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Science 339 (6126): 1436-1441

Copyright © 2013 by the American Association for the Advancement of Science

RNA Helicase DDX3 Is a Regulatory Subunit of Casein Kinase 1 in Wnt–β-Catenin Signaling

Cristina-Maria Cruciat1,*, Christine Dolde1,*, Reinoud E. A. de Groot2, Bisei Ohkawara1,{dagger}, Carmen Reinhard1, Hendrik C. Korswagen2, and Christof Niehrs1,3,{ddagger}

1 Division of Molecular Embryology, DKFZ-ZMBH Alliance, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.
2 Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences and University Medical Center Utrecht, Netherlands.
3 Institute of Molecular Biology, D-55128 Mainz, Germany.


Figure 1
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Fig. 1. DDX3 is required for Wnt–β-catenin signaling in mammalian cells and during a-p neural patterning in Xenopus. (A) Wnt luciferase reporter assay in HEK293T cells stimulated with Wnt3a-conditioned medium or by transfection with the indicated constructs, in the presence of the indicated siRNAs. RLA, relative luciferase activity. Error bars indicate SDs; n = 3, biological triplicates of one representative assay. (B) Western blot analysis of endogenous β-catenin from cytosolic and nuclear fractions of HEK293T cells stimulated with control or Wnt3a-conditioned medium in the presence of the indicated siRNAs. (C) Xenopus axis duplication assay by injection of the indicated mRNAs into the ventral blastomeres of four-cell-stage embryos. (D) Quantitative polymerase chain reaction (QPCR) analysis of siamois using VMZ explants from Xenopus embryos injected with the indicated mRNAs. Explants were excised and analyzed from gastrula-stage embryos. Error bars indicate SDs; n = 2 assays. VMZ, ventral marginal zone; sia, siamois. (E) Tadpole-stage Xenopus embryos that were injected at the two-cell stage in the animal hemisphere with DDX3 or LRP6 antisense Mo oligonucleotides in the absence or presence of human DDX3 mRNA (WT, wild type) as indicated. (F) Whole-mount in situ hybridization of neurula-stage embryos injected at four-cell stage in animal blastomeres with the indicated Mo plus β-galactosidase mRNA lineage tracer (red; arrow marks injected side).

 

Figure 2
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Fig. 2. DDX3 is required for Dvl2 phosphorylation and signalosome formation. (A) Western blot of endogenous Dvl2, DDX3, and CK1{varepsilon} from lysates of HEK293T cells treated with the indicated siRNAs. (B) Western blot analysis of Dvl2 phosphorylation status in lysates from HEK293T cells transfected as indicated. (C) QPCR analysis of siamois (sia) in animal cap explants from Xenopus embryos. Injected mRNA: hDDX3 (0.5 and 1 ng). Ornithine decarboxylase was used for normalization. Dvl2, CK1{varepsilon}, and β-catenin injected samples are set to 1. (D) Western blot of the indicated endogenous proteins from membrane lysates of NTERA2 cells treated with the indicated siRNAs and stimulated for 1 hour with Wnt3a-conditioned medium or control medium. tot. LRP6, total LRP6. (E) Confocal microscopy of HeLa cells transfected with Dvl2-HA or V5-CK1{varepsilon} in the absence or presence of DDX3-Myc. A low dose of CK1{varepsilon} was used, as high CK1{varepsilon} leads to nonvesicular Dvl due to decreased polymerization (41). (F) CoIP of V5-CK1{varepsilon} with Dvl2-Flag from lysates of transfected HEK293T cells in the presence or absence of DDX3-Myc.

 

Figure 3
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Fig. 3. DDX3 binds CK1{varepsilon} and Wnt promotes this interaction. (A) In vitro binding assays with the indicated recombinant proteins. IPs were performed with antibody against Myc (anti-Myc) and analyzed by Western blotting with anti-Dvl2 and anti-CK1{varepsilon}. Asterisk (*) denotes nonspecific band. (B) CoIPs of endogenous proteins from lysates of HEK293T cells. Anti-Dvl3 serves as negative control. (*) Nonspecific band. (C) CoIPs of endogenous proteins from lysates of HEK293T cells stimulated with control or Wnt3a-conditioned medium. (D) Schematic representation of C-terminally Myc-tagged DDX3 constructs used. Q and I to VI represent the conserved motifs of DEAD-box helicases, which are organized in domain 1 and 2 (17). Motifs Q, I, II, and VI bind ATP and are required for ATP hydrolysis. Motifs Ia, Ib, IV, and V are involved in RNA binding. Motif III is responsible for the communication between ATP-binding and RNA-binding sites. The Wnt signaling ability of DDX3 deletion mutants (from figs. S9 and S10) is summarized on the right. (E) CoIPs from lysates of HEK293T cells transfected with the indicated tagged constructs. DDX3 constructs as in (D).

 

Figure 4
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Fig. 4. DDX3 and other DEAD-box RNA helicases directly activate CK1 family members. (A) CK1{varepsilon} in vitro kinase assay with recombinant Dvl2 as substrate in the absence or presence of recombinant DDX3. Autoradiography after SDS–page polyacrylamide gel electrophoresis showing 32P incorporation. (B to D) In vitro kinase assays with the indicated recombinant kinases and CK1- or GSK3-specific peptide substrate in the absence or presence of DDX3. (E and F) In vitro kinase assays of endogenous CK1{varepsilon} (E) and CK1α (F) coimmunoprecipitated from lysates of HEK293T that were treated with the indicated siRNAs and stimulated with control or Wnt3a-conditioned medium. Kinase activity was normalized to total kinase (Western blot, lower panel). The experiment was performed three times with similar results. (G) CK1{varepsilon} in vitro kinase assay with CK1 peptide substrate in the absence or presence of the indicated recombinant proteins. Error bars indicate SDs; n = 3 assays. cpm, counts per minute.

 


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