Functional Analysis of Proteins regulated by Ubiquitin Ligase

2005 Fiscal Year Final Research Report Summary

• Project/Area Number: 16370084
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Cell biology
• 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)
• 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)
• Project Period (FY): 2004 – 2005
• Keywords: ubiquitin ligase / affinity chromatography / knock-out mouse

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.

Research Products

• [Journal Article] Two E3 ubiquitin ligases, SCF-Skp2 and DDB1-Cul4, target human Cdt1 for proteolysis. (2006)
  • Author(s): Nishitani, H. et al.
  • Journal Title: EMBO J. 25・5 Pages: 1126-36
  • Description: 「研究成果報告書概要(和文)」より
• [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.et al.
  • Journal Title: Arterioscler. Thromb. Vasc. Biol. 26・4 Pages: 780-6
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Identification of novel E2F1 target genes regulated in cell cycle-dependent and independent manners. (2006)
  • Author(s): Iwanaga, R.
  • Journal Title: Oncogene 25・12 Pages: 1786-98
  • Description: 「研究成果報告書概要(和文)」より
• [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.
  • Journal Title: Arterioscler.Thromb.Vase.Biol. 26 Pages: 780-786
  • Description: 「研究成果報告書概要(欧文)」より
• [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.
  • Journal Title: Oncogene 25 Pages: 1786-1798
  • Description: 「研究成果報告書概要(欧文)」より
• [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.
  • Journal Title: EMBO J. 25 Pages: 1126-1136
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Mammalian E4 is required for cardiac development and maintenance of the nervous system. (2005)
  • Author(s): Kaneko-Oshikawa, C. et al.
  • Journal Title: Mol. Cell. Biol. 25・24 Pages: 10953-64
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Phosphorylation of p27(KIP1) in the developing retina and retinoblastoma. (2005)
  • Author(s): Kase, S. et al.
  • Journal Title: Int. J. Mol. Med. 16・2 Pages: 257-62
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Suppression of cell migration by protein kinase Cdelta. (2005)
  • Author(s): Jackson, D. et al.
  • Journal Title: Oncogene 24・18 Pages: 3067-72
  • Description: 「研究成果報告書概要(和文)」より
• [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.et al.
  • Journal Title: J. Clin. Invest. 115・4 Pages: 1039-48
  • Description: 「研究成果報告書概要(和文)」より
• [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. et al.
  • Journal Title: Mol. Cell 18・6 Pages: 699-709
  • Description: 「研究成果報告書概要(和文)」より
• [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. et al.
  • Journal Title: J. Biol. Chem. 280・18 Pages: 17694-700
  • Description: 「研究成果報告書概要(和文)」より
• [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. et al.
  • Journal Title: Curr. Eye Res. 30・6 Pages: 437-42
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] p107 inhibits G1 to S phase progression by down-regulating expression of the F-box protein Skp2. (2005)
  • Author(s): Rodier, G. et al.
  • Journal Title: J. Cell Biol. 168・1 Pages: 55-66
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Role of serine 10 phosphorylation in p27 stabilization revealed by analysis of p27 knock-in mice harboring a serine 10 mutation. (2005)
  • Author(s): Kotake, Y. et al.
  • Journal Title: J. Biol. Chem. 280・2 Pages: 1095-102
  • Description: 「研究成果報告書概要(和文)」より
• [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.
  • Journal Title: Mol.Cell.Biol. 25 Pages: 10953-10964
  • Description: 「研究成果報告書概要(欧文)」より
• [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.
  • Journal Title: Int.J.Mol.Med. 16 Pages: 257-262
  • Description: 「研究成果報告書概要(欧文)」より
• [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.
  • Journal Title: Curr.Eye Res. 30 Pages: 437-442
  • Description: 「研究成果報告書概要(欧文)」より
• [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.
  • Journal Title: Mol.Cell 18 Pages: 699-709
  • Description: 「研究成果報告書概要(欧文)」より
• [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.
  • Journal Title: J.Biol.Chem. 280 Pages: 17694-17700
  • Description: 「研究成果報告書概要(欧文)」より
• [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.
  • Journal Title: J.Clin.Invest. 115 Pages: 1039-1048
  • Description: 「研究成果報告書概要(欧文)」より
• [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.
  • Journal Title: Oncogene. 24 Pages: 3067-3072
  • Description: 「研究成果報告書概要(欧文)」より
• [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.
  • Journal Title: J.Cell Biol. 168 Pages: 55-66
