Functional consequences of Wnt-induced dishevelled 2 phosphorylation in canonical and noncanonical Wnt signaling

Abstract

Dishevelled (Dvl) proteins are intracellular effectors of Wnt signaling that have essential roles in both canonical and noncanonical Wnt pathways. It has long been known that Wnts stimulate Dvl phosphorylation, but relatively little is known about its functional significance. We have previously reported that both Wnt3a and Wnt5a induce Dvl2 phosphorylation that is associated with an electrophoretic mobility shift and loss of recognition by monoclonal antibody 10B5. In the present study, we mapped the 10B5 epitope to a 16-amino acid segment of human Dvl2 (residues 594-609) that contains four Ser/Thr residues. Alanine substitution of these residues (P4m) eliminated the mobility shift induced by either Wnt3a or Wnt5a. The Dvl2 P4m mutant showed a modest increase in canonical Wnt/β-catenin signaling activity relative to wild type. Consistent with this finding, Dvl2 4Pm preferentially localized to cytoplasmic puncta. In contrast to wild-type Dvl2, however, the P4m mutant was unable to rescue Wnt3a-dependent neurite outgrowth in TC-32 cells following suppression of endogenous Dvl2/3. Earlier work has implicated casein kinase 1δ/ε as responsible for the Dvl mobility shift, and a CK1δ in vitro kinase assay confirmed that Ser(594), Thr(595), and Ser(597) of Dvl2 are CK1 targets. Alanine substitution of these three residues was sufficient to abrogate the Wnt-dependent mobility shift. Thus, we have identified a cluster of Ser/Thr residues in the C-terminal domain of Dvl2 that are Wnt-induced phosphorylation (WIP) sites. Our results indicate that phosphorylation at the WIP sites reduces Dvl accumulation in puncta and attenuates β-catenin signaling, whereas it enables noncanonical signaling that is required for neurite outgrowth.

FIGURE 1.

Deletion mapping of Wnt-induced phosphorylation sites in Dvl2 associated with mobility shift and mAb 10B5 epitope recognition. A, immunoblots showing Wnt-induced mobility shift of endogenous (left panels) and exogenous (right panels) Dvl2 proteins and the inability of mAb 10B5 to detect the mobility shifted forms. Rat2 fibroblasts transduced with control retroviral vector (left panels) or vector expressing Myc-tagged hDvl2 (right panels) were treated with Wnt3a CM for 2 h, and the lysates were processed for Western analysis with the indicated antibodies. Bands corresponding to hypophosphorylated (Dvl2) or phosphorylated and shifted Dvl2 (psDvl2) are indicated. The expected locations of psDvl and ps-hDvl2-Myc are indicated in the lower panels, but these bands are undetectable with mAb 10B5. Wnt treatment decreases the intensity of unshifted Dvl2 and hDvl2-Myc bands in the 10B5 immunoblots, reflecting the conversion of these to the phosphorylated forms detected by the other antibodies. B, schematic diagram of Dvl2 C-terminal deletion mutants. Sequences of full-length hDvl2 (1–736 residues) and truncated derivatives are represented, along with positions of the modular domains, DIX, b (basic region), PDZ, and DEP. Sequences present in the immunogen for mAb 10B5 (594–736) are indicated in gray. C, immunoblots of Myc-tagged hDvl2 full-length and deletion constructs transiently transfected in HEK293T cells. Proteins were detected with mAb 10B5 or anti-Myc. Molecular mass markers are indicated. Dvl2Δ127 is recognized by mAb 10B5, whereas Dvl2Δ135 is not. D, sequence comparison of human Dvl isoforms in region corresponding to deduced 10B5 epitope. The extent of epitope is demarcated by the box. Potential Ser/Thr phosphorylation sites within the epitope are shown as black letters. Epitope residues conserved in all three Dvl proteins are designated with asterisks. IB, immunoblotting.

FIGURE 2.

