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.