2005 Fiscal Year Final Research Report Summary
Real-time observation of interactions between trancription factors containing PAS domains
| Project/Area Number |
15207010
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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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 | Tohoku University |
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Principal Investigator |
SOGAWA Kazuhiro Tohoku University, Graduate School of Life Sciences, Professor, 大学院生命科学研究科, 教授 (80175421)
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| Co-Investigator(Kenkyū-buntansha) |
FUKUMURA Hiroshi Tohoku University, Graduate School of Science, Professor, 大学院理学研究科, 教授 (50208980)
YASUMOTO Ken-ichi Tohoku University, Graduate School of Life Sciences, Associate Professor, 大学院生命科学研究科, 助教授 (90241629)
KIKUCHI Yasuo Tohoku University, Graduate School of Life Sciences, Associate Professor, 大学院生命科学研究科, 助教授 (10004467)
TAKASAKI Chikahisa Tohoku University, Graduate School of Life Sciences, Instructor, 大学院生命科学研究科, 助手 (10004491)
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| Project Period (FY) |
2003 – 2005
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| Keywords | FRET / FLIM / living cells / protein-protein interaction / VBC complex / transcription factor |
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| Research Abstract |
We constructed a fluorescence lifetime imaging microscopy (FLIM) using a pulse diode laser and TSCSPC as an illuminator and a detector, respectively. Using the FLIM microscope, several protein-protein interactions were detected in living cells by FRET (Fluorescence Resonance Energy Transfer). First, interaction of GAPDH monomers in the tetramer was observed. Detailed analysis 1 of the FRET data suggested that GAPDH tetramers were formed posttranslationally by the random binding of monomers. Loose complex between GAPDH and PGK was observed by measuring FRET between them. The complex was also observed by measuring change in conformation of GAPDH by coexpression of PGK in living cells. We also observed conformational change of PGK by coexpression of PGK. Interaction between elongin B and elongin C was observed by FRET-FLIM. Although interaction between elongin C and pVHL was not observed, coexpression of elongin B largely induced interaction between elongin C and pVHL, suggesting that a conformational change of elongin C suitable for binding of pVHL was induced by binding of elongin B. In the LBP-1 protein family, homodimers of LBP-la and LBP-1c and heterodimers between LBP-1a and LBP-1c were observed in the cytosol. Homodimers of LBP-1b were observed in the nucleus of living cells using FRET-FLIM. Further, Arnt homodimer formation was observed in the PML body but not in the nucleoplasm by FRET-FLIM.
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