Dpr Acts as a Molecular Switch, Inhibiting Wnt Signaling when Unphosphorylated, but Promoting Wnt Signaling when Phosphorylated by Casein Kinase Iδ/ε

The Wnt pathway is a key regulator of development and tumorigenesis. Dpr (Dact/Frodo) influences Wnt signaling in part through the interaction of its PDZ-B domain with Dsh's PDZ domain. Studies have shown that XDpr1a and its close relative, Frodo, are involved in multiple steps of the Wnt pathway in either inhibitory or activating roles. We found that XDpr1a is phosphorylated by casein kinase Iδ/ε (CKIδ/ε), an activator of Wnt signaling, in the presence of XDsh. Abrogating XDpr1a's ability to bind XDsh through mutation of XDpr1a's PDZ-B domain blocks CK1δ/ε's phosphorylation of XDpr1a. Conversely, XDsh possessing a mutation in its PDZ domain that is unable to bind XDpr1a does not promote XDpr1a phosphorylation. Phosphorylation of XDpr1a and XDsh by CKIδ/ε decreases their interaction. Moreover, the phosphorylation of XDpr1a by CKIδ/ε not only abrogates XDpr1a's promotion of β-catenin degradation but blocks β-catenin degradation. Our data suggest that XDpr1a phosphorylation by CKIδ/ε is dependent on the interaction of XDpr1a's PDZ-B domain with XDsh's PDZ domain, and that the phosphorylation state of XDpr1a determines whether it inhibits or activates Wnt signaling

XDsh promotes the phosphorylation of XDpr1a by CKIδ both <i>in vitro</i> and <i>in vivo</i>.

<p>A. XDsh induces a CKIδ-mediated phosphorylation of XDpr1a <i>in vitro</i>. Phosphorylation reactions were carried out in the presence of [³⁵S]methionine-XDpr1a (lanes 1 and 2) or [γ-³³P]ATP (lanes 3 and 4) and in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of XDsh and CKIδ. Lanes 3 and 4 contain the immunopellet from an anti-Myc immunoprecipiation of [γ-³³P]ATP-labeled Myc:XDpr1a. XDpr1a undergoes a gel-shift and shows increased incorporation of [γ-³³P]ATP in the presence of XDsh and CKIδ. B. CKIε phosphorylates XDpr1a <i>in vivo</i>. HEK293 cells transfected with Flag:XDpr1a alone or with CKIε and XDsh were metabolically labeled with [³²P]orthophosphoric acid prior to XDpr1a immunoprecipitation with anti-Flag antibodies. The cotransfection of CKIε and XDsh with XDpr1a induces a gel-shift and increases [³²P]orthophosphoric acid incorporation into XDpr1a. This result is representative of experiments repeated three times with similar results.</p

Phosphorylation of XDpr1a and XDsh by CKIδ reduces their interaction.

<p>A. Myc-tagged XDpr1a was immunoprecipitated in the presence of HA-tagged XDsh in the absence or presence of CKIδ. The presence of CKIδ reduced the coimmunoprecipitation of XDsh with XDpr1a. B. Quantitation of the relative coimmunoprecipitation (coIP) of XDsh with XDpr. The quantitation of the coimmunoprecipitation of XDsh with XDpr1a revealed that the presence of CKIδ reduced the interaction between XDpr1a and XDsh by approximately one-half when compared to the control. Error bars signify standard deviation.</p

Unphosphorylated XDpr1a promotes, but CKIδ-phosphorylated XDpr1a blocks, β-catenin degradation.

<p>Myc:XDpr1a was added to an <i>in vitro</i> β-catenin degradation assay after preincubation with or without CKIδ followed by anti-Myc immunoprecipitation. β-galactosidase preincubated with or without CKIδ was used as a control. Untreated XDpr1a promoted β-catenin degradation, whereas XDpr1a preincubated with CKIδ blocked β-catenin degradation. The data shown represent assays repeated six times.</p

XDsh promotes a CKIδ-mediated mobility shift of XDpr1a.

<p>A. XDpr1a exhibits a mobility shift in the presence of CKIδ and XDsh. <i>In vitro</i> transcribed and translated XDpr1a exhibits a mobility shift in the presence of purified CKIδ, and in the presence of <i>in vitro</i> transcribed and translated XDsh. The mobility shift is greater in the presence of both CKIδ and XDsh. XDsh also exhibits a mobility shift in the presence of CKIδ. The XDpr1a mobility shift present in lanes 2 and 3 is likely due to limiting amounts of endogenous XDsh and CKIδ in the reticulocyte lysates used in the <i>in vitro</i> transcription and translation, respectively. B. XDsh-mediated CKIδ phosphorylation of XDpr1a has little effect on XDpr1a abundance. Phosphorylation reactions were carried out as in A., but with the inclusion of luciferase as a loading control. XDpr1a and luciferase bands were quantitated, and the XDpr1a signal was normalized to that of luciferase. The luciferase-normalized signals were then normalized to that of XDpr1a alone. Error bars signify standard deviation (n = 3 trials).</p