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, 402 (3), 515-23

Post-translational Palmitoylation and Glycosylation of Wnt-5a Are Necessary for Its Signalling

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Post-translational Palmitoylation and Glycosylation of Wnt-5a Are Necessary for Its Signalling

Manabu Kurayoshi et al. Biochem J.

Abstract

Wnt-5a is a representative ligand that activates a beta-catenin-independent pathway in Wnt signalling. In the present paper, the roles of the post-translational modifications in the actions of Wnt-5a were investigated. We found that Wnt-5a is modified with palmitate at Cys104 and glycans at Asn114, Asn120, Asn311 and Asn325. The palmitoylation was not essential for the secretion of Wnt-5a, but was necessary for its ability to suppress Wnt-3a-dependent T-cell factor transcriptional activity and to stimulate cell migration. Wnt-5a activated focal adhesion kinase and this activation also required palmitoylation. Wild-type Wnt-5a induced the internalization of Fz (Frizzled) 5, but a Wnt-5a mutant that lacks the palmitoylation site did not. Furthermore, the binding of Wnt-5a to the extracellular domain of Fz5 required palmitoylation of Wnt-5a. These results indicate that palmitoylation of Wnt-5a is important for the triggering of signalling at the cell surface level and, therefore, that the lipid-unmodified form of Wnt-5a cannot activate intracellular signal cascades. In contrast, glycosylation was necessary for the secretion of Wnt-5a, but not essential for the actions of Wnt-5a. Thus the post-translational palmitoylation and glycosylation of Wnt-5a are important for the actions and secretion of Wnt-5a.

