Molecular mechanisms of system control of NF-kappaB signaling by IkappaBalpha

Abstract

The NF-kappaB family of transcription factors responds to inflammatory cytokines with rapid transcriptional activation and subsequent signal repression. Much of the system control depends on the unique characteristics of its major inhibitor, IkappaBalpha, which appears to have folding dynamics that underlie the biophysical properties of its activity. Theoretical folding studies followed by experiments have shown that a portion of the ankyrin repeat domain of IkappaBalpha folds on binding. In resting cells, IkappaBalpha is constantly being synthesized, but most of it is rapidly degraded, leaving only a very small pool of free IkappaBalpha. Nearly all of the NF-kappaB is bound to IkappaBalpha, resulting in near-complete inhibition of nuclear localization and transcriptional activation. Combined solution biophysical measurements and quantitative protein half-life measurements inside cells have allowed us to understand how the inhibition occurs, why IkappaBalpha can be degraded quickly in the free state but remain extremely stable in the bound state, and how signal activation and repression can be tuned by IkappaB folding dynamics. This review summarizes results of in vitro and in vivo experiments that converge demonstrating the effective interplay between biophysics and cell biology in understanding transcriptional control by the NF-kappaB signaling module.

Figure 1

Schematic diagram of the NF-κB signaling pathway. The figure places emphasis on the role of IκBα, showing the different degradation pathways and transcriptional activation of new IκBα synthesis. The newly synthesized IκBα is either degraded, binds to an NF-κB in the cytoplasm, or enters the nucleus and binds nuclear NF-κB. This feedback part of the pathway is indicated by red arrows.

Figure 2

(A) Schematic diagram of NF-κB(p65) one of the most abundant NF-κB family members in the cell and of IκBα, the key member of the inhibitor family. (B) PONDR (21) analysis of the intrinsic disorder in the ankyrin repeat domain of IκBα. (C) LEFT: The crystal structure of IκBα (blue) bound to NF-κB (p50, green; p65, red) (19). RIGHT: The crystal structure of NF-κB (p50, green; p65, red) bound to κB site DNA (gold) (46). (Figure prepared using PyMOL (64)).

Figure 3

(A) Folding simulations of p16, an example ankyrin repeat protein. The folding of p16 was simulated with energetically unfrustrated models. The heat capacity as a function of temperature derived from several constant temperature runs is plotted. The peak in the plot corresponds to the folding temperature (T_f). (B) Similar analysis as in (A) for the ankyrin repeat domains of IκBα (residues 67-287). (C) Probability of contact formation during folding simulations of IκBα(67–287) at the first T_f (LEFT) and at the second T_f (RIGHT). The probability is plotted on a color scale with the most probable colored red. (D) Results from amide H/D exchange experiments on IκBα free in solution (LEFT) and when bound to NF-κB (RIGHT). The amount of exchange was measured after 2 min exposure to deuterated buffer followed by pepsin digestion and mass spectrometry. The extent of exchange is plotted on a color scale with the most exchanged colored red.

Figure 4

(A) Sequence of IκBα showing locations of some of the substitutions that stabilize the protein. (B) Equilibrium unfolding experiments with wild type (LEFT) and Y254L, T257A mutant IκBα. The insets show the change in fluorescence of W258, a naturally-occurring Trp in AR6. In the wild type protein, this residue does not change fluorescence appreciably with denaturant, however in the stabilized mutant, its fluorescence changes in a manner similar to the CD signal indicating it follows the major cooperative folding transition of the protein. (C) Plots of the thermodynamic parameters of binding of wild type (LEFT) and Y254L, T257A mutant (RIGHT) forms of IκBα to NF-κB(p50_248–350/p65_190–321) determined by ITC.

Figure 5

(A) NF-κB transcription activity was measured as a function of time after stimulation with tumor necrosis factor. Proteins were also measured by quantitative western blotting. TOP: NF-κB(p65, BOTTOM: IκB isoforms. (B) Quantitative Western blot showing the levels of IκBα(Y254L, T257A) and wild-type after cyclohexamide treatment in NF-κB −/− cells.

Figure 6

(A) Real-time binding and dissociation experiment monitored by SPR. Biotinylated κB-site DNA was bound to the streptavidin chip (t=0). NF-κB(p50_(19–363)/p65_(1–325)) was allowed to associate with the DNA until a pseudo-flowing equilibrium was reached (t=100 sec). Varying concentrations of IκBα were then injected through the second sample loop (co-inject experiment) and the dissociation rate constant (k_d) was measured. A schematic of the binding events is shown below the graph. (B) Plot of the k_d determined from experiments like that shown in (A) as a function of IκBα concentration. The error bars represent four independent experiments. The slope of the line is the pseudo-second order rate constant for IκBα-mediated dissociation, and its value of 10⁶ M⁻¹ s⁻¹ indicates that IκBα-mediated active dissociation is a very efficient process. (C) Dissociation was also monitored by stopped-flow fluorimetry using a pyrene-labeled DNA hairpin. Stopped-flow fluorescence experiment in which pyrene-labeled hairpin DNA (0.25 μM) complexed to NF-κB(p50_(19–363)/p65_(1–325)) (0.5 μM) in syringe 1 was rapidly mixed with a 50-fold excess (relative to NF-κB) of either unlabeled hairpin DNA (black curve, k_d = 0.41 s⁻¹) or IκBα (red curve, k_d = 18.2 s⁻¹).