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• W2001054344 abstract "Axin, a negative regulator of the Wnt signaling pathway, forms a complex with glycogen synthase kinase-3β (GSK-3β), β-catenin, adenomatous polyposis coli (APC) gene product, and Dvl, and it regulates GSK-3β-dependent phosphorylation in the complex and the stability of β-catenin. Using yeast two-hybrid screening, we found that regulatory subunits of protein phosphatase 2A, PR61β and -γ, interact with Axin. PR61β or -γ formed a complex with Axin in intact cells, and their interaction was direct. The binding site of PR61β on Axin was different from those of GSK-3β, β-catenin, APC, and Dvl. Although PR61β did not affect the stability of β-catenin, it inhibited Dvl- and β-catenin-dependent T cell factor activation in mammalian cells. Moreover, it suppressed β-catenin-induced axis formation and expression of siamois, a Wnt target gene, inXenopus embryos, suggesting that PR61β acts either at the level of β-catenin or downstream of it. Taken together with the previous observations that PR61 interacts with APC and functions upstream of β-catenin, these results demonstrate that PR61 regulates the Wnt signaling pathway at various steps. Axin, a negative regulator of the Wnt signaling pathway, forms a complex with glycogen synthase kinase-3β (GSK-3β), β-catenin, adenomatous polyposis coli (APC) gene product, and Dvl, and it regulates GSK-3β-dependent phosphorylation in the complex and the stability of β-catenin. Using yeast two-hybrid screening, we found that regulatory subunits of protein phosphatase 2A, PR61β and -γ, interact with Axin. PR61β or -γ formed a complex with Axin in intact cells, and their interaction was direct. The binding site of PR61β on Axin was different from those of GSK-3β, β-catenin, APC, and Dvl. Although PR61β did not affect the stability of β-catenin, it inhibited Dvl- and β-catenin-dependent T cell factor activation in mammalian cells. Moreover, it suppressed β-catenin-induced axis formation and expression of siamois, a Wnt target gene, inXenopus embryos, suggesting that PR61β acts either at the level of β-catenin or downstream of it. Taken together with the previous observations that PR61 interacts with APC and functions upstream of β-catenin, these results demonstrate that PR61 regulates the Wnt signaling pathway at various steps. glycogen synthase kinase-3β adenomatous polyposis coli T cell factor protein phosphatase 2A PP2A that consists of the C and A subunits rat Axin maltose-binding protein glutathione S-transferase hemagglutinin Xenopus globin 3-[(3-cholamidopropyl)dimethyl-ammonio] propanesulfonic acid dorso-anterior index reverse transcription-polymerase chain reaction Wnt proteins constitute a large family of cysteine-rich secreted ligands that control development in organisms ranging from nematode worms to mammals (1Wodarz A. Nusse R. Annu. Rev. Cell Dev. Biol. 1998; 14: 59-88Crossref PubMed Scopus (1716) Google Scholar). In vertebrates, the Wnt signaling pathway regulates axis formation, organ development, cell proliferation, morphology, motility, and fate (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, 4Miller J.R. Hocking A.M. Brown J.D. Moon R.T. Oncogene. 1999; 18: 7860-7872Crossref PubMed Scopus (601) Google Scholar, 5Huelsken J. Vogel R. Brinkmann V. Erdmann B. Birchmeier C. Birchmeier W. J. Cell Biol. 2000; 148: 567-578Crossref PubMed Scopus (509) Google Scholar). In the current model, the serine/threonine kinase GSK-3β1 targets cytoplasmic β-catenin for degradation in the absence of Wnt (6Yost C. Torres M. Miller J.R. Huang E. Kimelman D. Moon R.T. Genes Dev. 1996; 10: 1443-1454Crossref PubMed Scopus (1009) Google Scholar, 7Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1087) Google Scholar). As a result, the level of cytoplasmic β-catenin is low. Axin has been shown to be important for the degradation of β-catenin (8Kikuchi A. Cell. Signal. 1999; 11: 777-788Crossref PubMed Scopus (158) Google Scholar, 9Kikuchi A. Cytokine Growth Factor Rev. 1999; 10: 255-265Crossref PubMed Scopus (68) Google Scholar). It forms a complex with GSK-3β, β-catenin, and APC (7Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1087) Google Scholar, 8Kikuchi A. Cell. Signal. 1999; 11: 777-788Crossref PubMed Scopus (158) Google Scholar, 9Kikuchi A. Cytokine Growth Factor Rev. 1999; 10: 255-265Crossref PubMed Scopus (68) Google Scholar, 10Yamamoto 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, 11Kishida 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, 12Sakanaka C. Weiss J.B. Williams L.T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3020-3023Crossref PubMed Scopus (282) Google Scholar, 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, 14Hart 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, 15Itoh K. Krupnik V.E. Sokol S.Y. Curr. Biol. 1998; 8: 591-594Abstract Full Text Full Text PDF PubMed Google Scholar) and promotes GSK-3β-dependent phosphorylation of β-catenin and APC (7Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1087) Google Scholar, 10Yamamoto 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, 14Hart 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, 16Ikeda S. Kishida M. Matsuura Y. Usui H. Kikuchi A. Oncogene. 2000; 19: 537-545Crossref PubMed Scopus (161) Google Scholar, 17Hinoi T. Yamamoto H. Kishida M. Takada S. Kishida S. Kikuchi A. J. Biol. Chem. 2000; 275: 34399-34406Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Phosphorylated β-catenin forms a complex with Fbw1, a member of the F-box protein family, resulting in the degradation of β-catenin by the ubiquitin and proteasome pathways (18Kitagawa M. Hatakeyama S. Shirane M. Matsumoto M. Ishida N. Hattori K. Nakamichi I. Kikuchi A. Nakayama K.-I. Nakayama K. EMBO J. 1999; 18: 2401-2410Crossref PubMed Scopus (473) Google Scholar, 19Hart M. Concordet J.-P. Lassot I. Albert I. de los Santos R. Durand H. Perret C. Rubinfeld B. Margottin F. Benarous R. Polakis P. Curr. Biol. 1999; 9: 207-210Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar). Because expression of Axin indeed decreases the protein level of β-catenin (20Kishida 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), Axin is a negative regulator of the Wnt signaling pathway. In addition, Axin is phosphorylated by GSK-3β, and this phosphorylation stabilizes Axin in contrast to β-catenin (21Yamamoto H. Kishida S. Kishida M. Ikeda S. Takada S. Kikuchi A. J. Biol. Chem. 1999; 274: 10681-10684Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). When Wnt acts on its cell-surface receptor Frizzled, Dvl, a cytoplasmic protein, antagonizes the action of GSK-3β. Indeed, Dvl inhibits GSK-3β-dependent phosphorylation of β-catenin, APC, and Axin in vitro and the phosphorylation of Axin in intact cells (21Yamamoto H. Kishida S. Kishida M. Ikeda S. Takada S. Kikuchi A. J. Biol. Chem. 1999; 274: 10681-10684Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 22Kishida S. Yamamoto H. Hino S.-I. Ikeda S. Kishida M. Kikuchi A. Mol. Cell. Biol. 1999; 19: 4414-4422Crossref PubMed Google Scholar, 23Kadoya T. Kishida S. Fukui A. Hinoi T. Michiue T. Asashima M. Kikuchi A. J. Biol. Chem. 2000; 275: 37030-37037Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Although this mechanism has not yet been clarified, it has been suggested that Frat, which is a GSK-3-binding protein (24Yost C. Farr III, G.H. Pierce S.B. Ferkey D.M. Chen M.M. Kimelman D. Cell. 1998; 93: 1031-1041Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar), forms a complex with Dvl and that this complex induces the dissociation of GSK-3β from Axin (25Li L. Yuan H. Weaver C.D. Mao J. Farr III, G.H. Sussman D.J. Jonkers J. Kimelman D. Wu D. EMBO J. 1999; 18: 4233-4240Crossref PubMed Scopus (353) Google Scholar). We have recently identified the novel proteins Axam and Idax, which suppress the Wnt signaling (23Kadoya T. Kishida S. Fukui A. Hinoi T. Michiue T. Asashima M. Kikuchi A. J. Biol. Chem. 2000; 275: 37030-37037Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 26Hino S.