Stabilization of Basally Translated NF-κB-inducing Kinase (NIK) Protein Functions as a Molecular Switch of Processing of NF-κB2 p100

Guoliang Qing,Zhaoxia Qu,Gutian Xiao

doi:10.1074/jbc.m508776200

Guoliang Qing, Zhaoxia Qu + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m508776200

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Abstract

The non-canonical pathway based on processing of NF-kappaB2 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-kappaB-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-kappaB 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-kappaB 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-kappaB signaling is delayed and can be inhibited by protein synthesis inhibitor as well as why most classical NF-kappaB stimuli, including mitogens and tumor necrosis factor alpha, fail to induce p100 processing.

Highlights

The canonical NF-␬B pathway is required for fundamental functions of various cells and can be rapidly and transiently activated by a plethora of substances such as mitogens, cytokines, and microbial components [11]
P100 Processing Controlled by NF-␬Binducing kinase (NIK) Protein Stabilization induce p100 processing, we examined the kinetics of p100 processing induced by BAFF, CD40 ligand (CD40L), and NIK overexpression
Transfected or Purified NIK Can Mediate p100 Processing in the Absence of de Novo Protein Synthesis—Recent studies have indicated that p100 processing induced by receptor ligation requires de novo protein synthesis [15, 17] (Fig. 2A)

Summary

Introduction

The canonical NF-␬B pathway is required for fundamental functions of various cells and can be rapidly and transiently activated by a plethora of substances such as mitogens, cytokines, and microbial components [11] These stimuli lead to activation of a specific I␬B kinase (IKK) complex composed of two catalytic subunits, IKK␣ (IKK1) and IKK␤ (IKK2), and a regulatory subunit, IKK␥ (NEMO) [10]. Unlike I␬B degradation, the activation of the non-canonical NF-␬B pathway is strictly dependent on the IKK␣ and its activator, NF-␬Binducing kinase (NIK), but independent of IKK␤ and IKK␥ [9, 12, 13] This novel NF-␬B pathway only occurs in certain cell types at certain stages and responds to very limited stimuli, such as lymphotoxin ␤ [9, 14], B cell-activating factor (BAFF) [15, 16], and CD40 ligand (CD40L) [17]. These results provide the first evidence for explaining why the non-canonical NF-␬B pathway is delayed and can be inhibited by protein synthesis inhibitor

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