Akt Participation in the Wnt Signaling Pathway through Dishevelled

Inactivation of glycogen synthase kinase 3β (GSK3β) and the resulting stabilization of free β-catenin are critical steps in the activation of Wnt target genes. While Akt regulates GSK3α/β in the phosphatidylinositide 3-OH kinase signaling pathway, its role in Wnt signaling is unknown. Here we report that expression of Wnt or Dishevelled (Dvl) increased Akt activity. Activated Akt bound to the Axin-GSK3β complex in the presence of Dvl, phosphorylated GSK3β and increased free β-catenin levels. Furthermore, in Wnt-overexpressing PC12 cells, dominant-negative Akt decreased free β-catenin and derepressed nerve growth factor-induced differentiation. Therefore, Akt acts in association with Dvl as an important regulator of the Wnt signaling pathway. Inactivation of glycogen synthase kinase 3β (GSK3β) and the resulting stabilization of free β-catenin are critical steps in the activation of Wnt target genes. While Akt regulates GSK3α/β in the phosphatidylinositide 3-OH kinase signaling pathway, its role in Wnt signaling is unknown. Here we report that expression of Wnt or Dishevelled (Dvl) increased Akt activity. Activated Akt bound to the Axin-GSK3β complex in the presence of Dvl, phosphorylated GSK3β and increased free β-catenin levels. Furthermore, in Wnt-overexpressing PC12 cells, dominant-negative Akt decreased free β-catenin and derepressed nerve growth factor-induced differentiation. Therefore, Akt acts in association with Dvl as an important regulator of the Wnt signaling pathway. Dishevelled glycogen synthase kinase 3β lymphoid enhancer factor T cell factor phosphatidylinositide 3-OH kinase Dulbecco's modified Eagle's medium fetal bovine serum polymerase chain reaction glutathione S-transferase enhanced green fluorescent protein nerve growth factor Frizzled-related protein-1 Wnt signaling regulates several developmental processes in both insects and vertebrates. Aberrant Wnt signaling occurs both in oncogenic processes (1Cadigan K.M. Nusse R. Genes Dev. 1997; 11: 3286-3305Crossref PubMed Scopus (2228) Google Scholar, 2Smalley M.J. Dale T.C. Cancer Metastasis Rev. 1999; 18: 215-230Crossref PubMed Scopus (187) Google Scholar, 3Waltzer L. Bienz M. Cancer Metastasis Rev. 1999; 18: 231-246Crossref PubMed Scopus (57) Google Scholar) and in some cardiovascular diseases (4Mao C. Malek O.T. Pueyo M.E. Steg P.G. Soubrier F. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 43-51Crossref PubMed Scopus (39) Google Scholar, 5Ai Z. Fischer A. Spray D.C. Brown A.M. Fishman G.I. J. Clin. Invest. 2000; 105: 161-171Crossref PubMed Scopus (317) Google Scholar, 6Rezvani M. Liew C.C. J. Biol. Chem. 2000; 275: 18470-18475Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). The interaction between secreted Wnt glycoproteins and their cognate Frizzled receptors leads to the activation of Dvl1 protein, which transmits the Wnt signal to downstream effectors. By inhibiting phosphorylation of β-catenin by GSK3β, activated Dvl prevents the ubiquitination and subsequent proteosomal degradation of β-catenin. The resulting accumulation of free β-catenin enhances its interaction with transcription factors of the lymphoid enhancer factor-T cell factor (LEF/TCF) family and induces the transcription of target genes such as cyclin D1 and c-myc (1Cadigan K.M. Nusse R. Genes Dev. 1997; 11: 3286-3305Crossref PubMed Scopus (2228) Google Scholar, 2Smalley M.J. Dale T.C. Cancer Metastasis Rev. 1999; 18: 215-230Crossref PubMed Scopus (187) Google Scholar, 3Waltzer L. Bienz M. Cancer Metastasis Rev. 1999; 18: 231-246Crossref PubMed Scopus (57) Google Scholar). Two mechanisms have been advanced to account for the inhibition of GSK3β by Dvl: 1) the catalytic activity of GSK3β is blocked by serine phosphorylation (1Cadigan K.M. Nusse R. Genes Dev. 1997; 