Functional Analysis of Proteins regulated by Ubiquitin Ligase
Project/Area Number |
16370084
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Research Category |
Grant-in-Aid for Scientific Research (B)
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Allocation Type | Single-year Grants |
Section | 一般 |
Research Field |
Cell biology
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Research Institution | Tohoku University |
Principal Investigator |
NAKAYAMA Keiko Tohoku University, Graduate School of Medicine, Professor, 大学院・医学系研究科, 教授 (60294972)
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Co-Investigator(Kenkyū-buntansha) |
ISHIDA Noriko Tohoku University, Graduate School of Medicine, Research Associate, 大学院・医学系研究科, 助手 (10361073)
HARA Kentaro Tohoku University, Graduate School of Medicine, COE Fellow, 大学院・医学系研究科, COEフェロー (60400248)
三宅 智 東北大学, 大学院・医学系研究科, 講師 (00332346)
|
Project Period (FY) |
2004 – 2005
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Project Status |
Completed (Fiscal Year 2005)
|
Budget Amount *help |
¥15,400,000 (Direct Cost: ¥15,400,000)
Fiscal Year 2005: ¥5,300,000 (Direct Cost: ¥5,300,000)
Fiscal Year 2004: ¥10,100,000 (Direct Cost: ¥10,100,000)
|
Keywords | ubiquitin ligase / affinity chromatography / knock-out mouse / ユビキチン化 / 蛍光色素標識 / プロテオーム / ユビキチン / プロテオーム解析 |
Research Abstract |
We have tried to identify physiological substrates of ubiquitin ligase by establishment of comprehensive-analytical method for detection of difference between before and after genetic engineering in knock-out mice. First, we purified ubiquitinated protein specifically by anti-ubiquitin antibody. Ubiquitin is an abundant protein and exist as free form (not conjugated or binding form in mammalian cells. By affinity-chromatography using a conventional antibody, we recovered a free-ubiqitin mainly, and could not collect ubiquitin conjugated proteins. For improvement of the efficiency of collection, we assesed many kinds of antibodies and finally decided to use the FK2 monoclonal antibody, which binds only substrate-conjugated ubiqitin specifically. We succeeded in concentrating ubiquitinated proteins by affinity-chromatography using FK2 binding column. For comparison of proteins between knock-out mice and wild type mice, we have to choose the most adequate tissue and the most effective method for cell-extraction. We identified the tissue which expressed F-box protein we were interested in by in-situ hybridization. Based on these basic informations, we prepared Fbw7-deficient-embryonic fibroblasts and also wild-type fibroblast, and extracted whole cell lysate from each samples. Cell lysate loaded on column packed FK2-antibody conjugated beads for concentration of ubiquitinated proteins. We plan to compare the difference of the ubiquitinated proteins between Fbw7-deficient and wild-type embryonic fibroblasts.
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Report
(3 results)
Research Products
(69 results)
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[Journal Article] PKCdelta is necessary for Smad3 expression and transforming growth factorbeta-induced fibronectin synthesis in vascular smooth muscle cells.2006
Author(s)
Ryer, E.J., Hom, R.P., Sakakibara, K., Nakayama, K.I., Nakayama, K., Faries, P.L., Liu, B., Kent, K.C.
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Journal Title
Arterioscler.Thromb.Vase.Biol. 26
Pages: 780-786
Description
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[Journal Article] Identification of novel E2F1 target genes regulated in cell cycle-dependent and independent manners.2006
Author(s)
Iwanaga, R., Komori, H., Ishida, S., Okamura, N., Nakayama, K., Nakayama, K.I., Ohtani, K.
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Journal Title
Oncogene 25
Pages: 1786-1798
Description
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[Journal Article] Two E3 ubiquitin ligases, SCF-Skp2 and DDB1-Cul4, target human Cdt1 for proteolysis.2006
Author(s)
Nishitani, H., Sugimoto, N., Roukos, V., Nakanishi, Y., Saijo, M., Obuse, C., Tsurimoto, T., Nakayama, K.I., Nakayama, K., Fujita, M., Lygerou, Z., Nishimoto, T.
