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• W2013460988 abstract "Axin forms a complex with glycogen synthase kinase-3β (GSK-3β) and β-catenin and promotes GSK-3β-dependent phosphorylation of β-catenin, thereby stimulating the degradation of β-catenin. Because GSK-3β also phosphorylates Axin in the complex, the physiological significance of the phosphorylation of Axin was examined. Treatment of COS cells with LiCl, a GSK-3β inhibitor, and okadaic acid, a protein phosphatase inhibitor, decreased and increased, respectively, the cellular protein level of Axin. Pulse-chase analyses showed that the phosphorylated form of Axin was more stable than the unphosphorylated form and that an Axin mutant, in which the possible phosphorylation sites for GSK-3β were mutated, exhibited a shorter half-life than wild type Axin. Dvl-1, which was genetically shown to function upstream of GSK-3β, inhibited the phosphorylation of Axin by GSK-3β in vitro. Furthermore, Wnt-3a-containing conditioned medium down-regulated Axin and accumulated β-catenin in L cells and expression of Dvl-1ΔPDZ, in which the PDZ domain was deleted, suppressed this action of Wnt-3a. These results suggest that the phosphorylation of Axin is important for the regulation of its stability and that Wnt down-regulates Axin through Dvl. Axin forms a complex with glycogen synthase kinase-3β (GSK-3β) and β-catenin and promotes GSK-3β-dependent phosphorylation of β-catenin, thereby stimulating the degradation of β-catenin. Because GSK-3β also phosphorylates Axin in the complex, the physiological significance of the phosphorylation of Axin was examined. Treatment of COS cells with LiCl, a GSK-3β inhibitor, and okadaic acid, a protein phosphatase inhibitor, decreased and increased, respectively, the cellular protein level of Axin. Pulse-chase analyses showed that the phosphorylated form of Axin was more stable than the unphosphorylated form and that an Axin mutant, in which the possible phosphorylation sites for GSK-3β were mutated, exhibited a shorter half-life than wild type Axin. Dvl-1, which was genetically shown to function upstream of GSK-3β, inhibited the phosphorylation of Axin by GSK-3β in vitro. Furthermore, Wnt-3a-containing conditioned medium down-regulated Axin and accumulated β-catenin in L cells and expression of Dvl-1ΔPDZ, in which the PDZ domain was deleted, suppressed this action of Wnt-3a. These results suggest that the phosphorylation of Axin is important for the regulation of its stability and that Wnt down-regulates Axin through Dvl. Genetic and biochemical analyses have revealed that there are components that are structurally and functionally conserved in the Wnt signaling pathway among flies, frogs, and mammals (1Miller J.R. Moon R.T. Genes Dev. 1996; 10: 2527-2539Crossref PubMed Scopus (606) Google Scholar, 2Cadigan K.M. Nusse R. Genes Dev. 1997; 11: 3286-3305Crossref PubMed Scopus (2206) Google Scholar, 3Dale T.C. Biochem. J. 1998; 329: 209-223Crossref PubMed Scopus (433) Google Scholar). In mammals these include Wnt, frizzled, Dvl, GSK-3β, 1The abbreviations used are: GSK-3β, glycogen synthase kinase-3β; APC, adenomatous polyposis coli; GST, glutathioneS-transferase; MBP, maltose-binding protein; HA, hemagglutinin; PDZ, PSD95/Dlg/Zo-1; RalBP1, Ral-binding protein 1; kb, kilobase pair.1The abbreviations used are: GSK-3β, glycogen synthase kinase-3β; APC, adenomatous polyposis coli; GST, glutathioneS-transferase; MBP, maltose-binding protein; HA, hemagglutinin; PDZ, PSD95/Dlg/Zo-1; RalBP1, Ral-binding protein 1; kb, kilobase pair. β-catenin, and Lef/Tcf, which are homologous to the Drosophila proteins Wg, Dfz2, Dsh (Dishevelled), Shaggy, Armadillo, and Pangolin, respectively. The current model for the Wnt signaling pathway proposes