-I. Kishida S. Michiue T. Fukui A. Sakamoto I. Takada S. Asashima M. Kikuchi A. Mol. Cell. Biol. 2001; 21: 330-342Crossref PubMed Scopus (104) Google Scholar). Axam binds to Axin and inhibits the binding of Dvl to Axin. It stimulates the down-regulation of β-catenin in mammalian cells and inhibits the axis formation, which is regulated by Wnt in Xenopus embryos (23Kadoya T. Kishida S. Fukui A. Hinoi T. Michiue T. Asashima M. Kikuchi A. J. Biol. Chem. 2000; 275: 37030-37037Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Idax interacts with the PDZ domain of Dvl and inhibits the complex formation of Dvl and Axin. It inhibits Wnt-dependent accumulation of β-catenin in mammalian cells and Wnt-dependent axis duplication inXenopus embryos (26Hino S.-I. Kishida S. Michiue T. Fukui A. Sakamoto I. Takada S. Asashima M. Kikuchi A. Mol. Cell. Biol. 2001; 21: 330-342Crossref PubMed Scopus (104) Google Scholar). Therefore, Wnt signal may relieve the activities of Axam and Idax to inhibit the binding of Dvl to Axin. Once the phosphorylation of β-catenin is reduced, it dissociates from the Axin complex, and β-catenin is no longer degraded, resulting in its accumulation in the cytoplasm. Accumulated β-catenin is translocated into the nucleus where it binds to Tcf/lymphocyte enhancer binding factor, a transcription factor (27Behrens 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, 28Molenaar M. van de Wetering M. Oosterwegel M. Peterson-Maduro J. Godsave S. Korinek V. Roose J. Destrée O. Clevers H. 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Nature. 1999; 398: 422-426Crossref PubMed Scopus (3224) Google Scholar, 34Shtutman M. Zhurinsky J. Simcha I. Albanese C. D'Amico M. Pestell R. Ben-Ze'ev A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5522-5527Crossref PubMed Scopus (1891) Google Scholar, 35Crawford H.C. Fingleton B.M. Rudolph-Owen L.A. Goss K.J. Rubinfeld B. Polakis P. Matrisian L.M. Oncogene. 1999; 18: 2883-2891Crossref PubMed Scopus (610) Google Scholar). Thus, the Wnt signal stabilizes β-catenin, thereby regulating various gene expression. It has been shown that PP2A is a binding partner of Axin (16Ikeda S. Kishida M. Matsuura Y. Usui H. Kikuchi A. Oncogene. 2000; 19: 537-545Crossref PubMed Scopus (161) Google Scholar, 36Hsu W. Zeng L. Costantini F. J. Biol. Chem. 1999; 274: 3439-3445Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). PP2A is one of four major serine/threonine protein phosphatases (37Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2134) Google Scholar). The catalytic C subunit of PP2A is associated with the constant regulatory A subunit, and other regulatory subunits (B/PR55, B′/B56/PR61, or B“/PR72/PR59) associate with this dimeric core and modulate the enzymatic activity and substrate specificity (38Usui H. Imazu M. Maeta K. Tsukamoto H. Azuma K. Takeda M. J. Biol. Chem. 1988; 263: 3752-3761Abstract Full Text PDF PubMed Google Scholar, 39Zolnierowicz S. Van Hoof C. Andjelkovic N. Cron P. Stevens I. Merlevede W. Goris J. Hemmings B.A. Biochem. J. 1996; 317: 187-194Crossref PubMed Scopus (78) Google Scholar, 40McCright B. Rivers A.M. Audlin S. Virshup D.M. J. Biol. Chem. 1996; 271: 22081-22089Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 41Millward T.A. Zolnierowicz S. Hemmings B.A. Trends Biochem. Sci. 1999; 24: 186-191Abstract Full Text Full Text PDF PubMed Scopus (702) Google Scholar, 42Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1500) Google Scholar). PP2A that consists of the C and A subunits (PP2A(CA)) interacts with Axin and dephosphorylates Axin and APC (16Ikeda S. Kishida M. Matsuura Y. Usui H. Kikuchi A. Oncogene. 