Negative Regulation of LRP6 Function by Casein Kinase I ϵ Phosphorylation

Abstract

Wnt signaling acts in part through the low density lipoprotein receptor-related transmembrane proteins LRP5 and LRP6 to regulate embryonic development and stem cell proliferation. Up-regulated signaling is associated with many forms of cancer. Casein kinase I ϵ (CKIϵ) is a known component of the Wnt-β-catenin signaling pathway. We find that CKIϵ binds to LRP5 and LRP6 in vitro and in vivo and identify three CKIϵ-specific phosphorylation sites in LRP6. Two of the identified phosphorylation sites, Ser1420 and Ser1430, influence Wnt signaling in vivo, since LRP6 with mutation of these sites is a more potent activator of both β-catenin accumulation and Lef-1 reporter activity. Whereas Wnt3a regulates CKIϵ kinase activity, LRP6 does not, placing CKIϵ upstream of LRP6. Mutation of LRP6 Ser1420 and Ser1430 to alanine strengthens its interaction with axin, suggesting a mechanism by which CKIϵ may negatively regulate Wnt signaling. The role of CKIϵ is therefore more complex than was previously appreciated. Generation of active CKIϵ may induce a negative feedback loop by phosphorylation of sites on LRP5/6 that modulate axin binding and hence β-catenin degradation. Wnt signaling acts in part through the low density lipoprotein receptor-related transmembrane proteins LRP5 and LRP6 to regulate embryonic development and stem cell proliferation. Up-regulated signaling is associated with many forms of cancer. Casein kinase I ϵ (CKIϵ) is a known component of the Wnt-β-catenin signaling pathway. We find that CKIϵ binds to LRP5 and LRP6 in vitro and in vivo and identify three CKIϵ-specific phosphorylation sites in LRP6. Two of the identified phosphorylation sites, Ser1420 and Ser1430, influence Wnt signaling in vivo, since LRP6 with mutation of these sites is a more potent activator of both β-catenin accumulation and Lef-1 reporter activity. Whereas Wnt3a regulates CKIϵ kinase activity, LRP6 does not, placing CKIϵ upstream of LRP6. Mutation of LRP6 Ser1420 and Ser1430 to alanine strengthens its interaction with axin, suggesting a mechanism by which CKIϵ may negatively regulate Wnt signaling. The role of CKIϵ is therefore more complex than was previously appreciated. Generation of active CKIϵ may induce a negative feedback loop by phosphorylation of sites on LRP5/6 that modulate axin binding and hence β-catenin degradation. Wnts are secreted proteins that regulate morphogenesis by establishing cell polarity and stimulate cell proliferation and cell migration in response to Wnt concentration gradients in the embryo (1Moon R.T. Bowerman B. Boutros M. Perrimon N. Science. 2002; 296: 1644-1646Crossref PubMed Scopus (887) Google Scholar, 2Polakis P. Genes Dev. 2000; 14: 1837-1851Crossref PubMed Google Scholar). Wnt signaling is important both in development and adulthood, particularly for its role in regulating proliferation and differentiation of stem cells. Understanding Wnt signaling is also of interest due to its up-regulation in many cancers (2Polakis P. Genes Dev. 2000; 14: 1837-1851Crossref PubMed Google Scholar, 3Groden J. Thliveris A. Samowitz W. Carlson M. Gelbert L. Albertsen H. Joslyn G. Stevens J. Spirio L. Robertson M. Sargeant L. Krapcho K. et al.Cell. 1991; 66: 589-600Abstract Full Text PDF PubMed Scopus (2412) Google Scholar, 4Rubinfeld B. Robbins P. El-Gamil M. Albert I. Porfiri E. Polakis P. Science. 1997; 275: 1790-1792Crossref PubMed Scopus (1137) Google Scholar, 5Morin P.J. Sparks A.B. Korinek V. Barker N. Clevers H. Vogelstein B. Kinzler K.W. Science. 1997; 275: 1787-1790Crossref PubMed Scopus (3517) Google Scholar). Wnt signal transduction is initiated when the ligand interacts with two integral membrane proteins: a member of the frizzled family of seven pass transmembrane proteins and one of two co-receptors, the low density lipoprotein receptor-related proteins LRP5 and LRP6. This interaction recruits axin to LRP5/6 and leads to inhibition of β-catenin phosphorylation by GSK3 (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, 7Brennan K. Gonzalez-Sancho J.M. Castelo-Soccio L.A. Howe L.R. Brown A.M. Oncogene. 