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Regulation of the Protein Kinase Activity of ShaggyZeste-white3 by Components of the Wingless Pathway in Drosophila Cells and Embryos

1999; Elsevier BV; Volume: 274; Issue: 31 Linguagem: Inglês

10.1074/jbc.274.31.21790

1083-351X

Laurent Ruel, Vuk Stambolic, Adnan Hussein Ali, Armen S. Manoukian, James R. Woodgett,

Kruppel-like factors research

The protein-serine kinase ShaggyZeste-white3 (SggZw3) is theDrosophila homolog of mammalian glycogen synthase kinase-3 and has been genetically implicated in signal transduction pathways necessary for the establishment of patterning. SggZw3 is a putative component of the Wingless (Wg) pathway, and epistasis analyses suggest that SggZw3 function is repressed by Wg signaling. Here, we have investigated the biochemical consequences of Wg signaling with respect to the SggZw3 protein kinase in two types ofDrosophila cell lines and in embryos. Our results demonstrate that SggZw3 activity is inhibited following exposure of cells to Wg protein and by expression of downstream components of Wg signaling, Drosophila frizzled 2 and dishevelled. Wg-dependent inactivation of SggZw3 is accompanied by serine phosphorylation. We also show that the level of SggZw3 activity regulates the stability of Armadillo protein and modulates the level of phosphorylation of D-Axin and Armadillo. Together, these results provide direct biochemical evidence in support of the genetic model of Wg signaling and provide a model for dissecting the molecular interactions between the signaling proteins. The protein-serine kinase ShaggyZeste-white3 (SggZw3) is theDrosophila homolog of mammalian glycogen synthase kinase-3 and has been genetically implicated in signal transduction pathways necessary for the establishment of patterning. SggZw3 is a putative component of the Wingless (Wg) pathway, and epistasis analyses suggest that SggZw3 function is repressed by Wg signaling. Here, we have investigated the biochemical consequences of Wg signaling with respect to the SggZw3 protein kinase in two types ofDrosophila cell lines and in embryos. Our results demonstrate that SggZw3 activity is inhibited following exposure of cells to Wg protein and by expression of downstream components of Wg signaling, Drosophila frizzled 2 and dishevelled. Wg-dependent inactivation of SggZw3 is accompanied by serine phosphorylation. We also show that the level of SggZw3 activity regulates the stability of Armadillo protein and modulates the level of phosphorylation of D-Axin and Armadillo. Together, these results provide direct biochemical evidence in support of the genetic model of Wg signaling and provide a model for dissecting the molecular interactions between the signaling proteins. The product of the Drosophila wingless (wg) gene is a secreted protein homologous to vertebrate Wnts (1Klingensmith J. Nusse R. Dev. Biology. 1994; 166: 396-414Crossref PubMed Scopus (185) Google Scholar). Genetic analysis of wg has revealed roles in processes controlling embryonic segmentation, gut formation, and imaginal disc patterning (2Cohen S.M. Di Nardo S. Trends Genet. 1993; 9: 189-192Abstract Full Text PDF PubMed Scopus (8) Google Scholar, 3Diaz-Benjumea F.J. Cohen S.M. Development. 1994; 120: 1661-1670PubMed Google Scholar, 4Siegfried E. Chou T.B. Perrimon N. Cell. 1992; 71: 1167-1179Abstract Full Text PDF PubMed Scopus (344) Google Scholar). Additional genes have been implicated in the secretion, reception, or interpretation of the Wg 1The abbreviations used are: Wg, Wingless; Dsh, Dishevelled; Arm, Armadillo; Dfz2, Drosophila Frizzled 2; SggZw3, ShaggyZeste-white3, GSK-3, glycogen synthase kinase-3; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-1, 1-bis(hydroxymethyl)ethyl]glycine. signal:dishevelled (dsh) (5Klingensmith J. Nusse R. Perrimon N. Genes Dev. 1994; 8: 118-130Crossref PubMed Scopus (344) Google Scholar) and armadillo(arm) (6Peifer M. Wieschaus E. Cell. 1990; 63: 1167-1176Abstract Full Text PDF PubMed Scopus (385) Google Scholar). Dsh protein is a novel protein with a discs-large homology region, whereas the arm gene encodes theDrosophila homolog of β-catenin, a component of vertebrate adherens junctions. Drosophila frizzled 2 (Dfz2) was recently identified as a protein with an amino-terminal cysteine-rich extracellular domain followed by seven transmembrane domains (7Bhanot P. Brink M. Harryman Samos C. Hsieh J.