Stabilization of basally translated NF-κB-inducing kinase (NIK) protein functions as a molecular switch of processing of NF-κB2 p100

The non-canonical pathway based on processing of NF-kappa B2 precursor protein p100 to generate p52 plays a critical role in controlling B cell function and lymphoid organogenesis. Activation of this unique pathway by extracellular stimuli requires NF-kappa B-inducing kinase (NIK) and de novo protein synthesis. However, how NIK is regulated is largely unknown. Here, we systematically analyzed NIK expression at different levels in the presence or absence of different NF-kappa B stimuli. We found that NIK mRNA is relatively abundant and undergoes constitutive protein synthesis in resting B cells. However, NIK protein is undetectable. Interestingly, protein expression of NIK is steadily induced by B cell-activating factor or CD40 ligand, two major physiological inducers of p100 processing, but not by mitogen phorbol 12-myristate 13-acetate/ionomycin or cytokine tumor necrosis factor alpha, two well known inducers of the canonical NF-kappa B signaling. Remarkably, both B cell- activating factor and CD40 ligand do not significantly induce expression of NIK at translational or transcriptional level but rather rescue the basally translated NIK protein from undergoing degradation. Furthermore, overexpressed or purified NIK protein triggers p100 processing in the presence of protein synthesis inhibitor. Taken together, these studies define one important mechanism of NIK regulation and the central role of NIK stabilization in the induction of p100 processing. These studies also provide the first evidence explaining why activation of the non- canonical NF-kappa B signaling is delayed and can be inhibited by protein synthesis inhibitor as well as why most classical NF-kappa B stimuli, including mitogens and tumor necrosis factor alpha, fail to induce p100 processing.