Anatomy of a negative feedback loop: the case of I κ B α

Abstract

The magnitude, duration and oscillation of cellular signalling pathway responses are often limited by negative feedback loops, defined as an 'activator-induced inhibitor' regulatory motif. Within the NFkB signalling pathway, a key negative feedback regulator is IkBa. We show here that, contrary to current understanding, NFkB-inducible expression is not sufficient for providing effective negative feedback. We then employ computational simulations of NFkB signalling to identify IkBa molecular properties that are critical for proper negative feedback control and test the resulting predictions in biochemical and single-cell live-imaging studies. We identified nuclear import and nuclear export of IkBa and the IkBa-NFkB complex, as well as the free IkBa half-life, as key determinants of post-induction repression of NFkB and the potential for subsequent reactivation. Our work emphasizes that negative feedback is an emergent systems property determined by multiple molecular and biophysical properties in addition to the required 'activator-induced inhibitor' relationship.

Stochastic dynamics of genetic broadcasting networks

“…In the case of the NFκB network, molecular stripping by IκB solves the time-scale crisis for clearance and leads to fast responses and ultrasensitive oscillations with well defined periods. Our results are in harmony with recent in vivo single cell experiments where the stripping process has been perturbed specifically [34,35]. Finally, we predict that networks for which molecular stripping or similar mechanisms for assisted dissociation will be discovered will likewise show robustness at transitioning between steady states and oscillatory states with little to no dependence on the distribution of binding free energies and the number of broadcasting regulatory sites.…”

“…These time scales are very much in harmony with experiments of Dembinski et al [34] and Fagerlund et al [35]. These experiments carried out on single cells have probed the differences in nuclear clearance of NFκB for cells having wild type IκB versus a mutated [34] IκB or genetically engineered forms [35].…”

“…These experiments carried out on single cells have probed the differences in nuclear clearance of NFκB for cells having wild type IκB versus a mutated [34] IκB or genetically engineered forms [35]. Both experiments show that cells with wild type IκB rapidly and robustly clear NFκB from the nucleus within a 20-30-min window while cells in which natural IκBα has been replaced by IκBβ which is lacking a PEST sequence (amino acid sequence rich in P, E, S, T residues), which is crucial for molecular stripping [36], require more than ∼120-150 min in order to clear the NFκB from the nucleus.…”

Functional importance of stripping in NFκB signaling revealed by a stripping-impaired IκBα mutant

“…To ascertain the functional ramifications of the increased ternary complex stabilization, we generated IκBα −/− β −/− « −/− RelA −/− mouse embryonic fibroblasts (MEFs) that lack all three classical NFκB inhibitors and RelA and reconstituted them with a constitutively expressed fluorescent GFP-RelA and NFκB-responsively expressed IκBα(WT) or IκBα(5xPEST) as described previously (23). EMSA analysis showed the IκBα(5xPEST) normally was localized in resting cells and after TNF stimulation (Fig.…”

“…Intracellular NFκB Nuclear Export Experiments. Nuclear translocation experiments were performed as described previously (23). Briefly, NFκB-inducible IκBα constructs were derived from an MFG-derived self-inactivating retrovirus backbone (HRSpuro) modified to express the IκBα transgene under the control of five tandem κB sites upstream of a minimal promoter.…”