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J. Biol. Chem., Vol. 281, Issue 13, 8917-8926, March 31, 2006

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Dynamics of the Ras/ERK MAPK Cascade as Monitored by Fluorescent Probes^*

Aki Fujioka, Kenta Terai, Reina E. Itoh, Kazuhiro Aoki, Takeshi Nakamura, Shinya Kuroda, Eisuke Nishida^¶, and Michiyuki Matsuda¹

From the Department of Signal Transduction, Research Institute for Microbial Diseases, Osaka University, Yamadaoka, Suita-shi, Osaka 565-0871, Japan, the Undergraduate Program for Bioinformatics and Systems Biology, Graduate School of Information Science and Technology, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan, and the ^¶Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

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.

Received for publication, August 24, 2005 , and in revised form, December 27, 2005.

^* This work was supported by grants-in-aid for scientific research and for cancer research from the Ministry of Education, Science, Sports, and Culture of Japan and by grants from the New Energy and Industrial Technology Development Organization and from the Health Science Foundation (Japan). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental "Materials and Methods," Figs. 1-8, Tables 1-4, and Refs. 1-6.

¹ To whom correspondence should be addressed. Tel.: 81-6-6879-8316; Fax: 81-6-6879-8314; E-mail: matsudam{at}biken.osaka-u.ac.jp.

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