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• W2022309233 abstract "Glycogen synthase kinase 3β (GSK3β) is a key component in many biological processes including insulin and Wnt signaling. Since the activation of each signaling pathway results in a decrease in GSK3β activity, we examined the specificity of their downstream effects in the same cell type. Insulin induces an increased activity of glycogen synthase but has no influence on the protein level of β-catenin. In contrast, Wnt increases the cytosolic pool of β-catenin but not glycogen synthase activity. We found that, unlike insulin, neither the phosphorylation status of the serine9 residue of GSK3β nor the activity of protein kinase B is regulated by Wnt. Although the decrease in GSK3β activity is required, GSK3β may not be the limiting component for Wnt signaling in the cells that we examined. Our results suggest that the axin-conductin complexed GSK3β may be dedicated to Wnt rather than insulin signaling. Insulin and Wnt pathways regulate GSK3β through different mechanisms, and therefore lead to distinct downstream events. Glycogen synthase kinase 3β (GSK3β) is a key component in many biological processes including insulin and Wnt signaling. Since the activation of each signaling pathway results in a decrease in GSK3β activity, we examined the specificity of their downstream effects in the same cell type. Insulin induces an increased activity of glycogen synthase but has no influence on the protein level of β-catenin. In contrast, Wnt increases the cytosolic pool of β-catenin but not glycogen synthase activity. We found that, unlike insulin, neither the phosphorylation status of the serine9 residue of GSK3β nor the activity of protein kinase B is regulated by Wnt. Although the decrease in GSK3β activity is required, GSK3β may not be the limiting component for Wnt signaling in the cells that we examined. Our results suggest that the axin-conductin complexed GSK3β may be dedicated to Wnt rather than insulin signaling. Insulin and Wnt pathways regulate GSK3β through different mechanisms, and therefore lead to distinct downstream events. glycogen synthase kinase 3 glycogen synthase protein kinase B 4-hydroxytamoxifen adenomatous polyposis coli Dulbecco's modified Eagle's medium lymphoid enhancer factor T cell factor hemagglutinin GSK3-binding protein frequently rearranged in advanced T-cell lymphomas dishevelled Glycogen synthase kinase 3 (GSK3)1 was originally identified for its ability to phosphorylate and inhibit glycogen synthase (GS) (1Embi N. Rylatt D.B. Cohen P. Eur. J. Biochem. 1980; 107: 519-527Crossref PubMed Scopus (795) Google Scholar, 2Rylatt D.B. Aitken A. Bilham T. Condon G.D. Embi N. Cohen P. Eur. J. Biochem. 1980; 107: 529-537Crossref PubMed Scopus (173) Google Scholar). It is a serine/threonine kinase that recognizes the target sequence SXXXS with the second serine prephosphorylated (3Fiol C.J. Wang A. Roeske R.W. Roach P.J. J. Biol. Chem. 1990; 265: 6061-6065Abstract Full Text PDF PubMed Google Scholar). Many proteins other than GS also contain GSK3 recognition sequences, some of which can be phosphorylated by GSK3in vitro. These include ATP-citrate lyase, protein phosphatase 1, cAMP-dependent protein kinase, eIF2B, inhibitor-2, c-Jun, Myc, Myb, CREB, Tau, β-catenin, and IκB (4Welsh G.I. Wilson C. Proud C.G. Trends Cell Biol. 1996; 6: 274-279Abstract Full Text PDF PubMed Scopus (124) Google Scholar, 5Orford K. Crockett C. Jensen J.P. Weissman A.M. Byers S.W. J. Biol. Chem. 1997; 272: 24735-24738Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar, 6Woodgett J.R. Plyte S.E. Pulverer B.J. Mitchell J.A. Hughes K. Biochem. Soc. Trans. 1993; 21: 905-907Crossref PubMed Scopus (24) Google Scholar). GSK3 is also unusual in that its enzymatic activity remains high at resting state and decreases upon stimulation. GSK3 is conserved from yeast to mammals and has been implicated in strikingly versatile biological functions. However, how different signals regulate GSK3 is still unknown. GSK3 plays an important role in the cellular response to insulin (7Proud C.G. Denton R.M. Biochem. J. 1997; 328: 329-341Crossref PubMed Scopus (221) Google Scholar). The regulation of GSK3 by insulin has been shown to be mediated by protein kinase B (PKB). Upon insulin stimulation, threonine 308 (Thr-308) and serine 473 (Ser-473) residues of PKB are phosphorylated and PKB is activated (8Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2476) Google Scholar). Subsequently, both GSK3 isotypes (GSK3α and GSK3β) in mammalian cells are phosphorylated on a serine residue at the N terminus (serine 21 of GSK3α and serine 9 of GSK3β) (9Sutherland C. Leighton I.A. Cohen P. Biochem. J. 1993; 296: 15-19Crossref PubMed Scopus (740) Google Scholar, 10Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4292) Google Scholar), which leads to a decrease in GSK3 activity. Although this has usually been detected as a 50–70% drop, it is apparently sufficient to relieve the inhibition of GS and allow cells to complete glycogen synthesis. Another in vitro substrate of GSK3β is β-catenin, a protein involved in cell adhesion, oncogenesis and development (11Aberle H. Bauer A. Stappert J. Kispert A. Kemler R. EMBO J. 1997; 16: 3797-3804Crossref PubMed Scopus (2122) Google Scholar, 12Bullions L.C. Levine A.J. Curr. Opin. Oncol. 