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Journal Title
EMBO J. 25
Pages: 1126-1136
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[Journal Article] Mammalian E4 is required for cardiac development and maintenance of the nervous system.2005
Author(s)
Kaneko-Oshikawa, C., Nakagawa, T., Yamada, M., Yoshikawa, H., Matsumoto, M., Yada, M., Hatakeyama, S., Nakayama, K., Nakayama, K.I.
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Journal Title
Mol.Cell.Biol. 25
Pages: 10953-10964
Description
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[Journal Article] Phosphorylation of p27(KIP1) in the developing retina and retinoblastoma.2005
Author(s)
Kase, S., Nakayama, K.I., Nakayama, K., Ikeda, H., Harada, T., Harada, C., Ohgami, K., Shiratori, K., Ohno, S., Yoshida, K.
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Journal Title
Int.J.Mol.Med. 16
Pages: 257-262
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[Journal Article] Disappearance of p27(KIP1) and increase in proliferation of the lens cells after extraction of most of the fiber cells of the lens.2005
Author(s)
Kase, S., Ikeda, H., Harada, T., Harada, C., Imaki, J., Ohgami, K., Shiratori, K., Nakayama, K.I., Nakayama, K., Ohno, S., Yoshida, K.
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Journal Title
Curr.Eye Res. 30
Pages: 437-442
Description
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[Journal Article] Ubiquitylation of RAG-2 by Skp2-SCF links destruction of the V(D)J recombinase to the cell cycle.2005
Author(s)
Jiang, H., Chang, F.-C., Ross, A.E., Lee, J., Nakayama, K.I., Nakayama, K., Desiderio, S.
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Journal Title
Mol.Cell 18
Pages: 699-709
Description
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[Journal Article] Molecular dissection of the interaction between p27 and KPC, the ubiquitinligase that regulates proteolysis of p27 in G1 phase.2005
Author(s)
Kotoshiba, S., Kamura, T., Hara, T., Ishida, N., Nakayama, K.I.
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Journal Title
J.Biol.Chem. 280
Pages: 17694-17700
Description
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[Journal Article] Bcl-2-related protein A1 is an endogenous and cytokine-stimulated mediator of cytoprotection in hyperoxic acute lung injury.2005
Author(s)
He, C.H., Waxman, A.B., Lee, C.G., Link, H., Rabach, M.E., Ma, B., Chen, Q., Zhu, Z., Zhong, M., Nakayama, K., Nakayama, K.I., Homer, R., Elias, J.A.
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Journal Title
J.Clin.Invest. 115
Pages: 1039-1048
Description
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[Journal Article] Suppression of cell migration by protein kinase Cdelta.2005
Author(s)
Jackson, D., Zheng, Y., Lyo, D., Shen, Y., Nakayama, K., Nakayama, K.I., Humphries, M.J., Reyland, M.E., Foster, D.A.
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Journal Title
Oncogene. 24
Pages: 3067-3072
Description
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[Journal Article] p107 inhibits G1 to S phase progression by down-regulating expression of the F-box protein Skp2.2005
Author(s)
Rodier, G., Makris, C., Coulombe, P., Scime, A., Nakayama, K., Nakayama, K.I., Meloche, S.
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Journal Title
J.Cell Biol. 168
Pages: 55-66
Description
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[Journal Article] Cytoplasmic ubiquitin ligase KPC regulates proteolysis of p27^<Kip1> at G1 phase.2004
Author(s)
Kamura, T., Hara, T., Matsumoto, M., Ishida, N., Okumura, F., Hatakeyama, S., Yoshida, M., Nakayama, K., Nakayama, K.I.
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Journal Title
Nat.Cell Biol. 6
Pages: 1229-1235
Description
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[Journal Article] The ISG15 isopeptidase UBP43 is regulated by proteolysis via the SCFSkp2 ubiquitin ligase.2004
Author(s)
Tokarz, S., Berset, C., La Rue, J., Friedman, K., Nakayama, K.I., Nakayama, K., Zhang, D.E., Lanker, S.
