Functional analysis of a novel GTP-binding protein involved in apoptosis.
| Project/Area Number |
12480213
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Cell biology
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| Research Institution | Keio University |
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Principal Investigator |
INOUE Jun-ichiro Fac. Sci & Tech. Keio Univ. Professor, 理工学部, 教授 (70176428)
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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 |
¥7,600,000 (Direct Cost: ¥7,600,000)
Fiscal Year 2001: ¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2000: ¥3,900,000 (Direct Cost: ¥3,900,000)
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| Keywords | GTP-binding protein / Cell cycle / Apoptosis / RNP K homology domain / RNA-binding / protein, DT40 / 細胞増殖 / アポートシス / 遺伝子破壊 / Gタンパク質 / 翻訳後修飾 |
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| Research Abstract |
We have cloned cDNAs encoding the entire coding region of a human homologue (H-ERA) and a mouse homologue (M-ERA) of ERA. The mammalian homologue of ERA consists of a typical GTPase/GTP-binding domain and a putative K homology (KH) domain, which is known as an RNA binding domain. We performed transfection experiments with wild-type H-ERA or various H-ERA mutants. H-ERA possessing the amino acid substitution mutation into the GTPase domain induced apoptosis of HeLa cells, which was blocked by Bcl-2 expression. Deletion of the C-terminus, which contains a part of the KH domain, alleviated apoptosis by the H-ERA mutant, suggesting the importance of this domain in the function of H-ERA. We have also shown the RNA binding activity of H-ERA by pull-down experiments using RNA homopolymer immobilized on beads or recombinant H-ERA proteins. Thus, H-ERA may play an important role in the regulation of apoptotic signalling with its GTPase/GTP binding domain. To elucidate the physiological function of eukaryotic ERA, we have performed a genetic analysis of chicken ERA (GdERA) in DT40 cells. Depletion of GdERA diminished the growth rate of the cells, accompanied by an accumulation of apoptotic cells. The analysis of cell cycle indicates that the elimination of GdERA caused arrest at G1 phase, but not at M phase, which highlights the distinct role of vertebrate ERA in the cell cycle progression compared to prokaryotic ERA. Furthermore, human ERA (HsERA) rescued the phenotype of GdERA-deficient cells, whereas a mutant of HsERA deprived of RNA binding activity did not. These data suggest that vertebrate ERA regulates the G1 phase progression via an as yet unknown molecular mechanism, which involves RNA recognition by ERA.
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Report
(3 results)
Research Products
(11 results)