1998; 10: 81-87Crossref PubMed Scopus (185) Google Scholar, 13Polakis P. Curr. Opin. Genet. Dev. 1999; 9: 15-21Crossref PubMed Scopus (602) Google Scholar). Together with axin-conductin and APC, GSK3β is one of the components of a protein complex that regulates the stability of β-catenin (14Wodarz A. Nusse R. Annu. Rev. Cell Dev. Biol. 1998; 14: 59-88Crossref PubMed Scopus (1716) Google Scholar, 15Rubinfeld B. Albert I. Porfiri E. Fiol C. Munemitsu S. Polakis P. Science. 1996; 272: 1023-1026Crossref PubMed Scopus (1275) Google Scholar, 16Behrens J. Jerchow B.A. Wurtele M. Grimm J. Asbrand C. Wirtz R. Kuhl M. Wedlich D. Birchmeier W. Science. 1998; 280: 596-599Crossref PubMed Scopus (1095) Google Scholar, 17Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1087) Google Scholar). Phosphorylation of the GSK3β sites in the N terminus of β-catenin is believed to be a signal for degradation. When either APC or the GSK3β sites of β-catenin are mutated, as in 90% of colon cancer, levels of β-catenin are elevated (13Polakis P. Curr. Opin. Genet. Dev. 1999; 9: 15-21Crossref PubMed Scopus (602) Google Scholar). Excess β-catenin accumulates in the cytosol and nucleus, outside of cell adhesion complexes on cytoplasmic membrane where it normally resides. Nuclear β-catenin is capable of interacting with the LEF/TCF family DNA-binding proteins and activating transcription of genes containing LEF/TCF binding sites (18Behrens J. von Kries J.P. Kuhl M. Bruhn L. Wedlich D. Grosschedl R. Birchmeier W. Nature. 1996; 382: 638-642Crossref PubMed Scopus (2560) Google Scholar, 19Molenaar M. van de Wetering M. Oosterwegel M. Peterson-Maduro J. Godsave S. Korinek V. Roose J. Destree O. Clevers H. Cell. 1996; 86: 391-399Abstract Full Text Full Text PDF PubMed Scopus (1592) Google Scholar). Increased β-catenin levels can also be achieved through the activation of Wnt/wingless signaling pathway (20Willert K. Shibamoto S. Nusse R. Genes Dev. 1999; 13: 1768-1773Crossref PubMed Scopus (294) Google Scholar). GSK3β has been placed between Dishevelled (Dvl in mammalian cells, Dsh in other organisms) and β-catenin in the Wnt pathway based on a combination of genetic and biochemical evidence (21Hooper J.E. Nature. 1994; 372: 461-464Crossref PubMed Scopus (110) Google Scholar, 22Siegfried E. Wilder E.L. Perrimon N. Nature. 1994; 367: 76-80Crossref PubMed Scopus (282) Google Scholar, 23Smalley M.J. Sara E. Paterson H. Naylor S. Cook D. Jayatilake H. Fryer L.G. Hutchinson L. Fry M.J. Dale T.C. EMBO J. 1999; 18: 2823-2835Crossref PubMed Scopus (205) Google Scholar). It is not clear how the extracellular Wnt signal is transduced from the membrane receptor Frizzled to Dsh/Dvl, and then to GSK3β resulting in increased β-catenin levels. Decreases in the activity of GSK3β have been observed in mouse fibroblasts andDrosophila cells responding to wingless and Dsh (24Cook D. Fry M.J. Hughes K. Sumathipala R. Woodgett J.R. Dale T.C. EMBO J. 1996; 15: 4526-4536Crossref PubMed Scopus (343) Google Scholar, 25Ruel L. Stambolic V. Ali A. Manoukian A.S. Woodgett J.R. J. Biol. Chem. 1999; 274: 21790-21796Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Furthermore, inhibition of GSK3β activity by lithium salt or GSK3β-binding protein (GBP/FRAT) mimics Wnt signaling (26Stambolic V. Ruel L. Woodgett J.R. Curr. Biol. 1996; 6: 1664-1668Abstract Full Text Full Text PDF PubMed Google Scholar, 27Yost 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). Recently, it is reported that Dvl and GBP/FRAT are able to associate with the axin-conductin-APC-β-catenin complex (23Smalley M.J. Sara E. Paterson H. Naylor S. Cook D. Jayatilake H. Fryer L.G. Hutchinson L. Fry M.J. Dale T.C. EMBO J. 1999; 18: 2823-2835Crossref PubMed Scopus (205) Google Scholar, 28Li 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). Moreover, this entire complex is believed to dissociate in response to Wnt signaling (20Willert K. Shibamoto S. Nusse R. Genes Dev. 1999; 13: 1768-1773Crossref PubMed Scopus (294) Google Scholar, 25Ruel L. Stambolic V. Ali A. Manoukian A.S. Woodgett J.R. J. Biol. Chem. 1999; 274: 21790-21796Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 28Li 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). In this study, we investigated how different signals such as insulin and Wnt regulate GSK3β. Using mammalian cells that respond to both signals, we found that the downstream effect is specific to each