Title: Wnt Signal Amplification via Activity, Cooperativity, and Regulation of Multiple Intracellular PPPSP Motifs in the Wnt Co-receptor LRP6
Abstract: 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. Doble B. Woodgett J. Wynshaw-Boris A. Hsieh J.C. He X. Development (Camb.). 2008; 135: 367-375Crossref PubMed Scopus (363) Google Scholar) and results in the inhibition of β-catenin phosphorylation and degradation. This leads to β-catenin accumulates in the cytoplasm and translocation into the nucleus to bind the LEF/TCF transcription factor and activates the transcription of Wnt target genes (1Clevers H. Cell. 2006; 127: 469-480Abstract Full Text Full Text PDF PubMed Scopus (4555) Google Scholar, 8Nusse R. Cell Res. 2005; 15: 28-32Crossref PubMed Scopus (816) Google Scholar). LRP5 and LRP6 are of critical importance in human diseases. Loss-of-function and gain-of-function mutations in LRP5 result in osteoporosis-pseudoglioma and high bone mass disease, respectively (9Little R.D. Carulli J.P. Del Mastro R.G. Dupuis J. Osborne M. Folz C. Manning S.P. Swain P.M. Zhao S.C. Eustace B. Lappe M.M. Spitzer L. Zweier S. Braunschweiger K. Benchekroun Y. Hu X. Adair R. Chee L. FitzGerald M.G. Tulig C. Caruso A. Tzellas N. Bawa A. Franklin B. McGuire S. Nogues X. Gong G. Allen K.M. Anisowicz A. Morales A.J. Lomedico P.T. Recker S.M. Van Eerdewegh P. Recker R.R. Johnson M.L. Am. J. Hum. 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Warman M.L. Cell. 2001; 107: 513-523Abstract Full Text Full Text PDF PubMed Scopus (1873) Google Scholar). Recently a LRP6 mutation has been shown to be associated with coronary artery disease and osteoporosis (12Mani A. Radhakrishnan J. Wang H. Mani A. Mani M.A. Nelson-Williams C. Carew K.S. Mane S. Najmabadi H. Wu D. Lifton R.P. Science. 2007; 315: 1278-1282Crossref PubMed Scopus (498) Google Scholar). These LRP5 and LRP6 mutations result in abnormal Wnt/β-catenin signaling, which likely underlies the pathogenesis of these disorders. LRP5/6 functions 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 binding, and its deletion in a LRP6 mutant, LRP6ΔC, results in a dominant negative receptor that binds Wnt but is unable to bind Axin (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, 13Tamai K. Semenov M. Kato Y. Spokony R. Liu C. Katsuyama Y. Hess F. Saint-Jeannet J.P. He X. Nature. 2000; 407: 530-535Crossref PubMed Scopus (1103) Google Scholar). The LRP6 extracellular domain has auto-inhibitory activity because its deletion in LRP6ΔN results in a constitutively activated 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 previous study we identified a conserved PPPSP motif, which is reiterated five times in LRP5, LRP6, and their Drosophila homologue Arrow, as the docking site for Axin-binding (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 showed that a prototypic PPPSP motif (motif A) (supplemental Fig. S1) functions as a module that is sufficient to transfer signaling activity to a heterologous receptor, in this case a truncated LDL receptor (LDLRΔN) (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 further showed that this prototypic PPPSP motif is phosphorylated and is capable of binding Axin in a phosphorylation-dependent manner (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 following study we determined that this prototypic PPPSP site is phosphorylated by GSK3, which primes the phosphorylation of a neighboring S residue at the +3 position (PPPSPXS) by CKI, resulting in maximal Axin binding (14Zeng X. Tamai K. Doble B. Li S. Huang H. Habas R. Okamura H. Woodgett J. He X. Nature. 2005; 438: 873-877Crossref PubMed Scopus (667) Google Scholar). In the full-length LRP6 receptor we found that mutations of all five PPPSP motifs (LRP6 m5) results in a dominant negative receptor that is unable to bind Axin and thus is analogous to LRP6ΔC (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, 14Zeng X. Tamai K. Doble B. Li S. Huang H. Habas R. Okamura H. Woodgett J. He X. Nature. 