that in the absence of Wnt, GSK-3β phosphorylates β-catenin, resulting in the degradation of β-catenin. In response to Wnt, Dvl antagonizes GSK-3β activity through an as yet unknown mechanism. This leads to the stabilization and the accumulation of β-catenin. The accumulated β-catenin translocates to the nucleus, associates with the transcriptional enhancers of the Lef/Tcf family (4Yost C. Torres M. Miller J.R. Huang E. Kimelman D. Moon R.T. Genes Dev. 1996; 10: 1443-1454Crossref PubMed Scopus (1009) Google Scholar, 5Behrens J. von Kries J.P. Kühl M. Bruhn L. Wedlich D. Grosschedl R. Birchmeier W. Nature. 1996; 382: 638-642Crossref PubMed Scopus (2560) Google Scholar, 6Molenaar M. van de Wetering M. Oosterwegel M. Peterson-Maduro J. Godsave S. Korinek V. Roose J. Destrée O. Clevers H. Cell. 1996; 86: 391-399Abstract Full Text Full Text PDF PubMed Scopus (1592) Google Scholar), and stimulates gene expression such as Myc (7He T. Sparks A.B. Rago C. Hermeking H. Zawel L. da Costa L.T. Morin P.J. Vogelstein B. Kinzler K.W. Science. 1998; 281: 1509-1512Crossref PubMed Scopus (4026) Google Scholar). Axin was originally identified as a product of mouse fusedgene (8Zeng L. Fagotto F. Zhang T. Hsu W. Vasicek T.J. Perry III W.L. Lee J.J. Tilghman S.M. Gumbiner B.M. Costantini F. Cell. 1997; 90: 181-192Abstract Full Text Full Text PDF PubMed Scopus (779) Google Scholar). fused carries recessive mutations that are lethal and that cause a duplication of the embryonic axis (9Gluecksohn-Schoenheimer S. J. Exp. Zool. 1949; 110: 47-76Crossref PubMed Scopus (73) Google Scholar, 10Perry III W.L. Vasicek T.J. Lee J.J. Rossi J.M. Zeng L. Zhang T. Tilghman S.M. Costantini F. Genetics. 1995; 141: 321-332PubMed Google Scholar). Injection of Axin into Xenopus embryos causes strong axis defects, and coexpression of Axin inhibits the Xwnt8-dependent axis duplication (8Zeng L. Fagotto F. Zhang T. Hsu W. Vasicek T.J. Perry III W.L. Lee J.J. Tilghman S.M. Gumbiner B.M. Costantini F. Cell. 1997; 90: 181-192Abstract Full Text Full Text PDF PubMed Scopus (779) Google Scholar). Thus, Axin is a negative regulator of the Wnt signaling pathway and inhibits axis formation. We have identified rat Axin (rAxin) and its homolog, Axil (forAxin-like), as GSK-3β-interacting proteins (11Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1087) Google Scholar, 12Yamamoto H. Kishida S. Uochi T. Ikeda S. Koyama S. Asashima M. Kikuchi A. Mol. Cell. Biol. 1998; 18: 2867-2875Crossref PubMed Scopus (172) Google Scholar). Conductin has been identified as a β-catenin-binding protein (13Behrens J. Jerchow B.-A. Würtele M. Grimm J. Asbrand C. Wirtz R. Kühl M. Wedlich D. Birchmeier W. Science. 1998; 280: 596-599Crossref PubMed Scopus (1095) Google Scholar) and is identical to Axil. We have found that both Axin and Axil bind not only to GSK-3β but also to β-catenin and that they promote GSK-3β-dependent phosphorylation of β-catenin (11Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1087) Google Scholar, 12Yamamoto H. Kishida S. Uochi T. Ikeda S. Koyama S. Asashima M. Kikuchi A. Mol. Cell. Biol. 1998; 18: 2867-2875Crossref PubMed Scopus (172) Google Scholar). We have also shown that the regulators of G protein signaling (RGS) domain of rAxin directly interacts with APC and that expression of rAxin in COS and SW480 cells stimulates the degradation of β-catenin (14Kishida S. Yamamoto H. Ikeda S. Kishida M. Sakamoto I. Koyama S. Kikuchi A. J. Biol. Chem. 1998; 273: 10823-10826Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar, 15Kishida M. Koyama S. Kishida S. Matsubara K. Nakashima S. Higano K. Takada R. Takada S. Kikuchi A. Oncogene. 1999; 18: 979-985Crossref PubMed Scopus (114) Google Scholar). Other groups have reported similar results (13Behrens J. Jerchow B.