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/β-catenin signaling and (ii) suggest that CK1ϵ acts on Dvl via different mechanism than Fzd5.

• Keywords
• Casein Kinase 1ϵ, Cell Signaling, Dishevelled-3, Frizzled5, Mass Spectrometry (MS), Phosphorylation, Post-translational Modification (PTM), Wnt Pathway,
• MeSH
• Adaptor Proteins, Signal Transducing chemistry   metabolism   MeSH
• Chromatography, Liquid MeSH
• Phosphoproteins chemistry   metabolism   MeSH
• Phosphorylation MeSH
• Frizzled Receptors metabolism   MeSH
• Transcription, Genetic MeSH
• HEK293 Cells MeSH
• Casein Kinase 1 epsilon metabolism   MeSH
• Humans MeSH
• Molecular Sequence Data MeSH
• Dishevelled Proteins MeSH
• Xenopus Proteins MeSH
• Electrophoretic Mobility Shift Assay MeSH
• Protein Folding MeSH
• Amino Acid Sequence MeSH
• Subcellular Fractions metabolism   MeSH
• Tandem Mass Spectrometry MeSH
• Xenopus laevis MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adaptor Proteins, Signal Transducing MeSH
• DVL1 protein, Xenopus MeSH Browser
• DVL3 protein, human MeSH Browser
• Phosphoproteins MeSH
• Frizzled Receptors MeSH
• FZD5 protein, human MeSH Browser
• Casein Kinase 1 epsilon MeSH
• Dishevelled Proteins MeSH
• Xenopus Proteins MeSH

Notch and gp130 signaling are involved in the regulation of multiple cellular processes across various tissues during animal ontogenesis. In the developing nervous system, both signaling pathways intervene at many stages to determine cell fate-from the first neural lineage commitment and generation of neuronal precursors, to the terminal specification of cells as neurons and glia. In most cases, the effects of Notch and gp130 signaling in these processes are similar. The aim of the current review was to summarize the knowledge regarding the roles of Notch and gp130 signaling in the maintenance of neural stem and progenitor cells during animal ontogenesis, from early embryo to adult. Recent data show a direct crosstalk between these signaling pathways that seems to be specific for a particular type of neural progenitors.

• MeSH
• Cytokine Receptor gp130 metabolism   MeSH
• Receptor Cross-Talk MeSH
• Humans MeSH
• Neural Stem Cells metabolism   MeSH
• Neurogenesis MeSH
• Receptors, Notch metabolism   MeSH
• Signal Transduction * MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Cytokine Receptor gp130 MeSH
• Receptors, Notch MeSH

OBJECTIVES: This article is to study the role of G(1)/S regulators in differentiation of pluripotent embryonic cells. MATERIALS AND METHODS: We established a P19 embryonal carcinoma cell-based experimental system, which profits from two similar differentiation protocols producing endodermal or neuroectodermal lineages. The levels, mutual interactions, activities, and localization of G(1)/S regulators were analysed with respect to growth and differentiation parameters of the cells. RESULTS AND CONCLUSIONS: We demonstrate that proliferation parameters of differentiating cells correlate with the activity and structure of cyclin A/E-CDK2 but not of cyclin D-CDK4/6-p27 complexes. In an exponentially growing P19 cell population, the cyclin D1-CDK4 complex is detected, which is replaced by cyclin D2/3-CDK4/6-p27 complex following density arrest. During endodermal differentiation kinase-inactive cyclin D2/D3-CDK4-p27 complexes are formed. Neural differentiation specifically induces cyclin D1 at the expense of cyclin D3 and results in predominant formation of cyclin D1/D2-CDK4-p27 complexes. Differentiation is accompanied by cytoplasmic accumulation of cyclin Ds and CDK4/6, which in neural cells are associated with neural outgrowths. Most phenomena found here can be reproduced in mouse embryonic stem cells. In summary, our data demonstrate (i) that individual cyclin D isoforms are utilized in cells lineage specifically, (ii) that fundamental difference in the function of CDK4 and CDK6 exists, and (iii) that cyclin D-CDK4/6 complexes function in the cytoplasm of differentiated cells. Our study unravels another level of complexity in G(1)/S transition-regulating machinery in early embryonic cells.

• MeSH
• Models, Biological MeSH
• Cell Differentiation * MeSH
• Cell Lineage * MeSH
• Cyclin A metabolism   MeSH
• Cyclin D MeSH
• Cyclin E metabolism   MeSH
• Cyclin-Dependent Kinase 4 metabolism   MeSH
• Cyclin-Dependent Kinase 6 metabolism   MeSH
• Cyclins metabolism   MeSH
• Embryo, Mammalian cytology   metabolism   MeSH
• Embryonic Stem Cells metabolism   MeSH
• G1 Phase MeSH
• Cyclin-Dependent Kinase Inhibitor p27 metabolism   MeSH
• Intracellular Space metabolism   MeSH
• Humans MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Cell Proliferation MeSH
• S Phase MeSH
• Protein Transport MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CDK4 protein, human MeSH Browser
• Cyclin A MeSH
• Cyclin D MeSH
• Cyclin E MeSH
• Cyclin-Dependent Kinase 4 MeSH
• Cyclin-Dependent Kinase 6 MeSH
• Cyclins MeSH
• Cyclin-Dependent Kinase Inhibitor p27 MeSH