2000; 19: 537-545Crossref PubMed Scopus (161) Google Scholar). It has been recently reported that the B56 subunit of PP2A interacts with APC and that its expression reduces the level of β-catenin and inhibits the transcription of β-catenin target gene (43Seeling J.M. Miller J.R. Gil R. Moon R.T. White R. Virshup D.M. Science. 1999; 283: 2089-2091Crossref PubMed Scopus (362) Google Scholar). However, the mode of action of B56/PR61 in the Wnt signaling pathway is not known. In this study, we demonstrate that PR61 binds to Axin in intact cells and in vitro. We also show that PR61 does not affect the stability of β-catenin but that it inhibits Dvl- and β-catenin-induced Tcf activation in mammalian cells. Further, we demonstrate that PR61 induces ventralization and suppresses β-catenin-dependent axis duplication in Xenopus embryos, suggesting that PR61 functions either at the level of β-catenin or further downstream. Taken together with the previous observations that PR61 acts upstream of β-catenin (43Seeling J.M. Miller J.R. Gil R. Moon R.T. White R. Virshup D.M. Science. 1999; 283: 2089-2091Crossref PubMed Scopus (362) Google Scholar), these results suggest that PR61 negatively regulates the Wnt signaling pathway at various steps. Because PR61 and B56 denote the same molecule (41Millward T.A. Zolnierowicz S. Hemmings B.A. Trends Biochem. Sci. 1999; 24: 186-191Abstract Full Text Full Text PDF PubMed Scopus (702) Google Scholar), we use the name of “PR61” in this study. pCMV-Flag/PP2A(C), pcDNA3/Flag-rAxin, pcDNAI/hTcf-4E and pTOPFLASH, and pUC/EF-1α/β-cateninSA were supplied by Drs. K. Yonezawa (Kobe University, Kobe, Japan), K. Miyazono (Tokyo University, Tokyo, Japan), H. Clevers (University Hospital, Utrecht, The Netherlands), and A. Nagafuchi (Kumamoto University, Kumamoto, Japan), respectively. β-CateninSA is a β-catenin mutant in which serine and threonine residues of the GSK-3β phosphorylation sites are changed to alanine (6Yost C. Torres M. Miller J.R. Huang E. Kimelman D. Moon R.T. Genes Dev. 1996; 10: 1443-1454Crossref PubMed Scopus (1009) Google Scholar). The anti-MBP and anti-GST antibodies were kindly supplied by Dr. M. Nakata (Sumitomo Electronics, Yokohama, Japan). Wnt-3a conditioned medium was prepared as described (44Shibamoto S. Higano K. Takada R. Ito F. Takeichi M. Takada S. Genes Cells. 1998; 3: 659-670Crossref PubMed Scopus (230) Google Scholar). To generate the anti-phosphorylated Axin antibody, three phosphorylated peptides of rAxin, PA322 (Cys-Leu-Ala-Pro-Ala-Thr-Ser(P)322-Ala-Asn-Asp-Ser-Glu), PA326 (Cys-Thr-Ser-Ala-Asn-Asp-Ser(P)326-Glu-Gln-Gln-Ser-Leu), and PA330 (Cys-Asp-Ser-Glu-Gln-Gln-Ser(P)330-Leu-Ser-Ser-Asp-Ala), were synthesized, and polyclonal antibodies against PA322 (P322 antibody), PA326 (P326 antibody), and PA330 (P330 antibody) were produced in rabbits by immunization with these peptides by Dr. T. Hachiya (MBL, Nagoya, Japan). PP2A consisting of the catalytic and regulatory A subunits was purified from human erythrocytes (38Usui H. Imazu M. Maeta K. Tsukamoto H. Azuma K. Takeda M. J. Biol. Chem. 1988; 263: 3752-3761Abstract Full Text PDF PubMed Google Scholar). MBP- and GST-fused proteins were purified from Escherichia coli according to the manufacturer's instructions. The anti-Myc antibody was prepared from 9E10 cells. COS, 293 or L, and PC12 cells were cultured in Dulbecco's modified Eagle's medium containing 10% calf serum, 10% fetal calf serum, and 10% fetal calf serum and 5% horse serum, respectively. L cells stably expressing HA-PR61β (L/PR61β cells) were generated by selecting with G418 as described (20Kishida 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, 45Okazaki M. Kishida S. Murai H. Hinoi T. Kikuchi A. Cancer Res. 1996; 56: 2387-2392PubMed Google Scholar). L cells stably expressing wild-type Myc-rAxin (L/Axin cells), Myc-rAxinSA, and Myc-rAxinΔGSK-3β were obtained as described (17Hinoi