-A. Würtele M. Grimm J. Asbrand C. Wirtz R. Kühl M. Wedlich D. Birchmeier W. Science. 1998; 280: 596-599Crossref PubMed Scopus (1095) Google Scholar,16Sakanaka C. Weiss J.B. Williams L.T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3020-3023Crossref PubMed Scopus (282) Google Scholar, 17Hart M.J. de los Santos R. Albert I.N. Rubinfeld B. Polakis P. Curr. Biol. 1998; 8: 573-581Abstract Full Text Full Text PDF PubMed Google Scholar, 18Itoh K. Krupnik V.E. Sokol S.Y. Curr. Biol. 1998; 8: 591-594Abstract Full Text Full Text PDF PubMed Google Scholar, 19Nakamura T. Hamada F. Ishidate T. Anai K. Kawahara K. Toyoshima K. Akiyama T. Genes Cells. 1998; 3: 395-403Crossref PubMed Scopus (252) Google Scholar). Therefore, it appears that Axin family members down-regulate β-catenin. Axin enhances GSK-3β-dependent phosphorylation of APC in addition to β-catenin in vitro (17Hart M.J. de los Santos R. Albert I.N. Rubinfeld B. Polakis P. Curr. Biol. 1998; 8: 573-581Abstract Full Text Full Text PDF PubMed Google Scholar), and the phosphorylation of APC increases its binding to β-catenin (20Rubinfeld B. Albert I. Porfiri E. Fiol C. Munemitsu S. Polakis P. Science. 1996; 272: 1023-1026Crossref PubMed Scopus (1275) Google Scholar). Although Axin is also phosphorylated by GSK-3β directly, the phosphorylation of Axin does not affect its binding to GSK-3β and β-catenin in vitro (11Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1087) Google Scholar). These results indicate that β-catenin, APC, and Axin form a complex with GSK-3β and that the phosphorylation occurs efficiently in the complex. However, the physiological significance of the phosphorylation of Axin is not known. Therefore, we examined a role of the phosphorylation of Axin in the Wnt signaling pathway. Here we demonstrate that the phosphorylation of Axin by GSK-3β regulates its stability, that Dvl inhibits the GSK-3β-dependent phosphorylation of Axin, and that Wnt-3a down-regulates Axin through Dvl. Human Dvl-1 cDNA and a synthetic peptide substrate of GSK-3 (GSK peptide 1) were provided by Drs. B. Dallapiccola and G. Novelli (Vergata University, Rome, Italy) (21Pizzuti A. Amati F. Calabrese G. Mari A. Colosimo A. Silani V. Giardino L. Ratti A. Penso D.L.C. Palka G. Scarlato G. Novelli G. Dallapiccola B. Hum. Mol. Genet. 1996; 5: 953-958Crossref PubMed Scopus (61) Google Scholar) and C.W. Turck (University of California, San Francisco, CA) (22Murai H. Okazaki M. Kikuchi A. FEBS Lett. 1996; 392: 153-160Crossref PubMed Scopus (62) Google Scholar), respectively. The anti-Myc antibody was prepared from 9E10 cells. GST-GSK-3β was purified from Escherichia coli as described (22Murai H. Okazaki M. Kikuchi A. FEBS Lett. 1996; 392: 153-160Crossref PubMed Scopus (62) Google Scholar). GST fusion proteins and MBP fusion proteins were purified fromE. coli according to the manufacturer's instructions. L cells stably expressing HA-Dvl-1ΔPDZ(Dvl-1-(Δ283–336)) were produced by transfecting pCGN/Dvl-1ΔPDZ and pNeo. To prepare Wnt-3a-conditioned medium, L cells were transfected with pGK/Wnt-3a, and a number of stably transfected clones were established (23Shibamoto S. Higano K. Takada R. Ito F. Takeichi M. Takada S. Genes Cells. 1998; 3: 659-670Crossref PubMed Scopus (230) Google Scholar). The anti-Axin antibody was prepared in rabbits by immunization with a recombinant fragment of rAxin-(1–229). The anti-β-catenin and anti-GSK-3β antibodies were purchased from Transduction Laboratories (Lexington, KY). [γ-32P]ATP, [35S]methionine, and [35S]cysteine were purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK). Other materials were from commercial sources. pBSKS/rAxin, pMAL-c2/rAxin, pEF-BOS-Myc/rAxin, pBJ-Myc/rAxin, pGEX-2T/GSK-3β, and pBJ-Myc/RalBP1 were constructed as described (11Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1087) Google Scholar, 14Kishida S. Yamamoto H. Ikeda S. Kishida M. Sakamoto I. Koyama S. Kikuchi A. J. Biol. Chem. 1998; 273: 10823-10826Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar, 15Kishida M. Koyama S. Kishida S. Matsubara K. Nakashima S. Higano K. Takada R. Takada S. Kikuchi A. Oncogene. 