Computer-assisted simulation is usually a encouraging approach for clarifying complicated signaling networks. functions such as cell proliferation, differentiation, survival, and tumorigenesis (1,C3). This signaling pathway has been extensively analyzed, and vast amounts of proteins and regulations have been recognized, producing in an increase in the pathway’s complexity. Computer-assisted simulation is usually one of the most encouraging methods for the comprehensive understanding of the transmission transduction pathway as a system. Indeed, a number of simulation models of the EGFR-Ras-ERK MAP kinase pathway have been reported over the past 10 years (4,C7). In these simulation models, most of the kinetic parameters used for numerical simulations were not assessed experimentally but rather were thought by fitted the experimental data with the simulation data or just decided arbitrarily. Consequently, there are substantial differences in the parameters among these studies, making it hard to evaluate these simulation models quantitatively. The kinetic parameters used for the simulation 131438-79-4 manufacture of intracellular signal transduction include protein concentrations, enzymatic kinetics, diffusion coefficients, and dissociation constants of the protein-protein interactions, which are denoted is usually of central importance, because protein-protein interactions are a major constituent of signal transduction pathways (8). Under steady-state conditions, the of the simple binding between protein A and protein W is usually defined as and are association and dissociation rate constants, respectively, and [Free A], [Free W], and 131438-79-4 manufacture [AB] correspond to the concentrations of unbound free protein A, protein W, and protein AB complexes, respectively. According to these definitions, the smaller the values, the higher the affinity of the protein-protein conversation. The value has been decided by experiments such as coprecipitation experiments, sedimentation equilibrium using analytical ultracentrifugation, surface plasmon 131438-79-4 manufacture resonance (SPR), and isothermal titration calorimetry (ITC). All of these methods enable us to acquire the value (here referred to as the value displays the strength of the protein-protein conversation decided by the intrinsic properties of the two proteins. On the other hand, a few reports have assessed dissociation constants in living cells (here referred to as the can be affected mainly by two factors: competitive binding and molecular crowding (Fig. 1B). In the former case, non-fluorescently labeled proteins, including endogenous and other interacting protein, hole competitively to fluorescently labeled molecules and consequently appear to lead to an overestimation of the values comparative to the values (Fig. 1B, top). The overestimated is usually also known as the apparent confers a potential advantage to kinetic simulation models, because the authentically includes the effects of all intracellular environments, such as competitive bindings and molecular crowding, on protein-protein interactions within a cell. However, to date, only a few values have been made available for computer simulation for the EGFR-Ras-ERK MAP kinase pathway, possibly due to the technical troubles. FIG 1 Strategy for measuring by FCCS. (A and W) Comparison between (A) and (W) values. In general, the was affected by competitive binding protein (W, top) and molecular crowding (W, bottom), leading to increased … FCCS allows the measurement of protein mobility, protein concentrations, and protein-protein interactions by exploiting the temporal fluorescence fluctuations of two diffusing fluorescently labeled particles under a confocal laser scanning services microscope with a tiny focal volume, called the effective volume (16). As a unique number of fluorescently labeled molecules diffuse through the effective volume (approximately 1 fl), the fluorescence signals fluctuate in a manner dependent on the mobility and concentration. An autocorrelation function of the fluctuating fluorescence transmission provides the diffusion coefficient and concentration of molecules. FCCS utilizes two spectrally different fluorophores to label a pair of proteins. If the differently labeled particles are associated with each other, they pass through the effective volume in a synchronized way. Therefore, the simultaneous fluctuations of their fluorescence signals lead to an increase in the amplitude of the cross-correlation function. The amplitude provides the concentration of the protein-protein complex. In this BA554C12.1 study, we established a method for obtaining values in living cells by FCCS and decided >20 values for the EGFR-Ras-ERK MAP kinase pathway in HeLa cells. In addition, we built a simulation model of the EGFR-Ras-ERK MAP kinase pathway based on the values. This model suggested that multiple bindings of Shc to phosphorylated EGFR (pEGFR) are required for the peak activation of Ras, MEK, and ERK in response to EGF activation. Intriguingly, most of the values.