Direct Inhibition of GSK3β by the Phosphorylated Cytoplasmic Domain of LRP6 in Wnt/β-Catenin Signaling

Abstract

Wnt/β-catenin signaling plays a central role in development and is also involved in a diverse array of diseases. Binding of Wnts to the coreceptors Frizzled and LRP6/5 leads to phosphorylation of PPPSPxS motifs in the LRP6/5 intracellular region and the inhibition of GSK3β bound to the scaffold protein Axin. However, it remains unknown how GSK3β is specifically inhibited upon Wnt stimulation. Here, we show that overexpression of the intracellular region of LRP6 containing a Ser/Thr rich cluster and a PPPSPxS motif impairs the activity of GSK3β in cells. Synthetic peptides containing the PPPSPxS motif strongly inhibit GSK3β in vitro only when they are phosphorylated. Microinjection of these peptides into Xenopus embryos confirms that the phosphorylated PPPSPxS motif potentiates Wnt-induced second body axis formation. In addition, we show that the Ser/Thr rich cluster of LRP6 plays an important role in LRP6 binding to GSK3β. These observations demonstrate that phosphorylated LRP6/5 both recruits and directly inhibits GSK3β using two distinct portions of its cytoplasmic sequence, and suggest a novel mechanism of activation in this signaling pathway.

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

“…2) (Mao et al 2001b). This critical but unresolved issue has fueled an alternative model in which LRP6-Axin interaction may be indirectly mediated by other factors such as GSK3 (Piao et al 2008). …”

“…This region is likely phosphorylated by CK1, including the nearby T1479 by CK1g (Davidson et al 2005). The functional significance of this S/T cluster is unclear, although one study suggests it as a GSK3-binding site upon CK1 phosphorylation (Piao et al 2008). Secondly, there exist seven mammalian CK1s (a, b, g1, g2, g3, d, and 1) (Price 2006).…”

“…To integrate recent findings of LRP6 phosphorylation and DVL acting primarily through LRP6 phosphorylation, a newer model has emerged in which phosphorylated PPPSPxS motifs directly inhibit GSK3 (Cselenyi et al 2008;Piao et al 2008;Wu et al 2009). This model is based on findings that LRP6 binds to GSK3 (Zeng et al 2005;Mi et al 2006) and that phosphorylated recombinant LRP6 cytoplasmic domain and, in fact, phosphorylated PPPSPxS peptides bind to and inhibit b-catenin phosphorylation by GSK3 in vitro and activate b-catenin signaling in vivo (Cselenyi et al 2008;Piao et al 2008;Wu et al 2009).…”

Potential cross-talk between (pro)renin receptors and Wnt/frizzled receptors in cardiovascular and renal disorders

“…21 Binding of the axin-GSK-3b complex to phosphorylated LRP6 inhibits GSK-3b activity allowing cytoplasmic b-catenin to stabilize. 36,37 Thus, GSK-3b in the destruction complex promotes b-catenin degradation, whereas GSK-3b-mediated phosphorylation of LRP6 at the plasma membrane in response to Wnt binding to Fz receptors contributes to the stabilization of b-catenin. Taken together, Wnt binds to Fz-LRP5/6 complex and induces Fz recruitment of Dvl, which in turn recruits the axin-GSK-3b complex to the membrane, and thereby promotes LRP5/6 phosphorylation to initiate an active b-catenin signaling.…”

Antitumor effect of novel small chemical inhibitors of Snail-p53 binding in K-Ras-mutated cancer cells

“…To confirm the inhibition effect of GN chemical on p53-Snail binding, we designed following experiment; in previous, we have found that Snail middle region, which is also responsible for binding with p53 (Lee et al, 2009c), is phosphorylated by CK1/GSK3b (Cano et al, 2000;Yook et al, 2006). Thus, addition of recombinant p53 could block the CK1/GSK3b-mediated Snail phosphorylation, whereas p53 did not affect CK1/GSK3b-mediated b-catenin phosphorylation (Piao et al, 2008;Ha et al, 2004;Supplementary Figure S5E). However, addition of quercetin, specific inhibitor against p53-Snail binding (Lee et al, 2009a, c), or GN25 could recover the CK1/GSK3b-mediated Snail phosphorylation in the presence of p53 (Figures 3f and g).…”