A dual-kinase mechanism for Wnt co-receptor phosphorylation and activation

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Signalling by the Wnt family of secreted lipoproteins has essential functions in development and disease1. The canonical Wnt/β-catenin pathway requires a single-span transmembrane receptor, low-density lipoprotein (LDL)-receptor-related protein 6 (LRP6)2,3,4, whose phosphorylation at multiple PPPSP motifs is induced upon stimulation by Wnt and is critical for signal transduction5. The kinase responsible for LRP6 phosphorylation has not been identified. Here we provide biochemical and genetic evidence for a ‘dual-kinase’ mechanism for LRP6 phosphorylation and activation. Glycogen synthase kinase 3 (GSK3), which is known for its inhibitory role in Wnt signalling through the promotion of β-catenin phosphorylation and degradation, mediates the phosphorylation and activation of LRP6. We show that Wnt induces sequential phosphorylation of LRP6 by GSK3 and casein kinase 1, and this dual phosphorylation promotes the engagement of LRP6 with the scaffolding protein Axin. We show further that a membrane-associated form of GSK3, in contrast with cytosolic GSK3, stimulates Wnt signalling and Xenopus axis duplication. Our results identify two key kinases mediating Wnt co-receptor activation, reveal an unexpected and intricate logic of Wnt/β-catenin signalling, and illustrate GSK3 as a genuine switch that dictates both on and off states of a pivotal regulatory pathway.

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Figure 1: GSK3 involvement in PPPSP phosphorylation.
Figure 2: Sequential phosphorylation of the PPPSPxS motif is induced by Wnt3a and required for LRP6 signalling.
Figure 3: Site I and site II phosphorylation by GSK3 and CK1 promotes LRP6 recruitment of Axin.
Figure 4: Membrane-associated GSK3 phosphorylates LRP6 and activates LRP6 and TCF/β-catenin signalling.


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We thank M. Semenov and B. MacDonald for suggestions and discussion. This work is supported in part by a grant from the NIH to X.H., who is a W. M. Keck Foundation Distinguished Young Scholar and a Leukemia and Lymphoma Society Scholar. X.Z., K.T., H.H. and R.H. are or were in part supported by postdoctoral fellowships from the Children's Hospital Boston, Uehara Memorial Foundation (Japan) and CIHR (Canada), and a training grant from the NIH, respectively. B.D. and J.W. are supported by the CIHR. J.W. is a Howard Hughes Medical Institute International Scholar. H.O. was in part supported by a grant from the NIH to Anjana Rao.

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Correspondence to Xi He.

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Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

A PPPSP kinase is associated with GST-LRP6C. (DOC 602 kb)

Supplementary Figure 2

A PPPSP kinase is associated with Axin. (DOC 218 kb)

Supplementary Figure 3

The specificity of Ab1493 for site II. (DOC 319 kb)

Supplementary Figure 4

The specificity of Ab1493 is independent of site I. (DOC 277 kb)

Supplementary Figure 5

Ck1ɛ-/- MEFs. (DOC 89 kb)

Supplementary Methods

Additional details of the methods used in this study. (DOC 25 kb)

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