pathway, despite the indistinguishable decrease in GSK3β activity. We also generated the first inducible system to conditionally activate Dishevelled in mammalian cells as an independent method to turn on the Wnt signaling pathway. Serine 9 of GSK3β is not regulated in cells that are activated by Wnt or Dishevelled. Furthermore, we have evidence that the axin-conductin complexed GSK3β is not significantly phosphorylated at serine 9 upon insulin stimulation and, therefore, may be protected from insulin signaling. Human embryonic kidney 293 cells were purchased from ATCC. C57MG, Rat2-MV7, and Rat2-Wnt1 cell lines were generous gifts from Dr. Anthony Brown. These cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Inc.). CHOIR cells were kindly given by Drs. Richard Roth (29Dickens M. Chin J.E. Roth R.A. Ellis L. Denton R.M. Tavare J.M. Biochem. J. 1992; 287: 201-209Crossref PubMed Scopus (36) Google Scholar) and Ira Goldfine, maintained in Ham's F12 medium (Life Technologies, Inc.). All cell culture media were supplemented with 10% fetal calf serum (Life Technologies, Inc.) and 1% penicillin/streptomycin (Life Technologies, Inc.). For conditioned media, Rat2-MV7 and Rat2-Wnt1 were grown to 95% confluence. Cells were washed with phosphate-buffered saline and maintained in serum-free DMEM overnight. The conditioned media were filtered through 0.22-μm filter units, aliquoted, and stored at −80 °C until use. For insulin or Wnt stimulation, cells were grown to 70–80% confluence and serum-starved overnight, after which 5 μg/ml insulin or 0.2 ml/cm2 conditioned medium was added. Transfections of plasmids were performed by using LipofectAMINE Plus (Life Technologies, Inc.) or FuGENE6 (Roche Molecular Biochemicals) according to instructions from the manufacturer. To generate 293-D-ER cells, 293 cells were transfected with the Dvl-ER plasmid. Selection with 1 mg/ml Geneticin (Life Technologies, Inc.) started 24 h after transfection. Resistant cells were pooled together after two rounds of complete killing of the parental 293 cells. Once these cells were verified to have stable expression and inducible Dvl-ER, they were transfected with the His6-tagged conductin plasmid. 25 μg/ml blasticidin (Invitrogen) was used to select for 293-D-ER-His6-conductin cells. Mammalian expression plasmid encoding human Dishevelled 2 was a generous gift from Dr. Misha Semenov (30Semenov M.V. Snyder M. Genomics. 1997; 42: 302-310Crossref PubMed Scopus (66) Google Scholar). The coding region of Dishevelled 2 was also epitope-tagged with Glu-Glu (EE) tag and fused to the hormone binding domain of a modified version of the murine estrogen receptor (Dvl-ER). Conductin expression plasmid was from Dr. Walter Birchmeier (16Behrens J. Jerchow B.A. Wurtele M. Grimm J. Asbrand C. Wirtz R. Kuhl M. Wedlich D. Birchmeier W. Science. 1998; 280: 596-599Crossref PubMed Scopus (1095) Google Scholar), and was then epitope-tagged with the EE tag in pCDNA3 or His6 tag in pCDNA6. TOPTK reporter plasmid for TCF/LEF-dependent transcription was from Dr. Hans Clevers (31Korinek 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 (2896) Google Scholar). Dominant negative form of TCF4 (DNTCF4) expression plasmid was kindly provided by Dr. Osamu Tetsu (32Tetsu O. McCormick F. Nature. 1999; 398: 422-426Crossref PubMed Scopus (3225) Google Scholar). cDNA encoding human GSK3β in pBlueScriptSK+ was from Dr. James Woodgett (33Stambolic V. Woodgett J.R. Biochem. J. 1994; 303: 701-704Crossref PubMed Scopus (497) Google Scholar). GSK3β was amplified from this template by polymerase chain reaction and cloned into a pCDNA3-based plasmid with an N-terminal HA tag. Mutants of GSK3 were created by using QuickChange site-directed mutagenesis kit (Stratagene). Three versions of kinase-dead GSK3β were made with amino acid substitutions at the ATP binding site: lysine 85 to alanine, lysine 85 and 86 to arginines, and lysine 85 to methionine plus lysine 86 to alanine. Anti-GSK3β and anti-β-catenin antibodies were from Transduction Laboratory. Phosphotyrosine antibody 4G10 and anti-PKB antibody were from Upstate Biotechnology. Phosphospecific antibody against Ser-9 of GSK3β was from Quality Controlled Biochemicals. Phosphospecific antibodies against PKB were generously provided by Dr. David Stokoe. Anti-EE antibody was from Harlan Bioproducts. Anti-HA antibody was from Santa Cruz Biotechnology. To prepare cytosolic fractions, cells were washed and collected in ice-cold phosphate-buffered saline. Cell pellets were resuspended in ice-cold hypotonic buffer (25 mm Tris, pH 7.5, 1 mm EDTA, 25 mm NaF, 1 mmdithiothreitol) with Complete protease inhibitor mixture (Roche Molecular Biochemicals). Cells were lysed after incubating on ice for 10 min (verified by microscope). The lysates were subjected to ultracentrifugation at 100,000 × g for 30 min at 4 °C, and the supernatant was collected. For