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J. Biol. Chem., Vol. 283, Issue 23, 16115-16123, June 6, 2008

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Wnt Signal Amplification via Activity, Cooperativity, and Regulation of Multiple Intracellular PPPSP Motifs in the Wnt Co-receptor LRP6^*

From the F. M. Kirby Neurobiology Center, Children's Hospital Boston, Harvard Medical School, Boston, Massachusetts 02115

Low density lipoprotein receptor-related protein 6 (LRP6) and its homologue LRP5 serve as Wnt co-receptors that are essential for the Wnt/β-catenin pathway. Wnt activation of LRP6 leads to recruitment of the scaffolding protein Axin and inhibition of Axin-mediated phosphorylation/destruction of β-catenin. We showed that five conserved PPPSP motifs in the LRP6 intracellular domain are required for LRP6 function, and mutation of these motifs together abolishes LRP6 signaling activity. We further showed that Wnt induces the phosphorylation of a prototypic PPPSP motif, which provides a docking site for Axin and is sufficient to transfer signaling activity to a heterologous receptor. However, the activity, regulation, and functionality of multiple PPPSP motifs in LRP6 have not been characterized. Here we provide a comprehensive analysis of all five PPPSP motifs in LRP6. We define the core amino acid residues of a prototypic PPPSP motif via alanine scanning mutagenesis and demonstrate that each of the five PPPSP motifs exhibits signaling and Axin binding activity in isolation. We generated two novel phosphorylation-specific antibodies to additional PPPSP motifs and show that Wnt induces phosphorylation of these motifs in the endogenous LRP6 through glycogen synthase kinase 3. Finally, we uncover the critical cooperativity of PPPSP motifs in the full-length LRP6 by demonstrating that LRP6 mutants lacking a single PPPSP motif display compromised function, whereas LRP6 mutants lacking two of the five PPPSP motifs are mostly inactive. This cooperativity appears to reflect the ability of PPPSP motifs to promote the phosphorylation of one another and to interact with Axin synergistically. These results establish the critical role and a common phosphorylation/activation mechanism for the PPPSP motifs in LRP6 and suggest that the conserved multiplicity and cooperativity of the PPPSP motifs represents a built-in amplifier for Wnt signaling by the LRP6 family of receptors.

Received for publication, January 14, 2008 , and in revised form, March 17, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants F32 GM073357 (to B. T. M.) and RO1 GM74241 (to X. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S3.

¹ Present address: Dept. of Genetics, Case Western Reserve University, Cleveland, OH 44106.

² Scholar of the Leukemia and Lymphoma Society. To whom correspondence should be addressed: The F. M. Kirby Neurobiology Center, Children's Hospital Boston, Harvard Medical School, 320 Longwood Ave., Enders 461, Boston, MA 02115. Tel.: 617-919-2257; Fax: 617-730-1953; E-mail: xi.he{at}childrens.harvard.edu.

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