Bridging the gap between in silico and cell‐based analysis of the nuclear factor‐κB signaling pathway by in vitro studies of IKK2

Previously, we have shown by sensitivity analysis, that the oscillatory behavior of nuclear factor (NF-κB) is coupled to free IkappaB kinase-2 (IKK2) and IkappaBalpha(IκBα), and that the phosphorylation of IκBα by IKK influences the amplitude of NF-κB oscillations. We have performed further analyses of the behavior of NF-κB and its signal transduction network to understand the dynamics of this system. A time lapse study of NF-κB translocation in 10 000 cells showed discernible oscillations in levels of nuclear NF-κB amongst cells when stimulated with interleukin (IL-1α), which suggests a small degree of synchronization amongst the cell population. When the kinetics for the phosphorylation of IκBα by IKK were measured, we found that the values for the affinity and catalytic efficiency of IKK2 for IκBα were dependent on assay conditions. The application of these kinetic parameters in our computational model of the NF-κB pathway resulted in significant differences in the oscillatory patterns of NF-κB depending on the rate constant value used. Hence, interpretation of in silico models should be made in the context of this uncertainty.