2005; 438: 873-877Crossref PubMed Scopus (667) Google Scholar). Although these studies imply the importance of PPPSP motifs in LRP6 function, the activity and regulation of each of these PPPSP motifs and the relationship among them in mediating Wnt/LRP6 signaling have not been characterized and are the subjects of this study. Plasmids—All LRP6, LRP6ΔN, and LDLRΔN constructs were tagged with the vesicular stomatitis virus G (VSVG) epitope tag and cloned into pCS2+ as previously described (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). For PPPSP motif transfer into LDLRΔN, the last 11 amino acids of LDLRΔN (residues 781-860) were replaced with PPPSP motifs of 15-25 amino acid residues from LRP6. Transferred residues from LRP6 are: A (residues 1483-1497), B (residues 1521-1541), C (residues 1562-1586), D (residues 1580-1600), and E (residues 1596-1613). Single amino acid substitutions were generated using a QuikChange mutagenesis kit (Stratagene). LRP6 full-length and LRP6ΔN (residues 1370-1613) or LRP6m5 (all five sites mutated) and LRP6ΔNm5 were previously described (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, 13Tamai K. Semenov M. Kato Y. Spokony R. Liu C. Katsuyama Y. Hess F. Saint-Jeannet J.P. He X. Nature. 2000; 407: 530-535Crossref PubMed Scopus (1103) Google Scholar) and were used to generate further mutant constructs. Hemagglutinin-tagged GSK3 in pCS2+ was used in Axin co-IP experiments. Wnt1-LNCX and empty LNCX vector were used in Wnt co-transfections. Dual Luciferase Assay—Mammalian cell transfections were done in 293T cells using FuGENE 6 and performed in triplicate. The cells were plated at 1 × 105/ml in 24-well plates and transfected the following day with a total of 0.4 mg of DNA/well (0.1 μg of TOPFLASH, 0.01 μg of thymidine kinase promoter Renilla, 0.09 μg of pCS2+ (empty vector), and up to 0.2 μg of experimental DNA constructs). The lysates were collected 48 h post-transfection and used with the dual luciferase reporter system (Promega). Firefly and Renilla luciferase activity was measured using the Wallac 1420 multilabel counter in 96-well plates. Normalized data expressed in relative luciferase units was averaged from triplicate assays, and the error bars reflect the standard deviations. Immunoblotting and Antibodies—Cleared lysates in 1× PLB (Promega) or Triton lysis buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100, 10% glycerol, 10 mm NaF, 10 mm Na3VO4, and protease inhibitor mixture) were run on a SDS-PAGE and transferred to Immobilon-P membrane (Sigma). No detergents were added to the lysis buffer used to analyze the cytoplasmic levels of β-catenin. Indirect immunochemistry using a secondary antibody conjugated with horseradish peroxidase was visualized using ECL reagents on HyBlot CL film or LAS-3000 imager (FujiFilm). The following commercially available antibodies were used: VSVG (P5D4 and V4888; Sigma), FLAG (M2; Sigma), β-catenin (C2206; Sigma), GSK3 (4G-1E; Upstate Biotechnology, Inc.), and α-tubulin (sc8053, Santa Cruz). The LRP6 antibody to the phosphorylated A motif (Ab1490) was used as previously described (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 C motif phospho-specific antibody (Ab1572) was produced by Covance (Denver, PA) using a phosphopeptide CEPVPPPPT*PRSQY (* indicates phosphorylated residue) conjugated to keyhole limpet hemocyanin. Ab1607 was raised to the phosphopeptide LRP6 pE KHLYPPPPS*PCTDSS. The bleeds were passed through a phosphopeptide column, followed by at least two passes through a non-phosphopeptide column and concentrated to 150 mg/ml. For co-IP experiments, GSK3β was co-transfected to increase PPPSP site phosphorylation, and anti-FLAG (M2) was used to precipitate Axin. Cre-mediated deletion of Gsk3α was performed in mouse embryonic fibroblasts (MEFs) derived from Gsk3β-/-, Gsk3αflox/flox mice as previously described (14Zeng X. Tamai K. Doble B. Li S. Huang H. Habas R. Okamura H. Woodgett J. He X. Nature. 2005; 438: 873-877Crossref PubMed Scopus (667) Google Scholar, 15Wei Q. Yokota C. Semenov M.V. Doble B. Woodgett J. He X. J. Biol. Chem. 2007; 282: 15903-15911Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Xenopus Animal Cap Assay—Xenopus laevis embryos were injected with mRNAs at the two-cell stage in the animal pole. Animal explants (animal caps) were dissected from stage 9 embryos and cultured until stage 25 for marker gene analysis. Semi-quantitative reverse transcription-PCR was performed as previously described (16Kato Y. Shi Y. He X. J. Neurosci. 