Axin, a Negative Regulator of the Wnt Signaling Pathway, Directly Interacts with Adenomatous Polyposis Coli and Regulates the Stabilization of β-Catenin
1998; Elsevier BV; Volume: 273; Issue: 18 Linguagem: Inglês
10.1074/jbc.273.18.10823
ISSN1083-351X
AutoresShosei Kishida, Hideki Yamamoto, Satoshi Ikeda, Michiko Kishida, Ikuo Sakamoto, Shinya Koyama, Akira Kikuchi,
Tópico(s)Kruppel-like factors research
ResumoThe regulators of G protein signaling (RGS) domain of Axin, a negative regulator of the Wnt signaling pathway, made a complex with full-length adenomatous polyposis coli (APC) in COS, 293, and L cells but not with truncated APC in SW480 or DLD-1 cells. The RGS domain directly interacted with the region containing the 20-amino acid repeats but not with that containing the 15-amino acid repeats of APC, although both regions are known to bind to β-catenin. In the region containing seven 20-amino acid repeats, the region containing the latter five repeats bound to the RGS domain of Axin. Axin and β-catenin simultaneously interacted with APC. Furthermore, Axin stimulated the degradation of β-catenin in COS cells. Taken together with our recent observations that Axin directly interacts with glycogen synthase kinase-3β (GSK-3β) and β-catenin and that it promotes GSK-3β-dependent phosphorylation of β-catenin, these results suggest that Axin, APC, GSK-3β, and β-catenin make a tetrameric complex, resulting in the regulation of the stabilization of β-catenin. The regulators of G protein signaling (RGS) domain of Axin, a negative regulator of the Wnt signaling pathway, made a complex with full-length adenomatous polyposis coli (APC) in COS, 293, and L cells but not with truncated APC in SW480 or DLD-1 cells. The RGS domain directly interacted with the region containing the 20-amino acid repeats but not with that containing the 15-amino acid repeats of APC, although both regions are known to bind to β-catenin. In the region containing seven 20-amino acid repeats, the region containing the latter five repeats bound to the RGS domain of Axin. Axin and β-catenin simultaneously interacted with APC. Furthermore, Axin stimulated the degradation of β-catenin in COS cells. Taken together with our recent observations that Axin directly interacts with glycogen synthase kinase-3β (GSK-3β) and β-catenin and that it promotes GSK-3β-dependent phosphorylation of β-catenin, these results suggest that Axin, APC, GSK-3β, and β-catenin make a tetrameric complex, resulting in the regulation of the stabilization of β-catenin. Axin, which is a product of the mouse Fused locus, has been identified as a negative regulator of the Wnt signaling pathway (1Zeng 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 (793) Google Scholar). Fused is a mutation that causes dominant skeletal and neurological defects and recessive lethal embryonic defects including neuroectodermal abnormalities (2Reed S.C. Genetics. 1937; 22: 1-13Crossref PubMed Google Scholar, 3Gluecksohn-Schoenheimer S. J. Exp. Zool. 1949; 110: 47-76Crossref PubMed Scopus (73) Google Scholar, 4Jacobs-Cohen R.J. Spiegelman M. Cookingham J.C. Bennett D. Genet. Res. 1984; 43: 43-50Crossref PubMed Scopus (33) Google Scholar). Because dorsal injection of wild type Axin in Xenopus embryos blocks axis formation and coinjection of Axin inhibits Wnt8-, Dsh-, and kinase-negative GSK-3β 1The abbreviations used are: GSK-3β, glycogen synthase kinase-3β; APC, adenomatous polyposis coli; FAP, familial adenomatous polyposis; aa, amino acid(s); RGS, regulators of G protein signaling; G protein, GTP-binding protein; GST, glutathioneS-transferase; MBP, maltose-binding protein; HA, hemagglutinin. -induced axis duplication (1Zeng 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 (793) Google Scholar), Axin could exert its effects on axis formation by inhibiting the Wnt signaling pathway. However, the molecular mechanism by which Axin regulates axis formation has not been shown. We have recently identified rat Axin (rAxin) as a GSK-3β-interacting protein (5Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1101) Google Scholar). rAxin is phosphorylated by GSK-3β, directly binds to not only