Dendritic cells from humans with hypomorphic mutations in IKBKG/NEMO have impaired mitogen-activated protein kinase activity

Abstract

The covalent attachment of lysine 63-linked polyubiquitin to the zinc-finger domain of IKBKG/NEMO (also known as IKKγ) is necessary for full activation of NF-κB. Impairments of this biochemical mechanism explain the deleterious effects of hypomorphic NEMO mutations on NF-κB signaling function in humans suffering from X-linked ectodermal dysplasia and immunodeficiency. Nevertheless, the biological function of the NEMO zinc-finger domain in the regulation of mitogen-activated protein kinase (MAPK) activity is poorly understood. Here we show that dendritic cells from patients with EDI caused by a C-terminal E391X deletion of the zinc finger of NEMO exhibit impaired MAPK activation in response to lipopolysaccharide (LPS) stimulation. Interestingly, DCs from patients with a C417R missense mutation within the zinc finger domain of NEMO in which ubiquitination of NEMO is preserved are also defective in JNK and ERK activity following LPS stimulation. Our findings indicate that the structural integrity of the NEMO ZF domain is more important than its polyubiquitination for full activation of the MAPK. Furthermore, phosphorylation and polyubiquitination of upstream TAK1 were significantly reduced in the E391X zinc-finger deleted patients, indicating that the NEMO zinc finger may play an important role in assembling the proximal signaling complex for MAPK activation.

Fig 1. NEMO mutations and zinc finger structure in EDI patients

Left: domains of NEMO: αH, alpha-helix; C-C, coiled-coil; LZ, leucine zipper; ZF, zinc finger. The conserved CCHC residues coordinating zinc are in bold. The E391X mutation leads to ZF truncation. The C417R mutation destabilizes the ZF structure [Cordier, et al., 2008] but retains the polyubiquitination site K399 in the ZF domain. Right: proposed ZF secondary structure.

Fig 2. Cytokine production, NEMO protein expression, and NF-κB binding activity in dendritic cells with deleted or mutated zinc finger domain

(A) TNFα and (B) IL-12 production in patient monocytes after LPS/IFN-γ treatment. Open bar, normal; closed and hatched bars, two E391X siblings and two experiments on the same C417R patient. (C) Western analysis of NEMO protein in PBMCs. (D) EMSA analysis of NF-κB binding activity in DCs of two E391X siblings and C417R patient after LPS treatment. Fold inductions were quantitated using ImageJ software normalized by Sp1.

Fig 3. An intact NEMO ZF domain is more important than its polyubiquitination for full activation of MAPK in LPS-treated dendritic cells

Western blot analysis of MAPK activities in DCs from patients with the C417 missense mutation (A) and the E391X ZF deletion (B) after LPS treatment. Polyubiquitination of NEMO in E391X ZF deleted (C) and C417R missense (D) patients after LPS stimulation. (E) Bone marrow derived macrophages from NEMO K392R mice were stimulated with LPS and MAPK activities were analyzed by western blot.

Fig 4. Deletion of the ZF significantly impairs NEMO polyubiquitination but has a minimal effect on MAPK activities in PMA/ionomycin stimulated T-cells

(A) Western blot analysis of MAPK activities in EDI E391X T-cells after PMA/ionomycin treatment. (B) Polyubiquitination of NEMO in T-cells from E391X patient after PMA/ionomycin stimulation.

Fig 5. Deletion of NEMO ZF affects proximal TAK1 activity

(A) Polyubiquitination of TAK1 in E391X DCs after LPS treatment. (B) Phosphorylation of TAK1 in E391X DCs after LPS treatment. The film was scanned and signals were measured using ImageJ software; the numbers depict fold induction calculated based on the intensity of the corresponding zero time point.