1999; 19: 9364-9373Crossref PubMed Google Scholar). Anterior and posterior marker genes Otx2, En2, Krox20, HoxB9 (also known as XLHbox6), and EFIα were amplified using primer sequence obtained through the De Robertis lab home page or were previously used in another study (16Kato Y. Shi Y. He X. J. Neurosci. 1999; 19: 9364-9373Crossref PubMed Google Scholar). Alanine Scanning Mutational Analysis of the Prototypic PPPSP Motif (Motif A)—In our previous studies we showed that a 15-amino acid region of LRP6 encompassing the first PPPSP motif, referred to as motif A, is fully active when transferred to the truncated LDLR (LDLRΔN) in the absence of any Wnt ligand (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 this context we demonstrated that the PPPSP motif is phosphorylated, producing a distinct more slowly migrating band in electrophoresis followed by immunoblotting, and this phosphorylated PPPSP motif is specifically co-immunoprecipitated (co-IPed) with Axin (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). To investigate which residues might be critical for phosphorylation and activation of the Wnt/β-catenin pathway, we performed an alanine mutation scan (replacing each amino acid residue with alanine) using the prototypic LDLRΔN-PPPSP construct (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), which is referred to here as LDLRΔN-A to distinguish it from other PPPSP motifs (B to E); for residue Ala1492, glycine replacement was performed instead (Fig. 1A). We first examined the protein expression using an antibody to the VSVG tag and determined that they were expressed at comparable levels (Fig. 1B). In immunoblotting most of the LDLRΔN-A mutants exhibited a slower mobilizing band, which migrated similarly as the phosphorylated wild type (WT) LDLRΔN-A and was detected by the phospho-specific antibody Ab1490 (Fig. 1B, asterisks), although this upper band was greatly reduced in P1488A mutant. All LDLRΔN-A mutants, like the WT LDLRΔN-A, exhibited a more quickly migrating and presumably unphosphorylated band that differed in its position depending on the position of the alanine replacement. Consistent with our previous reports (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, 14Zeng X. Tamai K. Doble B. Li S. Huang H. Habas R. Okamura H. Woodgett J. He X. Nature. 2005; 438: 873-877Crossref PubMed Scopus (667) Google Scholar), S1490A and P1491A mutants did not display the slow migrating upper band and were not reactive to Ab1490. Therefore only the SP residues appear to be essential for phosphorylation at the PPPSP motif, consistent with GSK3 being a proline-directed kinase (17Frame S. Cohen P. Biochem. J. 2001; 359: 1-16Crossref PubMed Scopus (1282) Google Scholar). We also made two additional mutations in the prototypic motif A by replacing Ser1490 with threonine to resemble the conserved PPPTP motifs found in motifs B and C and replacing Pro1487 with cysteine to resemble the CPPSP of motif D (supplemental Fig. S1). A more slowly migrating band was present in both LDLRΔN-A variants; however, this upper band was greatly reduced in S1490T and was undetectable by Ab1490 (Fig. 1B), likely because of the change in the phosphorylated epitope. We examined the effect of alanine substitutions on the activation of the Wnt pathway using the TOPFLASH reporter, which is specifically activated by Wnt/β-catenin signaling (Fig. 1D). Using the WT LDLRΔN-A for comparison, we observed a significant reduction in activity for the alanine (or glycine) replacement of each of the core seven amino acid residues corresponding to PPPSPAT (Fig. 1D). Alanine replacements outside the core PPPSPXS motif had only minimal or small effects on the activity of the motif, with the exception of N1486A, which produced a hyperactive effect (Fig. 1D). Examination of the "B- or C-like" variant S1490T demonstrates that a Thr substitution of Ser activated the