1999; 18: 979-985Crossref PubMed Scopus (114) Google Scholar, 22Murai H. Okazaki M. Kikuchi A. FEBS Lett. 1996; 392: 153-160Crossref PubMed Scopus (62) Google Scholar, 24Matsubara K. Hinoi T. Koyama S. Kikuchi A. FEBS Lett. 1997; 410: 169-174Crossref PubMed Scopus (29) Google Scholar). To construct pMAL-c2/Dvl-1, pBSKS/Dvl-1 was digested with XbaI, and the 2.0-kb fragment encoding Dvl-1 was inserted into theXbaI-cut pMAL-c2. pBSKS/Dvl-1ΔPDZ was constructed as follows. The 0.42-kb fragment encoding Dvl-1-(337–476) with BamHI and PstI sites was synthesized by polymerase chain reaction, digested with BamHI andPstI, and inserted into the BamHI- andPstI-cut pBSKS to generate pBSKS/Dvl-1-(337–476). pBSKS/Dvl-1 was digested with PstI and HindIII, and the 0.6-kb fragment encoding Dvl-1-(477–670) was inserted into thePstI- and HindIII-cut pBSKS/Dvl-1-(337–476) to generate pBSKS/Dvl-1-(337–670). pBSKS/Dvl-1 was digested withXbaI and BamHI, and the 0.85-kb fragment encoding Dvl-1-(1–282) was inserted into the XbaI- andBamHI-cut pBSKS/Dvl-1-(337–670) to generate pBSKS/Dvl-1ΔPDZ. Thus, Dvl-1-(283–336) (the PDZ domain) was deleted in pBSKS/Dvl-1ΔPDZ. To construct pMAL-c2/Dvl-1ΔPDZ, pBSKS/Dvl-1ΔPDZ was digested with XbaI and HindIII, and the 1.9-kb fragment encoding Dvl-1ΔPDZ was inserted into theXbaI- and HindIII-cut pMAL-c2. pMAL-c2/Dvl-1ΔPDZ was digested with HindIII, blunted with Klenow fragment, and digested with XbaI. The 1.9-kb fragment encoding Dvl-1ΔPDZ was inserted into theXbaI- and SmaI-cut pCGN to generate pCGN/Dvl-1ΔPDZ. pBJ/Myc-rAxin322/326/330A was constructed as follows. The 0.65-kb fragment encoding rAxin-(182–401), in which Ser322, Ser326, and Ser330were mutated to Ala, was synthesized by polymerase chain reaction, digested with ClaI and XbaI, and inserted into the ClaI- and XbaI-cut pEF-BOS-Myc/rAxin-(1–181), which was obtained from pEF-BOS-Myc/rAxin to generate pEF-BOS-Myc/rAxin-(1–401,322/326/330A). To construct pEF-BOS-Myc/rAxin322/326/330A, pEF-BOS-Myc/rAxin was digested with XbaI, and the 1.3-kb fragment encoding rAxin-(402–832) was inserted into the XbaI-cut pEF-BOS-Myc/rAxin-(1–401,322/326/330A). To construct pBJ/Myc-rAxin322/326/330A, pEF-BOS-Myc/rAxin322/326/330A was digested withEcoRI, and the 2.6-kb fragment encoding Myc-rAxin322/326/330A was inserted into theEcoRI-cut pBJ-1. COS cells expressing Myc-rAxin or Myc-rAxin322/326/330A(35-mm-diameter dish) were metabolically labeled with32Pi (100 μCi/ml) in phosphate-free RPMI for 12 h in the presence or absence of 30 mm LiCl or 100 nm okadaic acid. The cells were lysed, and the lysates were immunoprecipitated with the anti-Myc antibody (11Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1087) Google Scholar). The immunoprecipitates were probed with the anti-Myc antibody and subjected to autoradiography. COS cells (35-mm-diameter dish) were transfected with pBJ-Myc/rAxin or pBJ/Myc-rAxin322/326/330A. After 48 h, pulse-chase analysis was performed as described (14Kishida S. Yamamoto H. Ikeda S. Kishida M. Sakamoto I. Koyama S. Kikuchi A. J. Biol. Chem. 1998; 273: 10823-10826Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar, 25Munemitsu S. Albert I. Souza B. Rubinfeld B. Polakis P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3046-3050Crossref PubMed Scopus (947) Google Scholar). Briefly, the cells were pulse-labeled with [35S]methionine and [35S]cysteine (50 μCi/ml) for 1 h at 37 °C. Then the cells were lysed immediately or at the indicated times following incubation with excess unlabeled methionine and cysteine in the presence or absence of 30 mm LiCl or 100 nmokadaic acid. The lysates were immunoprecipitated with the anti-Myc antibody, the precipitates were subjected to autoradiography, and then the densities of the labeled proteins were analyzed with a Fuji BAS 2000 image analyzer. 