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

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. 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. The canonical Wnt/β-catenin pathway controls cell proliferation and cell fate during embryogenesis and adult tissue homeostasis, and mutations that disrupt Wnt signaling contribute to a variety of diseases including cancer and osteoporosis (1Clevers H. Cell. 2006; 127: 469-480Abstract Full Text Full Text PDF PubMed Scopus (4555) Google Scholar). In the absence of an extracellular Wnt ligand, cytoplasmic β-catenin is phosphorylated and degraded by a complex that includes the scaffolding protein Axin, tumor suppressor protein APC, and the kinases GSK3 3The abbreviations used are: GSK, glycogen synthase kinase; LDL, low density lipoprotein; LRP, LDL receptor-related protein; CKI, casein kinase I; IP, immunoprecipitation; MEF, mouse embryonic fibroblast; WT, wild type; Ab, antibody; pE, phospho-motif E peptide; VSVG, vesicular stomatitis virus G. and CKI, preventing β-catenin-activated transcription in the nucleus (2Liu C. Li Y. Semenov M. Han C. Baeg G.H. Tan Y. Zhang Z. Lin X. He X. Cell. 2002; 108: 837-847Abstract Full Text Full Text PDF PubMed Scopus (1687) Google Scholar). The canonical pathway is initiated when a Wnt ligand binds to a member of the Frizzled serpentine receptor family and its co-receptor low density lipoprotein receptor-related protein 6 (LRP6) or a close relative such as LRP5 (3He X. Semenov M. Tamai K. Zeng X. Development (Camb.). 2004; 131: 1663-1677Crossref PubMed Scopus (873) Google Scholar, 4Macdonald B.T. Semenov M.V. He X. Cell. 2007; 131: 1204Abstract Full Text PDF PubMed Scopus (137) Google Scholar). This Wnt-induced Fz-LRP6 complex recruits Axin to the plasma membrane (5Mao J. Wang J. Liu B. Pan W. Farr 3rd, G.H. Flynn C. Yuan H. Takada S. Kimelman D. Li L. Wu D. Mol. Cell. 2001; 7: 801-809Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar, 6Tamai K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, 7Zeng X. Huang H. Tamai K. Zhang X. Harada Y. Yokota C. Almeida K. Wang J. B. J. He X. Development (Camb.). PubMed Scopus Google and results in the inhibition of β-catenin phosphorylation and This leads to β-catenin in the and the nucleus to the transcription and the transcription of Wnt (1Clevers H. Cell. 2006; 127: 469-480Abstract Full Text Full Text PDF PubMed Scopus (4555) Google Scholar, PubMed Scopus Google Scholar). LRP5 and LRP6 are of critical in and mutations in LRP5 in and J. M. C. B. L. S. K. Y. X. L. C. B. S. X. G. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. L. Wu D. K. J. 2002; PubMed Scopus Google Scholar, Y. G. S. Wang H. D. M. K. J. W. S. G. S. J. M. C. B. B. B. H. K. D. M. L. M. H. B. B. W. W. M. M. B. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). a LRP6 mutation been to with and osteoporosis J. Wang H. C. S. H. Wu D. 2007; PubMed Scopus Google Scholar). These LRP5 and LRP6 mutations in Wnt/β-catenin which the of these in the activation of the Wnt pathway via the recruitment of Axin to the plasma membrane (5Mao J. Wang J. Liu B. Pan W. Farr 3rd, G.H. Flynn C. Yuan H. Takada S. Kimelman D. Li L. Wu D. Mol. Cell. 2001; 7: 801-809Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar, 6Tamai K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar). The LRP6 cytoplasmic domain is essential for Axin and its in a LRP6 results in a receptor that binds Wnt is to Axin K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, K. Semenov M. Y. Liu C. Y. He X. PubMed Scopus Google Scholar). The LRP6 extracellular domain activity its in results in a receptor that binds Axin in the absence of Wnt ligand (5Mao J. Wang J. Liu B. Pan W. Farr 3rd, G.H. Flynn C. Yuan H. Takada S. Kimelman D. Li L. Wu D. Mol. Cell. 2001; 7: 801-809Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar, 6Tamai K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar). In a we a conserved PPPSP motif, which is five in and homologue as the docking site for K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar). We showed that a prototypic PPPSP motif as a that is sufficient to transfer signaling activity to a heterologous in a LDL receptor K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar). We further showed that prototypic PPPSP motif is phosphorylated and is of binding Axin in a K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar). In a we that prototypic PPPSP site is phosphorylated by which the phosphorylation of a the by CKI, in Axin binding X. Tamai K. B. Li S. Huang H. H. J. He X. PubMed Scopus Google Scholar). In the full-length LRP6 receptor we that mutations of all five PPPSP motifs results in a receptor that is to Axin and is to K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, X. Tamai K. B. Li S. Huang H. H. J. He X. PubMed Scopus Google Scholar). these the of PPPSP motifs in LRP6 function, the activity and of each of these PPPSP motifs and the in signaling have not been and are the of and with the vesicular stomatitis virus and as K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar). PPPSP