-C. Wang Y. Macke J.P. Andrew D. Nathans J. Nusse R. Nature. 1996; 382: 225-230Crossref PubMed Scopus (1235) Google Scholar). The Dfz2 protein functions as a Wg receptor in cultured cells, but as yet, there are no known Dfz2 mutants. Whereas the above-mentioned genes act positively in Wg signaling, an additional gene called shaggy or zeste-white3 (sgg zw3) plays an inhibitory role in this pathway (1Klingensmith J. Nusse R. Dev. Biology. 1994; 166: 396-414Crossref PubMed Scopus (185) Google Scholar, 4Siegfried E. Chou T.B. Perrimon N. Cell. 1992; 71: 1167-1179Abstract Full Text PDF PubMed Scopus (344) Google Scholar, 8Struhl G. Basler K. Cell. 1993; 72: 527-540Abstract Full Text PDF PubMed Scopus (727) Google Scholar). sgg zw3 encodes a protein-serine kinase that has been highly conserved throughout the eukaryotic kingdoms (4Siegfried E. Chou T.B. Perrimon N. Cell. 1992; 71: 1167-1179Abstract Full Text PDF PubMed Scopus (344) Google Scholar,9Ruel L. Pantesco V. Lutz Y. Simpson P. Bourouis M. EMBO J. 1993; 12: 1657-1669Crossref PubMed Scopus (63) Google Scholar, 10Ruel L. Bourouis M. Heitzler P. Pantesco V. Simpson P. Nature. 1993; 362: 557-560Crossref PubMed Scopus (165) Google Scholar). The mammalian homolog of sgg zw3 is glycogen synthase kinase-3 (GSK-3), which is encoded by two independent genes, GSK-3α and GSK-3β (11Woodgett J.R. EMBO J. 1990; 9: 2431-2438Crossref PubMed Scopus (1161) Google Scholar). By a combination of clonal analysis, genetic epistasis, and biochemical experiments, wg class genes have been ordered within the same pathway (12Hooper J.E. Nature. 1994; 372: 461-464Crossref PubMed Scopus (111) Google Scholar, 13Noordermeer J. Klingensmith J. Perrimon N. Nusse R. Nature. 1994; 367: 80-83Crossref PubMed Scopus (323) Google Scholar, 14Siegfried E. Wilder E.L. Perrimon N. Nature. 1994; 367: 76-80Crossref PubMed Scopus (284) Google Scholar, 15Yanagawa S. van Leeuwen F. Wodarz A. Klingensmith J. Nusse R. Genes Dev. 1995; 9: 1087-1097Crossref PubMed Scopus (342) Google Scholar). armadillo and dishevelledembryonic phenotypes are very similar to the wg embryonic phenotype (12Hooper J.E. Nature. 1994; 372: 461-464Crossref PubMed Scopus (111) Google Scholar, 13Noordermeer J. Klingensmith J. Perrimon N. Nusse R. Nature. 1994; 367: 80-83Crossref PubMed Scopus (323) Google Scholar, 14Siegfried E. Wilder E.L. Perrimon N. Nature. 1994; 367: 76-80Crossref PubMed Scopus (284) Google Scholar), whereas sgg zw3 has a mutant phenotype very similar to that of embryos in which wg has been expressed in all cells (12Hooper J.E. Nature. 1994; 372: 461-464Crossref PubMed Scopus (111) Google Scholar, 16Noordermeer J. Johnston P. Rijsewijk F. Nusse R. Lawrence P.A. Development. 1992; 116: 711-719PubMed Google Scholar, 17Peifer M. Sweeton M. Casey M. Wieschaus E. Development. 1994; 120: 369-380Crossref PubMed Google Scholar). Genetic data inDrosophila suggest that the functions ofsgg zw3 are antagonized by Wg signaling (4Siegfried E. Chou T.B. Perrimon N. Cell. 1992; 71: 1167-1179Abstract Full Text PDF PubMed Scopus (344) Google Scholar). In fact, mutations in wg and sgg zw3 have opposite effects on cell fate determination, and each mutation has an opposite effect on Arm protein levels (17Peifer M. Sweeton M. Casey M. Wieschaus E. Development. 1994; 120: 369-380Crossref PubMed Google Scholar, 18Peifer M. Pai L.M. Casey M. Dev. Biol. 1994; 166: 543-556Crossref PubMed Scopus (204) Google Scholar). In embryos, the normal segmental accumulation of Arm protein is absent in wg, whereassgg zw3 mutants have uniformly high levels of Arm protein. Recently, an additional protein called Axin has been implicated in the regulation of β-catenin/Arm (19Zeng 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 (797) Google Scholar). Axin and its Drosophilahomolog (D-Axin) act as scaffold proteins and bind GSK-3/SggZw3, β-catenin/Arm, and APC (adenomatous polyposis coli protein) in a complex (20Behrens 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 (1118) Google Scholar). In Drosophila cells, the overexpression of D-Axin results in Arm destabilization. 