T. Yamamoto H. Kishida M. Takada S. Kishida S. Kikuchi A. J. Biol. Chem. 2000; 275: 34399-34406Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 20Kishida 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). The anti-GSK-3β, anti-β-catenin, and anti-PP2A(C) antibodies were purchased from Transduction Laboratories (Lexington, KY). Other materials were from commercial sources. Standard recombinant DNA techniques were used to construct the following plasmids, pBTM116HA/Axil, pEF-BOS-HA/PR61β, pGEX-KG/PR61β, pSP64T-Myc/PR61β, pEF-BOS-HA/PR61γ, pBJ-1/PR61δ-3HA, pEF-BOS-Myc/rAxinΔDIX, pMAL-c2/rAxin-(229–506), and pMAL-c2/rAxin-(298–506)S322A. In these plasmids, some plasmid constructions were done by digesting the original plasmids with restriction enzymes and inserting the fragments into the vectors. The other constructions were done by inserting the fragments generated by Expand High Fidelity PCR system (Roche Diagnostics GmbH, Mannheim, Germany) into the vectors. The entire PCR products were sequenced, and the structures of all of the plasmids were confirmed by restriction analyses. pBJ-Myc/rAxin, pEF-BOS-Myc/rAxin, pEF-BOS-Myc/rAxin-(1–529), pBJ-Myc/rAxin-(1–229), pEF-BOS/Myc-rAxin-(298–506), pBJ-Myc/rAxin-(713–832), pMAL-c2/rAxin, pMAL-c2/rAxin-(1–529), pMAL-c2/rAxin-(1–229), pMAL-c2/rAxin-(508–832), pBJ-Myc/rAxinSA, pBJ-Myc/rAxinΔGSK-3β, pCGN/Dvl-1, pEF-BOS-HA/hTcf-4E, and pSP64T/Xglobin were constructed as described (7Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1087) Google Scholar, 10Yamamoto 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, 17Hinoi T. Yamamoto H. Kishida M. Takada S. Kishida S. Kikuchi A. J. Biol. Chem. 2000; 275: 34399-34406Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 20Kishida 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, 22Kishida S. Yamamoto H. Hino S.-I. Ikeda S. Kishida M. Kikuchi A. Mol. Cell. Biol. 1999; 19: 4414-4422Crossref PubMed Google Scholar). To determine whether PR61 interacts with Axin in intact cells, COS cells (60-mm-diameter dish) transfected with pEF-BOS- and pBJ-derived plasmids were lysed as described (46Kikuchi A. Williams L.T. J. Biol. Chem. 1996; 271: 588-594Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The lysates (200 µg of protein) were immunoprecipitated with the anti-Myc antibody, and then the precipitates were probed with the anti-Myc and anti-HA antibodies. To examine the direct interaction of PR61 with Axin using the purified proteins in vitro, MBP-rAxin and its deletion mutants (0.5 µm) were incubated with 10 pmol of GST-PR61β immobilized on glutathione-Sepharose 4B in 50 µl of reaction mixture (20 mm Tris/HCl, pH 7.5, 1 mmdithiothreitol, and 0.5% CHAPS) for 1 h at 4 °C. After GST-PR61β was precipitated by centrifugation, the precipitates were probed with the anti-MBP antibody. To examine whether the trimer of PP2A associates with Axin, GST-PR61β complexed with PP2A(CA) was prepared. After 2.4 µmGST-PR61β had been incubated with 3 µm purified PP2A(CA) in 1 ml of reaction buffer (20 mm Tris/HCl, pH 7.5, 1 mm dithiothreitol, 0.1 mm EDTA, and 1% glycerol) for 1 h at 4 °C, GST-PR61β was precipitated with glutathione-Sepharose 4B and washed three times with washing buffer (20 mm Tris/HCl, pH 7.5, and 1 mmdithiothreitol) to remove free PP2A(CA). About 0.25 mol of PP2A(CA) was complexed with 1 mol of GST-PR61β. 1.2 µmGST-PR61β, 0.3 µm PP2A(CA), and 1.2 µmGST-PR61β complexed with PP2A(CA) were incubated with 7.5 pmol of either MBP-rAxin or MBP immobilized on amylose resin in 25 µl of reaction mixture for 1 h at 4 °C. After MBP-rAxin and MBP were precipitated by centrifugation, the precipitates were probed with the anti-GST and anti-PP2A(C) antibodies. In another experiment, the lysates (100 µg of protein) of COS cells transfected with or without pEF-BOS-HA/PR61β were incubated with 30 pmol of MBP-rAxin or MBP immobilized on amylose resin in 100 µl