2004; 23: 4873-4884Crossref PubMed Scopus (91) Google Scholar, 8Mao J. Wang J. Liu B. Pan W. Farr III, 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). Consequently, β-catenin is not targeted to the proteasome and instead accumulates and translocates to the nucleus, where it interacts with TCF/LEF proteins to stimulate transcription of Wnt-responsive genes. Depending on cell type and type of receptors present, distinct Wnts can also activate additional signaling pathways, including cell migration via RhoA, and changes in calcium-mediated signaling (9Veeman M.T. Axelrod J.D. Moon R.T. Dev. Cell. 2003; 5: 367-377Abstract Full Text Full Text PDF PubMed Scopus (1163) Google Scholar, 10Endo Y. Wolf V. Muraiso K. Kamijo K. Soon L. Uren A. Barshishat-Kupper M. Rubin J.S. J. Biol. Chem. 2005; 280: 777-786Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 11Habas R. Dawid I.B. He X. Genes Dev. 2003; 17: 295-309Crossref PubMed Scopus (485) Google Scholar). The casein kinase I family plays both a positive and negative role in Wnt-β-catenin signaling. CKIα 4The abbreviations used are: CKI, casein kinase I; HA, hemagglutinin; VSVG, vesicular stomatitis virus G protein; LC, liquid chromatography; MS/MS, tandem mass spectrometry; HPLC, high pressure liquid chromatography; MBP, maltose-binding protein; Trx, thioredoxin; FL, full-length. interacts with axin and phosphorylates serine 45 of β-catenin. CKIα is a potent negative regulator of β-catenin, and its inhibition (e.g. by RNA interference) leads to decreased phosphorylation and marked accumulation of β-catenin, leading to activation of downstream signaling (12Amit S. Hatzubai A. Birman Y. Andersen J.S. Ben-Shushan E. Mann M. Ben-Neriah Y. Alkalay I. Genes Dev. 2002; 16: 1066-1076Crossref PubMed Scopus (595) Google Scholar, 13Liu 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, 14Yanagawa S. Matsuda Y. Lee J.S. Matsubayashi H. Sese S. Kadowaki T. Ishimoto A. EMBO J. 2002; 21: 1733-1742Crossref PubMed Scopus (158) Google Scholar). In overexpression screens, other members of the CKI family, CKIϵ and the closely related CKIδ, were found to be positive regulators of the pathway, acting upstream of axin and GSK3 and stabilizing β-catenin (15Matsubayashi H. Sese S. Lee J.S. Shirakawa T. Iwatsubo T. Tomita T. Yanagawa S. Mol. Cell. Biol. 2004; 24: 2012-2024Crossref PubMed Scopus (52) Google Scholar, 16Peters J.M. McKay R.M. McKay J.P. Graff J.M. Nature. 1999; 401: 345-350Crossref PubMed Scopus (385) Google Scholar, 17Sakanaka C. Leong P. Xu L. Harrison S.D. Williams L.T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12548-12552Crossref PubMed Scopus (189) Google Scholar, 18Kishida M. Hino Si S. Michiue T. Yamamoto H. Kishida S. Fukui A. Asashima M. Kikuchi A. J. Biol. Chem. 2001; 276: 33147-33155Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 19Gao Z.-H. Seeling J.M. Hill V. Yochum A. Virshup D.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1182-1187Crossref PubMed Scopus (192) Google Scholar). How CKIϵ regulates β-catenin accumulation is less clear. CKIϵ phosphorylates multiple proteins in the β-catenin degradation complex, including axin, Dvl (disheveled), and the APC (adenomatous polyposis coli) protein, and has previously been shown to destabilize the destruction complex, potentially through phosphorylation of axin or disheveled (19Gao Z.-H. Seeling J.M. Hill V. Yochum A. Virshup D.