2L. Ruel, N. Anthopoulos, J. Gonçalves, A. S. Manoukian, and J. R. Woodgett, submitted for publication. The presence of Axin is necessary for GSK-3 to efficiently phosphorylate β-catenin (19Zeng 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 (797) Google Scholar) and to inhibit β-catenin-mediated LEF-1 activation (22Sakanaka C. Weiss J. Williams L.T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3020-3023Crossref PubMed Scopus (283) Google Scholar). These data have been assembled into a model in which Wg protein is secreted and received by neighboring cells, where a signal transduction cascade is initiated (1Klingensmith J. Nusse R. Dev. Biology. 1994; 166: 396-414Crossref PubMed Scopus (185) Google Scholar). The Wg signal, at least in embryos and cultured cells, is transduced through Dsh and induces hyperphosphorylation of Dsh protein, possibly via casein kinase-2 (15Yanagawa S. van Leeuwen F. Wodarz A. Klingensmith J. Nusse R. Genes Dev. 1995; 9: 1087-1097Crossref PubMed Scopus (342) Google Scholar,23Willert K. Brink M. Wodarz A. Varmus H. Nusse R. EMBO J. 1997; 11: 3089-3096Crossref Scopus (203) Google Scholar). Through an unknown mechanism, activation of Dsh blocks the function of SggZw3 and D-Axin, resulting in decreased phosphorylation of Arm. Unphosphorylated Arm has increased stability and accumulates in the cytoplasm (15Yanagawa S. van Leeuwen F. Wodarz A. Klingensmith J. Nusse R. Genes Dev. 1995; 9: 1087-1097Crossref PubMed Scopus (342) Google Scholar, 24van Leeuwen F. Harryman Samos C. Nusse R. Nature. 1994; 368: 342-344Crossref PubMed Scopus (172) Google Scholar), where it interacts with an high mobility group-like factor, LEF-1/pangolin (25Brunner E. Peter O. Schweizer L. Basler K. Nature. 1997; 385: 829-833Crossref PubMed Scopus (447) Google Scholar, 26Riese J. Yu X. Munnerlyn A. Eresh S. Hsu S. Grosschedl R. Bienz M. Cell. 1997; 88: 777-787Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). Recently, the mammalian homolog of SggZw3, GSK-3, has been shown to be regulated by Drosophila Wg protein in fibroblasts (27Cook D. Fry M.J. Hughes K. Sumathipala R. Woodgett J.R. Dale T.C. EMBO J. 1996; 17: 4526-4536Crossref Scopus (344) Google Scholar), but direct biochemical evidence for inhibition of SggZw3 by Wg signaling has yet to be demonstrated. To address the mechanism by which Wg signals via SggZw3, the effect of the known components of Drosophila Wg signaling (Wg, Dfz2, and Dsh) on SggZw3 activity was investigated in cultured cells and embryos. We used an imaginal disc cell line (cl-8 (clone 8)) that responds to Wg signals and Schneider (S2) cells, which are unresponsive to Wg (15Yanagawa S. van Leeuwen F. Wodarz A. Klingensmith J. Nusse R. Genes Dev. 1995; 9: 1087-1097Crossref PubMed Scopus (342) Google Scholar, 24van Leeuwen F. Harryman Samos C. Nusse R. Nature. 1994; 368: 342-344Crossref PubMed Scopus (172) Google Scholar). Using Wg-conditioned medium, we show that the activity of SggZw3protein kinase is inhibited by Wg in cl-8 cells and that overexpression of Dfz2 or Dsh in cells reconstitutes Wg signaling in the absence of Wg as judged by inhibition of the kinase and accumulation of Arm protein. We also demonstrate that the regulation of SggZw3 activity, in turn, controls the stability of Arm protein by modulating the level of phosphorylation of D-Axin and Arm. These results provide direct biochemical evidence in support of previous genetic analyses. Rabbit antisera to Arm and Dsh were raised against glutathione S-transferase (GST) fusion proteins. GST-Dsh was constructed by cloning a 1256-base pair XhoI-NotI fragment of the dishevelled coding region, corresponding to amino acids 395–624, into XhoI-NotI sites in pGEX-4T-1 (Amersham Pharmacia Biotech). cDNA fragments encoding amino acids 1–367 of Arm protein and 1–514 of SggZw3protein were cloned into pGEX-4T-1 and pET15b (Novagen), respectively. Fusion proteins were produced in Escherichia coli strain BL21(DE3) and purified from bacterial lysates before immunization. DrosophilaSchneider line-2 and wing imaginal disc cl-8 cells were maintained as described (24van Leeuwen F. Harryman Samos C. Nusse R. Nature. 1994; 368: 342-344Crossref PubMed Scopus (172) Google Scholar). Wg protein assays were performed essentially as published (24van Leeuwen F. Harryman Samos C. Nusse R. Nature. 1994; 368: 342-344Crossref PubMed Scopus (172) Google Scholar, 28Cumberledge S. Krasnow M.A. Nature. 1993; 363: 549-552Crossref PubMed Scopus (44) Google Scholar). Selection of stably transformed cl-8 cell lines was performed using methotrexate (29Currie D.A. Milner M.J. Evans C.W. Development. 