immunoprecipitation, cells were washed twice in ice-cold phosphate-buffered saline, then lysed in IP buffer (125 mmNaCl, 25 mm NaF, 25 mm Tris, pH 7.5, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 10 mm β-glycerol phosphate, 5 mm sodium pyrophosphate, 1 mm NaVO3, 200 nmokadaic acid, 1 mm dithiothreitol) with Complete protease inhibitor mixture. Anti-GSK3β, anti-HA, or anti-EE antibody was added to clarified lysates for 1 h at 4 °C, and then Protein G beads (Sigma) were added for another 1 h. Immunoprecipitates were washed three times with IP buffer. To coimmunopricipitate GSK3β with His6-conductin, Ni-IP buffer was used. Ni-IP buffer was IP buffer without EDTA, EGTA, or dithiothreitol and supplemented with EDTA-free Complete protease inhibitor mixture (Roche Molecular Biochemicals). Nickel beads (ProBond resin, Invitrogen) were first blocked with 2 mg/ml bovine serum albumin in Ni-IP buffer for 2 h. After incubating with cleared lysates, nickel beads were washed three times with Ni-IP buffer supplemented with 200 mm immidazole and then once with Ni-IP buffer. Western blotting was carried out following standard procedures. 10% Tris-glycine polyacrylamide gels were used. For GSK3β kinase assays, GSK3β immunoprecipitates were washed once with kinase buffer (25 mm Tris, pH 7.5, 10 mm MgCl2) first. Kinase reactions were performed in kinase buffer with 100 μm [γ-32P]ATP and 100 μm2BSP peptide as the substrate (synthesized by the Biomedical Resource Center, University of California, San Francisco, CA). 2BSP is based on the GSK3 target site in eIF2B (34Welsh G.I. Patel J.C. Proud C.G. Anal. Biochem. 1997; 244: 16-21Crossref PubMed Scopus (56) Google Scholar). After 20 min at 30 °C, the reactions were spotted on phosphocellulose P81 paper (Whatman), washed four times with 100 mm phosphoric acid, and counted in scintillation counter. Luciferase assays were performed by using dual luciferase reporter assay system (Promega) in a Microplate Luminometer (EG&G Berthold). Transfection efficiency was normalized to the expression ofRenilla luciferase from the cotransfected pRL-TK plasmid. GS assays were performed as described previously (35Eldar-Finkelman H. Argast G.M. Foord O. Fischer E.H. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10228-10233Crossref PubMed Scopus (132) Google Scholar). Parallel assays were performed using low (0.1 mm) and high (10 mm) concentrations of glucose 6-phosphate to give active and total activities of GS. GS activity was calculated as the fraction of active from total activity. Cells in 10-cm dishes were grown to 70–80% confluence, serum-starved for 8 h in regular DMEM, then metabolically labeled with 2 mCi of [32P]orthophosphate (Amersham Pharmacia Biotech) in serum-free and phosphate-free DMEM for overnight. After 10 min of insulin or conditioned media stimulation, GSK3β was immunoprecipitated as described above and processed for phosphopeptide mapping as described previously (33Stambolic V. Woodgett J.R. Biochem. J. 1994; 303: 701-704Crossref PubMed Scopus (497) Google Scholar). To compare the regulation of GSK3β by insulin and Wnt, we needed to choose cell lines that respond to both signals. In this study, we used conditioned media from a stable Rat2 cell line expressing mouse Wnt-1 as a source of Wnt protein (36Bradley R.S. Brown A.M. Mol. Cell. Biol. 1995; 15: 4616-4622Crossref PubMed Scopus (73) Google Scholar). We first tested the effect of the Wnt conditioned media on human embryonic 293 cells as this epithelial cell line does respond to insulin (37Shaw M. Cohen P. Alessi D.R. FEBS Lett. 1997; 416: 307-311Crossref PubMed Scopus (213) Google Scholar). We observed a maximal decrease in GSK3β activity at 10 min after the addition of Wnt media to cells (Fig. 1 A). Wnt media also caused an accumulation of the cytosolic fraction of β-catenin, which peaked at about 3 h after stimulation (Fig.1 B). These results again place mammalian GSK3 upstream of β-catenin and downstream of Wnt. Similar results were also seen with C57MG (C57) cells, an immortalized mouse mammary gland epithelial cell line (data not shown). We then examined the relationship between Dsh/Dvl and GSK3β in 293 cells. Overexpression of Dsh/Dvl is known to activate the Wnt pathway and give rise to elevated levels of cytosolic β-catenin (23Smalley M.J. Sara E. Paterson H. Naylor S. Cook D. Jayatilake H. Fryer L.G. Hutchinson L. Fry M.J. Dale T.C. EMBO J. 1999; 18: 2823-2835Crossref PubMed Scopus (205) Google Scholar, 38Yanagawa S. van Leeuwen F. Wodarz A. Klingensmith J. Nusse R. Genes Dev. 1995; 9: 1087-1097Crossref PubMed Scopus (339) Google Scholar). We utilized a luciferase reporter driven by TCF/LEF binding sites (TOPTK) to measure the activity of transient overexpression of human Dishevelled 2 (hDvl2) (Fig. 1 C). Wild type GSK3β and a dominant negative form of the TCF4 transcription factor (DNTCF4) blocked