motif transfer the amino of with PPPSP motifs of amino acid residues LRP6. residues LRP6 are: and E amino acid generated a mutagenesis LRP6 full-length and or five and K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, K. Semenov M. Y. Liu C. Y. He X. PubMed Scopus Google and used to further GSK3 in used in Axin and used in Wnt cell in 6 and in The in and the with a of of of of kinase of and to of The and used with the and activity the in in relative and the reflect the and in or and a and to membrane to the used to the cytoplasmic of β-catenin. a with or The antibodies and β-catenin GSK3 and The LRP6 to the phosphorylated motif used as K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar). The LRP6 motif by a phosphorylated to to the LRP6 The through a by two through a and to to PPPSP site and used to of in mouse embryonic as X. Tamai K. B. Li S. Huang H. H. J. He X. PubMed Scopus Google Scholar, Yokota C. Semenov M.V. B. J. He X. J. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). with the in the and for as Y. Y. He X. J. PubMed Google Scholar). and as and through the or used in another Y. Y. He X. J. PubMed Google Scholar). of the PPPSP we showed that a acid of LRP6 the PPPSP motif, to as motif is when to the in the absence of Wnt ligand K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar). In we that the PPPSP motif is a in by and phosphorylated PPPSP motif is with Axin K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar). which residues critical for phosphorylation and activation of the Wnt/β-catenin we an alanine mutation each amino acid with the prototypic K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (452) Google which is to as to PPPSP motifs to for We the protein an to the and that In of the mutants a which as the phosphorylated wild and by the in the a and that in its the of the alanine with K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, X. Tamai K. B. Li S. Huang H. H. J. He X. PubMed Scopus Google and mutants not display the and not to the residues to essential for phosphorylation the PPPSP motif, with GSK3 a kinase S. J. 2001; PubMed Scopus Google Scholar). 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PubMed Scopus Google We further and in in X. is that Wnt/β-catenin signaling PubMed Scopus Google Scholar, C. 2004; PubMed Scopus Google which via the of endogenous We used to tissue in as by the of an and of as by the of and of and a and and activity and the cell fate to in a We that the cell fate to and the of the to the that is in the of and E that each of the five PPPSP motifs exhibits signaling to the prototypic motif that we K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (452) Google signaling the of the in these PPPSP that we have in motif K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, X. Tamai K. B. Li S. Huang H. H. J. He X. PubMed Scopus Google is critical for the activity of these PPPSP we to the for motifs and not for or E that phosphorylation of motifs or E not that in the of We generated which PPPSP phosphorylation in motif K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar). We to antibodies that PPPSP phosphorylation in as We to and which to the phosphorylated motif and motif In phospho-motif not the we that the phospho-motif E not the We that to pE, binding in with binding We to via through a such the not We and the and a that in and phosphorylation of motifs and E in with and not or whereas with not or These results not demonstrate that and are phosphorylated the PPPSP motif suggest that is for motif E whereas is for phosphorylation motifs and to the in Wnt and E via have that motif in the wild LRP6 is phosphorylated in to Wnt K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, X. Tamai K. B. Li S. Huang H. H. J. He X. PubMed Scopus Google Scholar). the and we that motif and motif as phosphorylated by in to motif phosphorylation by K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, X. Tamai K. B. Li S. Huang H. H. J. He X. PubMed Scopus Google phosphorylation motifs and E of with Wnt and for We have that motif phosphorylation is by GSK3 X. Tamai K. B. Li S. Huang H. H. J. He X. PubMed Scopus Google Scholar). We motifs and E phosphorylated by GSK3 by In these with a phosphorylation of the endogenous LRP6 motifs and E in to However, in with a the which the the and the protein we in phosphorylation motifs and E The phosphorylation is to of the two the of or suggest that Wnt induces PPPSP phosphorylation motifs and and the phosphorylation of these motifs are via of PPPSP in of the PPPSP motifs that each motif, when to or to to Axin and the Wnt/β-catenin that a single PPPSP motif in these is sufficient for Wnt signaling However, the that five PPPSP motifs are conserved in and in and to that in the of the full-length LRP6 receptor PPPSP motif, multiplicity is for Wnt via two of motif in provides signaling a single when to K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar). a single PPPSP motif in the full-length LRP6 is sufficient to Wnt we one PPPSP motif in LRP6 which the to alanine in all five PPPSP motifs in the full-length LRP6 and is K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar). the motif and the motifs are the we that each of the LRP6 single motif LRP6 in that each not to Wnt signaling as signaling by and These results that a single PPPSP motif is not sufficient to in the of the full-length LRP6. further the of each PPPSP motif in we a single motif in the full-length PPPSP LRP6 a that motif is to in These the of each motif in the full-length LRP6. 