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• W2023273217 abstract "Protein kinase CK2 (formerly casein kinase II) is a serine/threonine kinase overexpressed in many human tumors, transformed cell lines, and rapidly proliferating tissues. Recent data have shown that many cancers involve inappropriate reactivation of Wnt signaling through ectopic expression of Wnts themselves, as has been seen in a number of human breast cancers, or through mutation of intermediates in the Wnt pathway, such as adenomatous polyposis coli or β-catenin, as described in colon and other cancers. Wnts are secreted factors that are important in embryonic development, but overexpression of certain Wnts, such as Wnt-1, leads to proliferation and transformation of cells. We report that upon stable transfection of Wnt-1 into the mouse mammary epithelial cell line C57MG, morphological changes and increased proliferation are accompanied by increased levels of CK2, as well as of β-catenin. CK2 and β-catenin co-precipitate with the Dvl proteins, which are Wnt signaling intermediates. A major phosphoprotein of the size of β-catenin appears in in vitro kinase reactions performed on the Dvl immunoprecipitates. In vitro translated β-catenin, Dvl-2, and Dvl-3 are phosphorylated by CK2. The selective CK2 inhibitor apigenin blocks proliferation of Wnt-1-transfected cells, abrogates phosphorylation of β-catenin, and reduces β-catenin and Dvl protein levels. These results demonstrate that endogenous CK2 is a positive regulator of Wnt signaling and growth of mammary epithelial cells. Protein kinase CK2 (formerly casein kinase II) is a serine/threonine kinase overexpressed in many human tumors, transformed cell lines, and rapidly proliferating tissues. Recent data have shown that many cancers involve inappropriate reactivation of Wnt signaling through ectopic expression of Wnts themselves, as has been seen in a number of human breast cancers, or through mutation of intermediates in the Wnt pathway, such as adenomatous polyposis coli or β-catenin, as described in colon and other cancers. Wnts are secreted factors that are important in embryonic development, but overexpression of certain Wnts, such as Wnt-1, leads to proliferation and transformation of cells. We report that upon stable transfection of Wnt-1 into the mouse mammary epithelial cell line C57MG, morphological changes and increased proliferation are accompanied by increased levels of CK2, as well as of β-catenin. CK2 and β-catenin co-precipitate with the Dvl proteins, which are Wnt signaling intermediates. A major phosphoprotein of the size of β-catenin appears in in vitro kinase reactions performed on the Dvl immunoprecipitates. In vitro translated β-catenin, Dvl-2, and Dvl-3 are phosphorylated by CK2. The selective CK2 inhibitor apigenin blocks proliferation of Wnt-1-transfected cells, abrogates phosphorylation of β-catenin, and reduces β-catenin and Dvl protein levels. These results demonstrate that endogenous CK2 is a positive regulator of Wnt signaling and growth of mammary epithelial cells. glycogen synthase kinase-3β adenomatous polyposis coli casein kinase II casein kinase I mitogen-activated protein phosphatidylinositol 3-kinase T-cell factor lymphocyte enhancer binding factor Wnt-1 was first identified as a proto-oncogene activated in mammary carcinomas caused by mouse mammary tumor virus and soon after was identified as the mammalian homolog of the Drosophila wingless gene (1Nusse R. Varmus H.E. Cell. 1982; 31: 99-109Abstract Full Text PDF PubMed Scopus (1217) Google Scholar, 2Nusse R. Theunissen H. Wagenaar E. Rijsewijk F. Gennissen A. Otte A. Schuuring E. van Ooyen A. Mol. Cell. Biol. 1990; 10: 4170-4179Crossref PubMed Scopus (51) Google Scholar). Transgenic mice overexpressing