hDvl2 activity (Fig.1 C). GSK3β mutants that retain kinase activity, including serine 9 mutated to alanine or glutamic acid, and tyrosine 216 mutated to phenylalanine or glutamic acid, also retained the ability to block hDvl2 activity (see below and data not shown). In contrast, coexpression of kinase-dead mutants of GSK3β did not have any effect on the activity of hDvl2, nor did coexpression of active MEKK, an irrelevant protein kinase (Fig. 1 C). Similar results were also obtained from same experiments using CHOIR, a Chinese hamster ovary cell line stably expressing human insulin receptor (data not shown). Due to the low transfection efficiency of C57 cells, CHOIR and 293 cells were used for experiments involving transient transfections. These observations confirmed that a decrease in GSK3β activity is necessary to convey signals from Dvl to β-catenin and GSK3β is downstream of Dvl in mammalian cells. Since GSK3β is a major player in both insulin and Wnt signaling, we compared changes in GSK3β activity upon insulin and Wnt stimulation. A similar decrease in GSK3β activity was observed in 3 cell lines, 293, CHOIR and C57 (Fig. 2 A). We then examined the downstream events of activated insulin and Wnt pathways. The level of cytosolic β-catenin was increased in cells stimulated by Wnt but unchanged in insulin-treated cells (Fig. 2 B). GS activity was analyzed in CHOIR and C57 cells. Insulin-stimulated cells yielded higher GS activity, while Wnt conditioned media had no effect (Fig.2 C). 293 cells had high basal GS activity, and no significant activity increase was detected with insulin treatment (data not shown). These data represent an example of specificity in signaling, yet raised the question how different downstream effects were achieved through a seemingly indistinguishable change in the activity of GSK3β, a common component of the two signaling pathways. Serine 9 (Ser-9) is a key regulation site of GSK3β responding to insulin signaling (7Proud C.G. Denton R.M. Biochem. J. 1997; 328: 329-341Crossref PubMed Scopus (221) Google Scholar). Using a phosphospecific antibody against phospho-Ser-9 in GSK3β, we were able to detect a clear increase of phosphorylation on this residue upon insulin stimulation in CHOIR and C57 cells (Fig.3 A). However, we did not observe any obvious difference of phospho-Ser-9 reactivity in samples treated with Wnt media or control media. The phosphotyrosine content remained constant before and after either insulin or Wnt stimulation. Similar results were observed from 293 cells (data not shown). Since PKB is known to be the upstream regulator of GSK3 in insulin signaling, we analyzed the phosphorylation status of two key residues in PKB, Thr-308 and Ser-473, using phosphospecific antibodies. There was a strong increase in phosphorylation of both residues responding to insulin but not to Wnt (Fig. 3 B). We also metabolically labeled cells in vivo with [32P]orthophosphate before treatment with insulin or Wnt. We did not detect any significant changes in the total level of phosphorylation or phosphoamino acid analysis of GSK3β, although it confirmed that the majority of phosphorylation was on serine residues (data not shown). Phosphopeptide mapping was also performed (Fig.3 C). The phosphopeptide pattern for GSK3β from C57 cells treated with control or Wnt media appeared to be essentially the same. Nevertheless, GSK3β from insulin-treated cells elicited a distinctive increase in phosphorylation at the positions corresponding to peptides containing Ser-9 (33Stambolic V. Woodgett J.R. Biochem. J. 1994; 303: 701-704Crossref PubMed Scopus (497) Google Scholar). Similar patterns were obtained from 293 and CHOIR cells (data not shown). We then examined the response of a Ser-9 to alanine mutant of GSK3β (S9A-GSK3β) to insulin and Wnt signals. 293 or CHOIR cells transiently expressing an HA-epitope-tagged wild type or S9A mutant of GSK3β were stimulated with insulin or Wnt conditioned media. The kinase activity of S9A-GSK3β no longer decreased in response to insulin (Fig. 4 A), similar to what was reported previously (37Shaw M. Cohen P. Alessi D.R. FEBS Lett. 1997; 416: 307-311Crossref PubMed Scopus (213) Google Scholar). Upon Wnt stimulation, the S9A mutant exhibited an activity drop similar to that for the wild type kinase (Fig. 4 A). We also tested the ability of S9A to block Wnt signal by hDvl2 (Fig. 4 B). At higher expression level, both S9A mutant or wild type GSK3β efficiently blocked hDvl2-activated TCF/LEF-driven luciferase activity. Interestingly, at lower expression level, S9A was able to block hDvl2 activity roughly 2-fold better than the wild type GSK3β. This is probably due to the higher intrinsic kinase activity of S9A mutant that we and others have observed (35Eldar-Finkelman H. Argast G.M. Foord O. Fischer E.H. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10228-10233Crossref PubMed Scopus (132) Google Scholar). Similar effects were observed using CHOIR cells (data not shown). GSK3β has been found in the axin-conductin-APC-β-catenin complex (16Behrens J. Jerchow B.A. Wurtele M. Grimm J. Asbrand C. Wirtz R. Kuhl M. Wedlich D. Birchmeier W. Science. 1998; 280: 596-599Crossref PubMed Scopus (1095) Google Scholar, 17Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1087) Google Scholar). Because of the multitude of biological processes that GSK3β is involved in, it is reasonable to hypothesize that only a fraction of the total cellular GSK3β is in the axin-conductin complex. We investigated the response of GSK3β in the axin-conductin complex to insulin or Wnt. From CHOIR cells with insulin or Wnt treatment, endogenous GSK3β was coimmunoprecipitated with ectopically expressed EE-tagged conductin (EE-conductin). Only a small fraction of GSK3β was coimmunoprecipitated with EE-conductin and Ser-9 phosphorylation status of these GSK3β was not altered in response to Wnt media (Fig.5 A). After insulin treatment, we observed a much less significant increase in phosphorylation on Ser-9 of the GSK3β that was coimmunoprecipitated with EE-conductin. Phosphorylation on tyrosine residues were constant (data not shown).In vitro kinase assays were also performed on the pool of GSK3β that coimmunoprecipitated with EE-conductin (Fig.5 B). We observed decreased kinase activity of GSK3β responding to Wnt. Furthermore, in response to insulin, the decrease in kinase activity of this subpool of GSK3β seemed to be less prominent than the total pool of GSK3β. As the most upstream intracellular component of the Wnt pathway, Dvl is capable of activating downstream molecules independent of the extracellular ligand Wnt (23Smalley M.J. Sara E. Paterson H. Naylor S. Cook D. Jayatilake H. Fryer L.G. Hutchinson L. Fry M.J. Dale T.C. EMBO J. 1999; 18: 2823-2835Crossref PubMed Scopus (205) Google Scholar, 25Ruel L. Stambolic V. Ali A. Manoukian A.S. Woodgett J.R. J. Biol. Chem. 1999; 274: 21790-21796Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 38Yanagawa S. van Leeuwen F. Wodarz A. Klingensmith J. Nusse R. Genes Dev. 1995; 9: 1087-1097Crossref PubMed Scopus (339) Google Scholar). To further substantiate our findings described in the earlier sections, we also investigated the response of GSK3β to activated Dvl. We chose to generate a fusion protein between Dvl and a modified version of the hormone binding domain of the murine estrogen receptor (Dvl-ER) and use this as our inducible system to achieve rapid activation of the Wnt pathway (39Littlewood T.D. Hancock D.C. Danielian P.S. Parker M.G. Evan G.I. Nucleic Acids Res. 1995; 23: 1686-1690Crossref PubMed Scopus (695) Google Scholar). Expression of Dvl-ER activates TOPTK reporter activity in a hormone (4-hydroxytamoxifen (4-HT))-dependent manner (data not shown). A 293 cell line was then made to stably express Dvl-ER (293-D-ER). As shown in Fig.6 A, cytosolic β-catenin accumulated upon 4-HT treatment in 293-D-ER cells. We then examined the phosphorylation status of the Ser-9 residue of GSK3β. In this cell line, there remains a strong increase of phosphorylation of Ser-9 of GSK3β in response to insulin (Fig. 6 B). 4-HT treatment did not cause any significant change in Ser-9 phosphorylation (Fig.6 B). Furthermore, a His6-tagged conductin was integrated into 293-D-ER cells (293-D-ER-His6-conductin) so that the conductin-bound pool of GSK3β can be coimmunoprecipitated using nickel beads. Upon either insulin or 4-HT stimulation, the phosphorylation of Ser-9 on this pool of GSK3β was not significantly altered (Fig. 6 C). GSK3β has been implicated in mediating many diverse signals in various cell types. It is believed that many signals can down-regulate the kinase activity of GSK3β. How different signaling pathways achieve specificity through GSK3β remains unclear. In this study, we investigated the differential regulation of GSK3β by two extracellular signals, insulin and Wnt. Although both signals decrease GSK3β activity to a similar extent, we found that insulin and Wnt lead to very distinct downstream events. Furthermore, unlike in insulin signaling, Ser-9 of GSK3β is not phosphorylated by the Wnt signaling pathway. In any given organism, many cells will receive and respond to multiple extracellular signals. The cell lines that we chose for this study, for example, are able to respond to both insulin and Wnt. Insulin stimulation leads to increased glycogen synthesis, whereas Wnt causes accumulation of β-catenin. As expected, we observed a decrease in GSK3β activity in response to both insulin and Wnt. Decrease in GSK3β activity is sometimes sufficient to lead to downstream events. For example, lithium as a cell-permeable, noncompetitive inhibitor of GSK3β is able to mimic insulin in adipocytes (40Cheng K. Creacy S. Larner J. Mol. Cell. Biochem. 