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Journal Title
J.Biol Chem. 279
Pages: 46424-46430
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[Journal Article] Early embryonic death in mice lacking the beta-catenin-binding protein Duplin.2004
Author(s)
Nishiyama, M., Nakayama, K., Tsunematsu, R., Tsukiyama, T., Kikuchi, A., Nakayama, K.I.
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Journal Title
Mol.Cell.Biol. 24
Pages: 8386-8394
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[Journal Article] Distinct roles of c-Abl and Atm in oxidative stress response are mediated by protein kinase C delta.2004
Author(s)
Li, B., Wang, X., Rasheed, N., Hu, Y., Boast, S., Ishii, T., Nakayama, K., Nakayama, K.I., Goff, S.P.
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Journal Title
Genes Dev. 18
Pages: 1824-1837
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[Journal Article] Involvement of p27KIP1 in the proliferation of the developing corneal endothelium.2004
Author(s)
Yoshida, K., Kase, S., Nakayama, K., Nagahama, H., Harada, T., Ikeda, H., Harada, C., Imaki, J., Ohgami, K., Shiratori, K., Ilieva, I.B., Ohno, S., Nishi, S., Nakayama, K.I.
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Journal Title
Invest.Ophthalmol.Vis.Sci. 45
Pages: 2163-2167
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[Journal Article] Skp2-mediated degradation of p27 regulates into mitosis.2004
Author(s)
Nakayama, K., Nagahama, H., Minamishima, Y.A., Miyake, S., Ishida, N., Hatakeyama, S., Kitagawa, M., Iemura, S., Natsume, T., Nakayama, K.I.
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Journal Title
Dev.Cell 6
Pages: 661-672
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[Journal Article] The papillomavirus E7 oncoprotein is ubiquitinated by UbcH7 and Cullin 1- and Skp2-containin E3 ligase.2004
Author(s)
Oh, K.J., Kalinina, A., Wang, J., Nakayama, K., Nakayama, K.I., Bagchi, S.
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Journal Title
J.Virol. 78
Pages: 5338-5346
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[Journal Article] Phosphorylation-dependent degradation of c-Myc is mediated by the F-box protein Fbw7.2004
Author(s)
Yada, M., Hatakeyama, S., Kamura, T., Nishiyama, M., Tsunematsu, R., Imaki, H., Ishida, N., Okumura, F, Nakayama, K., Nakayama, K.I.
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Journal Title
EMBO J. 23
Pages: 2116-2125
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[Journal Article] Distribution of p27(KIP1), cyclin D1, and proliferating cell nuclear antigen after retinal detachment.2004
Author(s)
Yoshida, K., Kase, S., Nakayama, K., Nagahama, H., Harada, T., Ikeda, H., Harada, C., Imaki, J., Ohgami, K., Shiratori, K., Ohno, S., Nakayama, K.I.
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Journal Title
Graefes Arch.Clin.Exp.Ophthalmol 242
Pages: 437-441
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[Journal Article] Involvement of p27(KIP1) in proliferation of the retinal pigment epithelium and ciliary body.2004
Author(s)
Yoshida, K., Nakayama, K., Kase, S., Nagahama, H., Harada, T., Ikeda, H., Harada, C., Imaki, J., Ohgami, K., Shiratori, K., Ohno, S., Nishi, S., Nakayama, K.I.
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Journal Title
Anat.Embryol 208
Pages: 145-150
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[Journal Article] Mouse Fbw7/Sel-10/Cdc4 is required for Notch degradation during vascular development.2004
Author(s)
Tsunematsu, R., Nakayama, K., Oike, Y., Nishiyama, M., Ishida, N., Hatakeyama, S., Bessho, Y., Kageyama, R., Suda, T., Nakayama, K.I.
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Journal Title
J.Biol.Chem. 279
Pages: 9417-9423
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