Bridging the gap between in silico and cell-based analysis of the nuclear factor-kappaB signaling pathway by in vitro studies of IKK2.
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.
Ihekwaba
Adaoha E C
AE
School of Chemistry, The University of Manchester, Faraday Building, UK. ihekwaba@vbi.vt.edu
Wilkinson
Stephen J
SJ
Waithe
Dominic
D
Broomhead
David S
DS
Li
Peter
P
Grimley
Rachel L
RL
Benson
Neil
N
D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000255:Adenosine Triphosphate / Q000378:metabolism; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D003198:Computer Simulation; D003593:Cytoplasm / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051550:I-kappa B Kinase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020672:I-kappa B Proteins / Q000737:chemistry / Q000378:metabolism; D053582:Interleukin-1alpha / Q000494:pharmacology; D007700:Kinetics; D008954:Models, Biological; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013876:Thiophenes / Q000494:pharmacology
D020672:I-kappa B Proteins; D053582:Interleukin-1alpha; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D047428:Protein Kinase Inhibitors; D011993:Recombinant Fusion Proteins; C477523:SC 514; D013876:Thiophenes; D000072000:NF-KappaB Inhibitor alpha; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase; D051550:I-kappa B Kinase