A small number of mammalian signaling pathways mediate a myriad of distinct physiological responses to diverse cellular stimuli. Temporal control of the signaling module that contains IkappaB kinase (IKK), its substrate inhibitor of NF-kappaB (IkappaB), and the key inflammatory transcription factor NF-kappaB can allow for selective gene activation. We have demonstrated that different inflammatory stimuli induce distinct IKK profiles, and we examined the underlying molecular mechanisms. Although tumor necrosis factor-alpha (TNFalpha)-induced IKK activity was rapidly attenuated by negative feedback, lipopolysaccharide (LPS) signaling and LPS-specific gene expression programs were dependent on a cytokine-mediated positive feedback mechanism. Thus, the distinct biological responses to LPS and TNFalpha depend on signaling pathway-specific mechanisms that regulate the temporal profile of IKK activity.

Inflammatory NF-kappaB/RelA activation is mediated by the three canonical inhibitors, IkappaBalpha, -beta, and -epsilon. We report here the characterization of a fourth inhibitor, nfkappab2/p100, that forms two distinct inhibitory complexes with RelA, one of which mediates developmental NF-kappaB activation. Our genetic evidence confirms that p100 is required and sufficient as a fourth IkappaB protein for noncanonical NF-kappaB signaling downstream of NIK and IKK1. We develop a mathematical model of the four-IkappaB-containing NF-kappaB signaling module to account for NF-kappaB/RelA:p50 activation in response to inflammatory and developmental stimuli and find signaling crosstalk between them that determines gene-expression programs. Further combined computational and experimental studies reveal that mutant cells with altered balances between canonical and noncanonical IkappaB proteins may exhibit inappropriate inflammatory gene expression in response to developmental signals. Our results have important implications for physiological and pathological scenarios in which inflammatory and developmental signals converge.

TNF-induced NF-B activity shows complex temporal regulation whose different phases lead to distinct gene expression programs. Combining experimental studies and mathematical modeling, we identify two temporal amplification steps-one determined by the obligate negative feedback regulator IB␣-that define the duration of the first phase of NF-B activity. The second phase is defined by A20, whose inducible expression provides for a rheostat function by which other inflammatory stimuli can regulate TNF responses. Our results delineate the nonredundant functions implied by the knockout phenotypes of ib␣ and a20, and identify the latter as a signaling cross-talk mediator controlling inflammatory and developmental responses.[Keywords: NF-B signaling; negative feedback; computational modeling; temporal control; IB␣; A20] Supplemental material is available at http://www.genesdev.org.

The spliceosome, a dynamic assembly of proteins and RNAs, catalyzes the excision of intron sequences from nascent mRNAs. Recent work has suggested that the activity and composition of the spliceosome are regulated by ubiquitination, but the underlying mechanisms have not been elucidated. Here, we report that the spliceosomal Prp19 complex modifies Prp3, a component of the U4 snRNP, with nonproteolytic K63-linked ubiquitin chains. The K63-linked chains increase the affinity of Prp3 for the U5 snRNP component Prp8, thereby allowing for the stabilization of the U4/U6.U5 snRNP. Prp3 is deubiquitinated by Usp4 and its substrate targeting factor, the U4/U6 recycling protein Sart3, which likely facilitates ejection of U4 proteins from the spliceosome during maturation of its active site. Loss of Usp4 in cells interferes with the accumulation of correctly spliced mRNAs, including those for a-tubulin and Bub1, and impairs cell cycle progression. We propose that the reversible ubiquitination of spliceosomal proteins, such as Prp3, guides rearrangements in the composition of the spliceosome at distinct steps of the splicing reaction.[Keywords: Ubiquitin; K63-linked ubiquitin chains; splicing; Prp19 complex; Usp4] Supplemental material is available at http://www.genesdev.org.

NF-κB signaling is known to be critically regulated by the NF-κB–inducible inhibitor protein IκBα. The resulting negative feedback has been shown to produce a propensity for oscillations in NF-κB activity. We report integrated experimental and computational studies that demonstrate that another IκB isoform, IκBɛ, also provides negative feedback on NF-κB activity, but with distinct functional consequences. Upon stimulation, NF-κB–induced transcription of IκBɛ is delayed, relative to that of IκBα, rendering the two negative feedback loops to be in antiphase. As a result, IκBɛ has a role in dampening IκBα-mediated oscillations during long-lasting NF-κB activity. Furthermore, we demonstrate the requirement of both of these distinct negative feedback regulators for the termination of NF-κB activity and NF-κB–mediated gene expression in response to transient stimulation. Our findings extend the capabilities of a computational model of IκB–NF-κB signaling and reveal a novel regulatory module of two antiphase negative feedback loops that allows for the fine-tuning of the dynamics of a mammalian signaling pathway.