GSK-3β but also β-catenin, and promotes GSK-3β-dependent phosphorylation of β-catenin (5Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1101) Google Scholar). Because the phosphorylation of β-catenin by GSK-3β is essential for the down-regulation of β-catenin (6Miller J.R. Moon R.T. Genes Dev. 1996; 10: 2527-2539Crossref PubMed Scopus (607) Google Scholar, 7Yost C. Torres M. Miller J.R. Huang E. Kimelman D. Moon R.T. Genes Dev. 1996; 10: 1443-1454Crossref PubMed Scopus (1018) Google Scholar), our results suggest that rAxin may induce the degradation of β-catenin. These actions of rAxin are consistent with the observation that Axin inhibits dorsal axis formation in Xenopus embryos, because the accumulation of β-catenin induces the axis duplication (8Funayama N. Fagotto F. McCrea P. Gumbiner B.M. J. Cell Biol. 1995; 128: 959-968Crossref PubMed Scopus (500) Google Scholar). It has been shown that besides the phosphorylation by GSK-3β, the down-regulation of β-catenin requires APC, which is a tumor suppressor linked to FAP and to the initiation of sporadic human colorectal cancer (9Polakis P. Biochim. Biophys. Acta. 1997; 1332: F127-F147PubMed Google Scholar). The middle portion of APC contains three successive 15-amino acid (aa) repeats followed by seven related but distinct 20-aa repeats. Both types of repeats are able to bind independently to β-catenin (10Rubinfeld B. Souza B. Albert I. Müller O. Chamberlain S.H. Masiarz F.R. Munemitsu S. Polakis P. Science. 1993; 262: 1731-1734Crossref PubMed Scopus (1177) Google Scholar, 11Su L. Vogelstein B. Kinzler K.W. Science. 1993; 262: 1734-1737Crossref PubMed Scopus (1115) Google Scholar, 12Rubinfeld B. Souza B. Albert I. Munemitsu S. Polakis P. J. Biol. Chem. 1995; 270: 5549-5555Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). In FAP and colorectal cancers, most patients carry APC mutations that result in the expression of truncated proteins (9Polakis P. Biochim. Biophys. Acta. 1997; 1332: F127-F147PubMed Google Scholar). Almost all mutant proteins lack the C-terminal half including most of the 20-aa repeats but retain the 15-aa repeats. Colorectal carcinoma cells with mutant APC contain large amounts of monomeric β-catenin (13Munemitsu S. Albert I. Souza B. Rubinfeld B. Polakis P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3046-3050Crossref PubMed Scopus (955) Google Scholar). The accumulated β-catenin translocates to the nucleus, and this translocation involves the association of β-catenin with the transcription enhancers of the lymphocyte enhancer binding factor/T cell factor family (14Behrens J. von Kries J.P. Kühl M. Bruhn L. Wedlich D. Grosschedl R. Birchmeier W. Nature. 1996; 382: 638-642Crossref PubMed Scopus (2595) Google Scholar, 15Molenaar 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 (1618) Google Scholar). Because the APC mutants retain the β-catenin-binding activity, the interaction of APC with β-catenin is not sufficient for the down-regulation of β-catenin. How APC down-regulates β-catenin and the relationship between APC and Axin in the degradation of β-catenin are not clear. In addition to GSK-3β- and β-catenin-binding sites, rAxin has a domain that is homologous to RGS, and this domain is called the RGS domain (1Zeng 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 (793) Google Scholar, 5Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1101) Google Scholar). RGS has been originally identified as a protein that binds to the GTP- but not GDP-bound form of Gα and stimulates GTP hydrolysis of Gα (16Dohlman H.G. Thorner J. J. Biol. Chem. 1997; 272: 3871-3874Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). It has been shown that ΔRGS, a mutant of Axin in which the RGS domain is deleted, acts as a potent dorsalizer, producing a secondary axis and that Axin blocks the axis-inducing activity of ΔRGS (1Zeng 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 (793) Google Scholar). These results indicate that ΔRGS acts through a dominant-negative mechanism to inhibit an endogenous Axin activity and that it competes for binding to a protein with which Axin normally interacts. Therefore, the RGS domain may have an