1988; 102: 805-814Google Scholar). The expression vector pRmHa-1 is designed to express proteins under control of the metallothionein promoter. The 2.8-kilobase pair BamHI-HindIII fragment of dsh cDNA in pBluescript SK+(Stratagene) corresponding to the entire coding region was cloned into the BamHI-HindIII sites of pRmHa-1. Thedsh/pRmHa-1 or sgg zw3/HApRmHa-1 vector was introduced into cl-8 cells by cotransfection with a second vector, pHGCO, carrying a selectable dhfr gene, which confers resistance to methotrexate (0.5 μg/ml). Transformed cells were maintained between 1 × 106 and 1 × 107 cells/ml and examined for metal-inducible gene expression (by addition of 0.5 mm CuSO4) by immunoblotting. For expression in cl-8 cells, the D-axin-(332–642) fragment (amplified by polymerase chain) was subcloned into the pAc5.1/V5-His6 vector (Invitrogen) in frame with the His epitope. Transfected cells were washed with phosphate-buffered saline and lysed in 20 mm Tris-HCl (pH 8) and 100 mmNaCl. For purification of D-Axin-(330–642)-His6, 10 μl of nickel-Sepharose beads were added in lysates. The complexes were washed four times with 20 mm Tris-HCl (pH 8), 100 mm NaCl, and 10 mm imidazole and resolved by SDS-PAGE or incubated with [γ-32P]ATP for 30 min. Transfected Dsh S2 cells were treated with CuSO4 to induce Dsh expression and labeled overnight with 1 mCi of [32P]orthophosphate/ml of S2 phosphate-free medium + 10% dialyzed fetal calf serum. Radioimmune precipitation assay buffer cell lysates were normalized for incorporation by Cerenkov counting (30Woodgett J.R. Wood J. Cell Lines in Neurobiology: A Practical Approach. IRL Press, Oxford1992: 133-159Google Scholar). After immunoprecipitation of SggZw3 protein and separation by SDS-PAGE, proteins were transferred to polyvinylidene difluoride membranes.32P-Labeled SggZw3 was subjected to partial acid hydrolysis, and the phosphoamino acids were separated in two dimensions by thin-layer electrophoresis (31Boyle W.J. van der Geer P. Hunter T. Methods Enzymol. 1991; 201: 110-149Crossref PubMed Scopus (1276) Google Scholar). For overexpression of SggZw3, homozygous HS-SggZw3 Drosophila eggs were collected 3 h after laying, heat-shocked for 8 min at 37 °C, and allowed to recover for an additional 1.5 h at 25 °C. To generate sgg zw3 M11-1mutant embryos, germ line mosaics were produced using the yeast recombinase-base flippase-dominant female sterile system as described by Chou and Perrimon (32Chou T.B. Perrimon N. Genetics. 1992; 131: 643-653Crossref PubMed Google Scholar). Homozygous mutant embryos can be recognized morphologically by a lack of segmentation. For overexpression of Wg,Drosophila males homozygous for arm-Gal4 were crossed to virgin Drosophila females harboring pUAS-Wg, and their progeny embryos were collected at 3–6 h. Wild-type embryos of the same stage were used as controls. Embryos were lysed in Gentle Soft buffer (28Cumberledge S. Krasnow M.A. Nature. 1993; 363: 549-552Crossref PubMed Scopus (44) Google Scholar) and were subjected to immunoprecipitation analysis as described below. Cells lines were washed with phosphate-buffered saline and lysed in Gentle Soft buffer (28Cumberledge S. Krasnow M.A. Nature. 1993; 363: 549-552Crossref PubMed Scopus (44) Google Scholar). For SggZw3immunoprecipitation, 20 μl of protein A-Sepharose or 20 μl of protein G-Sepharose were pre-bound to rabbit polyclonal antiserum or to monoclonal antibodies (anti-SggZw3, 2G2C5), respectively, and were added to the clarified cell lysates at 4 °C for 2 h. Immunocomplexes were washed four times with Gentle Soft buffer (28Cumberledge S. Krasnow M.A. Nature. 1993; 363: 549-552Crossref PubMed Scopus (44) Google Scholar).In vitro SggZw3 kinase assays were performed for 30 min as described previously (33Stambolic V. Woodgett J.R. Biochem. J. 1994; 303: 701-704Crossref PubMed Scopus (512) Google Scholar, 34Hughes K. Nikolakaki E. Plyte S.E. Totty N.F. Woodgett J.R. EMBO J. 1993; 12: 803-808Crossref PubMed Scopus (525) Google Scholar). Phosphorylated peptide was separated from unincorporated [γ-32P]ATP by Tricine/SDS-PAGE and quantified using a PhosphorImager. To analyze the biochemical consequences of Wg signaling, we exploited an imaginal disc cell line (cl-8) that is responsive to Wg (24van Leeuwen F. Harryman Samos C. Nusse R. Nature. 