11: 3286-3305Crossref PubMed Scopus (2228) Google Scholar,7Cook D. Fry M.J. Hughes K. Sumathipala R. Woodgett J.R. Dale T.C. EMBO J. 1996; 15: 4526-4536Crossref PubMed Scopus (344) Google Scholar, 8Ruel 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, 9Brown J.D. Moon R.T. Curr. Opin. Cell Biol. 1998; 10: 182-187Crossref PubMed Scopus (99) Google Scholar, 10Stambolic V. Woodgett J.R. Biochem. J. 1994; 303: 701-704Crossref PubMed Scopus (508) Google Scholar); 2) the interaction of GSK3β and β-catenin is disrupted by a conformation change of the Axin-β-catenin-GSK3β-adenomatosis polyposis coli protein complex (Axin complex) (2Smalley M.J. Dale T.C. Cancer Metastasis Rev. 1999; 18: 215-230Crossref PubMed Scopus (187) Google Scholar, 11Li L. Yuan H. Weaver C.D. Mao J. Farr 3rd, G.H. Sussman D.J. Jonkers J. Kimelman D. Wu D. EMBO J. 1999; 18: 4233-4240Crossref PubMed Scopus (359) Google Scholar, 12Salic A. Lee E. Mayer L. Kirschner M.W. Mol. Cell. 2000; 5: 523-532Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 13Kikuchi A. Cytokine Growth Factor Rev. 1999; 10: 255-265Crossref PubMed Scopus (68) Google Scholar). However, in the first instance, the process whereby Dvl stimulates serine phosphorylation has not been elucidated; nor is it clear whether these two mechanisms function in a concerted manner to promote Wnt signaling. The serine/threonine kinase Akt (protein Kinase B/related to A and protein kinase C) is a major effector of the PI3K pathway and is activated by many polypeptide growth factors (14Alessi D.R. Cohen P. Curr. Opin. Genet. Dev. 1998; 8: 55-62Crossref PubMed Scopus (675) Google Scholar, 15Stambolic V. Mak T.W. Woodgett J.R. Oncogene. 1999; 18: 6094-6103Crossref PubMed Scopus (107) Google Scholar, 16Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3721) Google Scholar). The recruitment of Akt from the cytoplasm to the plasma membrane by the lipid products of PI3K leads to Akt phosphorylation by 3-phosphoinositide-dependent protein kinases. This phosphorylation of Akt at Thr308 and Ser473results in its activation. Akt acts in part through its phosphorylation of GSK3α/β, which in turn regulates cell metabolism. Recognizing the pivotal role of GSK3β in Wnt signaling, we tested the hypothesis that Akt functions in this pathway to control GSK3β activity. PC12, Int5 and Tni3 cells were cultured as described (17Shackleford G.M. Willert K. Wang J. Varmus H.E. Neuron. 1993; 11: 865-875Abstract Full Text PDF PubMed Scopus (49) Google Scholar). 293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Inc.) supplemented with 10% fetal bovine serum (FBS, Hyclone Laboratories), 20 mm HEPES (pH 7.35), 1 mm sodium pyruvate, and 1 mmnon-essential amino acids (all from Life Technologies, Inc.). NIH-3T3 cells were cultured in DMEM supplemented with 10% FBS. Mouse Dvl-1, Akt-1, Axin, and rat GSK3β were prepared by reverse transcriptase-polymerase chain reaction (PCR) using the high fidelity thermostable DNA polymerase PfuTurbo (Stratagene). Dvl-1 was subcloned in frame into pcDNA3.1(−)/Myc-His (Invitrogen) to introduce Myc-His epitope tags at the C terminus. GSK3β was subcloned into pcDNA3.1(−) (Invitrogen). Axin and Akt-1 were subcloned either in frame or with a stop codon into pFLAG-CMV™-5 (Sigma) to yield protein with or without FLAG tag at the C terminus, respectively. Deletion mutants of Dvl-1, N-Dvl-(1–497), and C-Dvl-(369–695) were generated by PCR and subcloned in frame into pcDNA3.1(−)/Myc-His. The C-terminal (β-catenin binding) region of E-cadherin (18Bafico A. Gazit A. Wu-Morgan S.S. Yaniv A. Aaronson S.A. Oncogene. 1998; 16: 2819-2825Crossref PubMed Scopus (71) Google Scholar) was cloned by reverse transcriptase-PCR in frame into pGEX-5X-1 (Amersham Pharmacia Biotech) for GST-E-cadherin pull-down assay. Four recombinant first-generation type 5 adenoviruses were used in these studies. Ad.EGFP has been described previously (19Matsui T. Li L. Del Monte F. Fukui Y. Franke T.F. Hajjar R.J. Rosenzweig A. Circulation. 