activity to transmit the signal by interacting with other protein(s). Here we report that the RGS domain of rAxin directly interacts with the region containing the 20-aa repeats of APC and that rAxin stimulates the down-regulation of β-catenin. Taken together with our recent observations (5Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1101) Google Scholar), these results indicate that Axin directly binds to APC, β-catenin, and GSK-3β and that it regulates the stabilization of β-catenin. APC cDNA, 293 cells, L cells, and SW480 and DLD-1 cells were kindly supplied from Drs. T. Akiyama (Osaka University, Suita, Japan), K. Morishita (Daiichi Pharmaceutical Co. Ltd., Tokyo, Japan), A. Nagafuchi and Sh. Tsukita (Kyoto University, Kyoto, Japan), and E. Tahara (Hiroshima University, Hiroshima, Japan), respectively. GST and MBP fusion proteins were purified from Escherichia coli according to the manufacturer's instructions. The anti-APC (Ab-1) and β-catenin antibodies were purchased from Oncogene Science Inc. (Cambridge, MA) and Transduction Laboratories (Lexington, KY), respectively. [35S]Methionine and [35S]cysteine were purchased from Amersham Inc. (Buckinghamshire, United Kingdom). Other materials and chemicals were from commercial sources. pEF-BOS-Myc/rAxin (full-length), pBSKS/rAxin (full-length), pBJ-Myc/rAxin-(1–229), pEF-BOS-Myc/rAxin-(1–713), pBJ-Myc/rAxin-(298–713), pEF-BOS/Myc-rAxin-(298–506), pBJ-Myc/rAxin-(713–832), pGEX-2T/β-catenin, and pMAL-c2/rAxin-(298–506) were constructed as described (5Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1101) Google Scholar). To construct pGEX-2T/RGS, the RGS cDNA fragment encoding rAxin-(89–216) was synthesized by polymerase chain reaction and inserted into pGEX-2T. To construct pMAL-c2 containing APC mutants, pMKITneo/APC was digested with various restriction enzymes, and the APC cDNA fragments were inserted into pMAL-c2. These procedures will be described in detail elsewhere. To construct pMAL-c2/rAxin (full-length), pBSKS/rAxin was digested with SmaI andEcoRV, and the rAxin cDNA fragment was inserted into pMALc-2, which was digested with XbaI and blunted with Klenow fragment. To construct pGEX-2T/rAxin-(1–529), pBSKS/rAxin was digested with SmaI and PvuII, and this fragment was inserted into SmaI cut pGEX-2T. To construct pCGN/β-catenin, pBSSK/β-catenin was digested with XhoI, blunted with Klenow fragment, and digested with XbaI. The β-catenin cDNA fragment was inserted into pCGN. COS cells (10-cm diameter dish) transfected with pBJ- and pEF-BOS-derived plasmids were lysed as described (17Kikuchi A. Williams L.T. J. Biol. Chem. 1996; 271: 588-594Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 18Hinoi T. Kishida S. Koyama S. Ikeda M. Matsuura Y. Kikuchi A. J. Biol. Chem. 1996; 271: 19710-19716Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 19Murai H. Ikeda M. Kishida S. Ishida O. Okazaki-Kishida M. Matsuura Y. Kikuchi A. J. Biol. Chem. 1997; 272: 10483-10490Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). rAxin and its deletion mutants were tagged with Myc epitope at their N termini. The lysates (160–800 μg of protein) were immunoprecipitated with the anti-Myc antibody, then the precipitates were probed with the anti-APC and β-catenin antibodies. When the interaction of the RGS domain of rAxin with APC was examined in vitro, 1 μm GST-RGS was incubated with the lysates (200 μg of protein) of COS, 293, L, SW480, and DLD-1 cells for 2 h at 4 °C. GST-RGS were precipitated with glutathione-Sepharose 4B, and the precipitates were probed with the anti-APC antibody. Various deletion mutants of MBP-APC (0.5–10 pmol) immobilized on the amylose resin were incubated with various concentrations of GST-RGS, GST-rAxin-(1–529), and GST-β-catenin in 100 μl of reaction mixture (20 mm Tris/HCl (pH 7.5) and 1 mm dithiothreitol) for 2 h at 4 °C. MBP fusion proteins were precipitated by centrifugation, and the precipitates were probed with the anti-GST antibody. When the effect of rAxin on the interaction of APC with β-catenin was examined, 50 nmGST-β-catenin was incubated with 250 nmMBP-APC-(959–1338) in the presence of various concentrations of