Site-directed mutagenesis identified individual residues that contribute to Dvl2 mobility shift. Each of the Ser/Thr residues and conserved Arg⁶⁰³ in the putative epitope of the Dvl2 mAb 10B5 were mutated as indicated, and Myc-tagged hDvl2 derivatives were stably expressed in Rat2 cells by retroviral transduction. After incubation for 2 h with Wnt3a CM or control CM, cell lysates were analyzed by immunoblotting with anti-Myc and Dvl2 10B5 antibodies. Bands corresponding to endogenous and exogenous Dvl2 and psDvl2 are indicated. The Wnt3a-induced mobility shift of Dvl2 was diminished for mutants S594A and S597A (upper panels), whereas in all cases recognition of endogenous and exogenous Dvl2 by 10B5 was reduced after Wnt3a treatment (lower panels). IB, immunoblotting.

FIGURE 3.

Wnt-dependent mobility shift of Dvl2 WT versus P4m and P3m mutants. A and B, Western blot analysis of Rat2 cells stably transduced with Myc-tagged hDvl2 WT, hDvl2 P4m, or empty retroviral vector and incubated overnight with Wnt3a CM (A) or Wnt5a CM (B). Exogenous Myc-tagged Dvl2 was detected with anti-Myc (upper panels). Dvl3 was probed for comparison (lower panels), providing a positive control for Wnt-induced phosphorylation of endogenous Dvl, and GSK3β served as a loading control. C, a similar analysis was performed with Rat2 cells stably expressing Myc-tagged hDvl2 WT, P4m, or P3m and treated with Wnt3a CM. D, immunoblot of FLAG-tagged mDvl2 WT and 4Pm in TC-32 cells with or without sFRP1 treatment. HSP70 served as a loading control. IB, immunoblotting.

FIGURE 4.

WIP mutant exhibits greater activity than hDvl2 WT in the β-catenin pathway. A, Topflash reporter assay in 293T cells. The cells were transiently transfected with Topflash reporter and empty vector (negative control), β-catenin cDNA (positive control), or varying amounts of hDvl2 WT or hDvl2 P4m expression constructs. The results were normalized to activity obtained with empty vector (50 ng/well). B, β-catenin stabilization in Rat2 cells stably transduced with hDvl2 WT or P4m vectors. The cells were treated with the indicated dilutions of Wnt3a CM for 2 h, and the lysates were blotted for β-catenin and GSK-3β; the latter was a loading control. At low doses of Wnt3a, cells expressing P4m were more sensitive to β-catenin stabilization than those expressing hDvl2 WT. The experiment was performed twice with similar results. C and D, C57MG cells were transiently transfected with empty vector, FLAG-tagged mDvl2 WT or P4m constructs and subsequently incubated with or without Wnt3a (50 ng/ml) for 6 h. Quantitative RT-PCR analysis of Axin2 (C) and RhoU transcripts (D) was normalized to β-actin transcript and expressed in arbitrary units where 1 unit = expression in cells transfected with empty vector and incubated in the absence of Wnt3a. The results are the means of triplicate measurements ± S.D. from one of two experiments that yielded similar data. IB, immunoblotting.

FIGURE 5.

Intracellular distribution of FLAG-tagged mDvl2 WT and mDvl2 P4m in TC-32 cells. A, representative micrographs of FLAG-mDvl2 distribution illustrating three patterns: cytoplasmic, cytoplasmic and puncta, and puncta predominant. B, cumulative quantitative analysis of distribution patterns for mDvl2 WT and P4m. N indicates the number of cells examined for each construct.

FIGURE 6.

Contrasting activity of FLAG-tagged mDvl2 WT versus mDvl2 P4m in neurite outgrowth assay. A, parental TC-32 cells and cells stably expressing FLAG-tagged mDvl2 WT or mDvl2 P4m were treated with siRNA reagents targeting luciferase (negative control) or Dvl2 and Dvl3, and 48 h later incubated with medium ± Wnt3a (100 ng/ml) for 3 h. Following fixation, neurite outgrowth was assessed in dozens of cells from each treatment group. The results from three separate experiments are indicated as the means ± S.D. Statistical significance of selected pairwise comparisons is indicated. B, Western blot analysis of Dvl2/3 expression in cells treated as described in A. The efficacy of knockdown of endogenous Dvl2/3 by siRNA is evident. HSP70 was a loading control. The positions of molecular mass markers are indicated. IB, immunoblotting.