Quantitative Dissection and Modeling of the NF-κB p100-p105 Module Reveals Interdependent Precursor Proteolysis

The mechanisms that govern proteolytic maturation or complete destruction of the precursor proteins p100 and p105 are fundamental to homeostasis and activation of NF-κB; however, they remain poorly understood. Using mass-spectrometry-based quantitative analysis of noncanonical LTβR-induced signaling, we demonstrate that stimulation induces simultaneous processing of both p100 and p105. The precursors not only form hetero-oligomers but also interact with the ATPase VCP/p97, and their induced proteolysis strictly depends on the signal response domain (SRD) of p100, suggesting that the SRD-targeting proteolytic machinery acts in cis and in trans. Separation of cellular pools by isotope labeling revealed synchronous dynamics of p105 and p100 proteolysis. The generation of p50 and p52 from their precursors depends on functional VCP/p97. We have developed quantitative mathematical models that describe the dynamics of the system and predict that p100-p105 complexes are signal responsive. Proteolytic precursor processing is a hallmark of the NF-κB system. Yilmaz etal. show that in lymphotoxin-stimulated cells p100 acts upstream of p105, resulting in concurrent production of p52 and p50. Both precursors form complexes and bind to segregase (p97/VCP), which promotes proteasomal processing. The findings are supported by mass spectrometry and incorporated in quantitative mathematical models.