Cellular signal transduction pathways are usually studied following administration of an external stimulus. However, disease-associated aberrant activity of the pathway is often due to misregulation of the equilibrium state. The transcription factor NF-jB is typically described as being held inactive in the cytoplasm by binding its inhibitor, IjB, until an external stimulus triggers IjB degradation through an IjB kinase-dependent degradation pathway. Combining genetic, biochemical, and computational tools, we investigate steady-state regulation of the NF-jB signaling module and its impact on stimulus responsiveness. We present newly measured in vivo degradation rate constants for NF-jB-bound and -unbound IjB proteins that are critical for accurate computational predictions of steady-state IjB protein levels and basal NF-jB activity. Simulations reveal a homeostatic NF-jB signaling module in which differential degradation rates of free and bound pools of IjB represent a novel cross-regulation mechanism that imparts functional robustness to the signaling module.

Dynamic properties of signaling pathways control their behavior and function. We undertook an iterative computational and experimental investigation of the dynamic properties of tumor necrosis factor (TNF)␣-mediated activation of the transcription factor NF-B. Surprisingly, we found that the temporal profile of the NF-B activity is invariant to the TNF␣ dose. We reverse engineered a computational model of the signaling pathway to identify mechanisms that impart this important response characteristic, thus predicting that the IKK activity profile must transiently peak at all TNF␣ doses to generate the observed NF-B dynamics. Experimental confirmation of this prediction emphasizes the importance of mechanisms that rapidly down-regulate IKK following TNF␣ activation. A refined computational model further revealed signaling characteristics that ensure robust TNF␣-mediated cell-cell communication over considerable distances, allowing for fidelity of cellular inflammatory responses in infected tissue.The transcription factor NF-B 3 is a key mediator of physiologic processes such as inflammation and adaptive immunity and has been implicated in numerous pathologic states such as cancer, rheumatoid arthritis, and sepsis (1). Consequently, understanding the mechanisms of NF-B activation and regulation is of prime importance. One major activator of NF-B is the potent inflammatory cytokine TNF␣. TNF␣ binds to and trimerizes its receptor, TNFR1, which leads to a receptorassociated signalosome that activates the kinase IKK (2). IKK phosphorylates IB proteins, which normally sequester NF-B in the cytoplasm; phosphorylated IBs are rapidly polyubiquitinated and proteasomally degraded, releasing free NF-B, which translocates to the nucleus and modulates gene expression (2).Detailed biochemical and genetic analyses over the past 25 years have helped elucidate the components that connect TNF␣ to NF-B. However, relatively little is known about how these molecular players act together as a signaling system, whose complex dynamics control the time-variable activity of NF-B and subsequent gene expression (3-6).Recently, it has become apparent that analysis of the systems properties of complex biochemical pathways can benefit from an integrated approach combining systematic experimental perturbations with an associated computational analysis of molecular interactions (5, 7, 8, 10 -13). This type of analysis applied to TNF␣-induced NF-B activity demonstrated that the ␣, ␤, and ⑀ isoforms of IB cooperate to produce a biphasic NF-B response (5). Varying the duration of the TNF␣ stimulus had no effect on the duration of the initial response, thus ensuring expression of some NF-B-regulated genes even in response to very short stimuli (5). This analysis, however, did not address the question of how other types of signaling inputs are processed.In this study, we analyze in detail a different type of inputs, constant stimulations at different TNF␣ doses, and experimentally and computationally analyze the resulting pathway characteristics. Surp...

Highly networked signaling hubs are often associated with disease, but targeting them pharmacologically has largely been unsuccessful in the clinic because of their functional pleiotropy. Motivated by the hypothesis that a dynamical signaling code confers functional specificity, we investigated whether dynamical features may be targeted pharmacologically to achieve therapeutic specificity. With a virtual screen we identified combinations of signaling hub topologies and dynamic signal profiles that are amenable to selective inhibition. Mathematical analysis revealed principles that may guide stimulus-specific inhibition of signaling hubs, even in the absence of detailed mathematical models. Using the NFκB signaling module as a test bed, we identified perturbations that selectively affect the response to cytokines or pathogen components. Together, our results demonstrate that the dynamics of signaling may serve as a pharmacological target, and we reveal principles that delineate the opportunities and constraints of developing stimulus-specific therapeutic agents aimed at pleiotropic signaling hubs.