Quantitative In Vivo Fluorescence Cross-Correlation Analyses Highlight the Importance of Competitive Effects in the Regulation of Protein-Protein Interactions

Abstract

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.

ACKNOWLEDGMENTS

We thank Kazunari Kaizu and Koichi Takahasi (QBiC, Japan) and the members of the Matsuda Laboratory for helpful discussions. We are also grateful to the Center for Meso-Bio Single Molecule Imaging and the Institute for Integrated Cell-Material Sciences at Kyoto University for the use of the microscope.

K.A. and M.M. were supported by the Research Program of Innovative Cell Biology by Innovative Technology (Cell Innovation) and the Platform for Dynamic Approaches to Living System from the Ministry of Education, Culture, Sports, and Science, Japan. K.A. was supported by the JST PRESTO program and by JSPS KAKENHI (23136504 and 25136706). W.S. was supported by the Yoshida Scholarship Foundation.