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First published online 23 January 2007
doi: 10.1242/jcs.03368

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Wnt-5a induces Dishevelled phosphorylation and dopaminergic differentiation via a CK1-dependent mechanism

Vítzslav Bryja^{* ¶,}, Gunnar Schulte^{* ¶,}, Nina Rawal^{* ¶}, Alexandra Grahn^{* ¶} and Ernest Arenas

Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden

View larger version (47K): [in this window] [in a new window]   Fig. 1. Wnt-5a and Wnt-3a activate Dvl in dopaminergic cells. (A) Time course of Wnt-5a- and Wnt-3a-induced activation of Dvl2 and Dvl3. SN4741 cells, treated with Wnt-3a (100 ng/ml) or Wnt-5a (100 ng/ml), were lysed after 0, 1, 5, 10, 15, 30, 60 and 120 minutes. (B) Both Wnt-5a and Wnt-3a activated Dvl2 and Dvl3, but only Wnt-3a activated -catenin after 2 hours of stimulation. (C) The effects of Wnt-5a or Wnt-3a (100 ng/ml) on Dvl were blocked by Fz8-CRD-conditioned medium. (A-C) Phosphorylation of Dvl isoforms were detected as phosphorylation-dependent mobility shifts of total Dvl2 and Dvl3. The position of dephosphorylated () and phosphorylated Dvl () is indicated. The activation of -catenin was determined by western blotting using antibodies against active (Ser33-Thr41-dephosphorylated) -catenin (ABC). Data are representative of at least three independent replicates.

View larger version (28K): [in this window] [in a new window]   Fig. 2. CK1 inhibition blocks Wnt-5a-induced phosphorylation of Dvl2 and Dvl3. SN4741 cells were treated with vehicle (control) or Wnt-5a (100 ng/ml) for 2 hours in the presence or absence of D4476 (100 µM) (A) or IC261 (10 µM) (C). Western blot analysis was performed as in Fig. 1. The position of dephosphorylated () and phosphorylated () Dvl is indicated. Antibodies against phospho-Ser45--catenin (CK1 target site), against active (Ser37-Thr41-dephosphorylated) -catenin (ABC) and total -catenin were used. (B) Scheme of phosphorylation sites of -catenin. Ser45 is a CK1 target site, which serves as a priming for sequential phosphorylation of Thr41, Ser37 and Ser33 by GSK3. (D) SN4741 cells were transfected with plasmids encoding Dvl2-Myc and either CK1 or kinase-dead CK1 (K>R) mutant. Phosphorylation of Dvl2-Myc was detected as a phosphorylation-dependent mobility shift by Myc- and Dvl2-specific antibodies. CK1 levels were monitored with a CK1 specific antibody. Data in A, C and D are representative of at least three independent replicates.

View larger version (55K): [in this window] [in a new window]   Fig. 3. Gene knockdown of CK1/ reduces the phosphorylation of Dvl2 induced by Wnt-5a. (A) SN4741 cells were transfected with control siRNA and three independent siRNAs against CK1 isoform. The efficiency of gene knockdown induced by individual siRNAs against CK1 was quantified in western blots. A non-specific protein, resulting in a band recognised by anti-CK1 antibody served as a loading control. Quantification (normalised to loading control) is shown below. (B,C) SN4741 cells were transfected with the indicated combinations of siRNAs, and treated with control, 50 and 100 ng/ml of Wnt-5a. The phosphorylation of Dvl isoforms was detected as phosphorylation-dependent mobility shift of total Dvl2. Data are representative of three (B) or two (C) independent experiments. The position of dephosphorylated () and phosphorylated Dvl () is indicated. The levels of CK1 and -actin were also determined to confirm the efficient gene knockout and the equal protein loading.

View larger version (25K): [in this window] [in a new window]   Fig. 4. CK1 is activated by Wnt-5a and binds Dvl2 in dopaminergic cells. (A) SN4741 cell lysates were immunoprecipitated with a control IgG or a CK1-specific antibody and endogenous or overexpressed CK1 and detected by western blotting. (B) Wnt-3a and Wnt-5a increased the phosphorylation of MBP as detected by western blotting using a specific antibody against phosphorylated serine. Cell lysates from control (1% CHAPS), Wnt-3a or Wnt-5a treated (100 ng/ml) SN4741 cells were immunoprecipitated with a CK1-specific antibody and subjected to kinase assay using MBP as a substrate. (C) Dvl2-Myc interacts with either CK1 (lane 2) or a kinase-dead CK1 (K>R) (lane 3) mutant in SN4741 cells, as assessed by immunoprecipitation and western blotting with Myc- and CK1-specific antibodies. Co-precipiated CK1 by the Myc antibody is indicated by . For all other panels: , phosphorylated Dvl2-Myc; , unphosphorylated Dvl2-Myc. (D) Dvl2-Myc interacts with endogenous CK1 as assessed by immunoprecipitation of SN4741 cells transfected with Dvl2-Myc only, by using Myc antibody-conjugated agarose beads (control, G-protein-conjugated beads). Data are representative of three independent replicates.