TOPFLASH as effectively (Fig. 1D), consistent with Ser/Thr conservation/equivalence at this position (Fig. 2A). However, the "D-like" P1487C substitution greatly diminished the activity similar to P1487A (Fig. 1D), indicating that a cysteine at this position (CPPSP) reduces the activity of the prototypic motif A. To validate our TOPFLASH reporter results, we measured the cytoplasmic β-catenin levels in cells expressing these LDLRΔN-A variant constructs (Fig. 1C). The increase in cytoplasmic β-catenin protein level corresponded well with the level of TOPFLASH activity, substantiating the importance of the core PPPSPXS motif. Functional Comparison of Individual PPPSP Motifs—Alignment of the five PPPSP motifs of LRP6 reveals the consensus of PPP(S/T)PX(S/T) (Fig. 2A), consistent with our experimental evidence from mutagenesis of motif A. However, the surrounding amino acids at each motif are unique and in some cases evolutionarily conserved (supplemental Fig. S1). To directly examine whether the other four PPPSP motifs have activities similar to the prototypic motif A, we transferred a short stretch of amino acid sequence spanning the LRP6 B, C, D, and E motifs to LDLRΔN, using the same approach as the LDLRΔN-A construct along with the alanine replacement of the Ser/Thr residue in each of the PPP(S/T)P motif. We examined the expression of LDLRΔN-B, -C, -D, and -E by immunoblotting and found that only the LDLRΔN-B construct yielded a more slowly migrating band, which presumably reflects phosphorylation (Fig. 2B). We suspected that LDLRΔN-C, -D, and -E were phosphorylated, but the phosphorylation in these contexts did not alter mobility during electrophoresis (see below). Motif B differs from the others by containing two internal threonines, although only the second (T1530) is conserved in the fly homologue Arrow (supplemental Fig. S1, arrowhead). To determine which threonine correlates with the upper band in LDLRΔN-B and whether motif B works similar as motif A, we made alanine mutations at each of the underlined residues PPTTPCS (Fig. 2C). We found that T1530A and P1531A mutants abolished the upper band, analogous to the SP mutations in LDLRΔN-A, whereas the T1529A mutant had no effect on the upper band (Fig. 2C). Using the TOPFLASH reporter assay we found that LDLRΔN-B, -C, and -E had comparable or greater signaling activities when compared with LDLRΔN-A (Fig. 2B). LDLRΔN-D had the weakest activity (Fig. 2B), consistent with the result from the D-like LDLRΔN-A P1487C mutant (Fig. 1, C and D). Mutation of the Ser/Thr residue inactivated each of the transferred PPPSP motifs (Fig. 2D), with the exception of motif B, whose mutation diminished but did not abolish its activity even when both Thr1529 and Thr1530 were simultaneously mutated to alanine (Fig. 2E). We showed previously that deletion of the LRP6 extracellular region (LRP6ΔN) results in a constitutively active receptor and that mutation of the Ser/Thr residue in all five PPPSP motifs (LRP6ΔNm5) abolishes its activity and prevents Axin binding (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, 14Zeng X. Tamai K. Doble B. Li S. Huang H. Habas R. Okamura H. Woodgett J. He X. Nature. 2005; 438: 873-877Crossref PubMed Scopus (667) Google Scholar). To independently compare the five PPPSP motifs in the context of the LRP6 intracellular region, we selectively restored the Ser/Thr residue in each of the five PPPSP motifs in LRP6ΔNm5. For example, LRP6ΔN-A contains only the active motif A, whereas the other four motifs remain mutated. We have noted previously that although LRP6ΔN expression levels are generally low (14Zeng X. Tamai K. Doble B. Li S. Huang H. Habas R. Okamura H. Woodgett J. He X. Nature. 