Figures

Figure 1
Figure 1. Palmitoylation of Wnt-5a protein
(A) Left-hand panel: Coomassie Brilliant Blue staining of an SDS/PAGE 10% (v/v) gel containing fractions from all steps of the purification. Right-hand panel: the purified Wnt-3a and Wnt-5a proteins were stained with Coomassie Brilliant Blue (CBB) or subjected to Western blotting and probed with anti-(Wnt-3a) or anti-(Wnt-5a) antibodies. (B) Purified Wnt-5a protein and CM containing Wnt-5a WT or Wnt-5a CA were subjected to the Triton X-114 phase-separation assay. An aliquot of each fraction was subjected to Western blotting and probed with anti-(Wnt-5a) antibody. Aq, aqueous phase; De, detergent phase. (C) Sequence alignment of Wnt proteins. The region of residues 98–119 of mouse Wnt-5a is aligned to mouse Wnt-1, Wnt-3a, Wnt-4, Wnt-5b, Wnt-7b, Wnt-11 and Drosophila Wg. The arrowhead shows the conserved cysteine.
Figure 2
Figure 2. Palmitoylation of Wnt-5a is required for its ability to suppress the β-catenin pathway
(A) After HEK-293 cells were stimulated with 40 ng/ml Wnt-3a or/and 150 ng/ml Wnt-5a for 1 h, the lysates were subjected to Western blotting and probed with the indicated antibodies. The blotting of GSK-3β was used as a loading control. (B) After NIH3T3 cells were stimulated with 40 ng/ml Wnt-3a or/and 150 ng/ml Wnt-5a for 1 h, the cells were stained with anti-(β-catenin) antibody. Scale bar, 10 μm. (C) HEK-293 (left-hand panel) and NIH3T3 (right-hand panel) cells stably expressing TOP-fos-Luc were incubated with 40 ng/ml Wnt-3a with or without the indicated amounts of Wnt-5a WT or Wnt-5a CA for 8 h. The luciferase activity was measured and expressed as the fold increase compared with the level observed in the cells without treatment. *, P<0.05. (D) After HEK-293 cells (293) or HEK-293 cells stably expressing β-cateninSA (293/SA-β-catenin) were transfected with pEF-BOS-hTcf-4E (0.1 μg) and TOP-fos-Luc (0.5 μg), they were incubated with the indicated amounts of Wnt-5a WT or Wnt-5a CA for 8 h. The luciferase activity was measured and expressed as the fold increase compared with the level observed in control HEK-293 cells. *, P<0.05. (E) NIH3T3 cells were incubated with serum-free (without FBS) or 2% (v/v) serum-containing (with 2% FBS) medium in the absence or presence of 100 ng/ml Wnt-3a and/or 300 ng/ml Wnt-5a, and then the cells were trypsinized and counted at each time point.
Figure 3
Figure 3. Palmitoylation of Wnt-5a is required for its ability to stimulate cell migration
(A) NIH3T3 cells were subjected to the transwell migration assay in the presence of purified Wnt-5a WT or Wnt-5a CA. *, P<0.05. (B) L cells containing empty vector or stably expressing Wnt-5a WT or Wnt-5a CA were subjected to the adhesion assay for the indicated time (*P<0.05). (C) and (D) L cells containing empty vector or stably expressing Wnt-5a WT or Wnt-5a CA were placed in non-coated (C) or Matrigel-coated (D) transwell chambers respectively. *, P<0.05.
Figure 4
Figure 4. Molecular mechanism by which Wnt-5a stimulates cell migration
(A) L cells stably expressing Wnt-5a WT or Wnt-5a CA were suspended in serum-free medium and were kept in suspension or replated onto fibronectin-coated dishes. After 1 h, the cells were lysed, subjected to Western blotting and probed with anti-(phospho-Tyr397-FAK) or anti-FAK antibodies. (B) Left-hand panel: HeLaS3 cells transfected with the control siRNA or Wnt-5a siRNA for 24 h were wounded. Scale bar, 200 μm. Right-hand panel: distance of migration was calculated by subtracting the width of the wound at 24 h from that at 0 h. The distance of migration with control siRNA was expressed as 100%. The relative migration distance with Wnt-5a siRNA was expressed as a percentage of control siRNA-treated cells. *, P<0.05. (C) Dynamics of GFP–paxillin in HeLaS3 cells treated with Wnt-5a siRNA were visualized by time-lapse fluorescence microscopy. Scale bar, 5 μm. (D) Rate constants of assembly and disassembly of GFP–paxillin shown in (C) were calculated. *, P<0.05. (E) HeLaS3 cells transfected with control siRNA or Wnt-5a siRNA were stained with FITC–phalloidin for visualizing F-actin 12 h after wounding. Then the cells (n=100) at the leading edge were counted and classified into three groups according to the appearance of F-actins: mainly stress fibres were observed; lamellipodium were visible clearly; neither stress fibres nor lamellipodium were clearly observed. Percentages of the respective groups in control siRNA- and Wnt-5a siRNA-treated cells are shown in the bar graph. *, P<0.05.
Figure 5
Figure 5. Palmitoylation is required for the binding to Fz5 and its internalization
(A) Left-hand panel: the indicated amounts of Wnt-5a WT and Wnt-5a CA were incubated with Fz5-CRD–IgG. Fz5-CRD–IgG was precipitated with Protein A–Sepharose, and the precipitates were subjected to Western blotting and probed with anti-(Wnt-5a) antibody. The arrows indicate non-specific bands of protein A crossreacted with anti-Wnt-5a antibody. The arrowhead indicates Wnt-5a. Right-hand panel: the amounts of precipitated Wnt-5a WT (○) and Wnt-5a CA (●) were quantified by densitometric tracing. The results are means of two independent experiments. (B) HEK-293 cells expressing Fz5–GFP were stimulated with CM containing Wnt-5a WT or Wnt-5a CA for the indicated periods of times and the fixed cells were directly processed for microscopy. Upper panel, confocal images; lower panel, quantification of internalized Fz5–GFP. Scale bar, 5 μm.
Figure 6
Figure 6. Glycosylation of Wnt-5a protein
(A) Purified Wnt-5a (75 ng) was treated with the indicated amounts of PNGase F and the samples were subjected to SDS/PAGE [10% (v/v) gels] and silver staining. (B) Upper panel: the indicated Wnt-5a mutants were expressed in HEK-293T cells and the lysates were subjected to Western blotting and probed with anti-(Wnt-5a) antibody. As a control, the cells expressing Wnt-5a WT were treated with tunicamycin. Lower panel: longer exposure of the same immunoblotting. The arrows indicate non-specific bands that were detected with anti-(Wnt-5a) antibody and the arrowhead a slowly migrating band of Wnt-5aN114Q/N311Q/N325Q. (C) Secretion of Wnt-5a. Wnt-5a WT or Wnt-5a NQ was expressed in HEK-293T cells and the lysates and ECM were probed by Western blotting with anti-(Wnt-5a) antibody. Wnt-5a in the CM was concentrated by precipitation with Blue Sepharose and the precipitates were probed with anti-(Wnt-5a) antibody. The expression levels of Wnt-5a in the lysate and ECM fractions can be compared directly and reflect the amount of Wnt-5a produced by confluent HEK-293T cells expressing Wnt-5a in a 60-mm-diameter dish. The expression level of Wnt-5a in the CM reflects the amount of Wnt-5a produced by one third of confluent HEK-293T cells expressing Wnt-5a. (D) HEK-293 cells stably expressing TOP-fos-Luc were incubated with 40 ng/ml Wnt-3a and 150 ng/ml Wnt-5a pre-treated or not pre-treated with PNGase F. The luciferase activity was measured and expressed as the fold increase as compared with the level observed in control cells. *, P<0.05. (E) NIH3T3 cells were subjected to the transwell migration assay in the presence of 300 ng/ml Wnt-5a pre-treated or not pre-treated with PNGase F. *, P<0.05.

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