Wnt Signaling Requires Sequestration of Glycogen Synthase Kinase 3 inside Multivesicular Endosomes

Abstract

SUMMARY Canonical Wnt signaling requires inhibition of Glycogen Synthase Kinase 3 (GSK3) activity, but the molecular mechanism by which this is achieved remains unclear. Here we report that Wnt signaling triggers the sequestration of GSK3 from the cytosol into multivesicular bodies (MVBs), so that this enzyme becomes separated from its many cytosolic substrates. Endocytosed Wnt co-localized with GSK3 in acidic vesicles positive for endosomal markers. After Wnt addition, endogenous GSK3 activity decreased in the cytosol, and GSK3 became protected from protease treatment inside membrane-bounded organelles. Cryoimmuno electron microscopy showed that these corresponded to multivesicular bodies. Two proteins essential for MVB formation, HRS/Vps27 and Vps4, were required for Wnt signaling. The sequestration of GSK3 extended the half-life of many other proteins in addition to β-Catenin, including an artificial Wnt-regulated reporter protein containing GSK3 phosphorylation sites. We conclude that multivesicular endosomes are essential components of the Wnt signal transduction pathway.

Frizzled and LRP5/6 Receptors for Wnt/ -Catenin Signaling

“…(2) Because GSK3 levels do not overtly decrease during Wnt signaling, it requires a mechanism for GSK3 subsequently to escape MVBs, which normally shuttles the cargo to lysosomes for degradation. (3) It suggests that genes involved in MVB formation are required for Wnt signaling, for which supporting evidence exists in Xenopus but not in Drosophila (Piddini et al 2005;Rives et al 2006;Seto and Bellen 2006;Taelman et al 2010). (4) It predicts that most GSK3 substrates (in fact, 20% of cellular proteins) in addition to b-catenin are under Wnt regulation (Taelman et al 2010), thus departing from the prevailing view that roles of GSK3 in Wnt and other signaling (such as insulin) pathways are insulated from one another.…”

“…DIX oligomerization/polymerization is proposed to provide a DVL platform for dynamic assembly of protein -protein interactions of low avidity, such as between DVL and Axin (Schwarz-Romond et al 2007b), and is a main underpinning for the receptor "signalosome" hypothesis (Bilic et al 2007) (see below). However, some have suggested that the DVL "aggregates" (or "dots" under microscopes) represent endocytic vesicles (Capelluto et al 2002;Taelman et al 2010). The simplest model states that DIX and PDZ domains, but not DEP, are required for Wnt/b-catenin signaling, as seen in some overexpression studies.…”

“…A drastically different model was proposed recently, in which multivesicular body (MVB) formation after endocytosis of the Wnt receptor wraps GSK3 into the deep interior of the MVB, thereby physically separating GSK3 from its cytoplasmic substrates including b-catenin (Fig. 5D) (Taelman et al 2010). This "MVB inclusion" model has several features: (1) It requires MVBs to round up most of the cellular GSK3 proteins upon Wnt signaling, because gene deletions of Gsk3a and/or Gsk3b suggest that cells can perform Wnt signaling when two (or less than three) of the four Gsk3 alleles are deleted (Doble et al 2007).…”

Les contes de la voie Wnt

“…Le tube intestinal présente pourtant une architecture fascinante, nettement conditionnée par sa fonction, qui est assurée par un agencement de villosités et de courbures servant à maximiser la surface d'absorption des nutriments. Dans un article récem-ment publié dans la revue Nature [2], nous avons étudié la morphogenèse des boucles intestinales. Une démarche interdisciplinaire en biologie et en physique, à la fois expérimentale et théorique, nous a permis de montrer que l'enroulement intestinal a pour origine un équilibre tissulaire de forces élastiques.…”

“…Cependant le mécanisme exact par lequel la voie Wnt inhibe l'activité de cette kinase était jusqu'à présent mal compris. Dans une étude récente [2], nous avons montré que l'activation de la voie Wnt induit la séquestration de GSK3 à l'intérieur de vésicules intracellulaires appelées endosomes multivésiculaires (MVE).…”