Wnt-1 develop mammary hyperplasia and carcinomas (3Tsukamoto A.S. Grosschedl R. Guzman R.C. Parslow T. Varmus H.E. Cell. 1988; 55: 619-625Abstract Full Text PDF PubMed Scopus (582) Google Scholar). Wnt-1 is one of the 16 known Wntgenes that encode secreted glycopeptide growth factors or morphogens (4Dale T.C. Biochem. J. 1998; 329: 209-223Crossref PubMed Scopus (433) Google Scholar). When expressed in C57MG, a normal mouse mammary epithelial cell line, many Wnts cause morphological transformation (5Brown A.M. Wildin R.S. Prendergast T.J. Varmus H.E. Cell. 1986; 46: 1001-1009Abstract Full Text PDF PubMed Scopus (140) Google Scholar, 6Wong G.T. Gavin B.J. McMahon A.P. Mol. Cell. Biol. 1994; 14: 6278-6286Crossref PubMed Scopus (284) Google Scholar, 7Shimizu H. Julius M.A. Giarre M. Zheng Z. Brown A.M. Kitajewski J. Cell Growth Differ. 1997; 8: 1349-1358PubMed Google Scholar). Normal C57MG cells that reach confluence in culture appear as a monolayer of regular cuboidal cells; however, C57MG cells transfected with Wnt-1 have an elongated refractile morphology. It has been shown that Wnts bind to seven transmembrane domain receptors of the “frizzled” family (8Bhanot P. Brink M. Samos C.H. Hsieh J.C. Wang Y. Macke J.P. Andrew D. Nathans J. Nusse R. Nature. 1996; 382: 225-230Crossref PubMed Scopus (1206) Google Scholar, 9Tomlinson A. Strapps W.R. Heemskerk J. Development. 1997; 124: 4515-4521Crossref PubMed Google Scholar, 10Slusarski D.C. Corces V.G. Moon R.T. Nature. 1997; 390: 410-413Crossref PubMed Scopus (540) Google Scholar, 11Yang-Snyder J. Miller J.R. Brown J.D. Lai C.J. Moon R.T. Curr. Biol. 1996; 6: 1302-1306Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar). Genetic studies have demonstrated that proteins of the “dishevelled” family (dsh in Drosophila, Dvl in mammals) are membrane-proximal signaling intermediates (12Sokol S.Y. Curr. 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In the absence of a Wnt signal, GSK3β phosphorylates β-catenin and induces its ubiquitination and proteolytic degradation (18Aberle H. Bauer A. Stappert J. Kispert A. Kemler R. EMBO J. 1997; 16: 3797-3804Crossref PubMed Scopus (2122) Google Scholar, 19Orford 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). Inhibition of GSK3β by the presence of a Wnt signal stabilizes β-catenin, allowing it to translocate into the nucleus (20Huber O. Korn R. McLaughlin J. Ohsugi M. Herrmann B.G. Kemler R. Mech. Dev. 1996; 59: 3-10Crossref PubMed Scopus (778) Google Scholar, 21Korinek V. Barker N. Willert K. Molenaar M. Roose J. Wagenaar G. Markman M. Lamers W. Destree O. Clevers H. Mol. Cell. Biol. 1998; 18: 1248-1256Crossref PubMed Scopus (291) Google Scholar, 22Yost C. Torres M. Miller J.R. Huang E. Kimelman D. Moon R.T. 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Goldschmeding R. Logtenberg T. Clevers H. Science. 1999; 285: 1923-1926Crossref PubMed Scopus (398) Google Scholar). Other proteins, such as the hereditary colon cancer gene product adenomatous polyposis coli (APC) (33Munemitsu S. Albert I. Souza B. Rubinfeld B. Polakis P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3046-3050Crossref PubMed Scopus (947) Google Scholar) and axin/axil (34Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1087) Google Scholar, 35Kishida S. Yamamoto H. Ikeda S. Kishida M. Sakamoto I. Koyama S. Kikuchi A. J. Biol. Chem. 1998; 273: 10823-10826Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar, 36Yamamoto H. Kishida S. Uochi T. Ikeda S. Koyama S. Asashima M. Kikuchi A. Mol. Cell. Biol. 1998; 18: 2867-2875Crossref PubMed Scopus (172) Google Scholar, 37Hart M.J. de los Santos R. Albert I.N. Rubinfeld B. Polakis P. Curr. Biol. 1998; 8: 573-581Abstract Full Text Full Text PDF PubMed Google Scholar, 38Sakanaka C. Weiss J.B. Williams