MBP-rAxin-(298–506) or MBP-rAxin (full-length) in 100 μl of reaction mixture for 2 h at 4 °C. GST-β-catenin was precipitated by glutathione-Sepharose 4B, and the precipitates were probed with the anti-MBP antibody. Where specified, the relative intensities of the precipitated GST and MBP fusion proteins were quantitated by densitometric tracing of the stained sheets using an NIH image program. COS cells (60–70% confluent on a 35-mm diameter dish) were transfected with pCGN/β-catenin alone or with pCGN/β-catenin and pEF-BOS-Myc/rAxin (full-length). After 60 h, pulse-chase analysis was performed as described (13Munemitsu S. Albert I. Souza B. Rubinfeld B. Polakis P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3046-3050Crossref PubMed Scopus (955) Google Scholar). Briefly, the cells were pulse-labeled with [35S]methionine and [35S]cysteine (50 μCi/ml) for 30 min at 37 °C. Then the cells were lysed immediately or at the indicated times following incubation with excess unlabeled methionine and cysteine. The lysates were immunoprecipitated with the anti-HA antibody, and the precipitates were probed with the anti-HA antibody and analyzed with a Fuji BAS 2000 image analyzer. We have recently found that rAxin directly binds to β-catenin (5Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1101) Google Scholar). Because it has been shown that β-catenin directly binds to APC (10Rubinfeld B. Souza B. Albert I. Müller O. Chamberlain S.H. Masiarz F.R. Munemitsu S. Polakis P. Science. 1993; 262: 1731-1734Crossref PubMed Scopus (1177) Google Scholar, 11Su L. Vogelstein B. Kinzler K.W. Science. 1993; 262: 1734-1737Crossref PubMed Scopus (1115) Google Scholar, 12Rubinfeld B. Souza B. Albert I. Munemitsu S. Polakis P. J. Biol. Chem. 1995; 270: 5549-5555Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar), we examined whether rAxin makes a complex with APC through β-catenin. Various deletion mutants of Myc-rAxin expressed in COS cells were immunoprecipitated with the anti-Myc antibody. Consistent with our recent observations (5Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1101) Google Scholar), β-catenin was coprecipitated with Myc-rAxin (full-length), Myc-rAxin-(1–713), Myc-rAxin-(298–713), and Myc-rAxin-(298–506) (Fig. 1 A). Among them, APC was detected in the Myc-rAxin (full-length) and Myc-rAxin-(1–713) immune complexes, but not in the Myc-rAxin-(298–713) and Myc-rAxin-(298–506) immune complexes (Fig. 1 A). Unexpectedly, APC but not β-catenin was detected in the Myc-rAxin-(1–229) immune complex. Neither β-catenin nor APC was coprecipitated with Myc-rAxin-(713–832). Because rAxin-(1–229) contains the RGS domain, we examined whether the RGS domain itself (amino acids 89–216) makes a complex with APC. APC in COS, 293, and L cells was coprecipitated with GST-RGS (Fig. 1 B). It is known that APC is truncated at amino acids 1337 and 1427 in SW480 and DLD-1 cells, respectively, and that these truncated forms of APC fail to down-regulate β-catenin (9Polakis P. Biochim. Biophys. Acta. 1997; 1332: F127-F147PubMed Google Scholar,13Munemitsu S. Albert I. Souza B. Rubinfeld B. Polakis P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3046-3050Crossref PubMed Scopus (955) Google Scholar). These APC mutants in SW480 and DLD-1 cells were not coprecipitated with GST-RGS (Fig. 1 B). Consistent with the previous observations (10Rubinfeld B. Souza B. Albert I. Müller O. Chamberlain S.H. Masiarz F.R. Munemitsu S. Polakis P. Science. 1993; 262: 1731-1734Crossref PubMed Scopus (1177) Google Scholar, 11Su L. Vogelstein B. Kinzler K.W. Science. 