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1182-1187Crossref PubMed Scopus (192) Google Scholar, 20Lee E. Salic A. Kirschner M.W. J. Cell Biol. 2001; 154: 983-993Crossref PubMed Scopus (128) Google Scholar). Notably, the kinase activity of CKIϵ is also regulated by Wnt. In resting cells, both endogenous and overexpressed CKIϵ are inhibited by autophosphorylation of their own carboxyl-terminal autoinhibitory domain. The addition of Wnt ligand leads to rapid dephosphorylation and activation of CKIϵ. Similarly, withdrawal of Wnt ligand rapidly induces phosphorylation and inactivation of CKIϵ. The relevant substrates of activated CKIϵ are not known. Interestingly, overexpression of a constitutively active variant of CKIϵ (CKIϵ MM2) only modestly increases β-catenin stabilization compared with wild type CKIϵ, suggesting that activated wild type CKIϵ may function in downstream events distinct from its role in activation of canonical Wnt signaling (21Swiatek W. Tsai I.C. Klimowski L. Pepler A. Barnette J. Yost H.J. Virshup D.M. J. Biol. Chem. 2004; 279: 13011-13017Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 22Cong F. L. H. Mol. Cell. Biol. 2004; 24: PubMed Scopus (128) Google Scholar). events may stem from the of wild type CKIϵ to regulate its activity. LRP5 and LRP6 are that Wnts in with members of the frizzled family and are to induce β-catenin stabilization and canonical Wnt signaling J. S. Nature. 2000; PubMed Scopus Google Scholar, K. Semenov M. Y. R. Liu C. Y. F. J.P. He X. Nature. 2000; PubMed Scopus Google Scholar, M. K. L. S. D. E. A. S. Nature. 2000; PubMed Scopus Google Scholar). The are transmembrane proteins with with to the low density lipoprotein family and a domain. these their signal the membrane is not of LRP5/6 that their are constitutively active in Wnt suggesting that plays role signaling in the of Wnt (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, 7Brennan K. Gonzalez-Sancho J.M. Castelo-Soccio L.A. Howe L.R. Brown A.M. Oncogene. 2004; 23: 4873-4884Crossref PubMed Scopus (91) Google Scholar, 8Mao J. Wang J. Liu B. Pan W. Farr III, 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). known for signaling is Wnt-responsive phosphorylation of a of in the domain. a of is for and mutation of the in LRP6 to of the 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, 8Mao J. Wang J. Liu B. Pan W. Farr III, 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, G. Wu W. J. J. U. P. A. C. Nature. 2005; PubMed Scopus Google Scholar, X. K. B. Li S. H. R. H. J. He X. Nature. 2005; PubMed Scopus Google Scholar). is that of the axin complex to the membrane via LRP5/6 β-catenin from phosphorylation events that it for destruction J. Wang J. Liu B. Pan W. Farr III, 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, X. Semenov M. K. Zeng X. 2004; PubMed Scopus Google Scholar). the of CKIϵ to with and LRP5 and LRP6. CKIϵ not CKIα with and phosphorylates the of LRP5/6 in vitro and in in vitro phosphorylation and mass two distinct from previously identified CKI sites in LRP6 were identified CKIϵ Mutation of these CKIϵ sites both β-catenin accumulation and downstream signaling and is associated with binding of axin to LRP6. are with that CKIϵ the β-catenin destruction complex and that CKIϵ can both and negatively regulate the canonical Wnt pathway. and was from D. Wu of and and were by K. and He (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, 8Mao J. Wang J. Liu B. Pan W. Farr III, 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). LRP6 and LRP6 are forms of LRP6 their domain. or the of CKIϵ were previously (21Swiatek W. Tsai I.C. Klimowski L. Pepler A. Barnette J. Yost H.J. Virshup D.M. J. Biol. Chem. 2004; 279: 13011-13017Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, Virshup D.M. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). and were by and The for was a from and Lin The maltose-binding and were by of via and The for and of LRP6 were by Ser1420 Ser1430 to alanine wild type or wild type and a were to the of the and were from β-catenin and CKIϵ were from was from The has been previously and the of CKIϵ and K. A. M. G. Virshup D.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and were from was from the autoinhibitory and were in and A. A. Virshup D.