of reaction mixture (20 mm Tris/HCl, pH 7.5, 1% Nonidet P-40, 137 mmNaCl, and 10% glycerol) for 1 h at 4 °C. After MBP-rAxin and MBP were precipitated by centrifugation, the precipitates were probed with the anti-HA and anti-PP2A(C) antibodies. To observe the phosphorylation state of Axin in intact cells, the lysates of COS, L, and 293 cells expressing wild-type Myc-rAxin, Myc-rAxinSA, or Myc-rAxinΔGSK-3β with or without HA-PR61β or Flag-PP2A(C) were probed with the anti-P322, anti-P326, or anti-P330 antibody. When necessary, Myc-rAxin was immunoprecipitated from L cells stably expressing Myc-rAxin with the anti-Myc antibody, and the immunoprecipitates were incubated with 6 units of alkaline phosphatase in 20 µl of phosphatase reaction mixture (50 mm Tris/HCl, pH 8.0, 10 mm MgCl2, and 0.1 mg/ml bovine serum albumin) for 30 min at 37 °C. After the incubation, the mixtures were probed with the anti-Myc or anti-P322 antibody. To show that the anti-P322 antibody detects the direct phosphorylation of Axin by GSK-3β, 0.2 µm MBP-rAxin, MBP-rAxin-(298–506), or MBP-rAxin-(298–506)S322A was incubated with 50 nm GST-GSK-3β in 30 µl of kinase reaction mixture (50 mm Tris/HCl, pH 7.5, 10 mmMgCl2, 1 mm dithiothreitol, and 50 µm ATP) for 30 min at 37 °C. After the incubation, the mixtures were probed with the anti-P322 antibody. To observe the effect of PR61β on the stabilization of β-catenin, wild-type L cells or L/PR61β cells (35-mm-diameter dish) were deprived of serum for 6 h, then treated with Wnt-3a conditioned medium for 12 h. The lysates were probed with the anti-β-catenin antibody. To examine whether PR61β affects the Wnt-3a-dependent Tcf-4 activation, wild-type L cells or L/PR61β cells (35-mm-diameter dish) were transfected with 0.5 µg of pTOPFLASH, 0.1 µg of pEF-BOS-HA/hTcf-4E, and 0.5 µg of pME18S/lacZ. After the cells were deprived of serum for 6 h and then treated with Wnt-3a conditioned medium for 12 h, they were lysed and the luciferase activity was measured (20Kishida 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). To observe the effect of PR61β on Dvl- and β-catenin-dependent Tcf-4 activation, 0.2 µg of pCGN/Dvl-1 or 30 ng of pUC/EF-1α/β-cateninSA and the indicated amounts of pCMV-Flag/PP2A(C) or pEF-BOS-HA/PR61β were transfected into 293 or PC12 cells (35-mm-diameter dish) with 0.5 µg of pTOPFLASH, 0.1 µg of pEF-BOS-HA/hTcf-4E, and 0.5 µg of pME18S/lacZ. After 46 h, the cells were lysed and luciferase activity was measured as described (20Kishida 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, 47Korinek V. Barker N. Morin P.J. van Wichen D. de Weger R. Kinzler K.W. Vogelstein B. Clevers H. Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2895) Google Scholar). To standardize the transfection efficiency, pME18S/lacZ carrying SRα-promoter linked to the coding sequence of β-galactosidase gene was used as an internal control. PR61β cDNA was subcloned into theBglII site of pSP64T. Sense mRNA was obtained byin vitro transcription of linearized templates using SP6-mMESSAGE mMACHINE kit (Ambion, Austin, TX). Fertilized eggs were dejellied using 4.5% cysteine acid, and the indicated mRNAs were injected into dorsal or ventral blastomeres at the four-cell stage. After injection, embryos were cultured for 3 days (at stage 40–41). UV light irradiation was performed as described (48Scharf S.R. Gerhart J.C. Dev. Biol. 1980; 79: 181-198Crossref PubMed Scopus (154) Google Scholar). The phenotypes of the injected embryos were evaluated by DAI (49Kao K.R. Elinson R.P. Dev. Biol. 1988; 127: 64-77Crossref PubMed Scopus (420) Google Scholar). To carry out RT-PCR, total RNAs of embryos at stage 10.5 were isolated. Oligo(dT)-primed cDNAs were synthesized using 5 µg of total RNA from 10 embryos. PCR analyses (35 cycles) were performed with ExTaq DNA polymerase (Takara). Primers for PCR are: EF-1α, 5′-CAG ATT GGT GCT GGA TAT GC-3′ and 