90 nm GST-GSK-3β was incubated with the indicated concentrations of MBP-rAxin and MBP-Dvl-1 in 30 μl of kinase reaction mixture (50 mm Tris/HCl, pH 7.5, 10 mm MgCl2, 1 mm dithiothreitol, and 50 μm [γ-32P]ATP (500–1500 cpm/pmol)) for 15 min at 30 °C. The samples were subjected to SDS-polyacrylamide gel electrophoresis followed by autoradiography, and then the radioactivities of the phosphorylated Axin were counted. The kinase activities of GSK-3β for GSK peptide 1 were measured as described (11Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1087) Google Scholar, 12Yamamoto H. Kishida S. Uochi T. Ikeda S. Koyama S. Asashima M. Kikuchi A. Mol. Cell. Biol. 1998; 18: 2867-2875Crossref PubMed Scopus (172) Google Scholar, 22Murai H. Okazaki M. Kikuchi A. FEBS Lett. 1996; 392: 153-160Crossref PubMed Scopus (62) Google Scholar). Confluent wild type L cells or L cells expressing HA-Dvl-1ΔPDZ (35-mm-diameter dish) were washed with Dulbecco's modified essential medium twice, and the indicated volume of Wnt-3a-conditioned medium, which was adjusted to a total volume of 700 μl with Dulbecco's modified essential medium, was added to the cells. After stimulation for 6 h, the cells were lysed in 100 μl of lysis buffer (11Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1087) Google Scholar), and the lysates (20 μg of protein) were probed with the anti-Axin and anti-β-catenin antibodies. rAxin was phosphorylated by GSK-3β directly in vitro, andSANDSEQQS330 of rAxin was one of the phosphorylation sites for GSK-3β (11Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1087) Google Scholar). First we examined whether rAxin is phosphorylated by GSK-3β in intact cells. Myc-rAxin was phosphorylated when COS cells were metabolically labeled with 32Pi (Fig.1 A). We tried to express Myc-rAxin322/326/330A, in which Ser322, Ser326, and Ser330 were mutated to Ala, in COS cells, but its protein level was lower than that of Myc-rAxin (wild type) (Fig. 1 A, lanes 3 and 4). Consistent with the protein level, the phosphorylation and apparent molecular weight of MycrAxin322/326/330A were reduced in comparison with Myc-rAxin (Fig. 1 A, lanes 1 and 2). Therefore, we used LiCl, which is known to be an inhibitor of GSK-3β (26Klein P.S. Melton D.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8455-8459Crossref PubMed Scopus (2056) Google Scholar, 27Stambolic V. Ruel L. Woodgett J.R. Curr. Biol. 1996; 6: 1664-1668Abstract Full Text Full Text PDF PubMed Google Scholar). It appeared that treatment of COS cells with LiCl decreased the phosphorylation of Myc-rAxin, whereas okadaic acid, a protein phosphatase 1 or 2A inhibitor, increased it (Fig. 1 A, lanes 5–7). However, these changes by LiCl and okadaic acid were also correlated with the protein level of Myc-rAxin (Fig. 1 A, lanes 8–10). LiCl decreased the protein level of Myc-rAxin in a dose-dependent manner (Fig. 1 B). Consistent with the previous observations (28Orford K. Crockett C. Jensen J.P. Weissman A.M. Byers S.W. J. Biol. Chem. 1997; 272: 24735-24738Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar), treatment of COS cells with LiCl resulted in the cytoplasmic accumulation of β-catenin (Fig. 1 B). Okadaic acid prevented the decrease of Myc-rAxin by LiCl (Fig. 1 B). LiCl did not affect the protein level of transfected Myc-RalBP1, an effector protein of