2006年3月31日
Dynamics of the Ras/ERK MAPK cascade as monitored by fluorescent probes.
The Journal of biological chemistry
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- 巻
- 281
- 号
- 13
- 開始ページ
- 8917
- 終了ページ
- 26
- 記述言語
- 英語
- 掲載種別
- 研究論文(学術雑誌)
- DOI
- 10.1074/jbc.M509344200
- 出版者・発行元
- AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
To comprehend the Ras/ERK MAPK cascade, which comprises Ras, Raf, MEK, and ERK, several kinetic simulation models have been developed. However, a large number of parameters that are essential for the development of these models are still missing and need to be set arbitrarily. Here, we aimed at collecting these missing parameters using fluorescent probes. First, the levels of the signaling molecules were quantitated. Second, to monitor both the activation and nuclear translocation of ERK, we developed probes based on the principle of fluorescence resonance energy transfer. Third, the dissociation constants of Ras.Raf, Raf.MEK, and MEK.ERK complexes were estimated using a fluorescent tag that can be highlighted very rapidly. Finally, the same fluorescent tag was used to measure the nucleocytoplasmic shuttling rates of ERK and MEK. Using these parameters, we developed a kinetic simulation model consisting of the minimum essential members of the Ras/ERK MAPK cascade. This simple model reproduced essential features of the observed activation and nuclear translocation of ERK. In this model, the concentration of Raf significantly affected the levels of phospho-MEK and phospho-ERK upon stimulation. This prediction was confirmed experimentally by decreasing the level of Raf using the small interfering RNA technique. This observation verified the usefulness of the parameters collected in this study.
- リンク情報
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- DOI
- https://doi.org/10.1074/jbc.M509344200
- PubMed
- https://www.ncbi.nlm.nih.gov/pubmed/16418172
- Web of Science
- https://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcAuth=JSTA_CEL&SrcApp=J_Gate_JST&DestLinkType=FullRecord&KeyUT=WOS:000236247100072&DestApp=WOS_CPL
- Scopus
- https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=33646852421&origin=inward 本文へのリンクあり
- Scopus Citedby
- https://www.scopus.com/inward/citedby.uri?partnerID=HzOxMe3b&scp=33646852421&origin=inward
- ID情報
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- DOI : 10.1074/jbc.M509344200
- ISSN : 0021-9258
- eISSN : 1083-351X
- PubMed ID : 16418172
- SCOPUS ID : 33646852421
- Web of Science ID : WOS:000236247100072