1983; 56: 183-189PubMed Google Scholar) and Wnt signaling in Drosophila cells (26Stambolic V. Ruel L. Woodgett J.R. Curr. Biol. 1996; 6: 1664-1668Abstract Full Text Full Text PDF PubMed Google Scholar). HGF was shown to down-regulate GSK3β activity and up-regulate β-catenin level (41Papkoff J. Aikawa M. Biochem. Biophys. Res. Commun. 1998; 247: 851-858Crossref PubMed Scopus (169) Google Scholar). However, we found that insulin did not cause β-catenin accumulation and Wnt did not increase glycogen synthase activity. Therefore, the effect of each signaling pathway is highly specific. A similar finding was reported by Staal et al. (42Staal F.J. Burgering B.M. van de Wetering M. Clevers H.C. Int. Immunol. 1999; 11: 317-323Crossref PubMed Scopus (67) Google Scholar) that T cell activation causes decrease in GSK3β activity but no change in β-catenin accumulation. A large body of evidence suggests that the regulation of GSK3β by insulin is a phosphorylation event at Ser-9 via activated PKB (7Proud C.G. Denton R.M. Biochem. J. 1997; 328: 329-341Crossref PubMed Scopus (221) Google Scholar, 10Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4292) Google Scholar). One way to achieve specificity via a common intermediate in different pathways could be through different posttranslational modifications. GSK3β activity can be down-regulated independent of Ser-9 phosphorylation or PKB in exercised muscle (43Markuns J.F. Wojtaszewski J.F. Goodyear L.J. J. Biol. Chem. 1999; 274: 24896-24900Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). We demonstrated that Wnt signaling did not cause Thr-308 or Ser-473 phosphorylation on PKB, nor was Ser-9 of GSK3β modified. Supporting our data, Yuan et al. (44Yuan H. Mao J. Li L. Wu D. J. Biol. Chem. 1999; 274: 30419-30423Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) reported that activation of PKB alone is not sufficient to mimic Wnt signaling. However, in their report, exogenous PKB had a synergistic effect on β-catenin with exogenously expressed Wnt1 or Frat1. We did not find any synergistic effect if we stimulated cells with insulin and Wnt simultaneously (data not shown). It is possible that, although activating Wnt signaling does not rely on the activity of PKB, addition of active PKB in higher amounts than ordinary insulin stimulation can still act on one of its substrates, GSK3β. This further decreased GSK3β activity may then contribute to the synergistic β-catenin accumulation effect. Our results are also in agreement with the report by Cook et al. (24Cook D. Fry M.J. Hughes K. Sumathipala R. Woodgett J.R. Dale T.C. EMBO J. 1996; 15: 4526-4536Crossref PubMed Scopus (343) Google Scholar), in which they showed that the decreased GSK3β activity in 10T1/2 cells responding to Drosophila wingless was insensitive to wortmannin. It was proposed that a phorbol ester-sensitive PKC may be the signaling molecule to GSK3β in the Wnt pathway (24Cook D. Fry M.J. Hughes K. Sumathipala R. Woodgett J.R. Dale T.C. EMBO J. 1996; 15: 4526-4536Crossref PubMed Scopus (343) Google Scholar). Certain isoforms of PKC are able to phosphorylate GSK3β in vitro (45Goode N. Hughes K. Woodgett J.R. Parker P.J. J. Biol. Chem. 1992; 267: 16878-16882Abstract Full Text PDF PubMed Google Scholar). Nevertheless, PKC may not be the sole signaling molecule since inhibitors of PKC do not completely block Wnt effects (46Chen R.H. Ding W.V. McCormick F. J. Biol. Chem. 2000; 275: 17894-17899Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Integrin-linked kinase is another kinase that was able to induce nuclear β-catenin accumulation (47Novak A. Hsu S.C. Leung-Hagesteijn C. Radeva G. Papkoff J. Montesano R. Roskelley C. Grosschedl R. Dedhar S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4374-4379Crossref PubMed Scopus (402) Google Scholar), activate PKB in vivoand phosphorylate GSK3β in vitro (48Delcommenne M. Tan C. Gray V. Rue L. Woodgett J. Dedhar S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11211-11216Crossref PubMed Scopus (937) Google Scholar). It is unclear whether GSK3β is regulated by phosphorylation during Wnt signaling. We were not able to detect any change in phosphorylation by in vivo labeling, phosphoamino acid analysis, and phosphopeptide mapping. Phosphorylation as a mode of regulating GSK3β in Wnt pathway cannot be ruled out although Ser-9 is not involved. Ruelet al. (25Ruel L. Stambolic V. Ali A. Manoukian A.S. Woodgett J.R. J. Biol. Chem. 1999; 274: 21790-21796Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) observed an increased phosphorylation on serine residues of Drosophila GSK3 (shaggy) in inducible Wnt or Dsh cells. The hormone-inducible Dvl-ER cells we generated will be useful for revisiting this issue and also probing for other biological activities of Dsh/Dvl. In addition, there are two forms of GSK3 in mammalian cells, GSK3α and GSK3β. The role of GSK3α in Wnt signaling is worthy of future investigation. Specificity in signaling could be achieved by specific complex formation and subcellular translocation. Ras is an example of such regulation (49Campbell S.L. Khosravi-Far R. Rossman K.L. Clark G.J. Der C.J. Oncogene. 