1994; 368: 342-344Crossref PubMed Scopus (172) Google Scholar). To determine the biological effects of Wg, cl-8 cells were exposed to the serum-free conditioned medium from either heat-shocked Schneider HS-wg (Wg-conditioned medium) or Schneider control cells (S2 control medium), and cytoplasmic extracts were prepared and immunoblotted with antibodies to Wg, Arm, and Dsh (Fig. 1A) (15Yanagawa S. van Leeuwen F. Wodarz A. Klingensmith J. Nusse R. Genes Dev. 1995; 9: 1087-1097Crossref PubMed Scopus (342) Google Scholar). Wg-containing medium increased Arm levels within 2 h, reaching a maximum after 6 h. By contrast, cellular levels of Dsh did not change in this time period. However, Wg induced the formation of electrophoretically retarded forms of Dsh. These modifications have been previously observed by Yanagawa et al. (15Yanagawa S. van Leeuwen F. Wodarz A. Klingensmith J. Nusse R. Genes Dev. 1995; 9: 1087-1097Crossref PubMed Scopus (342) Google Scholar) and Willertet al. (23Willert K. Brink M. Wodarz A. Varmus H. Nusse R. EMBO J. 1997; 11: 3089-3096Crossref Scopus (203) Google Scholar) and correspond to hyperphosphorylation of Dsh protein. Exposure of cells to medium conditioned by control S2 cells affected neither Arm levels nor the Dsh electrophoretic pattern. To determine whether Wg modulates SggZw3 activity, SggZw3 was immunoprecipitated from lysates of cl-8 cells treated with Wg-conditioned medium or S2 control medium. Protein kinase activity was measured using a peptide substrate specific for the GSK-3 family of protein kinases (GS-1 peptide (33Stambolic V. Woodgett J.R. Biochem. J. 1994; 303: 701-704Crossref PubMed Scopus (512) Google Scholar)). Incubation of cl-8 cells with Wg-conditioned medium caused a time-dependent inhibition of SggZw3 protein kinase activity (Fig. 1B). After 2–4 h of treatment with Wg-conditioned medium, total GS-1 peptide kinase activity was suppressed by 40–50% compared with the activity observed in cells treated with S2 control medium. Wg did not affect the expression of SggZw3 as judged by immunoblotting (Fig. 1A). To confirm the effect of Wg protein on the activity of SggZw3, we investigated how SggZw3 functions in Wg signaling during embryogenesis, analyzing SggZw3activity in embryos with a wild-type or sgg zw3mutant genotype, embryos overexpressing sgg zw3, and embryos expressing wg ubiquitously.sgg zw3 embryos were made homozygous for thesgg zw3 M11-1 allele, and SggZw3immunoprecipitates from these mutant embryos contained no detectable SggZw3 activity, which verified the specificity of the assay (Fig. 2B). Furthermore, SggZw3 immunoprecipitates from embryos overexpressing SggZw3 from a heat shock-inducible transgene (HS-SggZw3) exhibited 2.5-fold higher activity than immunoprecipitates from wild-type embryos (Fig. 2B). To determine the effect of Wg overexpression on SggZw3activity, Wg was ectopically expressed in early embryos using a line that carries a GAL4-driven wgtransgene (pUAS-Wg) crossed to a line that ubiquitously expresses GAL4 (arm-GAL4). The activity of SggZw3 from these embryos was determined to be ∼30% lower than that from wild-type control lysates (Fig. 2B). Immunoblotting of the embryonic extracts revealed equivalent SggZw3 levels in the wild-typesgg zw3 M11-1 allele and in the pUAS-Wg-expressing embryos, as expected (Fig. 2A). Armadillo immunoblots revealed accumulation of Arm protein in the SggZw3 M11-1and pUAS-Wg extracts. Overexpression of Dsh protein in cl-8 and S2 cells bypasses the need for Wg and mimics Wg signaling (15Yanagawa S. van Leeuwen F. Wodarz A. Klingensmith J. Nusse R. Genes Dev. 1995; 9: 1087-1097Crossref PubMed Scopus (342) Google Scholar). To investigate the effect of overexpression of Dsh on SggZw3 activity, we used S2 and cl-8 cell lines expressing Dsh under the control of an inducible metallothionein promoter. Treatment of these cell lines with CuSO4 led to a time-dependent increase in Dsh protein levels, as well as induction of forms of the protein with reduced electrophoretic mobility similar to the forms observed in untransfected cl-8 cells exposed to Wg protein (Fig. 3, A and C). Concomitant with the increase in Dsh protein levels was an increase in Arm levels (Fig. 3, A and C), indicating that overexpression of Dsh in S2 and cl-8 cells mimics Wg signaling. To determine whether Dsh protein inhibits SggZw3 activity, we examined SggZw3 protein kinase activity in the Dsh-inducible cl-8 and S2 cell lines (Fig. 3, B and D). Dsh overexpression in cl-8 and S2 cells revealed similar inhibition curves in both lines and induced a rapid decrease