small GTP-binding protein Ral (29Feig L.A. Urano T. Cantor S. Trends Biochem. Sci. 1996; 21: 438-441Abstract Full Text PDF PubMed Scopus (178) Google Scholar) or endogenous GSK-3β (Fig. 1 C). Therefore, the effect of LiCl that reduces rAxin is not nonspecific. These results suggest that the phosphorylation of rAxin is correlated with its stability. To investigate the stability of Axin by phosphorylation further, pulse-chase analysis was performed. Pulse-labeled Myc-rAxin in COS cells migrated slowly on SDS-polyacrylamide gel electrophoresis in a time-dependent manner (Fig.2 A), suggesting that Myc-rAxin was phosphorylated. Pulse-labeled Myc-rAxin did not exhibit a gel band shift and disappeared at 12 h in COS cells treated with LiCl (Fig.2 A). In contrast, okadaic acid enhanced the band shift and prevented the decay of pulse-labeled Myc-rAxin at 12 h (Fig.2 A). Pulse-labeled Myc-rAxin decreased gradually with a half-life of approximately 8 h, and pulse-labeled Myc-rAxin322/326/330A exhibited a shorter half-life (Fig.2 B). These results indicate that Axin is phosphorylated by GSK-3β in intact cells and that the phosphorylated form is more stable than the unphosphorylated form. Drosophila Dsh encodes a cytoplasmic protein of unknown biochemical function in the Wg signaling pathway (1Miller J.R. Moon R.T. Genes Dev. 1996; 10: 2527-2539Crossref PubMed Scopus (606) Google Scholar, 2Cadigan K.M. Nusse R. Genes Dev. 1997; 11: 3286-3305Crossref PubMed Scopus (2206) Google Scholar, 3Dale T.C. Biochem. J. 1998; 329: 209-223Crossref PubMed Scopus (433) Google Scholar). In mammals, dvl-1, -2, and -3genes have been isolated as homologs of Dsh (21Pizzuti A. Amati F. Calabrese G. Mari A. Colosimo A. Silani V. Giardino L. Ratti A. Penso D.L.C. Palka G. Scarlato G. Novelli G. Dallapiccola B. Hum. Mol. Genet. 1996; 5: 953-958Crossref PubMed Scopus (61) Google Scholar, 30Sussman D.J. Klingensmith J. Salinas P. Adams P.S. Nusse R. Perrimon N. Dev. Biol. 1994; 166: 73-86Crossref PubMed Scopus (151) Google Scholar, 31Klingensmith J. Yang Y. Axelrod J.D. Beier D.R. Perrimon N. Sussman D.J. Mech. Dev. 1996; 58: 15-26Crossref PubMed Scopus (96) Google Scholar). It has been shown that Dsh antagonizes shaggy, a fly homolog of GSK-3β, in the Wg signaling pathway (2Cadigan K.M. Nusse R. Genes Dev. 1997; 11: 3286-3305Crossref PubMed Scopus (2206) Google Scholar, 3Dale T.C. Biochem. J. 1998; 329: 209-223Crossref PubMed Scopus (433) Google Scholar), and that overexpression of Dvl-1 in Chinese hamster ovary cells inhibits GSK-3 activity as measured by the GSK-3-mediated phosphorylation of tau proteins (32Wagner U. Brownless J. Irving N.G. Lucas F.R. Salinas P.C. Miller C.C.J. FEBS Lett. 1997; 411: 369-372Crossref PubMed Scopus (49) Google Scholar). However, little is known about the biochemical pathway leading from Dvl to GSK-3β. Therefore, we examined whether Dvl-1 affects the phosphorylation of rAxin by GSK-3β in vitro. MBP-Dvl-1 itself was not phosphorylated by GST-GSK-3β (data not shown). GST-GSK-3β phosphorylated MBP-rAxin in a time-dependent manner (11Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1087) Google Scholar) (Fig. 3 A). MBP-Dvl-1 inhibited this phosphorylation of MBP-rAxin (Fig. 3 A). This inhibitory activity of MBP-Dvl-1 was dose-dependent, and MBP alone did not inhibit the GST-GSK-3β-dependent phosphorylation of MBP-rAxin (Fig. 3 B). Dvl has the PDZ domain, and disruption of the PDZ domain abolishes its activity in the Wg-Armadillo pathway and in the Xenopus axis induction assay (33Sokol S.Y. Curr. Biol. 1996; 6: 1456-1467Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar, 34Yanagawa S. van Leeuwen F. Wodarz A. Klingensmith J. Nusse R. Genes Dev. 1995; 9: 1087-1097Crossref PubMed Scopus (339) Google