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Science 339 (6126): 1441-1445

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

Phosphorylation of Dishevelled by Protein Kinase RIPK4 Regulates Wnt Signaling

XiaoDong Huang1,2, James C. McGann3,*, Bob Y. Liu4,*, Rami N. Hannoush5,*, Jennie R. Lill6, Victoria Pham6, Kim Newton1, Michael Kakunda2, Jinfeng Liu7, Christine Yu8, Sarah G. Hymowitz8, Jo-Anne Hongo9, Anthony Wynshaw-Boris10, Paul Polakis4, Richard M. Harland3, and Vishva M. Dixit1,{dagger}

1 Department of Physiological Chemistry, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA.
2 Department of Molecular Diagnostics and Cancer Cell Biology, Genentech, South San Francisco, CA 94080, USA.
3 Department of Molecular and Cell Biology and Center for Integrative Genomics, University of California, Berkeley, CA 94720, USA.
4 Department of Cancer Targets, Genentech, South San Francisco, CA 94080, USA.
5 Department of Early Discovery Biochemistry, Genentech, South San Francisco, CA 94080, USA.
6 Department of Protein Chemistry, Genentech, South San Francisco, CA 94080, USA.
7 Department of Bioinformatics, Genentech, South San Francisco, CA 94080, USA.
8 Department of Structural Biology, Genentech, South San Francisco, CA 94080, USA.
9 Department of Antibody Engineering, Genentech, South San Francisco, CA 94080, USA.
10 Department of Pediatrics and Institute for Human Genetics, School of Medicine, University of California, San Francisco, CA 94143, USA.


Figure 1
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Fig. 1. Stimulation of the canonical Wnt pathway by RIPK4. (A) Heat maps show gene expression patterns determined by RNA sequencing in PA-1 cells transfected with empty vector, RIPK1, RIPK2, RIPK3, or RIPK4 for 48 hours or treated with 200 ng/ml Wnt3a for 16 hours. (Left) Hierarchical clustering of variance-stabilized expression values for genes with substantial changes in the treatment groups compared with the vector group. (Right) Pearson’s correlation coefficients between samples. (B) Expression of a TOPbrite luciferase reporter in 293 cells transfected with the constructs indicated and then cultured in the absence or presence of Wnt3a for 5 hours. Error bars represent the SEM of triplicate measurements. Results are representative of three independent experiments. (C) Cytosolic β-catenin protein abundance (top) and CTNNB1 gene expression (bottom) in 293T cells transfected with RIPK4. Error bars represent the SEM of triplicate measurements. (D) Cytosolic β-catenin in 293T cells transfected with Bartsocas-Papas syndrome RIPK4 mutants. (E) Cytosolic β-catenin in A2780 or COV434 cells transfected with control (Ctrl) or RIPK4 siRNAs for 60 hours and then treated with vehicle or Wnt3a for 2 hours.

 

Figure 2
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Fig. 2. Modulation of Wnt signaling in Xenopus by Ripk4. (A) Secondary axis formation in Xenopus embryos injected ventral vegetally with Ripk4 (1 ng), Xwnt8 (1 pg), or both. Two hundred sixty-nine embryos were examined in three independent experiments. Representative embryos are shown. (B) Length of Chordin expression along the blastopore lip of embryos injected at the four-cell stage with the RNAs indicated. Error bars represent the mean ± SEM of 108 embryos. Vegetal views of representative embryos are shown. au, arbitrary units. (C) Reverse transcriptase (RT) PCR analyses show the effect of Ripk4 morpholinos on Xwnt8-induced expression of Xnr3 in Xenopus animal cap cells. Control reactions contained RNA from stage 10.5 Xenopus embryos with or without RT. Amplification of epidermal keratin and ornithine decarboxylase (ODC) confirmed RNA integrity. (D) Engrailed2 expression (blue) after blastomere injection at the two-cell stage (Ripk4-MO1, Ripk4-MO2, sorted by fluorescence of a coinjected fluorescein tracer) or the four-cell stage (all others). Right, injected side; top, anterior. Red staining is due to a nuclear β-galactosidase tracer.

 

Figure 3
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Fig. 3. Phosphorylation of DVL by RIPK4 promotes canonical Wnt signaling. (A) In 293T cells, endogenous RIPK4 coimmunoprecipitated with endogenous DVL2 in the presence or absence of 200 ng/ml Wnt3a for 30 min. Control (Ctrl) and RIPK4 siRNAs confirmed RIPK4 antibody specificity. WCL, whole-cell lysates; IP, immunoprecipitation. (B) Endogenous RIPK4 coimmunoprecipitated with endogenous LRP6 in 293T cells treated with Wnt3a. (C) In vitro kinase assays using the RIPK4 kinase domain and the DVL2 DEP (or PDZ) domain as a substrate. M.W., molecular weight. (D) RIPK4-dependent phoshorylation of endogenous DVL2 in 293T cells treated with Wnt3a for 10 min. Asterisks indicate nonspecific bands. (E) Effect of RIPK4-GFP on DVL2-FLAG cellular distribution in HeLa cells. Cells containing DVL2 puncta were enumerated by counting 250 cells per condition. Scale bars, 10 μm.

 

Figure 4
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Fig. 4. Delayed growth of Wnt-dependent xenograft tumors depleted of RIPK4. (A) Box-and-whisker plots show RIPK4 mRNA abundance measured by microarray in human colorectal, ovarian, and melanoma samples. N, noncancerous tissues; C, cancer. P values were derived from Student’s t tests. (B) Western blots show increased RIPK4 and cytosolic β-catenin in human ovarian adenocarcinomas compared to noncancerous ovarian tissue samples. (C and D) NTERA-2 and HCT116 cells were transduced with lentiviral particles encoding RIPK4 or control (Ctrl) shRNAs and injected subcutaneously into athymic nude mice. (C) Representative tumor-bearing mice and their dissected tumors after 47 days are shown. (D) Graphs show tumor volumes; error bars represent the SEM (n = 10 mice).

 


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