1999; 100: 2373-2379Crossref PubMed Scopus (338) Google Scholar). Ad.AAA-Akt and Ad.β-gal.EGFP, which also express EGFP, were constructed using the method described previously (20He T.C. Zhou S. Da Costa L.T., Yu, J. Kinzler K.W. Vogelstein B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2509-2514Crossref PubMed Scopus (3252) Google Scholar). The wild type Akt-expressing adenovirus was constructed using Akt-HA (kindly provided by T. Franke) in pAdRSV-4 (kindly provided by D. Dichek) and obtaining homologous recombinants through co-transfection with pJM17 (Microbix Biosystems) in 293 cells. Wnt-3a- and control-conditioned medium were prepared essentially as described (21Shibamoto S. Higano K. Takada R. Ito F. Takeichi M. Takada S. Genes Cells. 1998; 3: 659-670Crossref PubMed Scopus (230) Google Scholar). We verified that Wnt-3a medium specifically up-regulated the cellular β-catenin levels in L cells. 293T cells were transfected using LipofectAMINE™ (Life Technologies, Inc.) in 6-well plates with 30 ng/well of TOPFlash or FOPFlash luciferase reporter (Upstate Biotechnology) together with effector plasmids or empty pcDNA3 vector (amount added to keep the total DNA content at 1 μg/well) as described in the figure legends. Cell extracts were prepared 24 h after transfection by a detergent lysis method (Promega). Transfection efficiencies were normalized by pCMVβ-gal reporter activity (CLONTECH). Each construct was transfected at least three times in triplicate. Reporter activities (mean ± S.D.) are presented as the -fold increase of TOPFlash activity from the cells transfected with empty vector. Cell lysates were prepared in a buffer containing 20 mm Tris, pH 8.0, 140 mm NaCl, 1 mm EGTA, 1.5 mm MgCl2, 10% glycerol, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm β-glycerophosphate, 1 mm sodium vanadate, dithiothreitol, and protease inhibitors (Complete™-without EDTA, Roche). Cell extracts were clarified by centrifugation and subjected to SDS-polyacrylamide gel electrophoresis followed by immunoblotting with indicated antibodies. Cell lysates were precleared with protein A or G-Sepharose beads (Amersham Pharmacia Biotech or Calbiochem, respectively) for 1 h at 4 °C, and then incubated with the indicated antibody and protein A or G-Sepharose beads for 3.5 h at 4 °C. The immunocomplexes were pelleted and washed three times with cold lysis buffer. The samples were subjected to immunoblotting with the indicated antibodies. Akt activity was measured using an Akt kinase assay kit (New England BioLabs) with recombinant GSK3 as a substrate for immunoprecipitated Akt. Free β-catenin levels were detected by GST-E-cadherin pull-down assay as described (18Bafico A. Gazit A. Wu-Morgan S.S. Yaniv A. Aaronson S.A. Oncogene. 1998; 16: 2819-2825Crossref PubMed Scopus (71) Google Scholar). GST-E-cadherin fusion protein was expressed in bacteria and purified by binding to glutathione-Sepharose beads (Amersham Pharmacia Biotech). 1 mg of cell lysate was incubated with GST-E-cadherin-beads for 1 h at 4 °C. The beads were collected by centrifugation and washed, and proteins were dissolved in Laemmli buffer and subjected to SDS-polyacrylamide gel electrophoresis followed by immunoblotting with anti-β-catenin antibody. Cells were incubated in differentiation medium (DMEM supplemented with 1% horse serum and 100 ng/ml NGF (Austral Biologicals)) or growth medium (DMEM supplemented with 10% horse serum, 5% FBS). Medium was refreshed every 48 h. Neurite out-growth was defined as a phase dark process with a clearly defined growth cone that was at least 1.5 cell diameters in length. Experiments were carried out in triplicate and repeated at