期刊
MOLECULAR AND CELLULAR BIOLOGY
卷 34, 期 17, 页码 3272-3290出版社
AMER SOC MICROBIOLOGY
DOI: 10.1128/MCB.00087-14
关键词
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资金
- Research Program of Innovative Cell Biology by Innovative Technology (Cell Innovation)
- Ministry of Education, Culture, Sports, and Science, Japan
- JST PRESTO program
- JSPS KAKENHI [23136504, 25136706]
- Yoshida Scholarship Foundation
- Grants-in-Aid for Scientific Research [26290053, 23136504, 26286028, 26115708] Funding Source: KAKEN
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 (K-d) values of signaling molecule complexes in living cells (in vivo K-d). 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 K-d by FCCS. Using this pair, we determined 22 in vivo K-d 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 K-d values determined in this study were higher than the in vitro K-d values reported previously, suggesting the significance of competitive bindings inside cells. These in vivo K-d values will provide a sound basis for the quantitative understanding of signal transduction.
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