TY - JOUR
T1 - Bridging the gap between in silico and cell-based analysis of the nuclear factor-kappaB signaling pathway by in vitro studies of IKK2
AU - Ihekwaba, Adaoha E C
AU - Wilkinson, Stephen J
AU - Waithe, Dominic
AU - Broomhead, David S
AU - Li, Peter
AU - Grimley, Rachel L
AU - Benson, Neil
PY - 2007/2/20
Y1 - 2007/2/20
N2 - Previously, we have shown by sensitivity analysis, that the oscillatory behavior of nuclear factor (NF-kappaB) is coupled to free IkappaB kinase-2 (IKK2) and IkappaBalpha(IkappaBalpha), and that the phosphorylation of IkappaBalpha by IKK influences the amplitude of NF-kappaB oscillations. We have performed further analyses of the behavior of NF-kappaB and its signal transduction network to understand the dynamics of this system. A time lapse study of NF-kappaB translocation in 10,000 cells showed discernible oscillations in levels of nuclear NF-kappaB amongst cells when stimulated with interleukin (IL-1alpha), which suggests a small degree of synchronization amongst the cell population. When the kinetics for the phosphorylation of IkappaBalpha by IKK were measured, we found that the values for the affinity and catalytic efficiency of IKK2 for IkappaBalpha were dependent on assay conditions. The application of these kinetic parameters in our computational model of the NF-kappaB pathway resulted in significant differences in the oscillatory patterns of NF-kappaB depending on the rate constant value used. Hence, interpretation of in silico models should be made in the context of this uncertainty.
AB - Previously, we have shown by sensitivity analysis, that the oscillatory behavior of nuclear factor (NF-kappaB) is coupled to free IkappaB kinase-2 (IKK2) and IkappaBalpha(IkappaBalpha), and that the phosphorylation of IkappaBalpha by IKK influences the amplitude of NF-kappaB oscillations. We have performed further analyses of the behavior of NF-kappaB and its signal transduction network to understand the dynamics of this system. A time lapse study of NF-kappaB translocation in 10,000 cells showed discernible oscillations in levels of nuclear NF-kappaB amongst cells when stimulated with interleukin (IL-1alpha), which suggests a small degree of synchronization amongst the cell population. When the kinetics for the phosphorylation of IkappaBalpha by IKK were measured, we found that the values for the affinity and catalytic efficiency of IKK2 for IkappaBalpha were dependent on assay conditions. The application of these kinetic parameters in our computational model of the NF-kappaB pathway resulted in significant differences in the oscillatory patterns of NF-kappaB depending on the rate constant value used. Hence, interpretation of in silico models should be made in the context of this uncertainty.
KW - enzyme kinetics
KW - in silico
KW - in vitro
KW - nuclear factor-κB regulation
KW - signal transduction
U2 - 10.1111/j.1742-4658.2007.05713.x
DO - 10.1111/j.1742-4658.2007.05713.x
M3 - Article
C2 - 17313484
VL - 274
SP - 1678
EP - 1690
JO - European Journal of Biochemistry
JF - European Journal of Biochemistry
SN - 1742-464X
IS - 7
ER -