Scholar). Deletion of the PDZ domain from Dvl-1 (MBP-Dvl-1ΔPDZ) greatly reduced its activity to inhibit the phosphorylation of MBP-rAxin by GST-GSK-3β (Fig. 3 B). Inhibition of the phosphorylation of MBP-rAxin by MBP-Dvl-1 was not recovered even though the amounts of MBP-rAxin increased (Fig. 3 C). Lineweaver-Burk plots indicated that the K m andV max values of MBP-rAxin for GST-GSK-3β in the absence of MBP-Dvl-1 were 131 nm and 4.3 nmol/min/mg, respectively, and that those in the presence of MBP-Dvl-1 were 129 nm and 2.5 nmol/min/mg (Fig. 3 C). These results suggest that Dvl-1 inhibits the GSK-3β-dependent phosphorylation of Axin in a noncompetitive manner. This is the first demonstration showing that Dvl inhibits the function of GSK-3β directly. However, it is not likely that Dvl-1 inhibits GSK-3β activity itself, because MBP-Dvl-1 did not affect the phosphorylation of synthetic peptide substrate, which is designed from glycogen synthase, by GST-GSK-3β (data not shown). We have recently found that Dvl-1 directly binds to Axin and that the binding of Dvl-1 to Axin does not affect the interaction of GSK-3β with Axin. 2Kishida, S., Yamamoto, H., Hino, S., Ikeda, S., Kishida, M., and Kikuchi, A. (1999) Mol. Cell. Biol., in press. It is possible that the binding of Dvl to Axin induces the structural change of the Axin complex; therefore GSK-3β does not effectively phosphorylate Axin. However, higher concentrations (μm order) of Dvl-1 are required to inhibit the GSK-3β-dependent phosphorylation of Axin in our in vitro experiments. Therefore, modification of Dvl such as phosphorylation could be necessary to act on the Axin complex in intact cells. These results suggest that Dvl may regulate the stability of Axin. Finally we examined whether Wnt signal regulates the stability of endogenous Axin in intact cells. Although Wnt proteins are secretory, they predominantly bind to the cell surface or extracellular matrix. Small amounts of biologically active Wnt-1 or Wg can be found in culture medium conditioned by cells expressing these proteins (35van Leeuwen F. Samos C.H. Nusse R. Nature. 1994; 368: 342-344Crossref PubMed Scopus (172) Google Scholar, 36Bradley R.S. Brown A.M.C. Mol. Cell. Biol. 1995; 15: 4616-4622Crossref PubMed Scopus (73) Google Scholar). The Wg-conditioned medium from Schneider cells increases the level of Armadillo inDrosophila disc cells and inactivates GSK-3 in 10T1/2 fibroblasts (35van Leeuwen F. Samos C.H. Nusse R. Nature. 1994; 368: 342-344Crossref PubMed Scopus (172) Google Scholar, 37Cook D. Fry M.J. Hughes K. Sumathipala R. Woodgett J.R. Dale T.C. EMBO J. 1996; 15: 4526-4536Crossref PubMed Scopus (343) Google Scholar). Based on assays carried out with mammalian cell lines and Xenopus embryos, the Wnt proteins can be classified into two groups, Wnt-1 and Wnt-5a classes (38Wong G.T. Gavin B.J. McMahon A.P. Mol. Cell. Biol. 1994; 14: 6278-6286Crossref PubMed Scopus (284) Google Scholar, 39Du S.J. Purcell S.M. Christian J.L. McGrew L.L. Moon R.T. Mol. Cell. Biol. 1995; 15: 2625-2634Crossref PubMed Google Scholar, 40Shimizu H. Julius M.A. Giarre M. Zheng Z. Brown A.M. Kitajewski J. Cell Growth Differ. 1997; 8: 1349-1358PubMed Google Scholar). The Wnt-1 class includes Wnt-1, Wnt-2, Wnt-3, Wnt-3a, and Wnt-8, which have activities to transform the cells and to accumulate cytoplasmic β-catenin, whereas the Wnt-5a class includes Wnt-4, Wnt-5a, Wnt-5b, Wnt-7b, and Wnt-11, which do not exhibit the transformation and β-catenin accumulation activities. Because Wnt-3a displays characteristics similar to those of Wnt-1, we prepared Wnt-3a-containing conditioned medium. In these experiments we used mouse fibroblast L cells, because the changes in the expression level of β-catenin by Wnt are easily observed due to little expression of cadherin in the cells (15Kishida M. Koyama S. Kishida S. Matsubara K. Nakashima S. Higano K. Takada R. Takada S. Kikuchi A. Oncogene. 