1993; 262: 1734-1737Crossref PubMed Scopus (1115) Google Scholar), both full-length and truncated APC were coprecipitated with GST-β-catenin (Fig. 1 B). These results suggest that the RGS domain of rAxin makes a complex with the C-terminal half of APC in intact cells. Taken together with our observations (5Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1101) Google Scholar), rAxin has distinct binding sites for APC, β-catenin, and GSK-3β. It is notable that the APC mutants in colorectal carcinoma cell lines such as SW480 and DLD-1 cells do not associate with rAxin. To examine whether the RGS domain of rAxin directly interacts with APC, various deletion mutants of APC were purified as MBP fusion proteins (Fig. 2 A). GST-RGS bound to MBP-APC-(1211–2075), which contains seven 20-aa repeats, in a dose-dependent manner (Fig. 2 B). TheK d value was calculated to be 115 nm. However, GST-RGS did not bind to MBP-APC-(959–1338) which contains three 15-aa repeats and the first 20-aa repeat (Fig. 2 B). These results show that the RGS domain of rAxin directly interacts with the region containing the 20-aa repeats of APC. To characterize the interaction of APC with rAxin further, MBP-APC-(1211–1787), which contains the former four 20-aa repeats, and MBP-APC-(1788–2075), which contains the latter three 20-aa repeats, were purified. Both GST-RGS and GST-β-catenin bound to MBP-APC-(1211–1787), but they bound to MBP-APC-(1788–2075) less efficiently (Fig. 2 C). Furthermore, GST-β-catenin bound to both MBP-APC-(1211–1495) and MBP-APC-(1475–1787), whereas GST-RGS bound to MBP-APC-(1475–1787) but not to MBP-APC-(1211–1495) (Fig. 2 C). Therefore, the RGS domain does not interact with the region of APC containing the 15-aa repeats and the first and the second 20-aa repeats, which binds to β-catenin. These results are consistent with the observations that β-catenin but not the RGS domain of rAxin associated with the APC mutants in SW480 and DLD-1 cells. A family of RGS proteins has been identified in eukaryotic species ranging from yeast to mammals (16Dohlman H.G. Thorner J. J. Biol. Chem. 1997; 272: 3871-3874Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). The three-dimensional structure of a stable complex of RGS4 and Gαi1 has been determined (20Tesmer J.J.G. Berman D.M. Gilman A.G. Sprang S.R. Cell. 1997; 89: 251-261Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar). Residues that form the hydrophobic core of the RGS box of RGS4 are well conserved in the RGS domain of rAxin. However, 11 residues of RGS4 that make direct contact with Gαi1 are not conserved in the RGS domain of rAxin except for one amino acid. Therefore, it is conceivable that the RGS domain interacts with the proteins other than the α subunit of G proteins. Our results are the first demonstration that a member of the RGS protein family has a binding partner other than the α subunit of G proteins. We have found that rAxin-(298–506) directly binds to β-catenin-(175–423), which contains armadillo repeats 2–7 (5Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1101) Google Scholar). APC interacts with the armadillo repeats 2–10 of β-catenin (12Rubinfeld B. Souza B. Albert I. Munemitsu S. Polakis P. J. Biol. Chem. 1995; 270: 5549-5555Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar). Therefore, we next examined whether rAxin and APC share the binding site on β-catenin. GST-β-catenin bound to MBP-rAxin-(298–506) and MBP-APC-(959–1338) in a dose-dependent manner, and theirK d values were calculated to be 227 nmand 273 nm, respectively (data not shown). MBP-rAxin-(298–506) inhibited the binding of MBP-APC-(959–1338) to GST-β-catenin in a dose-dependent manner (Fig. 3 A). MBP-rAxin (full-length) also inhibited their binding although the inhibitory efficiency was less than MBP-rAxin-(298–506) (Fig. 3 A). Furthermore, we examined the effect of rAxin-(1–529), which contains the binding sites for APC and β-catenin, on the interaction of β-catenin with MBP-APC-(1211–1787), which binds to both β-catenin and rAxin. Although GST-rAxin-(1–529) bound to MBP-APC-(1211–1787) in a dose-dependent manner, it did not affect significantly the interaction of GST-β-catenin with MBP-APC-(1211–1787) (Fig. 3 B). These results are consistent with the results that in APC-(1211–1787) β-catenin prefers APC-(1211–1495) to APC-(1475–1787); inversely the RGS domain binds to APC-(1475–1787) but not to APC-(1211–1495). Taken together, although the β-catenin-binding sites of rAxin and APC do not simultaneously bind to β-catenin, β-catenin does not compete with rAxin for the binding to APC when they are full-length proteins. Furthermore, since rAxin has distinct binding sites for APC and β-catenin, these three proteins could make a complex. It has been