Coombs GS et al. (2010), WLS-dependent secretion of WNT3A requires Ser20...

XB-ART-42042

J Cell Sci 2010 Oct 01;123Pt 19:3357-67. doi: 10.1242/jcs.072132.

Show Gene links Show Anatomy links

WLS-dependent secretion of WNT3A requires Ser209 acylation and vacuolar acidification.

Coombs GS , Yu J , Canning CA , Veltri CA , Covey TM , Cheong JK , Utomo V , Banerjee N , Zhang ZH , Jadulco RC , Concepcion GP , Bugni TS , Harper MK , Mihalek I , Jones CM , Ireland CM , Virshup DM .

???displayArticle.abstract???
Wnt proteins are secreted post-translationally modified proteins that signal locally to regulate development and proliferation. The production of bioactive Wnts requires a number of dedicated factors in the secreting cell whose coordinated functions are not fully understood. A screen for small molecules identified inhibitors of vacuolar acidification as potent inhibitors of Wnt secretion. Inhibition of the V-ATPase or disruption of vacuolar pH gradients by diverse drugs potently inhibited Wnt/β-catenin signaling both in cultured human cells and in vivo, and impaired Wnt-regulated convergent extension movements in Xenopus embryos. WNT secretion requires its binding to the carrier protein wntless (WLS); we find that WLS is ER-resident in human cells and WNT3A binding to WLS requires PORCN-dependent lipid modification of WNT3A at serine 209. Inhibition of vacuolar acidification results in accumulation of the WNT3A-WLS complex both in cells and at the plasma membrane. Modeling predictions suggest that WLS has a lipid-binding β-barrel that is similar to the lipocalin-family fold. We propose that WLS binds Wnts in part through a lipid-binding domain, and that vacuolar acidification is required to release palmitoylated WNT3A from WLS in secretory vesicles, possibly to facilitate transfer of WNT3A to a soluble carrier protein.

???displayArticle.pubmedLink??? 20826466
???displayArticle.pmcLink??? PMC2939803
???displayArticle.link??? J Cell Sci
???displayArticle.grants??? [+]

Species referenced: Xenopus
Genes referenced: actl6a ctnnb1 fgf8 nodal3.2 odc1 porcn ptgds sia1 tbx2 wls wnt1 wnt3a wnt8a

???attribute.lit??? ???displayArticles.show???

Fig. 2. Inhibition of vesicular acidification mimics loss of function of canonical and non-canonical mediated Wnt effects in vivo. (A–D) Bafilomycin and concanamycin A treatment results in a loss of Fgf8 transcripts in chick mb–r2 explants. These explants were isolated from HH10 chick embryos, embedded in collagen and treated with drug for 24 hours. Explants were probed for the expression of Fgf8 mRNA (A and B, blue; C and D, red) and Wnt1 mRNA (C and D, blue). Explants A and B were processed simultaneously, as were explants C and D. The differences in explant size are consistent with drug-induced inhibition of Wnt secretion and Wnt-dependent loss of fibroblast growth factor (FGF), as these are well-established mitogens in neural development (Canning et al., 2007). (E–K) Bafilomycin and concanamycin lead to a loss of axial patterning in Xenopus laevis embryos. Two-cell embryos were cultured for 24 hours alone (E) or in 1 µM bafilomycin (F,J) or 2 µM concanamycin (G,K). Drug treatment had no effects on embryos assessed at the 32-cell stage (E–G), demonstrating they are healthy and viable during the early stages of development. Drug-induced Wnt-dependent defects are seen as the embryos undergo gastrulation, as seen at the 24-hour time point (H–K). Control embryos (H,I) develop normally, whereas those cultured in the presence of bafilomycin or concanamycin A have disrupted axial development. (L–N) Treatment of whole embryos with 20 mM LiCl rescues the effect of treatment (as above) with 1 µM bafilomycin. (O) The Wnt targets Siamois and Xnr3 are induced by Xenopus Wnt8 but are reduced to 45% and 48%, respectively, in the presence of 1 µM bafilomycin. Animal caps were injected with control (not shown) or Xenopus Wnt8. Gene expression is measured relative to that of ODC, an internal control. (P) Treatment of whole embryos with vesicular acidification inhibitors does not abrogate all signaling events in the early embryo, as assayed by phosphorylation of Smad-1, a read-out of activation of BMP signaling. Actin serves as a loading control. (Q–T) Bafilomycin and concanamycin A treatment inhibits non-canonical-mediated convergent extension movements in Xenopus embryos. (Q) Animal caps dissected from control embryos do not undergo convergent extension movements compared with those treated with activin alone (R). Bafilomycin (S) and concanamycin A (T) treatment inhibits activin-mediated convergence and extension.

References [+] :

Arnoux, A structural basis for the pH-dependent xanthophyll cycle in Arabidopsis thaliana. 2009, Pubmed