1999; 18: 979-985Crossref PubMed Scopus (114) Google Scholar, 23Shibamoto S. Higano K. Takada R. Ito F. Takeichi M. Takada S. Genes Cells. 1998; 3: 659-670Crossref PubMed Scopus (230) Google Scholar, 41Nagafuchi A. Shirayoshi Y. Okazaki K. Yasuda K. Takeichi M. Nature. 1987; 329: 341-343Crossref PubMed Scopus (575) Google Scholar). Furthermore, Western blot analyses with the anti-Axin antibody demonstrated that Axin is most abundant in L cells among various cell lines including SW480, NIH3T3, COS, and Chinese hamster ovary cells (data not shown). Wnt-3a conditioned medium induced the accumulation of β-catenin in L cells in a dose-dependent manner (Fig.4 A). In contrast, Wnt-3a decreased Axin (Fig. 4 A). Control conditioned medium did not affect the amounts of Axin and β-catenin (data not shown). To examine whether Dvl is involved in this action of Wnt-3a, we established L cells, which express HA-Dvl-1ΔPDZ stably. Wnt-3a-induced increase of β-catenin and decrease of Axin were suppressed in L cells expressing HA-Dvl-1ΔPDZ (Fig. 4 B). These results indicate that Wnt not only accumulates β-catenin but also down-regulates Axin through Dvl. We have recently found that in COS cells Axin interacts with GSK-3β, β-catenin, and APC in a high molecular mass complex with a molecular mass of more than 103 kDa on gel filtration column chromatography (15Kishida M. Koyama S. Kishida S. Matsubara K. Nakashima S. Higano K. Takada R. Takada S. Kikuchi A. Oncogene. 1999; 18: 979-985Crossref PubMed Scopus (114) Google Scholar). In L cells β-catenin is present in the high molecular mass complex in the absence of Wnt-3a, whereas addition of Wnt-3a to L cells increases β-catenin in a low molecular mass complex with a molecular mass of 200–300 kDa (15Kishida M. Koyama S. Kishida S. Matsubara K. Nakashima S. Higano K. Takada R. Takada S. Kikuchi A. Oncogene. 1999; 18: 979-985Crossref PubMed Scopus (114) Google Scholar). In L cells expressing Axin, Wnt-3a-induced increase of β-catenin in the low molecular mass complex is not observed (15Kishida M. Koyama S. Kishida S. Matsubara K. Nakashima S. Higano K. Takada R. Takada S. Kikuchi A. Oncogene. 1999; 18: 979-985Crossref PubMed Scopus (114) Google Scholar). These results suggest that a balance between the high and low molecular mass complexes containing β-catenin is closely regulated and that Axin plays a role in limiting the accumulation of β-catenin in the low molecular mass complex. Wnt may regulate the assembly of the complex consisting of Axin, APC, β-catenin, and GSK-3β and induce the dissociation of β-catenin from the complex. It is possible that β-catenin free from the complex is accumulated, binds to different partners such as Lef/Tcf, and transmits the signals. Our results suggest that the Wnt signal could act on the Axin complex through Dvl, resulting in the inhibition of the GSK-3β-dependent phosphorylation of Axin and the degradation of Axin. Degradation of Axin due to hypophosphorylation may induce the dissociation of β-catenin from the complex by decreasing the binding of β-catenin to Axin. Studies to clarify the mechanism of proteolysis of Axin are under way. We are grateful to Drs. B. Dallapiccola, G. Novelli, and C. Turck for reagents. We thank the Research Center for Molecular Medicine, Hiroshima University School of Medicine, for the use of their facilities." @default.
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• W2013460988 title "Phosphorylation of Axin, a Wnt Signal Negative Regulator, by Glycogen Synthase Kinase-3β Regulates Its Stability" @default.
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