L.T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3020-3023Crossref PubMed Scopus (282) Google Scholar), interact with β-catenin and regulate its turnover. Protein kinase CK2 (formerly casein kinase II) has recently been added to the cascade of proteins in the Drosophila wingless pathway. In Schneider 2 cells, it was shown that CK2 associates with and phosphorylates Dsh (39Willert K. Brink M. Wodarz A. Varmus H. Nusse R. EMBO J. 1997; 16: 3089-3096Crossref PubMed Scopus (201) Google Scholar). Transfection of mammalian Dvl-1 and Dvl-2 into clone-8 imaginal disc cell line results in hyperphosphorylation of Dvl proteins by CK2 (40Lee J.S. Ishimoto A. Yanagawa S. J. Biol. Chem. 1999; 274: 21464-21470Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). The Drosophila model suggests that CK2 could be an important regulator of Wnt signaling in other systems. CK2 is a serine/threonine kinase that is ubiquitously expressed in both the cytoplasm and nucleus of eukaryotic cells. It exists as a constitutively active tetramer that contains two catalytic subunits, α or α′ (37–44 kDa), and two regulatory β subunits (24–28 kDa) (41Pinna L.A. Meggio F. Prog. Cell Cycle Res. 1997; 3: 77-97Crossref PubMed Scopus (311) Google Scholar, 42Allende J.E. Allende C.C. FASEB J. 1995; 9: 313-323Crossref PubMed Scopus (584) Google Scholar). The two catalytic subunits are highly homologous, but the α′ subunit has a unique required role in spermatogenesis (43Xu X. Toselli P.A. Russell L.D. Seldin D.C. Nat. Genet. 1999; 23: 118-121Crossref PubMed Scopus (321) Google Scholar). CK2 phosphorylates serines or threonines in acidic domains, with (S/T)XX(D/E) being the canonical motif (44Hrubey T.W. Roach P.J. Biochem. Biophys. Res. Commun. 1990; 172: 190-196Crossref PubMed Scopus (26) Google Scholar, 45Litchfield D.W. Arendt A. Lozeman F.J. Krebs E.G. Hargrave P.A. 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Res. 1994; 40: 1-11PubMed Google Scholar). Dysregulated expression of CK2 in cells can be oncogenic, as transgenic expression of CK2α can promote lymphoma (52Seldin D.C. Leder P. Science. 1995; 267: 894-897Crossref PubMed Scopus (360) Google Scholar, 53Kelliher M.A. Seldin D.C. Leder P. EMBO J. 1996; 15: 5160-5166Crossref PubMed Scopus (246) Google Scholar, 54Landesman-Bollag E. Channavajhala P.L. Cardiff R.D. Seldin D.C. Oncogene. 1998; 16: 2965-2974Crossref PubMed Scopus (146) Google Scholar) and breast cancer. 2E. Landesman-Bollag, D. H. Song, R. D. Cardiff, and D. C. Seldin, unpublished results. 2E. Landesman-Bollag, D. H. Song, R. D. Cardiff, and D. C. Seldin, unpublished results. CK2 is characterized by the following biochemical properties: it is activated by polyamines (56Leroy D. Heriche J.K. Filhol O. Chambaz E.M. Cochet C. J. Biol. Chem. 1997; 272: 20820-20827Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 57Leroy D. Valero E. Filhol O. Heriche J.K. Goldberg Y. Chambaz E.M. Cochet C. Cell Mol. Biol. Res. 1994; 40: 441-453PubMed Google Scholar, 58Chaudhry P.S. Casillas E.R. Arch. Biochem. Biophys. 1989; 271: 98-106Crossref PubMed Scopus (14) Google Scholar), inhibited by apigenin (chrysin) and 6-dichloro-1-β-d-ribofuranosylbenzimidazole (59Zandomeni R. Zandomeni M.C. Shugar D. Weinmann R. J. Biol. Chem. 1986; 261: 3414-3419Abstract Full Text PDF PubMed Google Scholar, 60Critchfield J.W. Coligan J.E. Folks T.M. Butera S.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6110-6115Crossref PubMed Scopus (124) Google Scholar), and can utilize GTP as well as ATP as a phosphate donor (61Cochet C. Feige J.J. Pirollet F. Keramidas M. Chambaz E.M. Biochem. Pharmacol. 