least three times, and in each experiment cells were counted by at least two individuals who were blinded to the groups. Where indicated, comparisons between groups were made by factorial analysis of variance followed by Scheffe's test (Fig. 1 B) or by unpaired Student's t test (Fig.5 C) when appropriate. Significance was accepted atp < 0.05.Figure 5Dominant-negative Akt restores neuronal differentiation of Int5 cells. A, Int5 cells were incubated in differentiation medium including vehicle (control) or sFRP-1 (15 μg/ml) for 4 days. Cells were observed using phase contrast microscopy (original magnification, 200×). B, Int5 cells were infected with control Ad.β-gal.EGFP (left panels) or Ad.AAA-Akt (right panels) at MOI 50 for 2 , and then were maintained for 3 days in the differentiation medium. Cells were observed using phase contrast panels) and microscopy panels) (original magnification, Cells infected by the adenovirus were by The of cells with total cells is indicated as differentiation. are presented as the ± < PC12 and Int5 cells were infected with control Ad.β-gal.EGFP or and cells were maintained in growth medium or differentiation medium for 3 days. were with a antibody. protein the was also with an We first the that Wnt signaling stimulates Akt activity. a PC12 cell (17Shackleford G.M. Willert K. Wang J. Varmus H.E. Neuron. 1993; 11: 865-875Abstract Full Text PDF PubMed Scopus (49) Google not in either the PC12 or in an PC12 cell Akt was phosphorylated at both Thr308 and The phosphorylation of both is and for Akt activation S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3721) Google Scholar). Akt measured by its to recombinant GSK3β, was in Int5 cells in control cells (Fig. 1 Furthermore, phosphorylation of GSK3β at which in its V. Woodgett J.R. Biochem. J. 1994; 303: 701-704Crossref PubMed Scopus (508) Google was in Int5 in control cells (Fig. A of Akt and GSK3β was by the Wnt Frizzled-related protein-1 C) by a in free β-catenin as we previously A. F. V. K. S. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus (317) Google Scholar). indicated that phosphorylation of Akt and GSK3β, and free β-catenin levels in Int5 cells, were secreted these to cells, we also Akt phosphorylation in NIH-3T3 cells transfected with Dvl-1, and a β-catenin that of these products an increase in free of Wnt signaling (2Smalley M.J. Dale T.C. Cancer Metastasis Rev. 1999; 18: 215-230Crossref PubMed Scopus (187) Google Scholar, 8Ruel 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, K. C. A.M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, H. Mao J. Li L. Wu D. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, A. S. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). expression of or Dvl-1, not increased Akt phosphorylation indicated that Akt activation is a of Wnt signaling that occurs downstream of whether activated Akt is for the Wnt signaling, we used a dominant-negative of Akt R. Z. J. Woodgett J.R. A. Mol. Cell. Biol. 1999; PubMed Scopus Google Scholar). Int5 cells infected with an adenovirus in phosphorylated GSK3β and free β-catenin protein levels (Fig. 2 not in cells infected with control transfection using 293T cells, Dvl-1, and β-catenin each enhanced reporter activity (Fig. 2 TOPFlash activity by or Dvl-1 not the β-catenin activity (Fig. 2 The activity of the reporter was not in these not and that Akt is in Wnt signaling, at a in the pathway between Dvl and enhances Wnt signaling in the presence of A, Akt expression free β-catenin levels in Int5 cells not in Tni3 cells. Tni3 and Int5 cells were infected with Ad.EGFP or and cells were after 3 days. The cell lysates were with Akt the expression of a Wnt target in the presence of 293T cells were transfected with the TOPFlash or the FOPFlash reporter together with plasmids of Akt and Dvl-1 or pcDNA3 as TOPFlash