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Wu N. K. Nature. 2005; PubMed Scopus Google Scholar). were from Science. was from Cell and embryonic and with reporter Wang Y. A. Williams J. C. M.W. L. W. Zhang K. J. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google a of K. were in with and and were in a and were in of a with were and to the was to with of CKIϵ, or were of LRP6 and of CKIϵ, and were in was where for to to cell was and the were with and in of of two of was used with I or were with and were and the were for and in were by a and were and and proteins were with of of or of Cell and were in for on to of were with in for or with the were by of in a and with and was previously (21Swiatek W. Tsai I.C. Klimowski L. Pepler A. Barnette J. Yost H.J. Virshup D.M. J. Biol. Chem. 2004; 279: 13011-13017Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, Virshup D.M. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). with was to that of were in In LRP6 type and were in E. of was to kinase and with or The kinase were for and by the addition of were on to was by was when and of were and with or and were to A. M. Mann M. Chem. PubMed Scopus Google Scholar). The were to with for liquid was a with The was to a with with J. Chem. 2000; PubMed Scopus Google Scholar). was to for J. 2002; PubMed Scopus Google Scholar). were by a mass a of The used was in in were the and of of β-catenin was C. Leong P. Xu L. Harrison S.D. Williams L.T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12548-12552Crossref PubMed Scopus (189) Google to the of Wnt activation in response to LRP6 in of a were with of LRP6. were with that of was the were with and with of with and was and were on for were and and were by for of was for β-catenin by and their were in with a was used to were with and and activity were the in a to the for activity was to activity. The of activity was by activity by that from with are S.D. of in CKIϵ with LRP5 and LRP6 in and the Wnt LRP5 and are both positive regulators of Wnt signaling. CKIϵ acts on LRP5/6 to regulate Wnt the of the proteins to in vivo was In CKIϵ with both LRP5 and LRP6 and The interaction with CKIϵ was since LRP5/6 not CKIϵ in the of and CKIϵ not LRP5/6 in the of interaction of LRP5/6 with CKIα was The interaction CKIϵ and LRP5/6 not to phosphorylation of the by CKIϵ, since of the CKIϵ on the interaction and CKIϵ wild type CKIϵ not CKIϵ with and LRP6 in of the of the of LRP5/6 a CKIϵ phosphorylation of the The H. Wang P.J. J. Biol. Chem. Full Text PDF PubMed Google has been found in the and in the transcription H. C. H. J. Virshup D.M. A. Mol. Cell. Biol. 2004; 24: PubMed Scopus Google Scholar, He Y. Virshup D.M. Science. 2001; PubMed Scopus Google Scholar). the of CKIϵ to LRP6 and to the interaction in the of LRP6 of was and from E. We also and a of LRP6 in which of the sites been to LRP6 and LRP6 concentration and in when with and kinase is constitutively active due to of the autoinhibitory of the of in vitro phosphorylation that CKIϵ of of when the is to suggesting that the interaction CKIϵ and LRP6 is and sites are In not with CKIϵ and was not a not LRP6 was with and to of the wild type in that CKIϵ not phosphorylation of the that LRP6 by GSK3 phosphorylation the can be by G. Wu W. J. J. U. P. A. C. Nature. 2005; PubMed Scopus Google Scholar, X. K. B. Li S. H. R. H. J. He X. Nature. 