1982; 31: 1357-1361Crossref PubMed Scopus (94) Google Scholar). CK2 has been implicated in the regulation of many cellular processes, including DNA replication, basal and inducible transcription, and the regulation of cell growth and metabolism (62Pinna L.A. Biochim. Biophys. Acta. 1990; 1054: 267-284Crossref PubMed Scopus (805) Google Scholar, 63Litchfield D.W. Luscher B. Mol. Cell. Biochem. 1993; 127–128: 187-199Crossref PubMed Scopus (160) Google Scholar, 64Issinger O.G. Pharmacol. Ther. 1993; 59: 1-30Crossref PubMed Scopus (248) Google Scholar). Here, we demonstrate that CK2 is up-regulated by the presence of a Wnt-1 signal in mouse mammary epithelial cells and that endogenous CK2 in mammalian cells associates with the Dvl proteins. Furthermore, β-catenin can also be found in this complex and is phosphorylated by CK2. Inhibition of CK2 activity by apigenin accelerates the degradation of β-catenin and Dvl proteins and causes cell cycle arrest. These results demonstrate that CK2 is a key regulator of the Wnt signaling pathway in mammalian cells. pMV7-Wnt-1 (kindly provided by Dr. Anthony M. C. Brown) and control pMV7 empty vector were transfected into C57MG cells via electroporation. Stable clones were generated by selection in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2% l-glutamine, 1% penicillin/streptomycin (Cellgro, Mediatech Inc., Herndon, VA), 10 μg/ml insulin (Sigma), and 400 μg/ml G418 (Life Technologies, Inc.). Clones were screened for Wnt-1 expression, and results for several Wnt-1-positive clones were compared with those for neomycin-resistant clones transfected with the empty vector in each experiment. In addition to the stably transfected clones, we also analyzed pools of cells transiently infected with retrovirus expressing Wnt-1 to confirm our results. Retrovirally mediated gene transfer was performed essentially as described (65Serrano M. Lin A.W. McCurrach M.E. Beach D. Lowe S.W. Cell. 1997; 88: 593-602Abstract Full Text Full Text PDF PubMed Scopus (3860) Google Scholar) with the following modifications: Phoenix cells (obtained from Dr. Gary Nolan) were plated at 1 × 106 cells/60-mm dish and transfected overnight using the CLONTECH calcium phosphate transfection kit. The pBabe retroviral vector (66Morgenstern J.P. Land H. Nucleic Acids Res. 1990; 18: 3587-3596Crossref PubMed Scopus (1886) Google Scholar) and pBabe-Wnt-1 expression vector (a generous gift from Brian Elenbaas) were used at a concentration of 6 μg/400 μl of calcium phosphate solution. C57MG cells were plated at 5 × 105 cells/60-mm dish 1 day prior to infection. 24 h after infection, C57MG cells were split and maintained in medium containing 2 μg/ml puromycin. To measure changes in mammary epithelial cell growth, Wnt-1-transfected clones were cultured with varying concentrations of apigenin (Sigma) for up to 2 days in microtiter wells. At 24 and 48 h, cells were treated with 50 μl of freshly made 1 mg/ml 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (Polyscience Inc., Warrington, PA) and 25 μmphenazine methosulfate (Aldrich) solution for 4 h at 37 °C, at which time, the absorbance at 450 nm was determined with subtraction of background absorbance at 650 nm, providing quantitation of cell number (67Scudiero D.A. Shoemaker R.H. Paull K.D. Monks A. Tierney S. Nofziger T.H. Currens M.J. Seniff D. Boyd M.R. Cancer Res. 1988; 48: 4827-4833PubMed Google Scholar). The absorbance at 450–650 nm for medium alone was subtracted from each value. To determine the mechanism by which apigenin inhibits proliferation of the cells, the effects on cell cycle progression were determined. Cells were treated with apigenin overnight, harvested, resuspended in Nicoletti buffer (0.1% Triton X-100 and 0.1% sodium citrate) containing 0.5 mg/ml propidium iodide (Sigma), and analyzed on a flow cytometer (Becton Dickinson, Mountain View, CA) using the Cellquest program. RNA was isolated using Ultraspec RNA (Biotecx Laboratories Inc. Houston, TX) and quantitated by spectrophotometer. 