or FOPFlash reporter activities are presented as the -fold increase of TOPFlash activity from the cells transfected with empty test whether Akt activity is to an increase in free we infected Int5 and Tni3 cells with either an wild type Akt or a control adenovirus the and activity of Akt protein were increased in both infected with a in free β-catenin levels was in the Int5 cells (Fig. 3 that Akt activity was not to free β-catenin that it the activity of a Wnt that a dominant-negative of Akt Wnt signaling by Dvl, we that Akt in with Dvl to promote Wnt signaling. of Akt in 293T cells a increase in Akt activity observed with PC12 cell Akt activity was not to Wnt signaling in the reporter assay. However, Akt enhanced TOPFlash activity that was by Dvl-1 (Fig. 3 B) not by β-catenin not Akt nor Dvl-1 control FOPFlash activity (Fig. 3 that Dvl is for Akt to the of Akt and Dvl signal we their interaction with the Axin have that activated Dvl Axin and protein to the Axin This binding induces a change in the resulting in the inhibition of GSK3β phosphorylation of β-catenin (2Smalley M.J. Dale T.C. Cancer Metastasis Rev. 1999; 18: 215-230Crossref PubMed Scopus (187) Google Scholar, 12Salic A. Lee E. Mayer L. Kirschner M.W. Mol. Cell. 2000; 5: 523-532Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 13Kikuchi A. Cytokine Growth Factor Rev. 1999; 10: 255-265Crossref PubMed Scopus (68) Google Scholar). We first the association between Akt and to medium induces the Wnt signaling pathway (2Smalley M.J. Dale T.C. Cancer Metastasis Rev. 1999; 18: 215-230Crossref PubMed Scopus (187) Google Scholar, 9Brown J.D. Moon R.T. Curr. Opin. Cell Biol. 1998; 10: 182-187Crossref PubMed Scopus (99) Google Scholar, A. S. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). Axin was with Akt in 293T cells for 24 h with medium not control medium the association of Akt with Axin a Wnt with using PC12 cells (Fig. 1 these 293T cells increased phosphorylation levels of Akt and GSK3β (Fig. 4 A, Cell We also total GSK3β in cell lysates and GSK3β in the Axin the defined as the GSK3β protein with transfected 293T cells, Dvl expression the phosphorylation of both total GSK3β (Fig. 4 and GSK3β in the Axin complex B, Akt expression not the phosphorylation of GSK3β in the Axin complex (Fig. 4 it the phosphorylation of total GSK3β (Fig. 4 B, Cell However, in the presence of Dvl, Akt increased the of phosphorylated GSK3β in the Axin complex (Fig. 4 B, that Dvl was for phosphorylation of GSK3β in the Axin Furthermore, phosphorylated Akt was in the Axin complex in the presence of Dvl (Fig. 4 Dvl protein family three an a and a C-terminal A. Cytokine Growth Factor Rev. 1999; 10: 255-265Crossref PubMed Scopus (68) Google Scholar, M. M. Dev. 1999; PubMed Scopus Google Scholar). the of each have not been either the or is for Axin binding L. Yuan H. Weaver C.D. Mao J. Farr 3rd, G.H. Sussman D.J. Jonkers J. Kimelman D. Wu D. EMBO J. 1999; 18: 4233-4240Crossref PubMed Scopus (359) Google Scholar). that Dvl binding to Axin is for Akt in the Axin Dvl mutants were with L. Yuan H. Weaver C.D. Mao J. Farr 3rd, G.H. Sussman D.J. Jonkers J. Kimelman D. Wu D. EMBO J. 1999; 18: 4233-4240Crossref PubMed Scopus (359) Google Scholar, S. H. S. S. M. A. Mol. Cell. Biol. 1999; PubMed Google which both the and not to Axin, N-Dvl-(1–497), which the C-terminal region to the bound to Axin (Fig. 4 Dvl and N-Dvl-(1–497), not Akt interaction with the Axin complex (Fig. 4 the of Akt association with the Axin complex by was that with Dvl-1, this was with the of the expressed Dvl proteins (Fig. 4 C-Dvl-(369–695) and were expressed at levels (Fig. 4 that the of Akt in the Axin complex is not to C-Dvl-(369–695) expression levels. that the region of Dvl, which to Axin, is to Akt association with the Axin Akt not with Axin or Dvl (Fig. 4 and not