in SggZw3 activity that was detectable after 2 h and reached a maximum (70%) after 4–6 h, whereas SggZw3expression levels were not affected (Fig. 3, A and C). The decrease in SggZw3 activity observed in the Dsh experiments in cl-8 cells coincided with the effects of Wg on SggZw3 activity in cl-8 cells and supports the genetic model in which Wg repression of SggZw3 is mediated via Dsh. Unlike cl-8 cells, S2 cells do not respond to extracellular Wg as judged by Dsh modification and Arm stabilization (data not shown) (15Yanagawa S. van Leeuwen F. Wodarz A. Klingensmith J. Nusse R. Genes Dev. 1995; 9: 1087-1097Crossref PubMed Scopus (342) Google Scholar, 24van Leeuwen F. Harryman Samos C. Nusse R. Nature. 1994; 368: 342-344Crossref PubMed Scopus (172) Google Scholar). Transfection of the transmembrane proteinDrosophila Frizzled 2 (Dfz2) into S2 cells enables the cells to accumulate Arm in response to Wg, suggesting that Dfz2 acts as a receptor for Wg and that the reason for the lack of responsiveness of these cells to Wg is simply due to lack of Dfz2 expression (7Bhanot P. Brink M. Harryman Samos C. Hsieh J.-C. Wang Y. Macke J.P. Andrew D. Nathans J. Nusse R. Nature. 1996; 382: 225-230Crossref PubMed Scopus (1235) Google Scholar). To investigate whether Dfz2 expression affected SggZw3activity, we used S2 cell lines expressing Dfz2 under the control of an inducible metallothionein promoter. Addition of CuSO4 to the medium of these cells induced an increase in the levels ofDfz2 RNA (Fig. 4A), leading to the appearance of slower migrating forms of Dsh and an increase in cytoplasmic Arm levels within 2 h, whereas SggZw3 protein levels were unaffected (Fig. 4A). However, immunoprecipitates of SggZw3 exhibited a time-dependent decrease in protein kinase activity upon induction of Dfz2 expression, similar to the effects of overexpression of Dsh in S2 cells (Fig. 4B). Together, these data demonstrate that overexpression of Dfz2 in S2 cells is sufficient to trigger the Wg pathway, including modification of Dsh, repression of SggZw3, and stabilization of Arm. To probe the mechanism via which Wg, Dfz2, and Dsh inactivate SggZw3, S2 cell lines harboring inducible Dsh were metabolically labeled with [32P]phosphate, and SggZw3 was immunoprecipitated and resolved by SDS-PAGE. Induction of Dsh expression caused a 2–2.5-fold increase in [32P]phosphate associated with SggZw3(Fig. 5A). Subsequent phosphoamino acid analysis revealed the presence of only phosphoserine in the S2 cell sample (Fig. 5B). These data suggest that Dsh induces a specific increase in serine phosphorylation of SggZw3, which may mediate the reduction in protein kinase activity. Surprisingly, SggZw3 in S2 cells does not contain detectable phosphotyrosine (34Hughes K. Nikolakaki E. Plyte S.E. Totty N.F. Woodgett J.R. EMBO J. 1993; 12: 803-808Crossref PubMed Scopus (525) Google Scholar). SggZw3 contained both phosphotyrosine and phosphoserine in cl-8 cells. Since induction of the Wg pathway resulted in equal -fold inhibition in both S2 and cl-8 cells, we conclude that Wg-mediated regulation of SggZw3 is independent of tyrosine phosphorylation. We have shown that negative regulation of SggZw3 activity leads to Arm accumulation in Drosophila embryos and cells. Biochemical analysis has indicated that D-Axin/Axin negatively regulates β-catenin/Arm by interacting with GSK-3β/SggZw3 (19Zeng 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 (797) Google Scholar).2 D-Axin is structurally related to vertebrate Axins, with the regions of highest identity corresponding to previously defined binding domains of Axin.2 Armadillo contains "consensus" phosphorylation site sequences for GSK-3/SggZw3 (35Aberle H. Bauer A. Stappert J. Kispert A. Kemler R. EMBO J. 1997; 16: 3797-3804Crossref PubMed Scopus (2172) Google Scholar). D-Axin also contains such sequences (19Zeng 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 (797) Google Scholar).2 However, it has been reported that mammalian GSK-3 phosphorylates β-catenin significantly only in the presence of the Axin protein (19Zeng 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 (797) Google Scholar). Therefore, we examined whether SggZw3could phosphorylate Arm and D-Axin under conditions in which these proteins formed a complex. To determine whether D-Axin and Arm are substrates for SggZw3, we purified D-Axin or various deletion mutants of D-Axin and