10 μg of RNA was loaded onto a formaldehyde-based 1% agarose gel, electrophoresed, and blotted onto a nylon membrane (Gene screen Plus, NEN Life Science Products). The membrane was baked for 2 h at 80 °C and prehybridized in Church buffer (7% SDS, 1% bovine serum albumin, 1 mm EDTA, 0.25M Na2HPO4, 0.17% H3PO4). Full-length Wnt-1 and CK2 cDNAs were labeled with Klenow DNA polymerase (New England Biolabs Inc., Beverly, MA) and used to screen for Wnt-1 and CK2 expression. To extract cellular proteins, Wnt-1-expressing cells were lysed in buffer supplemented with protease and phosphatase inhibitors (50 mm Tris-HCl, pH 8.0, 1% Nonidet P-40, 125 mmNaCl, 1 mm NaF, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml pepstatin, 1 μg/ml leupeptin, 1 mm Na3VO4, and 10 mm sodium pyrophosphate). The BCA protein assay (Pierce) was used to determine the protein concentrations of the whole cell lysates. Equal amounts of protein were loaded onto polyacrylamide gels, electrophoresed, and transferred onto nitrocellulose membranes (Schleicher & Schuell Inc., Keene, NH) using a semidry electroblotter (Owl Scientific, Woburn, MA). After transfer, the membranes were blocked overnight in 5% dry milk and probed for protein expressions using antibodies against CK2α (Upstate Biotechnology Inc., Lake Placid, NY), GSK3β (Transduction Laboratories, Lexington, KY), or β-catenin (Transduction Laboratories). Dvl proteins were detected with monoclonal antibodies: The monoclonal antibody to Dvl-1 has been described (68Lijam N. Paylor R. McDonald M.P. Crawley J.N. Deng C.X. Herrup K. Stevens K.E. Maccaferri G. McBain C.J. Sussman D.J. Wynshaw-Boris A. Cell. 1997; 90: 895-905Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar). Mouse monoclonals against Dvl-2 and 3 were generated against glutathione S-transferase fusion proteins containing either the C-terminal amino acids of Dvl-2 (2–10B5) or Dvl-3 (3–4D3) using the ClonaCell-HY hybridoma cloning kit (StemCell Technologies, Inc., Vancouver, British Columbia, Canada). The specificity of the three Dvl monoclonal antibodies was confirmed by Western blotting of extracts of COS cells transiently transfected with Dvl-1, Dvl-2, or Dvl-3 expression constructs, in which each antibody recognized its cognate Dvl only (data not shown). Immunoreactive bands using appropriate secondary antibodies coupled to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA) were visualized by chemiluminescence in signaling solution (Pierce). Ponceau S (Sigma) staining and/or a monoclonal β-actin antibody (Sigma) were used to confirm equal loading of all Western blot membranes. For immunoprecipitation, 300 μg of protein extracts was precleared with protein A-agarose beads (Sigma). 100 μl of Dvl-1, Dvl-2, or Dvl-3 hybridoma supernatant or 1 μl of 68 mg/ml β-catenin antibody (Sigma) was added to precleared lysates and incubated overnight at 4 °C. 40 μl of protein A-agarose beads was added to each of the incubations for 2 h at 4 °C. Control immunoprecipitations with irrelevant antibody or beads alone were also performed. The immunoprecipitated complexes were washed five times in phosphate-buffered saline, and Western blotting was performed as described. 12 μg of protein was incubated in buffer (100 mm Tris, pH 8.0, 20 mm MgCl2, 100 mm NaCl, 50 mm KCl, and 100 μm Na3VO4) with 5 μCi of [γ-32P]GTP and 1 μg/μl CK2 substrate peptide (RRREEETEEE, Promega, Madison, WI) for 15 min at 30 °C (69Kuenzel E.A. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 737-741Crossref PubMed Scopus (167) Google Scholar). Control kinase reactions without the peptide were also done for each of the samples. After 15 min of incubation, the kinase assays were stopped with 10 mm cold ATP and 0.4 n HCl. The samples were spotted onto a P81 Whatman filter circle and washed four times, 5 min each, with 150 mm H3PO4 to elute unincorporated counts. Incorporated counts were quantified in an automatic scintillation counter. The samples were assayed in triplicate. Following immunoprecipitation of the whole cell lysates with Dvl-1, Dvl-2, and Dvl-3 antibodies as described above, the bead-bound proteins were incubated with 5 μCi of [γ-32P]GTP at 37 °C for 20 min alone or in the presence of apigenin (80 μm) or wortmannin (25 nm). The kinase reactions were stopped by the addition of 2× sample loading buffer. The samples were boiled, centrifuged, and subsequently loaded onto a denaturing SDS-polyacrylamide gel. The gel was transferred onto a nitrocellulose membrane and autoradiography of the membrane was performed to visualize [γ-32P]GTP-labeled proteins. Western blotting was performed to identify the phosphorylated target using specific antibodies. pCI-β-catenin (kindly provided by Dr. Bert Vogelstein), pSVK-Dvl-1,pBSK-Dvl-2, and pSVK-Dvl-3 plasmid DNA (1 μg each) were in vitro transcribed and translated using the TNT-coupled reticulocyte lysate system (Promega). Plasmid DNAs were incubated for 75 min with appropriate polymerase in the presence of 20 μCi of [35S]methionine. A negative control (no DNA) was prepared in the same manner. After the coupled transcription and translation reaction, 35S-labeled protein products were calf intestinal phosphatase-treated as described (70Kasahara H. Izumo S. Mol. Cell. Biol. 1999; 19: 526-536Crossref PubMed Scopus (82) Google Scholar) and immunoprecipitated with respective antibodies. These protein products were incubated with 10 units of recombinant CK2 enzyme (New England Biolabs Inc.) in the presence of 5 μCi of [γ-32P]GTP at 37 °C for 20 min. Products were loaded onto an acrylamide gel and transferred onto a nitrocellulose membrane. The phosphorylated proteins were visualized by autoradiography. The same membrane was cut, and Western blotting analysis was done to confirm the presence of the translated proteins. Wnt-1-expressing subclones of C57MG cells were generated to study the role of CK2 in Wnt signaling. C57MG cells were transfected with the pMV7 vector alone or pMV7-Wnt-1, and clones were selected in G418. Northern blotting (Fig.1 A) and reverse transcription-polymerase chain reaction were employed to identifyWnt-1 mRNA positive clones. Morphological differences between the Wnt-1-expressing clones and the control neomycin-transfected clones were observed, as previously reported (5Brown A.M. Wildin R.S. Prendergast T.J. Varmus H.E. Cell. 1986; 46: 1001-1009Abstract Full Text PDF PubMed Scopus (140) Google Scholar), and the proliferation rate of the Wnt-1 transfectants was uniformly greater than that of the controls. One high expressing clone was studied in detail, and the results were confirmed in other clones and in pools of cells infected with pBABE-Wnt-1 retrovirus. Northern analysis demonstrated that CK2 mRNA expression was elevated 2.5-fold in the Wnt-1-expressing clones (Fig.1 B); two different specific transcripts for CK2 are typically seen. CK2 activity was also up-regulated in the Wnt-1 transfectants, as determined using a synthetic CK2 peptide substrate and [γ-32P]GTP as a phosphate donor.Wnt-1-expressing cells exhibited 2-fold higher CK2 activity than the vector controls (Fig. 1 D). As expected in Wnt-1-overexpressing cells, β-catenin protein levels were up-regulated, as were CK2 protein levels. These results were seen in both the pMV7-Wnt-1-transfected and pBABE-Wnt-1-infected cells (Fig.2).Figure 2β-Catenin and CK2 α proteins are up-regulated in Wnt-1-overexpressing C57MG cells. Protein extracts were prepared from clones stably transfected with Wnt-1 or neomycin plasmid vector alone (left panel) or from cells transiently infected with a Wnt-1retrovirus or puromycin retrovirus control (right panel). 