1998; 17: 1395-1413Crossref PubMed Scopus (918) Google Scholar). At the focal point of a multitude of signals and mitogens, it has many downstream effectors. Ras interacts with different effectors via different groups of residues. Upon activation, Ras binds Raf and relocalizes Raf to the plasma membrane for further activation. GSK3β has been shown to form complexes with APC and axin-conductin (15Rubinfeld B. Albert I. Porfiri E. Fiol C. Munemitsu S. Polakis P. Science. 1996; 272: 1023-1026Crossref PubMed Scopus (1275) Google Scholar, 16Behrens J. Jerchow B.A. Wurtele M. Grimm J. Asbrand C. Wirtz R. Kuhl M. Wedlich D. Birchmeier W. Science. 1998; 280: 596-599Crossref PubMed Scopus (1095) Google Scholar, 17Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1087) Google Scholar). Recently, ectopically expressed Dvl, GBP/FRAT, and PP2A were found in the axin complex (23Smalley M.J. Sara E. Paterson H. Naylor S. Cook D. Jayatilake H. Fryer L.G. Hutchinson L. Fry M.J. Dale T.C. EMBO J. 1999; 18: 2823-2835Crossref PubMed Scopus (205) Google Scholar, 28Li 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, 50Seeling 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). Dvl was able to relocalize axin to the cell membrane (23Smalley M.J. Sara E. Paterson H. Naylor S. Cook D. Jayatilake H. Fryer L.G. Hutchinson L. Fry M.J. Dale T.C. EMBO J. 1999; 18: 2823-2835Crossref PubMed Scopus (205) Google Scholar). Peptides derived from Frat1 specifically inhibit kinase activity of GSK3 on axin and β-catenin but not GS (51Thomas G.M. Frame S. Goedert M. Nathke I. Polakis P. Cohen P. FEBS Lett. 1999; 458: 247-251Crossref PubMed Scopus (201) Google Scholar). Several groups suggested that Wnt signaling dissociated this complex (20Willert K. Shibamoto S. Nusse R. Genes Dev. 1999; 13: 1768-1773Crossref PubMed Scopus (294) Google Scholar, 25Ruel L. Stambolic V. Ali A. Manoukian A.S. Woodgett J.R. J. Biol. Chem. 1999; 274: 21790-21796Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 28Li 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). Recently, overexpressed casein kinase I was found to interact with Dsh and to mimic Wnt signaling (52Peters J.M. McKay R.M. McKay J.P. Graff J.M. Nature. 1999; 401: 345-350Crossref PubMed Scopus (381) Google Scholar). Are there different pools of GSK3β complexes in different signaling pathways? Several findings by us and others imply that GSK3β may not be limiting in cells. First, kinase-dead GSK3β mutants failed to elicit any effect on Wnt signaling in 293 or CHOIR cell lines based on the unaltered β-catenin-dependent luciferase activity (data not shown and 23). Although in Xenopus overexpression of kinase-dead GSK3β did cause axis duplication, mimicking activated Wnt signaling pathway (53He X. Saint-Jeannet J.P. Woodgett J.R. Varmus H.E. Dawid I.B. Nature. 1995; 374: 617-622Crossref PubMed Scopus (446) Google Scholar), this effect has not been observed consistently in mammalian cell systems. Second, in cells that contain high amounts of β-catenin, such as SW480 cell line or 293 cells overexpressing β-catenin, exogenous GSK3β was not able to decrease the level of β-catenin-dependent transcription, whereas axin-conductin or APC did so efficiently (data not shown and Ref. 16Behrens J. Jerchow B.A. Wurtele M. Grimm J. Asbrand C. Wirtz R. Kuhl M. Wedlich D. Birchmeier W. Science. 1998; 280: 596-599Crossref PubMed Scopus (1095) Google Scholar). It is not certain whether the levels of overexpression of GSK3β and axin-conductin are comparable. Nevertheless, one explanation is that GSK3 is not the limiting factor, thus supporting the idea that a subpopulation of GSK3β is dedicated to form complexes with axin-conductin and only this pool of GSK3β participates in Wnt signaling. Furthermore, we observed that the GSK3β complexed to transiently or stably expressed conductin was significantly protected from Ser-9 phosphorylation by insulin. Further analysis through the investigation of this complex will shed light on our understanding of how GSK3 is regulated in the Wnt signaling pathway. We thank Drs. W. Birchmeier, A. Brown, H. Clevers, I. Goldfine, M. Roth, M. Semenov, D. Stokoe, O. Tetsu, and J. Woodgett for reagents. We also thank Drs. Art Alberts, Mike Fried, Peter Sabbatini, and David Stokoe for critically reading the manuscript and members of the McCormick laboratory for support." @default.
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• W2022309233 title "Differential Regulation of Glycogen Synthase Kinase 3β by Insulin and Wnt Signaling" @default.
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