shown that APC down-regulates the level of β-catenin (9Polakis P. Biochim. Biophys. Acta. 1997; 1332: F127-F147PubMed Google Scholar,13Munemitsu S. Albert I. Souza B. Rubinfeld B. Polakis P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3046-3050Crossref PubMed Scopus (955) Google Scholar). This APC activity was localized to the central region of the protein which contains at least three of the 20-aa repeat sequence (9Polakis P. Biochim. Biophys. Acta. 1997; 1332: F127-F147PubMed Google Scholar). The fragment containing the 15-aa repeats and the first 20-aa repeat does not down-regulate β-catenin (9Polakis P. Biochim. Biophys. Acta. 1997; 1332: F127-F147PubMed Google Scholar). Our results indicate that this region binds to β-catenin but not to the RGS domain of rAxin. Therefore, the binding of APC to β-catenin is not sufficient for decreasing the β-catenin level, and the binding to Axin may be necessary. To investigate whether rAxin regulates the stabilization of β-catenin, pulse-chase analysis in COS cells expressing HA-β-catenin was performed. Although equivalent amounts of HA-β-catenin were immunoprecipitated with the anti-HA antibody from the lysates of COS cells expressing HA-β-catenin alone and coexpressing HA-β-catenin and Myc-rAxin as assessed by immunoblot analysis (data not shown), pulse-labeled. HA-β-catenin gradually decreased with a half-life of 4 h (Fig. 4). When Myc-rAxin was cotransfected, HA-β-catenin exhibited a shorter half-life (Fig. 4). These results indicate that rAxin has an activity to stimulate the down-regulation of β-catenin. To down-regulate β-catenin, its phosphorylation by GSK-3β is required, and the mutations of the phosphorylation site stabilize β-catenin (7Yost C. Torres M. Miller J.R. Huang E. Kimelman D. Moon R.T. Genes Dev. 1996; 10: 1443-1454Crossref PubMed Scopus (1018) Google Scholar). It has been reported recently that β-catenin is ubiquitinated and that the ubiquitination of β-catenin is abolished when the GSK-3β phosphorylation site in β-catenin is mutated (21Aberle H. Bauer A. Stappert J. Kispert A. Kemler R. EMBO J. 1997; 16: 3797-3804Crossref PubMed Scopus (2160) Google Scholar). Therefore, the degradation of β-catenin could be regulated by the ubiquitination-proteasome pathway. Taken together with our observations that rAxin promotes GSK-3β-dependent phosphorylation of β-catenin (5Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1101) Google Scholar), the present results strongly suggest that rAxin stimulates the down-regulation of β-catenin in cooperation with APC. Literally hundreds of APC mutants have been reported in FAP and cancer patients (9Polakis P. Biochim. Biophys. Acta. 1997; 1332: F127-F147PubMed Google Scholar). Almost all of these mutations are confined to the 5′-half of the APC coding sequence and result in truncation of APC, which lacks the C-terminal half containing most of the 20-aa repeats. Our results indicate that these APC mutants do not interact with rAxin. Therefore, the reason why mutations of APC cause cancer may be due to its inability to bind to Axin. It has been reported that there are mutations of serine in consensus sequence of the phosphorylation site of β-catenin for GSK-3β in melanoma and colon cancer that have normal APC protein (22Korinek 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 (2935) Google Scholar, 23Morin P.J. Sparks A.B. Korinek V. Barker N. Clevers H. Vogelstein B. Kinzler K.W. Science. 1997; 275: 1787-1790Crossref PubMed Scopus (3504) Google Scholar, 24Rubinfeld B. Robbins P. El-Gamil M. Albert I. Porfiri E. Polakis P. Science. 1997; 275: 1790-1792Crossref PubMed Scopus (1135) Google Scholar). Thus, there are at least two ways to increase levels of β-catenin due to mutations in APC and β-catenin itself. Therefore, mutations in APC-, GSK-3β-, and β-catenin-binding sites on Axin may cause human cancer. We thank Drs. T. Akiyama, K. Morishita, A. Nagafuchi, Sh. Tsukita, and E. Tahara for their plasmids and cell lines. We wish to thank the Research Center for Molecular Medicine, Hiroshima University School of Medicine, for the use of their facilities.
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