View larger version (40K): [in this window] [in a new window]   Fig. 5. CK1 and Wnt-5a mediate the changes in phosphorylation and cytoplasmic distribution of Dvl2. (A) The subcellular distribution of Dvl2-Myc in transfected SN4741 cells was assessed through an anti-Myc antibody by confocal microscopy. Four patterns were detected: (1) even distribution, and punctae of (2) small, (3) intermediate or, (4), large size. For a detailed description see the results section. (B) Co-transfection of Dvl2-Myc and CK1 or the kinase-dead CK1 (K>R) mutant resulted in a predominant even or punctate distribution of Dvl2-Myc, respectively. The arrow points to a cell transfected with Dvl2-Myc (Cy3, red) with no or low levels of exogenous CK1 (Cy2, green), serving as an internal control of the experiment. (C) Quantification of changes in the intracellular localization of Dvl2-Myc upon coexpression of CK1 or CK1 (K>R) as described in A and B. (D) D4476 (100 µM) blocks the phosphorylation dependent shift of Dvl2-Myc induced by different amounts (ng/well) of transfected CK1 in SN4741 cells. The position of dephosphorylated () and phosphorylated () Dvl2 is indicated. Data are representative of at least three independent replicates. (E-G) SN4741 cells were transfected with Dvl2-Myc and treated as indicated: (E) The general CK1 inhibitor D4476 (100 µM) and (F) the CK1/-specific inhibitor IC261 (20 µM) changed the subcellular localization of Dvl2-Myc to a predominant punctate distribution. This is in contrast with Wnt-5a treatment (100 ng/ml) (G), which leads to a more even distribution of Dvl2-Myc than in control. Data in C,E,F,G (n3) were statistically evaluated, Data represent the mean ± s.d., one-way ANOVA with Bonferroni post-tests (*P<0.05, **P<0.01, ***P<0.001).

View larger version (36K): [in this window] [in a new window]   Fig. 6. Wnt-5a cooperates with Wnt-3a in the phosphorylation of Dvl2, but inhibits Wnt-3a-induced activation of -catenin. (A) Increasing doses of Wnt-5a (50, 100, 300, 500, 1000 ng/ml) increase Dvl2 phosphorylation in vehicle (DMSO)-treated SN4741 cells and compete in the blockade of CK1 by D4476. (B-E) SN4741 cells were pretreated with 100 ng/ml of Wnt-5a (B,D) or 20 ng/ml of Wnt-3a (C,E) in the presence (D,E) or absence (B,C) of D4476 (100 µM) for 5 minutes. Subsequently, Wnt-3a (50, 100 and 200 ng/ml) or Wnt-5a (100, 200 and 500 ng/ml), was added for 2 hours. In A-E the phosphorylation of Dvl2 was detected by western blotting as phosphorylation-dependent mobility shift (dephosphorylated, ; phosphorylated, ). In B-E the activation of -catenin (ABC) was determined using an antibody against Ser33/37-dephosphorylated -catenin. Results shown in B-E were quantified using densitometry and either normalised to untreated control for ABC or shown as a ratio of phosphorylated:dephosphorylated for Dvl2. Duplicate experiments showed comparable results.

View larger version (52K): [in this window] [in a new window]   Fig. 7. CK1 inhibitors block the effects of Wnt-5a on dopaminergic differentiation. (A) Wnt-5a (100 ng/ml) increased the number of tyrosine-hydroxylase-positive (TH+) neurons in rat E14.5 ventral midbrain precursor cultures. However, addition of increasing doses of the chemical inhibitor of CK1 (D4476) reduced the numbers of TH+ neurons. (B) Double TH-Hoechst33258 immunostaining shows an increase in the number of TH immunoreactive neurons after treatment with Wnt-5a (100 ng/ml). Addition of 25 µM D4476 to Wnt-5a-treated cultures decreases the number of TH+ neurons after 3 days. Bar, 25 µm.

View larger version (15K): [in this window] [in a new window]   Fig. 8. Schematic model illustrating the role of CK1/ in the activation of Dvl by Wnt-3a and Wnt-5a. Both Wnt-3a and Wnt-5a bind Frizzled that by unknown mechanism activates CK1/, which in turn phosphorylates Dvl. The interaction of Dvl with additional pathway-specific signalling components (including co-receptors), would direct downstream signalling. In case of Wnt-3a the co-receptor might be Lrp5/6, whereas Ror2 or Knypek might be the co-receptors for Wnt-5a. This model predicts that, when both Wnt-3a and Wnt-5a are present, Frizzled and phosphorylated Dvl will compete for binding and activation of additional signalling components. The prevalent signalling direction would thus be determined by the recruitment of the additional signalling units by phosphorylated Dvl.