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

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.

• 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

Casein kinase 1δ/ε (CK1δ/ε) is a key component of noncanonical Wnt signaling pathways, which were shown previously to drive pathogenesis of chronic lymphocytic leukemia (CLL). In this study, we investigated thoroughly the effects of CK1δ/ε inhibition on the primary CLL cells and analyzed the therapeutic potential in vivo using 2 murine model systems based on the Eµ-TCL1-induced leukemia (syngeneic adoptive transfer model and spontaneous disease development), which resembles closely human CLL. We can demonstrate that the CK1δ/ε inhibitor PF-670462 significantly blocks microenvironmental interactions (chemotaxis, invasion and communication with stromal cells) in primary CLL cells in all major subtypes of CLL. In the mouse models, CK1 inhibition slows down accumulation of leukemic cells in the peripheral blood and spleen and prevents onset of anemia. As a consequence, PF-670462 treatment results in a significantly longer overall survival. Importantly, CK1 inhibition has synergistic effects to the B-cell receptor (BCR) inhibitors such as ibrutinib in vitro and significantly improves ibrutinib effects in vivo. Mice treated with a combination of PF-670462 and ibrutinib show the slowest progression of disease and survive significantly longer compared with ibrutinib-only treatment when the therapy is discontinued. In summary, this preclinical testing of CK1δ/ε inhibitor PF-670462 demonstrates that CK1 may serve as a novel therapeutic target in CLL, acting in synergy with BCR inhibitors. Our work provides evidence that targeting CK1 can represent an alternative or addition to the therapeutic strategies based on BCR signaling and antiapoptotic signaling (BCL-2) inhibition.

• MeSH
• chronická lymfatická leukemie farmakoterapie   enzymologie   genetika   MeSH
• HEK293 buňky MeSH
• kaseinkinasa Idelta antagonisté a inhibitory   genetika   metabolismus   MeSH
• kaseinkinasa Iepsilon antagonisté a inhibitory   genetika   metabolismus   MeSH
• lékové transportní systémy MeSH
• lidé MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádorové proteiny antagonisté a inhibitory   genetika   metabolismus   MeSH
• pyrazoly farmakologie   MeSH
• pyrimidiny farmakologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• MeSH
• adaptorové proteiny signální transdukční chemie   metabolismus   MeSH
• chromatografie kapalinová MeSH
• fosfoproteiny chemie   metabolismus   MeSH
• fosforylace MeSH
• frizzled receptory metabolismus   MeSH
• genetická transkripce MeSH
• HEK293 buňky MeSH
• kaseinkinasa Iepsilon metabolismus   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• retardační test MeSH
• sbalování proteinů MeSH
• sekvence aminokyselin MeSH
• subcelulární frakce metabolismus   MeSH
• tandemová hmotnostní spektrometrie MeSH
• Xenopus laevis MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Dishevelled (Dvl) is a key component in the Wnt/β-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/β-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/β-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/β-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/β-catenin pathway that uncovers a built-in de-activation mechanism that is triggered by activating phosphorylation of Dvl by CK1δ/ε.

• MeSH
• adaptorové proteiny signální transdukční genetika   metabolismus   MeSH
• beta-katenin genetika   metabolismus   MeSH
• fosfoproteiny genetika   metabolismus   MeSH
• fosforylace fyziologie   MeSH
• HEK293 buňky MeSH
• kaseinkinasa Idelta genetika   metabolismus   MeSH
• kaseinkinasa Iepsilon genetika   metabolismus   MeSH
• kaseinkinasa II genetika   metabolismus   MeSH
• lidé MeSH
• myši MeSH
• peptidové mapování MeSH
• proteiny Wnt genetika   metabolismus   MeSH
• receptor PAR-1 genetika   metabolismus   MeSH
• signální transdukce fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

We are using a candidate gene approach to identify genes contributing to cancer through somatic mutation. Somatic mutations were found in breast cancer samples in the human casein kinase I epsilon (CKIepsilon) gene, a homolog of the Drosophila gene dco in which certain point mutations lead to imaginal disc overgrowth. We therefore created fly genotypes in which the dco gene carried point mutations homologous to those discovered in CKIepsilon, and tested them in vivo. The results show that the most frequent mutation discovered in breast cancer, L39Q, causes a striking overgrowth phenotype in flies. Further experiments show that this mutation affects the newly recognized Fat/Warts signaling pathway, which controls organ size and shape in both flies and mammals. Another mutation, S101R, modifies the mutant phenotype so that the affected tissue disintegrates, mimicking more aggressive forms of breast cancer. Our results thus strongly support the conclusion that CKIepsilon mutations play important roles in breast carcinogenesis.

• MeSH
• alely MeSH
• Drosophila embryologie   genetika   růst a vývoj   MeSH
• fenotyp MeSH
• kaseinkinasa Iepsilon chemie   genetika   fyziologie   MeSH
• larva genetika   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• nádory prsu genetika   MeSH
• proliferace buněk MeSH
• proteiny Drosophily chemie   genetika   fyziologie   MeSH
• sekvence aminokyselin MeSH
• signální transdukce MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• MeSH
• beta-katenin metabolismus   MeSH
• buněčná adheze MeSH
• duktální karcinom prsu genetika   metabolismus   patologie   MeSH
• fosforylace MeSH
• imunoprecipitace MeSH
• kaseinkinasa Iepsilon chemie   genetika   metabolismus   MeSH
• konformace proteinů MeSH
• lidé MeSH
• MAP kinasa-kinasa 4 metabolismus   MeSH
• messenger RNA genetika   MeSH
• mutace genetika   MeSH
• nádorové buněčné linie MeSH
• nádory prsu genetika   metabolismus   patologie   MeSH
• pohyb buněk MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• proliferace buněk MeSH
• proteiny Wnt metabolismus   MeSH
• rac1 protein vázající GTP metabolismus   MeSH
• transkripční faktory NFATC metabolismus   MeSH
• western blotting MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH