MAGE-A4 Interacts with the Liver Oncoprotein Gankyrin and Suppresses Its Tumorigenic Activity

Hepatocellular carcinoma ranks among the most common malignancies in Southeast Asia and South Africa. Although there are many modalities of treatment, the recurrence and metastasis rates are high, and the prognosis is unsatisfactory. Gankyrin, a recently found oncoprotein, is a promising target for drug therapy because it is overexpressed in all studied hepatocellular carcinomas. Gankyrin contains six ankyrin repeats and interacts with Rb, Cdk4, and the S6 ATPase of the 26 S proteasome. In this study, a yeast two-hybrid screen with gankyrin has identified MAGE-A4 as another interacting protein. The interaction, mediated by the C-terminal half of MAGE-A4, was reproduced in mammalian cells. The interaction was specific to MAGE-A4, because other MAGE family proteins structurally similar to MAGE-A4,i.e. MAGE-A1, MAGE-A2, and MAGE-A12, did not bind to gankyrin. MAGE-A4 partially suppressed both anchorage-independent growth in vitro and tumor formation in athymic mice of gankyrin-overexpressing cells. The ability of mutant MAGE-A4 to interact with gankyrin correlated with the ability to suppress the anchorage-independent growth. These results demonstrate that MAGE-A4 binds to gankyrin and suppresses its oncogenic activity. So far, the major focus of studies on the MAGE proteins has been on their potential for cancer immunotherapy. Our results may also shed light on novel functions for MAGE-A proteins. Hepatocellular carcinoma ranks among the most common malignancies in Southeast Asia and South Africa. Although there are many modalities of treatment, the recurrence and metastasis rates are high, and the prognosis is unsatisfactory. Gankyrin, a recently found oncoprotein, is a promising target for drug therapy because it is overexpressed in all studied hepatocellular carcinomas. Gankyrin contains six ankyrin repeats and interacts with Rb, Cdk4, and the S6 ATPase of the 26 S proteasome. In this study, a yeast two-hybrid screen with gankyrin has identified MAGE-A4 as another interacting protein. The interaction, mediated by the C-terminal half of MAGE-A4, was reproduced in mammalian cells. The interaction was specific to MAGE-A4, because other MAGE family proteins structurally similar to MAGE-A4,i.e. MAGE-A1, MAGE-A2, and MAGE-A12, did not bind to gankyrin. MAGE-A4 partially suppressed both anchorage-independent growth in vitro and tumor formation in athymic mice of gankyrin-overexpressing cells. The ability of mutant MAGE-A4 to interact with gankyrin correlated with the ability to suppress the anchorage-independent growth. These results demonstrate that MAGE-A4 binds to gankyrin and suppresses its oncogenic activity. So far, the major focus of studies on the MAGE proteins has been on their potential for cancer immunotherapy. Our results may also shed light on novel functions for MAGE-A proteins. hepatocellular carcinoma retinoblastoma tumor suppressor protein cyclin-dependent kinase melanoma antigen 3-aminotriazole hemagglutinin doxycycline enhanced green fluorescent protein fluorescein isothiocyanate tetramethylrhodamine isothiocyanate Gankyrin (gann ankyrin repeat protein, also known as PSMD10 and p28) is an oncoprotein, the expression of which is increased (1Higashitsuji H. Itoh K. Nagao T. Dawson S. Nonoguchi K. Kido T. Mayer R.J. Arii S. Fujita J. Nat. Med. 2000; 6: 96-99Crossref PubMed Scopus (281) Google Scholar, 2Fu X.Y. Wang H.Y. Tan L. Liu S.Q. Cao H.F. Wu M.C. World J. Gastroenterol. 2002; 8: 638-643Crossref PubMed Scopus (90) Google Scholar) in hepatocellular carcinomas (HCCs).1 Gankyrin consists of six ankyrin repeats and a 38-amino acid N-terminal extension and binds to the retinoblastoma tumor suppressor protein (Rb), the S6 ATPase subunit of the 26 S proteasome (PSMC4, RPT3, TBP7), and cyclin-dependent kinase 4 (Cdk4) (1Higashitsuji H. Itoh K. Nagao T. Dawson S. Nonoguchi K. Kido T. Mayer R.J. Arii S. Fujita J. Nat. Med. 2000; 6: 96-99Crossref PubMed Scopus (281) Google Scholar, 3Dawson D. Apcher S. Mee M. Higashitsuji H. Baker R. Uhle S. Dubiel W. Fujita J. Mayer R.J. J. Biol. Chem. 2002; 277: 10893-10902Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 4Li J. Tsai M.-D. Biochemistry. 2002; 41: 3977-3983Crossref PubMed Scopus (83) Google Scholar). Overexpression of gankyrin increases both the phosphorylation and degradation of Rbin vivo and oncogenically transforms NIH/3T3 cells. Gankyrin binds to Cdk4 and counteracts the inhibitory function of the tumor suppressors p16INK4A and p18INK4C (4Li J. Tsai M.-D. Biochemistry. 2002; 41: 3977-3983Crossref PubMed Scopus (83) Google Scholar). In a rodent model of hepatocarcinogenesis, gankyrin is overexpressed from the earliest stage of tumor development (5Park T.J. Kim H.S. Byun K.H. Jang J.J. Lee Y.S. Lim I.K. Mol. Carcinog. 2001; 30: 138-150Crossref PubMed Scopus (55) Google Scholar). These findings suggest that gankyrin is a major player in cell cycle control and tumorigenesis in HCCs. The MAGE (melanoma antigen) genes were initially identified because they encode tumor antigens that can be recognized by cytolytic T lymphocytes derived from the blood lymphocytes of cancer patients (6van der Bruggen P. Traversari C. Chomoz P. Lurquin C. De Plaen E. van den Eynde B. Knuth A. Boon T. Science. 1991; 254: 1643-1647Crossref PubMed Scopus (3126) Google Scholar). The MAGE gene family is composed of more than 25 genes in humans and are classified as type I MAGE genes (including MAGE-A, MAGE-B, and MAGE-Cgenes) and type II MAGE genes, which include those that reside outside of the MAGE-A, MAGE-B, andMAGE-C genomic clusters (7Chomez P. De Backer O. Bertrand M. De Plaen E. Boon T. Lucas S. Cancer Res. 2001; 61: 5544-5551PubMed Google Scholar, 8Baker P.A. Salehi A. J. Neurosci. Res. 2002; 67: 705-712Crossref PubMed Scopus (263) Google Scholar). The MAGE-Asubfamily comprises 12 genes (MAGE-A1 toMAGE-A12), and is expressed in various types of tumors but not in normal adult tissues, except for testis and placenta. The MAGE-A antigens are of particular interest for antitumor immunotherapy because they are strictly tumor specific and are shared by many tumors. Despite the isolation of growing numbers ofMAGE genes, their function in normal tissues remains mostly unknown. To further characterize the molecular mechanism underlying the oncogenic activity of gankyrin and facilitate development of a therapeutic agent against HCCs, we have used a yeast two-hybrid screen to identify further gankyrin interactions. We report here that MAGE-A4 binds to human gankyrin and suppresses its oncogenicity. A full-length human gankyrin cDNA was cloned into pAS2–1 vector (BD Biosciences) and co-transformed into Y190 yeast cells with a placenta or a U-2 OS cDNA library in pACT2 vector (BD Biosciences). Yeast clones containing interacting proteins were identified by growth on media lacking tryptophan, leucine, and histidine, followed by assaying for β-galactosidase activity. The interaction was confirmed by co-transformation into Y190 cells with pAS2–1-gankyrin and their growth on the selection medium with 25 mm 3-aminotriazole (3AT). For analysis of interactions, full-length and various mutant cDNAs of gankyrin (GenBankTM accession number D83197) and MAGE-A4 (GenBankTM accession number U10687) were generated by the polymerase chain reaction and cloned into a pAS2–1 or pACT2 vector, respectively. cDNAs corresponding to the C-terminal regions of MAGE-A1 (amino acids 203–309, GenBankTMaccession number NM_004988), MAGE-A2 (amino acids 210–314, GenBankTM accession number NM_005361), and MAGE-A12 (amino acids 210–314, GenBankTM accession number XM_010079) were also cloned into pACT2. FLAG-tagged MAGE-A4, HA-tagged MAGE-A4, and HA-tagged gankyrin cDNAs were cloned into the eukaryotic expression vectors pMKit-neo, pMKit-hygro, and pCMV4. pEGFP-C1 vector (BD Biosciences) was used to express EGFP-S6 and EGFP-MAGE-A4 fusion proteins. For conditional expression of FLAG-MAGE-A4, we used the tetracycline-regulated system as described (9Higashitsuji H. Higashitsuji H. Nagao T. Nonoguchi K. Fujii S. Itoh K. Fujita J. Cancer Cell. 2002; 2: 335-346Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The pMACS4-IRES vector (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) was used to express MAGE-A4 or C-terminal 107 or 55 amino acids of MAGE-A4 with truncated human CD4. The CD4-expressing transfectants were enriched by using the MACSelect 4 system (Miltenyi Biotec GmbH). Mouse NIH/3T3 embryonal fibroblasts and its derivatives, monkey COS-7 kidney cells, human U-2 OS osteosarcoma cells, Huh-7 hepatoma cells, PLC/PRF/5 hepatoma cells, and HEK293 human embryonal kidney cells and its derivatives were cultured in Dulbecco's modified Eagle medium supplemented with 10% calf or fetal calf serum at 37 °C in a humidified atmosphere of 5% CO2 in air. Transfection was performed by the calcium phosphate method. pl16-GK-6 cells were generated from NIH/3T3 cells so that they constitutively expressed HA-tagged human gankyrin and the tetracycline activator, rtTA (9Higashitsuji H. Higashitsuji H. Nagao T. Nonoguchi K. Fujii S. Itoh K. Fujita J. Cancer Cell. 2002; 2: 335-346Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). In the presence of 2 μg/ml doxycycline (Dox) in culture medium, they expressed FLAG-tagged MAGE-A4. GK-S25 cells were derived from NIH/3T3 cells and constitutively expressed gankyrin. To assess the anchorage dependence of growth in vitro, 5 × 102 cells were plated in 0.3% agarose on top of 0.6% agarose in the presence or absence of Dox in a 35-mm dish. Four weeks later, the number of colonies (>20 cells) was counted microscopically. Statistical differences between sample means were calculated by analysis of variance, followed by an unpaired Student's ttest. Western blot analysis and immunoprecipitation were performed as described previously (1Higashitsuji H. Itoh K. Nagao T. Dawson S. Nonoguchi K. Kido T. Mayer R.J. Arii S. Fujita J. Nat. Med. 2000; 6: 96-99Crossref PubMed Scopus (281) Google Scholar, 9Higashitsuji H. Higashitsuji H. Nagao T. Nonoguchi K. Fujii S. Itoh K. Fujita J. Cancer Cell. 2002; 2: 335-346Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Antibodies used were mouse monoclonal anti-HA antibody (Roche Diagnostics), anti-FLAG antibody (Sigma), anti-GFP antibody (BD Biosciences), anti-actin antibody (Chemicon International, Inc., Temecula, CA), rabbit polyclonal anti-gankyrin antibody (Santa Cruz Biotechnology), anti-MAGE antibody (FL-309; Santa Cruz Biotechnology), horseradish peroxidase-conjugated goat anti-mouse antibody (DAKO, Kyoto, Japan), and anti-rabbit antibody (DAKO). To analyze interaction between endogenous MAGE-A4 and endogenous gankyrin, U-2 OS cell lysates were immunoprecipitated using rabbit anti-MAGE antibody (FL-309) immobilized to the protein G support (Seize X mammalian immunoprecipitation kit; Pierce). The antibody is broadly reactive with all MAGE family members according to the manufacturer. The immobilized antibody was incubated consecutively with four aliquots of cell lysates (total of 1 ml), the first aliquot at 4 °C overnight and the others at 20 °C sequentially for 1 h. The bound proteins were eluted with the elution buffer supplied in the kit. The elution was repeated three times, and each fraction was analyzed by Western blotting using rabbit polyclonal anti-gankyrin antibody (Santa Cruz Biotechnology.). Interaction of transiently expressed exogenous MAGE-A4 or its mutant with exogenous gankyrin was examined by co-transfecting COS-7 or U-2 OS cells with plasmids expressing FLAG-tagged MAGE-A4, FLAG-tagged C-terminal 107 amino acids of MAGE-A4, HA-tagged full-length gankyrin, FLAG alone, or HA alone in various combinations. Interaction of stably transfected gankyrin with MAGE-A4 was examined by using pl16-GK-6 cells, which were derived from mouse NIH/3T3 cells, constitutively expressed HA-tagged human gankyrin, and inducibly expressed FLAG-tagged MAGE-A4 in the presence of Dox. To analyze the effects of MAGE-A4 on binding of gankyrin to Rb, S6, and Cdk4, U-2 OS cells and 293T cells were co-transfected with plasmids expressing FLAG-gankyrin, HA-tagged Rb, EGFP-S6 fusion protein, HA-tagged Cdk4, HA-tagged MAGE-A4, EGFP-MAGE-A4 fusion protein, EGFP alone, or FLAG alone in various combinations. Immunofluorescence staining was performed essentially as described (9Higashitsuji H. Higashitsuji H. Nagao T. Nonoguchi K. Fujii S. Itoh K. Fujita J. Cancer Cell. 2002; 2: 335-346Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 10Hopwood D. Celis J.E. Cell Biology, A Laboratory Handbook. 2nd Ed. Academic Press, San Diego, CA1998: 221-231Google Scholar). COS-7 cells were replated on chamber slides after transfection, fixed with phosphate-buffered saline containing 4% paraformaldehyde for 30 min, and then rendered permeable with phosphate-buffered saline containing 0.2% Triton X-100 for 30 min at room temperature. After blocking nonspecific antibody-binding sites with bovine serum albumin, the cells were incubated with mouse monoclonal anti-FLAG antibody (Sigma) and rabbit polyclonal anti-HA antibody (Berkeley Antibody, Richmond, CA). Then the bound antibodies were reacted with FITC-linked anti-mouse and TRITC-linked anti-rabbit IgGs (Amersham Biosciences) and observed under a confocal laser microscope (Olympus, Tokyo, Japan). Twenty-four female BALB/c Slc-nu/nu athymic mice (4 weeks old) were injected subcutaneously with pl16-GK-6 cells or GK-S25 cells (8 × 106 cells each) and divided into two groups. Twelve were given Dox in the was calculated by the and with We performed yeast two-hybrid to for proteins of interacting with human gankyrin. full-length gankyrin as a we identified clones of × 106 yeast clones with human placenta or U-2 OS cell cDNA on media containing the and was for β-galactosidase staining 1 and not analysis of the plasmids that two of the C-terminal 107 amino acids of MAGE-A4. with findings D. Apcher S. Mee M. Higashitsuji H. Baker R. Uhle S. Dubiel W. Fujita J. Mayer R.J. J. Biol. Chem. 2002; 277: 10893-10902Abstract Full Text Full Text PDF PubMed Scopus (113) Google the clones C-terminal of the S6 ATPase subunit of the 26 S proteasome. MAGE-A4 did not interact with the binding alone 1 that it with gankyrin. To that gankyrin interacts with the MAGE-A4 in mammalian cells, COS-7 cells were co-transfected with expressing HA-tagged human gankyrin and FLAG-tagged C-terminal 107 amino acids of MAGE-A4. cell lysates were immunoprecipitated with an anti-FLAG was in but not in from cells co-transfected with FLAG vector and 1 truncated MAGE-A4 was in the anti-HA from cells co-transfected with plasmids expressing and FLAG-tagged truncated MAGE-A4 1 We examined full-length MAGE-A4 interacts with gankyrin in COS-7 cells. in 1 HA-tagged gankyrin was immunoprecipitated with anti-FLAG antibody in lysates from cells co-transfected and expressing FLAG-tagged full-length MAGE-A4 and HA-tagged gankyrin. FLAG-tagged MAGE-A4 was immunoprecipitated with an anti-HA were observed in U-2 OS cells as not in the COS-7 cells expressing and staining that both proteins were in the 1 COS-7 cells are known to transfected which it a to that between expressed proteins. we generated an in which was stably overexpressed and was inducibly expressed in the presence of Dox. in Dox increased the of but not that of in pl16-GK-6 cells. in the presence of Dox was with by the anti-HA antibody and with by anti-FLAG antibody two of MAGE-A4 and 5% of gankyrin in pl16-GK-6 cells after were to be in a not Western blot analysis that the of stably overexpressed gankyrin in pl16-GK-6 cells was than those observed in human U-2 OS osteosarcoma and Huh-7 hepatoma cells top The of MAGE-A4 in the presence of Dox was than that in U-2 OS osteosarcoma and PLC/PRF/5 hepatoma cells 2 is for this because the anti-MAGE antibody used was reactive with MAGE proteins other than MAGE-A4. We examined the interaction between endogenous gankyrin and MAGE-A4 in U-2 OS cells from which we the truncated MAGE-A4 cDNA in 2 the endogenous gankyrin was with MAGE proteins by anti-MAGE results suggest that gankyrin and MAGE-A4 interacts in human cancer cells. gankyrin binds to Rb, the S6 subunit of the 26 S and Cdk4 (1Higashitsuji H. Itoh K. Nagao T. Dawson S. Nonoguchi K. Kido T. Mayer R.J. Arii S. Fujita J. Nat. Med. 2000; 6: 96-99Crossref PubMed Scopus (281) Google Scholar, 3Dawson D. Apcher S. Mee M. Higashitsuji H. Baker R. Uhle S. Dubiel W. Fujita J. Mayer R.J. J. Biol. Chem. 2002; 277: 10893-10902Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 4Li J. Tsai M.-D. Biochemistry. 2002; 41: 3977-3983Crossref PubMed Scopus (83) Google the effects of of MAGE-A4 on the binding of gankyrin to proteins were U-2 OS cells were co-transfected with plasmids expressing FLAG-tagged gankyrin and HA-tagged Rb, was immunoprecipitated with anti-FLAG antibody as Overexpression of EGFP-MAGE-A4 fusion protein by did not the of immunoprecipitated U-2 OS cells were co-transfected with plasmids expressing FLAG-tagged gankyrin and EGFP-S6 fusion protein, EGFP-S6 was immunoprecipitated with anti-FLAG antibody as Overexpression of HA-tagged MAGE-A4 by did not the of immunoprecipitated of EGFP-MAGE-A4 was observed on the binding of gankyrin to Cdk4 not results were with 293T cells not To characterize the interacting of gankyrin and MAGE-A4, we various and analyzed their in the yeast two-hybrid We previously that full-length is for the interaction with (1Higashitsuji H. Itoh K. Nagao T. Dawson S. Nonoguchi K. Kido T. Mayer R.J. Arii S. Fujita J. Nat. Med. 2000; 6: 96-99Crossref PubMed Scopus (281) Google Scholar). mutant of gankyrin with MAGE-A4, that all ankyrin repeats and the N-terminal extension are for the binding and not The MAGE-A4 by the two-hybrid screen the C-terminal 107 amino acids to A and and 4 N-terminal to did not the interaction with gankyrin. a of amino acids from the of MAGE-A4 its binding to gankyrin The interaction was specific to MAGE-A4, because the corresponding regions of other MAGE structurally similar to MAGE-A4, did not interact with gankyrin and These results that the C-terminal of MAGE-A4 containing the P. Lurquin C. Boon T. van der Bruggen P. J. PubMed Scopus Google Scholar, S. O. P. A. S. K. J. 2002; PubMed Scopus Google Scholar) and interacts with gankyrin. To the effects of the interaction of gankyrin and MAGE-A4, we used the pl16-GK-6 cells in which gankyrin was stably overexpressed and MAGE-A4 expression be with Dox 2 These cells colonies in the cells were cultured in the presence of the numbers of colonies was to of those in the absence of Dox 5 the mouse GK-S25 cells stably gankyrin were transfected with plasmids expressing the C-terminal 55 amino acids of MAGE-A4 that did not bind to gankyrin 4 in the number of colonies were observed 5 the C-terminal 107 amino acids of the MAGE-A4 that binds to gankyrin the that the ability to bind to gankyrin with the ability to suppress anchorage-independent growth in The anchorage-independent in cell culture has been correlated with the ability of cells to tumors in R. R. S. A. PubMed Scopus Google Scholar). We the of MAGE-A4 on tumor formation of cells in athymic After subcutaneously with pl16-GK-6 cells, the were divided into two of which was Dox in In the absence of all mice tumors 5 weeks after the of pl16-GK-6 cells the tumors and in mice given Dox. Dox by effects on tumor formation by GK-S25 cells not results demonstrate that MAGE-A4 binds to gankyrin and suppresses its activity. and in a of growth inhibitory to the cell cycle control B. D. 2000; PubMed Scopus Google Scholar, K.H. 2001; PubMed Scopus Google Scholar, Nat. 2002; 2: PubMed Scopus Google Scholar). The of two in human and that into the cell cycle and from cell cycle are two that a cell cycle to Gankyrin and is overexpressed in (1Higashitsuji H. Itoh K. Nagao T. Dawson S. Nonoguchi K. Kido T. Mayer R.J. Arii S. Fujita J. Nat. Med. 2000; 6: 96-99Crossref PubMed Scopus (281) Google Scholar, 2Fu X.Y. Wang H.Y. Tan L. Liu S.Q. Cao H.F. Wu M.C. World J. Gastroenterol. 