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ファイル | 記述 | サイズ | フォーマット | |
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MCB.00087-14.pdf | 3.25 MB | Adobe PDF | 見る/開く |
タイトル: | Quantitative in vivo fluorescence cross-correlation analyses highlight the importance of competitive effects in the regulation of protein-protein interactions. |
著者: | Sadaie, Wakako Harada, Yoshie Matsuda, Michiyuki https://orcid.org/0000-0002-5876-9969 (unconfirmed) Aoki, Kazuhiro https://orcid.org/0000-0001-7263-1555 (unconfirmed) |
著者名の別形: | 青木, 一洋 |
発行日: | 23-Jun-2014 |
出版者: | American Society for Microbiology |
誌名: | Molecular and cellular biology |
巻: | 34 |
号: | 17 |
開始ページ: | 3272 |
終了ページ: | 3290 |
抄録: | Computer-assisted simulation is a promising approach for clarifying complicated signaling networks. However, this approach is currently limited by a deficiency of kinetic parameters determined in living cells. To overcome this problem, we applied fluorescence cross-correlation spectrometry (FCCS) to measure dissociation constant (Kd) values of signaling molecule complexes in living cells (in vivo Kd). Among the pairs of fluorescent molecules tested, that of monomerized enhanced green fluorescent protein (mEGFP) and HaloTag-tetramethylrhodamine was most suitable for the measurement of in vivo Kd by FCCS. Using this pair, we determined 22 in vivo Kd values of signaling molecule complexes comprising the epidermal growth factor receptor (EGFR)-Ras-extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase pathway. With these parameters, we developed a kinetic simulation model of the EGFR-Ras-ERK MAP kinase pathway and uncovered a potential role played by stoichiometry in Shc binding to EGFR during the peak activations of Ras, MEK, and ERK. Intriguingly, most of the in vivo Kd values determined in this study were higher than the in vitro Kd values reported previously, suggesting the significance of competitive bindings inside cells. These in vivo Kd values will provide a sound basis for the quantitative understanding of signal transduction. |
著作権等: | Copyright © 2015 by the American Society for Microbiology, Mol. Cell. Biol. September 2014 vol.34 no.17 3272-3290, doi:10.1128/MCB.00087-14 |
URI: | http://hdl.handle.net/2433/198825 |
DOI(出版社版): | 10.1128/MCB.00087-14 |
PubMed ID: | 24958104 |
出現コレクション: | 学術雑誌掲載論文等 |
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