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Role of serine 10 phosphorylation in p27 stabilization revealed by analysis of p27 knock-in mice harboring a serine 10 mutation. (2005)
  • Author(s): Kotake, Y., Nakayama, K., Ishida, N., Nakayama, K.I.
  • Journal Title: J.Biol.Chem. 280 Pages: 1095-1102
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Skp2-mediated degradation of p27 regulates progression into mitosis. (2004)
  • Author(s): Nakayama, K. et al.
  • Journal Title: Dev. Cell 6 Pages: 661-672
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Cytoplasmic ubiquitin ligase KPC regulates proteolysis of p27Kip1 at G1 phase. (2004)
  • Author(s): Kamura, T. et al.
  • Journal Title: Nature Cell Biol. 6 Pages: 1229-1235
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] The ISG15 isopeptidase UBP43 is regulated by proteolysis via the SCF(Skp2) ubiquitin ligase. (2004)
  • Author(s): Tokarz, S.A. et al.
  • Journal Title: J. Biol. Chem. 279 Pages: 46424-46430
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Early embryonic death in mice lacking the beta-catenin-binding protein Duplin. (2004)
  • Author(s): Nishiyama, M. et al.
  • Journal Title: Mol. Cell. Biol. 24 Pages: 8386-8394
  • Description: 「研究成果報告書概要(和文)」より
• [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.et al.
  • Journal Title: Genes Dev. 18 Pages: 1824-1837
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Involvement of p27KIP1 in the proliferation of the developing corneal endothelium. (2004)
  • Author(s): Yoshida, K. et al.
  • Journal Title: Invest. Ophthalmol. Vis. Sci. 45 Pages: 2163-2167
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] The papillomavirus E7 oncoprotein is ubiquitinated by UbcH7 and Cullin 1- and Skp2-containing E3 ligase. (2004)
  • Author(s): Oh, K.J.et al.
  • Journal Title: J. Virol. 78 Pages: 5338-5346
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Involvement of p27(KIP1) in proliferation of the retinal pigment epithelium and ciliary body. (2004)
  • Author(s): Yoshida, K. et al.
  • Journal Title: Anat. Embryol. 208 Pages: 145-150
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Distribution of p27(KIP1), cyclin D1, and proliferating cell nuclear antigen after retinal detachment. (2004)
  • Author(s): Yoshida, K. et al.
  • Journal Title: Graefes Arch. Clin. Exp. Ophthalmol. 242 Pages: 437-441
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Phosphorylation-dependent degradation of c-Myc is mediated by the F-box protein Fbw7. (2004)
  • Author(s): Yada, M. et al.
  • Journal Title: EMBO J. 23 Pages: 2116-2125
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Mouse Fbw7/Sel-10/Cdc4 is required for Notch degradation during vascular development. (2004)
  • Author(s): Tsunematsu, R. et al.
  • Journal Title: J. Biol. Chem. 279 Pages: 9417-9423
  • Description: 「研究成果報告書概要(和文)」より
• [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.
  • Journal Title: Nat.Cell Biol. 6 Pages: 1229-1235
  • Description: 「研究成果報告書概要(欧文)」より
• [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.
  • Journal Title: J.Biol Chem. 279 Pages: 46424-46430
  • Description: 「研究成果報告書概要(欧文)」より
• [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.
  • Journal Title: Mol.Cell.Biol. 24 Pages: 8386-8394
  • Description: 「研究成果報告書概要(欧文)」より
• [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.
  • Journal Title: Genes Dev. 18 Pages: 1824-1837
  • Description: 「研究成果報告書概要(欧文)」より
• [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.
  • Journal Title: Invest.Ophthalmol.Vis.Sci. 45 Pages: 2163-2167
  • Description: 「研究成果報告書概要(欧文)」より
• [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.
  • Journal Title: Dev.Cell 6 Pages: 661-672
  • Description: 「研究成果報告書概要(欧文)」より
• [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.
  • Journal Title: J.Virol. 78 Pages: 5338-5346
  • Description: 「研究成果報告書概要(欧文)」より
• [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.
  • Journal Title: EMBO J. 23 Pages: 2116-2125
  • Description: 「研究成果報告書概要(欧文)」より
• [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.
  • Journal Title: Graefes Arch.Clin.Exp.Ophthalmol 242 Pages: 437-441
  • Description: 「研究成果報告書概要(欧文)」より
• [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.
  • Journal Title: Anat.Embryol 208 Pages: 145-150
  • Description: 「研究成果報告書概要(欧文)」より
• [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.
  • Journal Title: J.Biol.Chem. 279 Pages: 9417-9423
  • Description: 「研究成果報告書概要(欧文)」より