The Wnt canonical ligands elicit the activation of β-catenin transcriptional activity, a response dependent on, but not limited to, β-catenin stabilization through the inhibition of GSK3 activity. Two mechanisms have been proposed for this inhibition, one dependent on the binding and subsequent block of GSK3 to LRP5/6 Wnt coreceptor and another one on its sequestration into multivesicular bodies (MVBs). Here we report that internalization of the GSK3-containing Wnt-signalosome complex into MVBs is dependent on the dissociation of p120-catenin/cadherin from this complex. Disruption of cadherin-LRP5/6 interaction is controlled by cadherin phosphorylation and requires the previous separation of p120-catenin; thus, p120-catenin and cadherin mutants unable to dissociate from the complex block GSK3 sequestration into MVBs. These mutants substantially inhibit, but do not completely prevent, the β-catenin upregulation caused by Wnt3a. These results, besides elucidating how GSK3 is sequestered into MVBs, support this mechanism as cause of β-catenin stabilization by Wnt.

p120-catenin is an E-cadherin-associated protein that modulates E-cadherin function and stability. We describe here that p120-catenin is required for Wnt pathway signaling. p120-catenin binds and is phosphorylated by CK1ε in response to Wnt3a. p120-catenin also associates to the Wnt co-receptor LRP5/6, an interaction mediated by E-cadherin, showing an unexpected physical link between adherens junctions and a Wnt receptor. Depletion of p120-catenin abolishes CK1ε binding to LRP5/6 and prevents CK1ε activation upon Wnt3a stimulation. Elimination of p120-catenin also inhibits early responses to Wnt, such as LRP5/6 and Dvl-2 phosphorylation and axin recruitment to the signalosome, as well as later effects, such as β-catenin stabilization. Moreover, since CK1ε is also required for E-cadherin phosphorylation, a modification that decreases the affinity for β-catenin, p120-catenin depletion prevents the increase in β-catenin transcriptional activity even in the absence of β-catenin degradation. Therefore, these results demonstrate a novel and crucial function of p120-catenin in Wnt signaling and unveil additional points of regulation by this factor of β-catenin transcriptional activity different of β-catenin stability.

Activation of the Wnt pathway promotes the progressive phosphorylation of coreceptor LRP5/6 (low-density lipoprotein receptor-related proteins 5 and 6), creating a phosphorylated motif that inhibits glycogen synthase kinase 3␤ (GSK-3␤), which in turn stabilizes ␤-catenin, increasing the transcription of ␤-catenin target genes. Casein kinase 1 (CK1) kinase family members play a complex role in this pathway, either as inhibitors or as activators. In this report, we have dissected the roles of CK1 isoforms in the early steps of Wnt signaling. CK1 is constitutively bound to LRP5/6 through its interaction with p120-catenin and E-cadherin or N-cadherin and is activated upon Wnt3a stimulation. CK1␣ also associates with the LRP5/6/p120-catenin complex but, differently from CK1, only after Wnt3a addition. Binding of CK1␣ is dependent on CK1 and occurs in a complex with axin. The two protein kinases function sequentially: whereas CK1 is required for early responses to Wnt3a stimulation, such as recruitment of Dishevelled 2 (Dvl-2), CK1␣ participates in the release of p120-catenin from the complex, which activates p120-catenin for further actions on this pathway. Another CK1, CK1␥, acts at an intermediate level, since it is not necessary for Dvl-2 recruitment but for LRP5/6 phosphorylation at Thr1479 and axin binding. Therefore, our results indicate that CK1 isoforms work coordinately to promote the full response to Wnt stimulus.The Wnt pathway plays diverse roles in embryonic development and has been implicated in human diseases, including cancer (9). A key element in this pathway is the Ecadherin-associated protein ␤-catenin. When released from the junctional complex, ␤-catenin translocates to the nucleus, where it interacts with the Tcf family of transcriptional factors and regulates the expression of a variety of genes. The translocation of ␤-catenin to the nucleus is tightly controlled by the activity of a complex involved in ␤-catenin degradation. This complex includes the product of the tumor suppressor adenomatous polyposis coli (APC) gene, axin, and the associated Thr/Ser protein kinases, CK1␣ and glycogen synthase kinase 3␤ (GSK-3␤) (12). As a result of the activity of this complex, ␤-catenin is phosphorylated and degraded by the proteasome. The activity of the degradation complex is blocked by canonical Wnt factors, which activate a signaling pathway leading to the stabilization of cytosolic ␤-catenin (12, 13).Wnt ligands form a complex with low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) and Frizzled (Fz) receptors (7). Upon Wnt ligand binding, the LRP5/6 cytosolic domain gets phosphorylated in different residues by the action of several protein kinases (15). For, instance Thr1479 is phosphorylated by casein kinase 1␥ (CK1␥), a modification that is also dependent on the Fz-associated protein Dishevelled (Dvl) (1,4,28). Other members of this family, such as CK1ε and CK1␣, also contribute to the phosphorylation of LRP5/6 and Dvl (17,21,27). Phosphorylation of LRP5/6 promotes the recruitment of ...

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