1998 Fiscal Year Final Research Report Summary
Role of Phosphorylation on Intracellular Localization of Interleukin 1alpha
| Project/Area Number |
09680633
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Functional biochemistry
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| Research Institution | Toho University |
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Principal Investigator |
WATANABE Naoko Toho Univ., Fac.Sci., Associate Professor, 理学部, 助教授 (80230978)
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| Co-Investigator(Kenkyū-buntansha) |
KOBAYASHI Yoshiro Toho Univ., Fac.Sci., Professor, 理学部, 教授 (10134610)
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| Project Period (FY) |
1997 – 1998
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| Keywords | interleukin 1alpha / inthacellular localization / phosphorylation / nuclear localization signal / fusion protein |
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| Research Abstract |
A precursor portion of a precursor form of IL-1alpha (pre-IL-1alpha) contains nuclear localization signal sequence (NLS) the vicinity to which is phosphorylated. Cleavage of pre-IL-1alpha is accompanied by release of a mature form of IL-1alpha (mature IL-1alpha). Here we investigated the relationship between phosphorylation and intracellular location of pre-IL-1alpha using expression vectors harboring a fusion protein with beta-galactosidase (beta-gal) or green fluorescence protein (GFP). These vectors were transfected into NIH/3T3 cells with calcium phosphate, followed by incubation for 2 days and examination under a microscope.
(1) We first confirmed that beta-gal containing the NLS was localized in nuclei, and that beta-gal fused with mature IL-1alpha was localized in cytoplasm.
(2) When beta-ga1 is fused with a sequence containing the NLS and its phosphorylatable or mutated (unphosphorylatable) vicinity, the fused proteins were localized in nuclei. The same was true for beta-gal fused with a precursor portion of pre-IL-1alpha.
(3) beta-gal fused with mutated entire pre-IL-1alpha (unphosphorylatable) was localized in nuclei, whereas that with wild-type entire pre-IL-1alpha was localized in nuclei and cytoplasm. When GFP was fused with wild-type or mutated entire pre-IL-1alpha, we obtained the similar results.
Although we didn't detect phosphorylation of pre-IL-1alpha in the cells, these findings suggest that phosphorylation partially suppresses nuclear transport of pre-IL-1alpha. Since the suppression occurred only in the fusion protein with entire pre-IL-1alpha but not with a precursor portion, it is possible that phosphorylation may induce conformational change of pre-IL-1alpha.
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[Publications] Kobayashi, Y., Yamashiro, T., Jinnai, T., Nakano, A., Watanabe, N., Kawanishi, T., Tanaka, H.and Shigenobu, K.: "Target cell-induced calcium signals in human natural killer leukemia cells as revealed by confocal fluorescence microscopy." Exp.Cell Res.232. 42-64 (1997)
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「研究成果報告書概要(欧文)」より
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[Publications] Yamashiro, T., Watanabe, N.and Kobayashi, Y.: "Reduction of intracellular pH by inhibitors of natural killer cell activity, nicardipine, methyl 2-(N-benzyl-N-methylamino)ethyl-2,6-dimethyl-4-(2-isopropyl-pyrazolo[1,5-a]pyridine-3-y1)-1,4-dihydropyridine-3,5-dicarboxylate(AHC-52)and 4,4'-diisothiocyano-2,2'-disulfonic acid stildene(DIDS)." Biochem.Pharmacol.54. 143-148 (1997)
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「研究成果報告書概要(欧文)」より
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[Publications] Miyachi, H., Azuma, A., Ogasawara, A., Uchimura, E., Watanabe, N., Kobayashi, Y., Kato, F., Kato, M.and Hashimoto, Y.: "Novel biological response modifiers : phthalimides with tumor necrosis factor-alpha production-regulating activity." J.Med.Chem.40. 2858-2865 (1997)
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[Publications] Uchimura, E., Kodaira, T., Kurosaka, K., Yang, D., Watanabe, N.and Kobayashi, Y.: "Interaction of phagocytes with apoptotic cell leads to production of pro-inflammatory cytokines." Biochem.Biophys.Res.Comm.239. 799-803 (1997)
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[Publications] Yamashiro, T., Watanabe, N., Yokoyama, K.K., Koga, C., Tsuruo, T.and Kobayashi, Y.: "Requirement of expression of P-glycoprotein on human natural killer leukemia cells for cell-mediated cytotoxicity." Biochem.Pharmacol.55. 1385-1390 (1998)
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