2005; 438: 873-877Crossref PubMed Scopus (667) Google Scholar), LRP6ΔNm5 expresses well, as do LRP6ΔN-A, -B, -C, -D, and -E (Fig. 3C). We tested the activity of these LRP6ΔN constructs and found that LRP6ΔN-A, -B, -C, and -E strongly activated the TOPFLASH reporter, whereas the LRP6ΔN-D was significantly weaker (Fig. 3A). These results are fully consistent with those derived from the motif transfer to LDLRΔN and indicate that in isolation motifs A, B, C, and E are highly active, whereas motif D is significantly less active, in two distinct contexts in the stimulation of the Wnt/β-catenin pathway. We have previously demonstrated that LRP6ΔN is highly phosphorylated, resulting in multiple slower mobilizing bands that can be reduced with phosphatase treatment (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 examined the LRP6ΔN-A single active motif using VSVG antibody and an observed 33-kDa band that corresponds to the predicted molecular mass and two prominent upper bands at 40 and 47 kDa (Fig. 3B). Phosphatase treatment of LRP6ΔN-A reduced the 47 kDa down to a faster migrating species and removed phosphorylation at the A motif as assayed by Ab1490. To determine whether the observed TOPFLASH activity correlated with Axin binding, we examined the binding between Axin (FLAG-tagged) and each of LRP6ΔN-A, -B, -C, -D, and -E via co-IP. In these experiments LRP6ΔN-A, LRP6ΔN-B, and LRP6ΔNm5 were expressed at similar levels, whereas LRP6ΔN-C, LRP6ΔN-D, and LRP6ΔN-E were present at higher levels, and LRP6ΔN was expressed at a significantly lower level (Fig. 3C). Axin co-IPed LRP6ΔN strongly (lane 5) and each of LRP6ΔN-A, -B, -C, -D, and -E (lanes 6-10), but not LRP6ΔNm5 (Fig. 3C, lane 11); Axin co-IPed significantly less LRP6ΔN-D than LRP6ΔN-C and -E (lane 9 versus lanes 8 and 10), demonstrating that the weak signaling activity of LRP6ΔN-D correlates with poor Axin binding. We note that LRP6 intracellular domain is highly enriched with Ser/Thr residues (28%) and is heavily phosphorylated both within and outside PPPSP motifs (i.e. even when all Ser/Thr residues in PPPSP motifs are mutated (14Zeng X. Tamai K. Doble B. Li S. Huang H. Habas R. Okamura H. Woodgett J. He X. Nature. 2005; 438: 873-877Crossref PubMed Scopus (667) Google Scholar)). The smeary and multiple bands of LRP6ΔN and mutants are primarily caused by the heavy phosphorylation (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, 14Zeng X. Tamai K. Doble B. Li S. Huang H. Habas R. Okamura H. Woodgett J. He X. Nature. 2005; 438: 873-877Crossref PubMed Scopus (667) Google Scholar) (Fig. 3B). We further tested LRP6ΔN-A and -D activities in anterior-posterior patterning in X. laevis embryos. It is well established that graded Wnt/β-catenin signaling causes gradual posteriorization (18McGrew L.L. Lai C.J. Moon R.T. Dev. Biol. 1995; 172: 337-342Crossref PubMed Scopus (199) Google Scholar, 19Niehrs C. Nat. Rev. Genet. 2004; 5: 425-434Crossref PubMed Scopus (229) Google Scholar), which can be assayed via the expression of endogenous anterior-posterior markers. We used noggin (a BMP inhibitor) to induce anterior neural tissue in animal pole explants, as shown by the expression of an anterior marker Otx2 (supplemental Fig. S2, compare lanes 1 and 10). Injection of Wnt3a mRNA caused posteriorization, as demonstrated by the suppression of Otx2 and induction of Krox20 and HoxB9, a hindbrain and spinal cord marker, respectively (supplemental Fig. S2, compare lanes 1 and 9). LRP6ΔN mRNA had similar activity and shifted the cell fate to posterior in a dose-dependent manner (supplemental Fig. S2, lanes 2-5). We observed that LRP6ΔN-A shifted the cell fate to express posterior genes En2, Krox20 and HoxB9 while suppressing Otx2 expression (supplemental Fig. S2, lane 6). LRP6ΔN-D induced the expression of the posterior genes; however, it failed to suppress the anterior marker Otx2 (supplemental Fig. S2, lane 7), indicating that LRP6ΔN-D is also less active than LRP6ΔN-A in the Xenopus embryo. Generation of Phospho-motif C and -motif E Antibodies—Our study thus far suggests that each of the five PPPSP motifs exhibits signaling activities similar to the prototypic motif A that we initially characterized (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), although signaling strength varies. Given the functional importance of the Ser/Thr residue in these PPPSP motifs, it seems likely that Ser/Thr phosphorylation, like what we have observed in motif A (6Tamai K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. Mol. Cell. 2004; 13: 149-156Abstract Full Text Ful