However, Akt binding to the Axin complex by GSK3β, from the of Akt and GSK3β levels in the complex (Fig. 4 in the of GSK3β and Akt protein in the Axin complex was in cells in control cells. that Dvl binding to the Axin complex Akt to with and to GSK3β in the Wnt signaling is for in cell and differentiation (1Cadigan K.M. Nusse R. Genes Dev. 1997; 11: 3286-3305Crossref PubMed Scopus (2228) Google Scholar, 2Smalley M.J. Dale T.C. Cancer Metastasis Rev. 1999; 18: 215-230Crossref PubMed Scopus (187) Google Scholar, 3Waltzer L. Bienz M. Cancer Metastasis Rev. 1999; 18: 231-246Crossref PubMed Scopus (57) Google Scholar). instance, the Wnt-overexpressing Int5 cells have a and not with NGF the PC12 cells are and in to NGF (17Shackleford G.M. Willert K. Wang J. Varmus H.E. Neuron. 1993; 11: 865-875Abstract Full Text PDF PubMed Scopus (49) Google Scholar). Int5 cells in to NGF (Fig.5 this that the of Int5 cells Wnt signaling. test the of Akt this cellular we infected Int5 cells with either a dominant-negative Akt or a control contrast to the cells with control Int5 cells infected with Ad.AAA-Akt a with in to NGF (Fig. 5 and differentiation of Int5 cells was by in levels. with a report Cell Growth 1998; Google in to PC12 cells not protein PC12 cells (Fig. 5 However, Int5 cells increased levels of when cultured in differentiation medium (Fig. 5 levels of are the of Int5 cell differentiation not of a of levels of PC12 and Int5 cells in growth medium were not enhanced by Ad.AAA-Akt (Fig. 5 that inhibition of Akt activity the of Int5 cells, the that Akt has an important role in the Wnt signaling pathway. Here we that Wnt signaling stimulates Akt and that activated in association with Dvl, enhances the phosphorylation of GSK3β in the Axin The concerted of Dvl and Akt is a of Wnt signaling, which phosphorylation of the GSK3β that is for phosphorylation of β-catenin and its subsequent This such as and growth that Akt through the PI3K pathway not Dvl to an increase in β-catenin F. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, F. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). We that Dvl interaction with Axin induces a GSK3β to phosphorylated by Akt. This phosphorylated GSK3β β-catenin which in the and to the it with family to have that Akt is not in Wnt signaling F. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, H. Moon R.T. Mol. Cell. Biol. 1999; PubMed Google Scholar). to these we observed that Akt by was not to a Wnt with this of Akt (14Alessi D.R. Cohen P. Curr. Opin. Genet. Dev. 1998; 8: 55-62Crossref PubMed Scopus (675) Google Scholar) not TOPFlash in the presence of Dvl not Akt is to with the Axin complex in the and GSK3β activity in this critical contrast to these wild type Akt is activated at the plasma membrane and then to the cellular V. Mak T.W. Woodgett J.R. Oncogene. 1999; 18: 6094-6103Crossref PubMed Scopus (107) Google Scholar). not Akt activation and resulting GSK3β phosphorylation at when cells were with medium for free β-catenin was by this 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, F. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). we Akt activation and in Wnt signaling using by either or Wnt that the of Akt is to or not the signaling is Akt is activated by a Wnt kinase was to with activated Dvl J. Cell Biol. 2000; Scopus Google to Akt P. M. J. D. Z. A. S. J. Biochem. 2000; PubMed Scopus Google and to Wnt target genes P. M. J. D. Z. A. S. J. Biochem. 2000; PubMed Scopus Google Scholar, M. J. C. B. M. Moon R.T. R. M. J. A. S. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). whether Dvl Akt through the kinase pathway. We M. E. Greenberg and A. E. for PC12 cells, M. for Int5 and Tni3 cells, H. for 293T cells, J. R. Woodgett for the expression S. for the β-catenin expression K. Willert and R. Nusse for the and S. Takada for the expression