2002; 8: 638-643Crossref PubMed Scopus (90) Google Scholar). Gankyrin an in and is a promising target for therapeutic drug we that MAGE-A4 binds to gankyrin and suppresses its oncogenic activity. The interaction was identified by the yeast two-hybrid and reproduced in mammalian cells. endogenous gankyrin is with endogenous MAGE in human cancer cells in which MAGE-A4 were it is that the interaction is MAGE-A4 is a of the MAGE a of proteins that a amino acid known as the MAGE (7Chomez P. De Backer O. Bertrand M. De Plaen E. Boon T. Lucas S. Cancer Res. 2001; 61: 5544-5551PubMed Google Scholar, 8Baker P.A. Salehi A. J. Neurosci. Res. 2002; 67: 705-712Crossref PubMed Scopus (263) Google Scholar). Although its function is the MAGE of binds to H. K. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the study, the of MAGE-A4, in to the C-terminal half of MAGE was found to be for MAGE-A4 to bind to gankyrin. binding was specific for MAGE-A4, because similar amino acid from other MAGE-A family members did not bind to gankyrin. Despite their the MAGE genes are expressed in a of cancer cells. In the of more than of the carcinomas which have been as K. M. S. M. PubMed Scopus Google Scholar, Wang H.S. J. 2002; PubMed Scopus Google Scholar). of family MAGE-A4, has also been in Lee H.S. PubMed Scopus Google Scholar). overexpressed in the of cancer cells, MAGE proteins are to the and then on the cell as major P.A. Salehi A. J. Neurosci. Res. 2002; 67: 705-712Crossref PubMed Scopus (263) Google Scholar). has been on the potential of MAGE as a target for cancer immunotherapy. The C-terminal of MAGE-A4 that binds to gankyrin contains by P. Lurquin C. Boon T. van der Bruggen P. J. PubMed Scopus Google Scholar, S. O. P. A. S. K. J. 2002; PubMed Scopus Google Scholar). binding of gankyrin degradation of MAGE-A4 the of to be The by the MAGE gene family are with a P.A. Salehi A. J. Neurosci. Res. 2002; 67: 705-712Crossref PubMed Scopus (263) Google Scholar). has an in development of cells in the Overexpression of cell cycle that may with or H. K. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). which binds to proteins the and of proteins cell cycle and C. A. P.A. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, J. R. L. M. S. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). is as cell and its expression suppresses cell S. D. K. K. K. H. C. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). In to and which to the type II MAGE genes, MAGE-A4 did not the cell cycle by Nagao and J. effects on gankyrin-overexpressing cells. this was observed in vitro as vivo in athymic it is cell and not mediated by T cells. In the of Cdk4, and is for of the S Cell. Full Text PDF PubMed Scopus Google Scholar). A major target for the is of to of the that the expression of genes for of the S B. D. 2000; PubMed Scopus Google Scholar). Although there to be cell and differences in the control of a has that the and major cell cycle for the of the by anchorage and oncogenic S. M. A. H. 2002; PubMed Scopus Google Scholar). Gankyrin binds to Cdk4, by D. Apcher S. Mee M. Higashitsuji H. Baker R. Uhle S. Dubiel W. Fujita J. Mayer R.J. J. Biol. Chem. 2002; 277: 10893-10902Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 4Li J. Tsai M.-D. Biochemistry. 2002; 41: 3977-3983Crossref PubMed Scopus (83) Google increases phosphorylation and degradation of Rb, and transforms cells to in an anchorage-independent (1Higashitsuji H. Itoh K. Nagao T. Dawson S. Nonoguchi K. Kido T. Mayer R.J. Arii S. Fujita J. Nat. Med. 2000; 6: 96-99Crossref PubMed Scopus (281) Google Scholar). the of MAGE-A4 on anchorage-independent growth correlated with its binding to gankyrin, it is mediated by gankyrin on with gankyrin. we were to effects of MAGE-A4 on the degradation of MAGE-A4 did not the binding of gankyrin to Rb, S6, or in the activity of MAGE-A4 are to be activity of a specific has been found to be to the cancer in cancer cells, which a that of gankyrin alone has a therapeutic in Science. 2002; PubMed Scopus Google Scholar). of the underlying the effects of MAGE-A4 on gankyrin and anchorage-independent growth facilitate development of novel against and also shed light on normal functions of MAGE-A proteins. We for