Arm from E. coli as histidine fusion proteins (Fig. 6). Baculovirus-expressed GST-SggZw3 (36Stambolic V. Ruel L. Woodgett J.R. Curr. Biol. 1996; 6: 1664-1668Abstract Full Text Full Text PDF PubMed Google Scholar) phosphorylated D-Axin, D-Axin-(302–746), D-Axin-(356–565), and D-Axin-(356–746), but not D-Axin-(383–565) and D-Axin-(34–356) (Fig. 6). In the absence of D-Axin, no significant phosphorylation of Armadillo was observed, whereas in its presence, the phosphorylation was greatly increased (Fig. 6). These data indicate that Sgg phosphorylation of Armadillo is directed via D-Axin. We found that D-Axin is phosphorylated by SggZw3 and binds to both SggZw3 and Arm.2 We therefore examined whether the inhibition of SggZw3 activity by Wg affects its interaction with D-Axin and monitored the level of phosphorylation of D-Axin. To test this possibility, in vitro binding and phosphorylation assays were carried out using a D-Axin-(330–642) fusion protein containing SggZw3-binding sites and consensus sites of phosphorylation for SggZw3. D-Axin-(330–642)-His6 was transfected as a histidine fusion protein into cl-8 cells, cl-8 cells treated with Wg, and cl-8 cells expressing SggZw3. The histidine-tagged complexes from the cl-8 cell lysates were purified using nickel-Sepharose beads, and the amount of SggZw3 captured on the beads was determined by immunoblotting. In addition, the phosphorylation of D-Axin-(330–642)-His6 by SggZw3 was determined by addition of [γ-32P]ATP. In the lysates from cells treated with Wg, SggZw3 was found in association with D-Axin-(330–642)-His6. However, the degree of binding was reduced ∼2-fold compared with the amount of SggZw3 associated with Axin in lysates of untreated cl-8 cells (Fig. 7). The negative effect of Wg signal on the binding of SggZw3 correlated with a decrease in phosphorylation of D-Axin. By contrast, Axin complexes within lysates expressing SggZw3 contained more SggZw3protein as well as higher Axin kinase activity (Fig. 7). These results indicate that D-Axin physically interacts with SggZw3 and that Wg signaling leads to a reduction of both SggZw3activity and its interaction with D-Axin. Previous studies have shown that treatment of cl-8 cell lines with Wg leads to hyperphosphorylation of Dsh protein and to cytoplasmic accumulation of Armadillo (15Yanagawa S. van Leeuwen F. Wodarz A. Klingensmith J. Nusse R. Genes Dev. 1995; 9: 1087-1097Crossref PubMed Scopus (342) Google Scholar, 23Willert K. Brink M. Wodarz A. Varmus H. Nusse R. EMBO J. 1997; 11: 3089-3096Crossref Scopus (203) Google Scholar, 24van Leeuwen F. Harryman Samos C. Nusse R. Nature. 1994; 368: 342-344Crossref PubMed Scopus (172) Google Scholar). Here, we report that Wg signaling as initiated by Wg, Dfz2, or Dsh expression causes enzymatic inactivation of SggZw3 activity in concert with stabilization of Arm. These data indicate that Wg or overexpression of "upstream" components of this pathway mimics Wingless signaling by specifically inhibiting the activity of SggZw3. We have demonstrated that regulation of kinase activity, rather than protein levels, is the main determinant of the effects of Wg on SggZw3, suggesting post-translational modification of this protein kinase activity. In support of this, induction of Dsh expression increased the levels of SggZw3 phosphorylation 2-fold (Fig. 6), and the presence of phosphoserine in SggZw3 protein from S2 cells suggested that the mechanism of repression of SggZw3 activity is mediated by serine phosphorylation. Previous studies have shown that members of the GSK-3 family are inhibited by phosphorylation at an amino-terminal serine residue (serine 9 in GSK-3β and serine 21 in GSK-3α) (33Stambolic V. Woodgett J.R. Biochem. J. 1994; 303: 701-704Crossref PubMed Scopus (512) Google Scholar, 37Sutherland C. Leighton I.A. Cohen P. Biochem. J. 1993; 296: 15-19Crossref PubMed Scopus (758) Google Scholar). Phosphorylation of the SggZw3 residue equivalent to serine 9 does not appear to be the mechanism via which the Wg pathway inhibits SggZw3 for several reasons. In mammals, this site is targeted by agents acting via phosphatidylinositol 3′-kinase, and the residue can be phosphorylated in vitro and in transfected cells by protein kinase B/AKT (38Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4397) Google Scholar). However, Wg inhibition of GSK-3 in 10-T1/2 cells is not sensitive to inhibitors of phosphatidylinositol 3′-kinase, nor is Drosophila protein kinase B activity stimulated by Wg (27Cook D. Fry M.J. Hughes K. Sumathipala R. Woodgett J.R. Dale T.C. EMBO J. 1996; 17: 4526-4536Crossref Scopus (344) Google Scholar). 