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Tamai K. B. Li S. Huang H. H. J. He X. PubMed Scopus Google LRP6 a and LRP6 showed Axin binding to that of whereas LRP6 LRP6 and LRP6 binding to Axin LRP6 signaling with the Axin binding activity. in the activation of the Wnt/β-catenin pathway is Wnt-induced phosphorylation of LRP6 and (5Mao J. Wang J. Liu B. Pan W. Farr 3rd, G.H. Flynn C. Yuan H. Takada S. Kimelman D. Li L. Wu D. Mol. Cell. 2001; 7: 801-809Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar, 6Tamai K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar). we a PPPSP motif, which is five to in and homologue as a for Axin binding and signaling K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar). However, are the analysis of a single prototypic motif and the activity and of PPPSP motifs and have been or not In the we have all five PPPSP motifs and the alanine scanning mutagenesis we that the core of motif is of whereas amino acid residues core to we showed that each of the five PPPSP motifs in is to as a signaling in two to transfer Wnt signaling activity to a heterologous receptor and to signaling activity to the we generated antibodies to motifs and E and showed that to motif motifs and E of the endogenous LRP6 are phosphorylated in to Wnt in a we the of each PPPSP motif and in the full-length LRP6. we that LRP6 mutants lacking a single PPPSP motif have signaling whereas LRP6 mutants lacking two of the five PPPSP motifs are mostly and we that Axin binding with the signaling in the of a single PPPSP motif in or the full-length LRP6. These results together uncover and common of all five PPPSP motifs and the critical for in LRP6 signaling the these PPPSP motifs in and in the full-length LRP6. PPPSP we that motif the activity. This is by low activity of motif and the exhibits Axin binding and to in motif and the motifs is its the is conserved in LRP5 and motif in the full-length LRP6 appears to the critical its single mutation or mutation and another motif results in a LRP6 with compromised or activity. the motif exhibits signaling activity in to the full-length its single mutation results in LRP6 activity. However, motif with motifs mutations and another motif results in LRP6 signaling and results in suggest that the of of PPPSP motifs is E in E full-length LRP6. phosphorylation-specific and have of Wnt-induced phosphorylation of LRP6 motifs K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, X. Tamai K. B. Li S. Huang H. H. J. He X. PubMed Scopus Google and E the for motif GSK3 appears to Wnt-induced PPPSP phosphorylation motifs and E We have not been to antibodies that are for PPPSP phosphorylation motifs and D. However, motif is in the K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (452) Google in a that is when is to or we have that and of the five PPPSP motifs are not display a that we with phosphorylation such a is in the that a motif and is phosphorylated the to alanine in motif LRP6 in LRP6 signaling and motif in full-length LRP6 is we have that all five PPPSP motifs are phosphorylated by a common mechanism that is by Wnt is that alanine scanning mutagenesis the core amino acid residues for motif as This with X. Tamai K. B. Li S. Huang H. H. J. He X. 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In such an a phosphorylated PPPSP motif, by of its binding to the the phosphorylation of additional PPPSP and its mutation have the phosphorylation of PPPSP with which is and Wnt phosphorylation motifs and E and that the of PPPSP motifs a or E The the cooperativity PPPSP motifs in the full-length LRP6 appears to the results that a single PPPSP motif is sufficient to transfer signaling to and to the K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (452) Google that a to J. Huang G. M. C. 2007; PubMed Scopus Google Scholar). We that and is to the and is of the PPPSP Zhang and X. together multiple of PPPSP motifs in to the the cooperativity of multiple PPPSP motifs in in the full-length LRP6. We that is as to in to multiplicity of PPPSP motifs in which in LRP6 Wnt-induced as J. Huang G. M. C. 2007; PubMed Scopus Google Scholar). In results suggest that LRP6 cooperativity of PPPSP motifs in the phosphorylation of one another and in the recruitment of Axin, and cooperativity represents a built-in amplifier that is essential for Wnt signaling the receptor X. Huang H. Tamai K. Zhang X. Harada Y. Yokota C. Almeida K. Wang J. B. J. He X. Development (Camb.). PubMed Scopus Google Scholar, S. W. M. 2007; PubMed Scopus Google Scholar). in J. J. PubMed Scopus Google PPPSP in LRP6 We He and Zhang for and with