One distinction of IKK activation in microglia, however, appears to be the severe attenuation of IKK activity by 10 min following stimulation, only 5 min removed from peak activation levels. Despite the general similarities in NF B then and IKK activation between microglia and other cell types, a recently published mathematical model of the signaling network was unable to recapitulate the nuances of the rapid attenuation of IKK activity simultaneously with the brief delay in the onset of NF B activity in microglia. Noting that the largest discrepancies between the data and model simulations occurred within the first 20 min of activation, we used this information together with insight gained from sensitivity analysis to develop a new model that is able to match both IKK and NF B activity in this cell type.

The new model was developed in a modular fashion, which was made possible by collecting ELISA based measurements of IKK in addition to measurements of NF B activity and by exploiting the multiple feedback structure of the network. First the IKK data set from microglia was used to develop the downstream signaling module independently of the outer feedback loop, then the upstream signaling pathway was modified to fit IKK activation data, and finally the two modules were integrated to form the full model for which the para meter estimates were refined. The novel downstream signaling pathway includes additional reactions preced ing stimulus induced I Ba degradation, which are suffi cient to capture the delayed onset of NF B activity observed in microglia.

The mathematical representation we use to describe the additional dynamics is rather basic, yet captures effects that are likely significant at the biomolecular level. We attribute the intermediate model reactions to key steps in the ubiquitination pathway that implicitly have been lumped together in prior models. Ubiquitination of I Ba is typically thought to occur almost instantaneously following its phosphorylation by IKK. Consistent with this view, recent in vitro kinetic studies revealed in exquisite detail that the SCF bTrCP E3 ligase sequentially adds ubiquitin molecules to phosphorylated substrate to form a polyubiquitin chain able to be recognized by the proteasome in a process last ing only seconds after the first Ub molecule has been added.

However, the same study also demon strated that the addition of the first Ub to the substrate is the rate limiting step and occurs with low efficiency dur ing a single encounter between enzyme and substrate, suggesting that any cellular differences affecting how effi Brefeldin_A ciently the initial Ub is conjugated will contribute appre ciably to the dynamics. One such possibility for the differential ubiquitination dynamics is cell type specific expression of the E3 ligase components, such as the F box protein, bTrCP, which recognizes phosphorylated I Ba.