30 μg of protein was transferred onto a nitrocellulose membrane, and Western blotting was performed with antibodies against β-catenin, CK2α, or β-actin as a loading control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine the functional importance of CK2 activity upon growth of the Wnt-1-transfected cells, we used the selective CK2 inhibitor apigenin. Apigenin rapidly inhibited CK2 activity in a dose-dependent manner (Fig.3 A) and dramatically blocked cell proliferation over the course of a 2-day treatment (Fig.3 B). [3H]Thymidine incorporation was inhibited (not shown), and cells treated with apigenin accumulated in the G2/M phase of the cell cycle (Fig. 3 C). Short term treatment with apigenin did not alter the morphology of the Wnt-1-expressing cells. To determine whether CK2 associates with Dvl in Wnt-1-expressing cells, immunoprecipitations with specific monoclonal antibodies raised against Dvl-1, Dvl-2, and Dvl-3 were performed, followed by Western blotting. Lysates prepared from whole cells were Western blotted with the different primary antibodies to ascertain the electrophoretic mobilities of the corresponding proteins (Fig. 4 A). Each of the Dvl proteins was detected in Western blots of immunoprecipitates performed with the cognate antibody. In addition, we detected immunoreactive CK2 and β-catenin protein in the complexes precipitated by each of the Dvl antibodies (Fig. 4 B). GSK3β was not detected in the Dvl immunoprecipitates. Control immunoprecipitates using nonspecific antibodies did not precipitate any of the proteins. We attempted to perform the reciprocal immunoprecipitates; however, the CK2 antibody failed to work for immunoprecipitation. The β-catenin antibody was capable of inefficiently immunoprecipitating a minor fraction of the β-catenin in the cell lysate, but neither CK2 nor Dvl proteins could be detected in this material. This may be a quantitative problem, because most β-catenin is involved in the formation of adhesion complexes with E-cadherin; alternatively, the polyclonal antibody may preferentially precipitate monomeric β-catenin, or in fact it may compete with CK2 and Dvl for binding to the β-catenin. To determine whether the CK2 in the Dvl immunoprecipitates was capable of phosphorylating its partners, an in vitro kinase reaction was performed on the immunoprecipitates. We found that a single major phosphoprotein of approximately 97 kDa appeared in all three Dvl immunoprecipitates (Fig. 5 A). This protein migrated in polyacrylamide gels slightly more slowly than immunoreactive β-catenin detected on Western blotting of the same gel, consistent with the phosphorylated form of β-catenin (71Serres M. Grangeasse C. Haftek M. Durocher Y. Duclos B. Schmitt D. Exp. Cell Res. 1997; 231: 163-172Crossref PubMed Scopus (51) Google Scholar). Dvl-1 migrates slightly more slowly than the 97-kDa phosphoprotein, whereas Dvl-2 and Dvl-3 run faster. These data suggested that the phosphoprotein might be β-catenin; the fact that the 97-kDa phosphoprotein a" @default.
• W2023273217 created "2016-06-24" @default.
• W2023273217 creator A5026644077 @default.
• W2023273217 creator A5042697980 @default.
• W2023273217 creator A5046864369 @default.
• W2023273217 date "2000-08-01" @default.
• W2023273217 modified "2023-10-16" @default.
• W2023273217 title "Endogenous Protein Kinase CK2 Participates in Wnt Signaling in Mammary Epithelial Cells" @default.
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• W2023273217 doi "https://doi.org/10.1074/jbc.m909107199" @default.
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