| Project/Area Number |
12670114
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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 |
General medical chemistry
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| Research Institution | Osaka University |
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Principal Investigator |
TANIURA Hideo Osaka University, Institute for Protein Research, Instructor, 蛋白質研究所, 助手 (80263325)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2001: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2000: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Keywords | Necdin / NEFA / hnRNP U / ca^<++> binding protein / neuron / ER / nuclear matrix / cell growth / 核マトリックス / 細胞増殖 / ニューロン / necdin / cell cycle / E2F / p53 / neural differentiation / transcription / growth arrest / postmitotic cell |
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| Research Abstract |
Necdin, a growth suppressor expressed predominantly in postmitotic neurons, interacts with viral oncoproteins and cellular transcription factors B2F1 and p53. In search of other cellular targets of necdin, we screened cDNA library from neurally differentiated embryonal carcinoma P19 cells by the yeast two-hybrid assay. We isolated two positive cDNA clones encoding NEFA and hnRNP U. Necdin interacted with NEFA via a domain encompassing two EF hand motifs, which had Ca binding activity. NEFA was widely distributed in mouse organs including the brain. By immunoelectron microscopy, NEFA was localized to the cisternae of the endoplasmic reticulumn and nuclear envelope in brain neurons. NEFA-GFP fusion protein expressed in NIE-115 cells was retained in the cytoplasm and partly secreted into the culture medium. Necdin enhanced the cytoplasmic retention of NEFA-GFP and potentiated the effect of NEFA-GFP on caffeine-evoked elevation of cytosolic Ca levels. Thus, necdin and NEFA might be involved in Ca homeostasis in neuronal cytoplasm. hnRNP U is a nuclear matrix-associated protein that interacts with chromosomal DNA. The necdin-binding site of hnRNP U was localized near a COOH-terminal region that mediated the association between hnRNP U and the nuclear matrix. In postmitotic neurons, endogenously expressed necdin and hnRNP U were detected in the nuclear matrix and formed a stable complex. Ectopically expressed necdin was concentrated in the nucleoli, but coexpressed hnRNP U recruited necdin to the nucleoplasmic compartment of the nuclear matrix. Furthermore, under the same conditions necdin and hnRNP U cooperatively suppressed the colony formation of transfected SAOS-2 cells. These results suggest that necdin suppresses cell proliferation through its interaction with hnRNP U in the specific subnuclear structure.
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