5′-ACT GCC TTG ATG ACT CCT AG-3′;siamois, 5′-AAG ATA ACT GGC ATT CCT GAG C-3′ and 5′-GGT AGG GCT GTG TAT TTG AAG G-3′. Yeast two-hybrid screening was carried out as described (7Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1087) Google Scholar, 10Yamamoto 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). Protein concentrations were determined with bovine serum albumin as a standard (50Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211941) Google Scholar). To identify proteins that physically interact with Axil (10Yamamoto 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), an Axin homolog, we conducted a mouse brain cDNA library screening by the yeast two-hybrid method. From 1.7 × 105 initial transformants, two clones, PR61β and PR61γ, were found to confer both the His+ and LacZ+ phenotypes on L40 expressing Axil. Both proteins are known to be regulatory subunits of PP2A and to regulate its phosphatase activity and subcellular localization (41Millward T.A. Zolnierowicz S. Hemmings B.A. Trends Biochem. Sci. 1999; 24: 186-191Abstract Full Text Full Text PDF PubMed Scopus (702) Google Scholar, 42Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1500) Google Scholar). In the two-hybrid assay, Axin also interacted with PR61β and PR61γ (data not shown), suggesting that PR61 might regulate the functions of both Axin and Axil. Because Axin has been well characterized (8Kikuchi A. Cell. Signal. 1999; 11: 777-788Crossref PubMed Scopus (158) Google Scholar, 9Kikuchi A. Cytokine Growth Factor Rev. 1999; 10: 255-265Crossref PubMed Scopus (68) Google Scholar), we used Axin to examine this possibility. Various deletion mutants of rAxin in this study are shown in Fig. 1. First we examined whether PR61 forms a complex with Axin in intact cells. Myc-rAxin was co-expressed with HA-PR61β or HA-PR61γ in COS cells (Fig. 2 A, lanes 1–5). When the lysates co-expressing Myc-rAxin with HA-PR61β were immunoprecipitated with the anti-Myc antibody, HA-PR61β was detected in the Myc-rAxin immune complex (Fig. 2 A,lane 8). Similarly, when the lysates co-expressing Myc-rAxin with HA-PR61γ were immunoprecipitated with the anti-Myc antibody, HA-PR61γ was detected in the Myc-rAxin immune complex (Fig.2 A, lane 9). When the lysates expressing either HA-PR61β or HA-PR61γ alone were immunoprecipitated with the anti-Myc antibody, neither HA-PR61β nor HA-PR61γ was detected in the immunoprecipitates (Fig. 2 A, lanes 6 and7). Further, HA-PR61δ also formed a complex with Myc-rAxin in L cells (data not shown). To determine which region of rAxin forms a complex with PR61β in intact cells, various deletion mutants of Myc-rAxin were co-expressed with HA-PR61β in COS cells (Fig.2 B, lanes 1–7). When the lysates expressing Myc-rAxin mutants were immunoprecipitated with the anti-Myc antibody, HA-PR61β was coprecipitated with Myc-rAxin (full-length), Myc-rAxinΔDIX, and Myc-rAxin-(1–529) but not with Myc-rAxin-(1–229), Myc-rAxin-(298–506), or Myc-rAxin-(713–832) (Fig. 2 B, lanes 8–13). These results indicate that Axin forms a complex with PR61 in intact cells and that the N-terminal region of Axin (1–529 amino acids) containing the RGS (regulators of the G protein signaling) domain and the GSK-3β- and β-catenin-binding sites is necessary for the interaction with PR61, but none of these individual sites is sufficient. To examine whether the interaction of Axin with PR61 is direct, MBP-rAxin, its deletion mutants, and GST-PR61β w" @default.
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• W2001054344 date "2001-07-01" @default.
• W2001054344 modified "2023-09-28" @default.
• W2001054344 title "Inhibition of the Wnt Signaling Pathway by the PR61 Subunit of Protein Phosphatase 2A" @default.
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• W2001054344 doi "https://doi.org/10.1074/jbc.m100443200" @default.
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