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.

• MeSH
• analýza jednotlivých buněk metody   MeSH
• biosenzitivní techniky MeSH
• enzymatické testy metody   MeSH
• fluorescenční mikroskopie metody   MeSH
• fosforylace fyziologie   MeSH
• frizzled receptory metabolismus   MeSH
• genový knockout MeSH
• HEK293 buňky MeSH
• kaseinkinasa Iepsilon genetika   metabolismus   MeSH
• lidé MeSH
• mutageneze cílená MeSH
• oocyty MeSH
• PDZ domény fyziologie   MeSH
• protein dishevelled genetika   metabolismus   MeSH
• rezonanční přenos fluorescenční energie MeSH
• signální dráha Wnt fyziologie   MeSH
• simulace molekulární dynamiky MeSH
• Xenopus laevis MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DVL3 protein, human MeSH Prohlížeč
• frizzled receptory MeSH
• FZD6 protein, human MeSH Prohlížeč
• kaseinkinasa Iepsilon MeSH
• protein dishevelled MeSH

Intrinsically disordered regions (IDRs) are protein regions that lack persistent secondary or tertiary structure under native conditions. IDRs represent >40% of the eukaryotic proteome and play a crucial role in protein-protein interactions. The classical approach for identification of these interaction interfaces is based on mutagenesis combined with biochemical techniques such as coimmunoprecipitation or yeast two-hybrid screening. This approach either provides information of low resolution (large deletions) or very laboriously tries to precisely define the binding epitope via single amino acid substitutions. Here, we report the use of a peptide microarray based on the human scaffold protein AXIN1 for high-throughput and -resolution mapping of binding sites for several AXIN1 interaction partners in vitro For each of the AXIN1-binding partners tested, i.e. casein kinase 1 ϵ (CK1ϵ); c-Myc; peptidyl-prolyl cis/trans isomerase, NIMA-interacting 1 (Pin1); and p53, we found at least three different epitopes, predominantly in the central IDR of AXIN1. We functionally validated the specific AXIN1-CK1ϵ interaction identified here with epitope-mimicking peptides and with AXIN1 variants having deletions of short binding epitopes. On the basis of these results, we propose a model in which AXIN1 competes with dishevelled (DVL) for CK1ϵ and regulates CK1ϵ-induced phosphorylation of DVL and activation of Wnt/β-catenin signaling.

• Klíčová slova
• Myc (c-Myc), Wnt pathway, axin, casein kinase 1ϵ, dishevelled, intrinsically disordered region, p53, peptide array, scaffold protein, serine/threonine protein kinase,
• MeSH
• axin protein metabolismus   MeSH
• beta-katenin metabolismus   MeSH
• čipová analýza proteinů metody   MeSH
• fosforylace MeSH
• interakční proteinové domény a motivy * MeSH
• kaseinkinasa Iepsilon metabolismus   MeSH
• kompetitivní vazba MeSH
• lidé MeSH
• peptidy metabolismus   MeSH
• protein dishevelled metabolismus   MeSH
• proteiny Wnt metabolismus   MeSH
• signální dráha Wnt MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• axin protein MeSH
• AXIN1 protein, human MeSH Prohlížeč
• beta-katenin MeSH
• kaseinkinasa Iepsilon MeSH
• peptidy MeSH
• protein dishevelled MeSH
• proteiny Wnt MeSH

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.

• Klíčová slova
• Dishevelled, NEK2, Wnt signaling, centrosome, linker proteins,
• MeSH
• autoantigeny metabolismus   MeSH
• centrozom metabolismus   MeSH
• fosforylace MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• intracelulární signální peptidy a proteiny metabolismus   MeSH
• kinasy NEK fyziologie   MeSH
• lidé MeSH
• protein dishevelled fyziologie   MeSH
• proteiny buněčného cyklu metabolismus   MeSH
• proteiny nervové tkáně metabolismus   MeSH
• signální dráha Wnt MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• autoantigeny MeSH
• CDK5RAP2 protein, human MeSH Prohlížeč
• CEP250 protein, human MeSH Prohlížeč
• intracelulární signální peptidy a proteiny MeSH
• kinasy NEK MeSH
• NEK2 protein, human MeSH Prohlížeč
• protein dishevelled MeSH
• proteiny buněčného cyklu MeSH
• proteiny nervové tkáně MeSH

Alkaline phosphatase is an enzyme commonly expressed in almost all living organisms. In humans and other mammals, determinations of the expression and activity of alkaline phosphatase have frequently been used for cell determination in developmental studies and/or within clinical trials. Alkaline phosphatase also seems to be one of the key markers in the identification of pluripotent embryonic stem as well as related cells. However, alkaline phosphatases exist in some isoenzymes and isoforms, which have tissue specific expressions and functions. Here, the role of alkaline phosphatase as a stem cell marker is discussed in detail. First, we briefly summarize contemporary knowledge of mammalian alkaline phosphatases in general. Second, we focus on the known facts of its role in and potential significance for the identification of stem cells.

• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

LDL-related protein 6 (LRP6) is a coreceptor of WNTs and a key regulator of the WNT/β-catenin pathway. Upon activation, LRP6 is phosphorylated within its intracellular PPPS/TP motifs. These phosphorylated motifs are required to recruit axin and to inhibit glycogen synthase kinase 3 (GSK3), two basic components of the β-catenin destruction complex. On the basis of a kinome-wide small interfering RNA (siRNA) screen and confirmative biochemical analysis, we show that several proline-directed mitogen-activated protein kinases (MAPKs), such as p38, ERK1/2, and JNK1 are sufficient and required for the phosphorylation of PPPS/TP motifs of LRP6. External stimuli, which control the activity of MAPKs, such as phorbol esters and fibroblast growth factor 2 (FGF2) control the choice of the LRP6-PPPS/TP kinase and regulate the amplitude of LRP6 phosphorylation and WNT/β-catenin-dependent transcription. Our findings suggest that cells not only recruit one dedicated LRP6 kinase but rather select their LRP6 kinase depending on cell type and the external stimulus. Moreover, direct phosphorylation of LRP6 by MAPKs provides a unique point for convergence between WNT/β-catenin signaling and mitogenic pathways.

• MeSH
• aminokyselinové motivy MeSH
• beta-katenin metabolismus   MeSH
• buněčné linie MeSH
• fosforylace MeSH
• krysa rodu Rattus MeSH
• LDL receptor related protein 6 MeSH
• LDL-receptory metabolismus   MeSH
• lidé MeSH
• malá interferující RNA genetika   MeSH
• MAP kinasový signální systém MeSH
• mitogenem aktivovaná proteinkinasa 8 metabolismus   MeSH
• mitogenem aktivované proteinkinasy p38 metabolismus   MeSH
• mitogenem aktivované proteinkinasy antagonisté a inhibitory   genetika   metabolismus   MeSH
• nádorové buňky kultivované MeSH
• proteiny související s LDL-receptory chemie   genetika   metabolismus   MeSH
• proteiny Wnt metabolismus   MeSH
• signální transdukce MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, N.I.H., Intramural MeSH
• Názvy látek
• beta-katenin MeSH
• CTNNB1 protein, human MeSH Prohlížeč
• LDL receptor related protein 6 MeSH
• LDL-receptory MeSH
• LRP6 protein, human MeSH Prohlížeč
• malá interferující RNA MeSH
• mitogenem aktivovaná proteinkinasa 8 MeSH
• mitogenem aktivované proteinkinasy p38 MeSH
• mitogenem aktivované proteinkinasy MeSH
• proteiny související s LDL-receptory MeSH
• proteiny Wnt 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
• biologické modely MeSH
• buněčná diferenciace * MeSH
• buněčný rodokmen * MeSH
• cyklin A metabolismus   MeSH
• cyklin D MeSH
• cyklin E metabolismus   MeSH
• cyklin-dependentní kinasa 4 metabolismus   MeSH
• cyklin-dependentní kinasa 6 metabolismus   MeSH
• cykliny metabolismus   MeSH
• embryo savčí cytologie   metabolismus   MeSH
• embryonální kmenové buňky metabolismus   MeSH
• G1 fáze MeSH
• inhibitor p27 cyklin-dependentní kinasy metabolismus   MeSH
• intracelulární prostor metabolismus   MeSH
• lidé MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• proliferace buněk MeSH
• S fáze MeSH
• transport proteinů MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CDK4 protein, human MeSH Prohlížeč
• cyklin A MeSH
• cyklin D MeSH
• cyklin E MeSH
• cyklin-dependentní kinasa 4 MeSH
• cyklin-dependentní kinasa 6 MeSH
• cykliny MeSH
• inhibitor p27 cyklin-dependentní kinasy MeSH