2005; PubMed Scopus Google The in vivo with the in vitro phosphorylation a and interaction CKIϵ and the of LRP6. was with CKIϵ, and the interaction was of the with and CKIϵ both with and LRP6 that the interaction is and that the of CKIϵ is not for the The was on kinase activity, since it was by of the CKIϵ in vitro phosphorylation of LRP6 and LRP6 with kinase or casein kinase a distinct and less with the that GSK3 may be the kinase that LRP5 J. Wang J. Liu B. Pan W. Farr III, 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 the mutation the Whereas the of LRP6 a with kinase or casein kinase the was not by mutation of the of LRP6 and the with CKIϵ with kinase or casein kinase to that by CKIϵ that the is due to CKIϵ and is not phosphorylation of LRP6. The of also that CKIϵ does not LRP6 for phosphorylation by kinase or casein kinase LRP6 in in a that CKIϵ and the of LRP6 in vitro and in vivo and that LRP6 is a for CKIϵ in CKIϵ also LRP6 phosphorylation in vivo, were with LRP6 of or with CKIϵ, or were with from multiple to of LRP6 of LRP6 with CKIα and not induce The of was on the of CKIϵ and was by the CKIϵ of the that CKIϵ phosphorylates LRP6 both in vivo and in vitro on sites distinct from the previously identified identify CKIϵ phosphorylation sites, LRP6 was in vitro with CKIϵ and to phosphorylation sites were Ser1430, and or and are to the sites targeted by GSK3 and the pass transmembrane of of the in LRP5/6 of Ser1430 in from to is in LRP6 is in LRP5 The high of of phosphorylation these The LRP5 and LRP6 is with distinct in vivo for the two Mutation of and the on LRP6 function and was not by for the the phosphorylation sites were in CKIϵ phosphorylation sites, the of serine alanine in LRP6 was type and LRP6 a in the of CKIϵ, the mutation of Ser1420 and Ser1430 to the these sites are it is that are of the is that these sites both phosphorylation other Wnt has multiple substrates in the Wnt pathway, and overexpression of CKIϵ has the of stabilizing β-catenin and signaling. We therefore the of mutation of these CKIϵ phosphorylation sites on LRP6 type and phosphorylation LRP6 were in a type LRP6 a in both the and the activity in the and wild type a of LRP6 of the was the wild type LRP6 was a more potent activator of the Wnt pathway, and its activity was and by and mutation of the identified phosphorylation the of the reporter LRP6 β-catenin more than wild type The in β-catenin and downstream signaling was not due to of the on LRP6 since the phosphorylation not the of LRP6 wild type and LRP6 This that phosphorylation of and negatively regulates Wnt signaling and a previously role for CKIϵ in of Wnt signaling. CKIϵ of and shown and that CKIϵ acts downstream of and the low activity of CKIϵ is by canonical Wnt (21Swiatek W. Tsai I.C. Klimowski L. Pepler A. Barnette J. Yost H.J. Virshup D.M. J. Biol. Chem. 2004; 279: 13011-13017Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, F. Virshup D.M. P. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). Wnt through interaction with cell receptors including members of the frizzled family and LRP6 also CKIϵ activity. type and phosphorylation of LRP6 were with CKIϵ, and kinase activity was in (21Swiatek W. Tsai I.C. Klimowski L. Pepler A. Barnette J. Yost H.J. Virshup D.M. J. Biol. Chem. 2004; 279: 13011-13017Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). of wild type and phosphorylation of LRP6 on CKIϵ activity LRP6 which is to activate also to activate CKIϵ. In to the of of LRP6 on CKIϵ activity, of the known CKIϵ activator Wnt3a with CKIϵ CKIϵ activity to (21Swiatek W. Tsai I.C. Klimowski L. Pepler A. Barnette J. Yost H.J. Virshup D.M. J. Biol. Chem. 2004; 279: 13011-13017Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). In a of CKIϵ with wild type and phosphorylation of LRP6 activated signaling to the reporter of with CKIϵ and wild type LRP6 activity that of with CKIϵ of the phosphorylation from to more than wild type LRP6. previously the LRP6 was to activate signaling in signaling were not due to changes in The of influence of LRP6 and on CKIϵ activation that LRP6 is downstream or to CKIϵ in the Wnt signaling pathway. The of Wnt signaling other to activate CKIϵ and LRP6 function may be additional mechanism to the of signaling. of Ser1420 and Ser1430 that axin is to the of LRP6 by Wnt signaling. CKIϵ is activated and can multiple including axin, and LRP6. CKIϵ activation can of (19Gao Z.-H. Seeling J.M. Hill V. Yochum A. Virshup D.