3L. Ruel, unpublished observation.Furthermore, Dsh-induced tryptic phosphopeptides of SggZw3are inconsistent with phosphorylation of the site analogous to serine 9 in GSK-3β.3 Identification of the Wg/Dsh-inducible serine residue(s) on SggZw3 is underway. Although our data provide biochemical support for the genetically defined Wg pathway, at least three gaps remain in this signaling cascade: the mechanism via which Dsh is activated by Dfz2, the mechanism by which Dsh inhibits SggZw3, and the means by which SggZw3 induces turnover of Arm. We found a correlation between the modification of the phosphorylation state of Dsh protein and an increase in Arm stability, in agreement with the studies of Yanagawa et al. (15Yanagawa S. van Leeuwen F. Wodarz A. Klingensmith J. Nusse R. Genes Dev. 1995; 9: 1087-1097Crossref PubMed Scopus (342) Google Scholar) and Willert et al. (23Willert K. Brink M. Wodarz A. Varmus H. Nusse R. EMBO J. 1997; 11: 3089-3096Crossref Scopus (203) Google Scholar). A similar correlation was observed between the decrease in SggZw3 activity and accumulation of hypophosphorylated Arm protein. Willert et al. (23Willert K. Brink M. Wodarz A. Varmus H. Nusse R. EMBO J. 1997; 11: 3089-3096Crossref Scopus (203) Google Scholar) found that whereas Dfz2 expression induced Dsh hyperphosphorylation, it did not induce stabilization of Arm. In our hands, Dfz2 expression was sufficient for both of these processes in S2 cells. The reason for the discrepancy is unclear, but may relate to the degree of overexpression of Dfz2. Yost et al. (39Yost C. Torres M. Miller J.R. Huang E. Kimelman D. Moon R.T. Genes Dev. 1996; 10: 1443-1454Crossref PubMed Scopus (1020) Google Scholar) proposed that β-catenin is directly phosphorylated by GSK-3, consistent with the finding that phosphorylation of Arm protein is decreased with the inhibition of SggZw3 activity. However, Arm is a poor in vitrotarget of SggZw3. Phosphorylation of Arm is enormously increased in the presence of D-Axin. We have demonstrated that D-Axin is phosphorylated by SggZw3 and that the binding of SggZw3 to D-Axin is dependent upon the level of SggZw3 activity. Repression of SggZw3 activity by Wg signaling induced dissociation of the SggZw3·D-Axin·Arm complex, leading to an accumulation of Arm protein. Together, these data suggest that Sgg binding is dependent upon or stimulated by its phosphorylation of Axin. Once bound to Axin, it can access the Arm molecule that is associated with Axin and phosphorylate it. Inactivation of Sgg results in dephosphorylation of Axin and release of the kinase, compartmentalizing it away from Arm. Mammalian studies have suggested that a more complex mechanism for the regulation of β-catenin levels by GSK-3 involved another player, APC. In this case, Axin forms a complex with GSK-3, β-catenin, and APC (19Zeng 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 (797) Google Scholar, 20Behrens 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 (1118) Google Scholar). APC is directly phosphorylated by GSK-3 via Axin, which increases binding of APC to β-catenin and its subsequent degradation (40Rubinfield B. Souza B. Albert I. Muller O. Chamberlain S.H. Masiarz F.R. Munemitsu S. Polakis P. Science. 1993; 262: 1731-1734Crossref PubMed Scopus (1183) Google Scholar, 41Papkoff J. Rubinfield B. Schryver B. Polakis P. Mol. Cell. Biol. 1996; 16: 2128-2134Crossref PubMed Scopus (312) Google Scholar). Mutation of a DrosophilaAPC homolog did not affect Wg function, suggesting either divergence of the molecular mechanisms of Arm stabilization or the existence of additional APC-like molecules in flies (21Hayashi S. Rubinfield B. Souza B. Polakis P. Wieschaus E. Levine A.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 242-247Crossref PubMed Scopus (116) Google Scholar). Resolution of these mechanisms will require identification of the serine kinase acting to inhibit SggZw3 and the means by which it is, in turn, controlled by Dsh. We thank R. Nusse for kindly providing cl-8 cells and Drosophila frizzled 2 cDNA. We thank A. Martinez Arias for Wg antibodies, L. Cherbas and P. Cherbas for pHGCO and pRmHa-1 vectors, and M. Barber for animal assistance.