação , Serina/metabolismo , Transdução de SinaisRESUMO
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 1 épsilon/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 Gênica , Proteínas de Xenopus , Xenopus laevisRESUMO
Frizzleds (FZDs) are classified as G-protein-coupling receptors, but how signals are initiated and specified through heterotrimeric G proteins is unknown. FZD6 regulates convergent extension movements, and its C-terminal Arg511Cys mutation causes nail dysplasia in humans. We investigated the functional relationship between FZD6, Disheveled (DVL), and heterotrimeric G proteins. Live cell imaging combined with fluorescence recovery after photobleaching (FRAP) revealed that inactive human FZD6 precouples to Gαi1 and Gαq but not to GαoA,Gαs, and Gα12 proteins. G-protein coupling is measured as a 10-20% reduction in the mobile fraction of fluorescently tagged G proteins on chemical receptor surface cross-linking. The FZD6 Arg511Cys mutation is incapable of G-protein precoupling, even though it still binds DVL. Using both FRAP and Förster resonance energy transfer (FRET) technology, we showed that the FZD6-Gαi1 and FZD-Gαq complexes dissociate on WNT-5A stimulation. Most important, G-protein precoupling of FZD6 and WNT-5A-induced signaling to extracellular signal-regulated kinase1/2 were impaired by DVL knockdown or overexpression, arguing for a strict dependence of FZD6-G-protein coupling on DVL levels and identifying DVL as a master regulator of FZD/G-protein signaling. In summary, we propose a mechanistic connection between DVL and G proteins integrating WNT, FZD, G-protein, and DVL function.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Receptores Frizzled/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Fosfoproteínas/metabolismo , Membrana Celular/metabolismo , Proteínas Desgrenhadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Mutação , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Wnt/metabolismo , Proteína Wnt-5aRESUMO
In this study we use comparative genomics to uncover a gene with uncharacterized function (1700011H14Rik/C14orf105/CCDC198), which we hereby name FAME (Factor Associated with Metabolism and Energy). We observe that FAME shows an unusually high evolutionary divergence in birds and mammals. Through the comparison of single nucleotide polymorphisms, we identify gene flow of FAME from Neandertals into modern humans. We conduct knockout experiments on animals and observe altered body weight and decreased energy expenditure in Fame knockout animals, corresponding to genome-wide association studies linking FAME with higher body mass index in humans. Gene expression and subcellular localization analyses reveal that FAME is a membrane-bound protein enriched in the kidneys. Although the gene knockout results in structurally normal kidneys, we detect higher albumin in urine and lowered ferritin in the blood. Through experimental validation, we confirm interactions between FAME and ferritin and show co-localization in vesicular and plasma membranes.
Assuntos
Metabolismo Energético , Estudo de Associação Genômica Ampla , Animais , Humanos , Peso Corporal , Metabolismo Energético/genética , Ferritinas/genética , Rim , Homem de NeandertalRESUMO
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 1 épsilon/genética , Caseína Quinase 1 épsilon/metabolismo , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Caseína Quinase Idelta/genética , Caseína Quinase Idelta/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/genéticaRESUMO
Primary cilia play critical roles in development and disease. Their assembly and disassembly are tightly coupled to cell cycle progression. Here, we present data identifying KIF14 as a regulator of cilia formation and Hedgehog (HH) signaling. We show that RNAi depletion of KIF14 specifically leads to defects in ciliogenesis and basal body (BB) biogenesis, as its absence hampers the efficiency of primary cilium formation and the dynamics of primary cilium elongation, and disrupts the localization of the distal appendage proteins SCLT1 and FBF1 and components of the IFT-B complex. We identify deregulated Aurora A activity as a mechanism contributing to the primary cilium and BB formation defects seen after KIF14 depletion. In addition, we show that primary cilia in KIF14-depleted cells are defective in response to HH pathway activation, independently of the effects of Aurora A. In sum, our data point to KIF14 as a critical node connecting cell cycle machinery, effective ciliogenesis, and HH signaling.
Assuntos
Aurora Quinase A/metabolismo , Ciclo Celular/genética , Cílios/metabolismo , Proteínas Hedgehog/metabolismo , Cinesinas/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Aurora Quinase A/antagonistas & inibidores , Aurora Quinase A/genética , Corpos Basais/metabolismo , Cromatografia Líquida , Cílios/genética , Cílios/patologia , Células HEK293 , Humanos , Interfase/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cinesinas/genética , Mitose/genética , Proteínas Oncogênicas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Transdução de Sinais/genética , Canais de Sódio/metabolismo , Espectrometria de Massas em TandemRESUMO
Understanding the mechanisms that drive the differentiation of dopaminergic (DA) neurons is crucial for successful development of novel therapies for Parkinson's disease, in which DA neurons progressively degenerate. However, the mechanisms underlying the differentiation-promoting effects of Wnt5a on DA precursors are poorly understood. Here, we present the molecular and functional characterization of a signaling pathway downstream of Wnt5a, the Wnt/Dvl/Rac1 pathway. First, we characterize the interaction between Rac1 and Dvl and identify the N-terminal part of Dvl3 as necessary for Rac1 binding. Next, we show that Tiam1, a Rac1 guanosine exchange factor (GEF), is expressed in the ventral midbrain, interacts with Dvl, facilitates Dvl-Rac1 interaction, and is required for Dvl- or Wnt5a-induced activation of Rac1. Moreover, we show that Wnt5a promotes whereas casein kinase 1 (CK1), a negative regulator of the Wnt/Dvl/Rac1 pathway, abolishes the interactions between Dvl and Tiam1. Finally, using ventral midbrain neurosphere cultures, we demonstrate that the generation of DA neurons in culture is impaired after Tiam1 knockdown, indicating that Tiam1 is required for midbrain DA differentiation. In summary, our data identify Tiam1 as a novel regulator of DA neuron development and as a Dvl-associated and Rac1-specific GEF acting in the Wnt/Dvl/Rac1 pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Neuropeptídeos/metabolismo , Fosfoproteínas/metabolismo , Proteínas Wnt/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Caseína Quinase I/metabolismo , Diferenciação Celular , Proteínas Desgrenhadas , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/imunologia , Humanos , Imunoprecipitação , Mesencéfalo/citologia , Mesencéfalo/embriologia , Mesencéfalo/metabolismo , Camundongos , Neuropeptídeos/genética , Fosfoproteínas/genética , Transdução de Sinais , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T , Proteínas Wnt/genética , Via de Sinalização Wnt , Proteína Wnt-5a , Proteínas rac de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTPRESUMO
The lipoglycoproteins of the WNT family act on seven transmembrane-spanning Class Frizzled receptors. Here, we show that WNT-5A evokes a proliferative response in a mouse microglia-like cell line (N13), which is sensitive to pertussis toxin, thus implicating the involvement of heterotrimeric G proteins of the G(i/o) family. We continue to show that WNT-5A stimulation of N13 membranes and permeabilized cells evokes the exchange of GDP for GTP of pertussis toxin-sensitive G proteins employing [γ-(35)S]GTP assay and activity state-specific antibodies to GTP-bound G(i) proteins. Our functional analysis of the PTX-sensitivity of WNT-induced G protein activation and PCR analysis of G protein and FZD expression patterns suggest that WNT-5A stimulation leads to the activation of G(i2/3) proteins in N13 cells possibly mediated by FZD(5), the predominant FZD expressed. In summary, we provide for the first time molecular proof that WNT-5A stimulation results in the activation of heterotrimeric G(i2/3) proteins in mammalian cells with physiological protein stochiometry.