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1182-1187Crossref PubMed Scopus (192) Google Scholar). We therefore the of CKIϵ and mutation of CKIϵ phosphorylation sites on the interaction of both and LRP6 with LRP6 LRP6 and axin were identified (e.g. and were by mutation of CKIϵ phosphorylation Ser1420 or Ser1430 and and by mutation of these sites and The of axin to LRP6 by mutation of these CKIϵ phosphorylation sites is with the stabilizing of the on β-catenin and their of reporter activation The of axin was not due to changes in LRP6 since of LRP6 were in of axin was also The of CKIϵ on the interaction was also In the of overexpressed CKIϵ, less axin with LRP6. The of CKIϵ on the complex was not by mutation of the CKI sites in previously that CKIϵ phosphorylates axin and additional proteins in the axin complex to modulate The for activation of the Wnt-β-catenin signaling is that Wnt with both a frizzled and via a with a LRP5/6 complex is the of LRP5/6 on multiple of a signaling that recruits axin to the membrane and the degradation of β-catenin. and CKIϵ and disheveled are and distinct downstream signaling regulating and cell are that a role of LRP5/6 is to regulate the β-catenin of the Wnt disheveled proteins important role in In the role of CKIϵ in regulating LRP5/6 phosphorylation and activity. We find that LRP5/6 and CKIϵ both in vivo and in suggesting that the interaction is in vitro phosphorylation of LRP6 by three distinct CKIϵ phosphorylation sites on LRP6 were of which were distinct from the previously identified signaling Whereas phosphorylation of by a CKIϵ phosphorylation of in vitro phosphorylation sites not their Mutation of two of the identified CKIϵ phosphorylation sites the activity of LRP6 by both β-catenin stabilization and activation of a with the also the binding of axin to LRP6. Whereas Wnt signaling CKIϵ, find that LRP6 does not activate CKIϵ. the that activated CKIϵ phosphorylates sites on LRP6 and that CKIϵ may be regulated by one function of the is of the of the Wnt LRP6 β-catenin pathway. CKIϵ activation negative feedback on other signaling that CKIϵ also to LRP6 phosphorylation and decreased Wnt that members of the CKI family Wnt signaling and other the of also that of phosphorylation are to and of phosphorylation sites on of the phosphorylation sites, Ser1430, was in both LRP5 and a was only in LRP6. This is with the distinct of LRP5 and LRP6. LRP5 and LRP6 are with distinct and of these has been associated with LRP6 is in and the LRP6 and that are a of Wnt J. S. Nature. 2000; PubMed Scopus Google Scholar). the LRP5 density and in and in LRP5 are associated with T. H. Y. H. Takada S. M. H. M. T. A. Yamamoto J. K. S. Y. H. M. M. S. K. M. J. Yamamoto Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar, J. J. L. A. Wu D. K. N. J. 2002; PubMed Scopus Google Scholar, Y. N. G. S. A.M. Wang H. T. D. M. K. J. W. S. G. S. J. M. P. C. B. A. B. B. T. A. H. K. A. D. M. E. T. L. M. H. E. B. B. A. W. W. M. M. B. T. R. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, M. W. Williams Mol. Cell. Biol. 2005; PubMed Scopus Google Scholar). The may not be due to that a phosphorylation is in LRP6 and that the of is in LRP5 is with other that these receptors are The negative role of that LRP5 and LRP6 may in their to by negative feedback via activation of CKIϵ. CKIϵ in that it has been to in multiple its including Wnt and signaling and of J. J. Dev. Biol. Google Scholar). the Wnt signaling pathway, CKIϵ is known to with and a of substrates β-catenin, axin, and that CKIϵ is a positive of CKIϵ has been to to that is and that CKIϵ can also function a negative regulator of Wnt signaling. of LRP5/6 on sites negatively regulates LRP5/6 activity by axin overexpression of CKIϵ also the interaction of axin with LRP6. (19Gao Z.-H. Seeling J.M. Hill V. Yochum A. Virshup D.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1182-1187Crossref PubMed Scopus (192) Google also that axin were by CKIϵ activity. CKIϵ in the Wnt in that a and in the with which it