Inhibition of GSK3 by Wnt signalling – two contrasting models

Ciara Metcalfe 0 1 Mariann Bienz 0 0 MRC Laboratory of Molecular Biology , Hills Road, Cambridge CB2 0QH , UK 1 Present address: Genentech Inc , South San Francisco, CA 94080 , USA LRP CKI TCF Fig. 1. Signalosome-based Wnt/b-catenin signalling. According to the signalosome hypothesis, a Wnt ligand binds simultaneously to Frizzled (Fz) and LRP, clustering these transmembrane receptors and, thereby, triggering the recruitment and DIX-dependent polymerisation of Dishevelled (Dvl). Owing to an increase in avidity, polymerisation of Dishevelled enables it to bind to Axin (through direct heterotypic DIX interactions) and to recruit Axin together with GSK3 to LRP. LRP thus becomes a substrate for GSK3 and CK1 (- and -isoforms), which then phosphorylate the PPPSPxS motifs in the LRP cytoplasmic tail, enabling this motif to bind to the catalytic pocket of GSK3 (see Fig. 3), thereby blocking its activity towards b-catenin (b-cat). ce Unphosphorylated b-catenin is released and escapes ubiquitylation and n proteasomal degradation, which allows it to accumulate in the cytoplasm and ice nucleus. There, it functions as a co-activator of TCF to promote Wnt-induced S transcription which is facilitated by the chromatin-binding PygoBCL9 complex. l l e C fo Signalosome assembly l Wnt-induced signalosome formation at the plasma membrane is a run tshpeeckifeiycasltley,pittshahtigphrleycecdoensseinrvheibditDioInX odfomGSaiKn3(.BDiliicsheetvaell.l,e2d0a0n7d),, Jo have a pivotal role in the assembly of the signalosome. The DIX domain has a remarkable property in that it mediates reversible head-to-tail polymerisation, which is crucial for Dishevelled signalling activity (Schwarz-Romond et al., 2007a; SchwarzRomond et al., 2005). DIX-dependent homo-polymerisation of Dishevelled allows it to assemble large, yet highly dynamic, protein clusters that are detectable by immunofluorescence in live and fixed cells as distinct cytoplasmic puncta (Fig. 2Ai). These DIXdependent protein assemblies were initially identified as membranecontaining vesicle-like structures, and a putative phospholipid-binding VKEEIS motif was found to be required for formation and activity of Dishevelled puncta (Capelluto et al., 2002). Subsequently, however, two independent studies using live imaging and photobleaching discovered that Dishevelled puncta represent dynamic protein assemblies and ruled out that they are endocytic vesicles by demonstrating that they failed to colocalise with a large number of endocytic markers, membrane lipids and endocytic cargo (Schwarz-Romond et al., 2005; Smalley et al., 2005). A more recent study, which focused on non-canonical Wnt signalling, similarly failed to colocalise Dishevelled puncta with various vesicle and membrane markers (Nishita et al., 2010). Indeed, the VKEEIS motif turned out to be located on the DIX DIX interaction surface, as revealed by X-ray crystallography; this interface is essential for DIX-dependent homo-polymerisation of Dishevelled in vitro, and Dishevelled puncta formation and signalling in vivo (Liu et al., 2010; Schwarz-Romond et al., 2007a). The same interface also mediates heterotypic interaction with the DIX domain of Axin, which closely mimics the homotypic interaction (Fiedler et al., 2010); this heterotypic interaction between the two domains mediates recruitment of Axin by Dishevelled into the signalosome and, consequently, results in the activation of b-catenin (Schwarz-Romond et al., 2007b). Notably, the affinity of Dishevelled for its PDZ- and DEPdomain-binding partners, including Frizzled, is weak, typically with a Kd in the low micromolar range (Simons et al., 2009; Wong et al., 2003; Yu et al., 2010). It was, therefore, proposed that the DIX-dependent polymerisation of Dishevelled results in a high binding avidity for its signalling partners, owing to a transient high local concentration of protein interaction sites, which allows Dishevelled to bind efficiently to its low-affinity interacting partners (Fiedler et al., 2010; Schwarz-Romond et al., 2007a). DIXDIX interactions themselves are even weaker (with a Kd in the midmicromolar range) (Fiedler et al., 2010; Schwarz-Romond et al., 2007a), and are, thus, unlikely to occur spontaneously at physiological concentrations. Therefore, DIX-dependent polymerisation is likely to require an event triggered by Frizzled and is possibly amplified by Frizzled-dependent regulatory factors, as outlined in the next paragraph. The exact sequence of events during signalosome formation is still unclear but analysis of a non-canonical signalling pathway (involving Wnt5a, Fz7, Ror2 and Dishevelled) suggested that engagement (and possibly also the clustering) of Wnt receptors and co-receptors by their Wnt ligands triggers the polymerisation of Dishevelled (Nishita et al., 2010). Dishevelled polymers therefore acquire the necessary avidity to co-polymerise with Axin (and associated proteins), thereby overcoming the low affinity between the two DIX domains (Fiedler et al., 2010). Clustered Dishevelled then promotes the polymerisation-dependent phosphorylation of LRP6 by CK1 [- and -isoforms (Davidson et al., 2005; Zeng et al., 2005)], which is crucial for the functioning of the signalosome (Bilic et al., 2007; Zeng et al., 2008). The stability of Wnt signalosomes is essential for their signalling activity, and appears to depend on the interaction of additional proteins with the cytoplasmic tail of LRP6 (Metcalfe et al., 2010). Such stability factors might include phosphatidylinositol (4,5)bisphosphate, production of which is stimulated by Dishevelled acting on phosphatidylinositol kinases (Pan et al., 2008); in mammalian cells, this phospholipid is recognised by the signalosome-promoting protein AMER1 (also known as Amer1/WTX) (Tanneberger et al., 2011). DEP-domain-mediated interactions of Dishevelled with peripheral membrane proteins such as clathrin adaptors [in the case of non-canonical signalling (Yu et al., 2010)], and/or with lipid head groups of the plasma membrane itself (Simons et al., 2009), might also contribute to signalosome stability, although this has not been tested explicitly. Additional potential signalosome-stabilising factors have been reviewed elsewhere (Wu and Pan, 2010). Direct catalytic inhibition of GSK3 in the LRP signalosome: the biochemical model How signalosomes lead to a block of b-catenin phosphorylation by GSK3 remains an open question. A major advance towards answering this question was the discovery that, following Wnt signalling, the multiple conserved PPPSPxS motifs within the cytoplasmic tail of LRP which are crucial for Wnt signal transduction (Mao et al., 2001; Tamai et al., 2004) become dually phosphorylated by CK1 (- and -isoforms) following priming by GSK3 (Davidson et al., 2005; Zeng et al., 2005) (this dual Fig. 2. Protein assemblies capable of activating b-catenin signaling in the absence of Wnt. (AC)HeLa cells transfected with various Wnt signalling components (...truncated)