Mechanism Underlying IκB Kinase Activation Mediated by the Linear Ubiquitin Chain Assembly Complex

Abstract

N uclear factor B (NF-B) is a family of transcription factors that play essential roles in many biological phenomena, including inflammatory responses, cell survival, and innate and acquired immune responses (1). Because aberrant activation of NF-B signaling is associated with many pathological conditions, such as autoinflammatory diseases and malignancies (2, 3), signalinduced activation of NF-B has been studied extensively (4). In resting cells, inactive NF-B resides in the cytoplasm bound to its inhibitor proteins, the inhibitors of B (IBs). Stimulation by inflammatory cytokines activates the IB kinase (IKK) complex, composed of IKK1, IKK2, and NF-B essential modulator (NEMO). Following phosphorylation by activated IKK, IBs are degraded by the proteasome, leading to the release of NF-B, which then translocates to the nucleus to induce transcription of its target genes (5).The ubiquitin (Ub) conjugation system is deeply involved in the regulation of NF-B pathway (6). Recent studies showed that the linear ubiquitin chain assembly complex (LUBAC) ligase, which specifically generates linear polyubiquitin chains, is involved in NF-B activation (7,8). LUBAC is composed of three subunits: HOIP, HOIL-1L, and SHARPIN. Patients lacking HOIL-1L and mice lacking SHARPIN exhibit immunodeficiency and chronic inflammation, demonstrating the physiological significance of LUBAC-mediated linear polyubiquitination (9-12). In cells from mice lacking HOIL-1L or SHARPIN, the level of the residual LUBAC complex (consisting of the remaining two components) is reduced, and tumor necrosis factor alpha (TNF-␣)-induced NF-B activation is sharply attenuated (9-12). Although NEMO is a target of linear polyubiquitination by LUBAC, it is not yet clear how linear polyubiquitination of NEMO triggers IKK activation.In this study, using an in vitro LUBAC-mediated IKK activation assay, we found that linear diubiquitin conjugation to NEMO potently induces IKK activation. We then dissected the molecular mechanism underlying linear polyubiquitination of NEMO by LUBAC and found that the NPL4 zinc finger 1 (NZF1) domain of HOIP is responsible for recognition of a region in the coiled-coil 2 and leucine zipper (CoZi) domains of NEMO. Mutational analyses based on a cocrystal structure of HOIP NZF1 and NEMO CoZi revealed that HOIP NZF1 binds to NEMO and ubiquitin simultaneously and that both interactions are involved in linear polyubiquitination of NEMO, IKK activation, and subsequent activation of NF-B. Finally, we showed that homodimerization of IKK2 is involved in linear ubiquitin chain-mediated IKK activation. Taken together, our results suggest that recognition of linear polyubiquitins conjugated to NEMO, possibly by NEMO in another IKK complex, triggers activation of IKK2 by trans autophosphorylation. MATERIALS AND METHODS RT-PCR and plasmids.The open reading frames (ORFs) of mouse HOIP and NEMO were amplified by reverse transcription-PCR (RT-PCR) of total RNA from C57BL/6 mouse liver. Other cDNAs used in this study were described previously (8, 1...

Differential Involvement of the Npl4 Zinc Finger Domains of SHARPIN and HOIL-1L in Linear Ubiquitin Chain Assembly Complex-Mediated Cell Death Protection

“…Also, we previously showed that the recruitmentpromoting HOIP NZF1 domain recognizes K63-linked chains (11). Our present results suggest that the SHARPIN NZF functions in concert with the HOIP NZF1 to direct LUBAC to the TNFR complex.…”

“…LUBAC participates in canonical NF-B activation by conjugating linear ubiquitin chains onto the NF-B essential modulator (NEMO) (9,10). This appears to be a critical step in the activation of the IKK complex, the principal regulator of the NF-B cascade (9)(10)(11)(12). In addition, LUBAC exhibits a novel function to antagonize programmed cell death provoked by various stimuli, including tumor necrosis factor receptor (TNFR) signaling, although the precise molecular mechanisms remain unknown (13)(14)(15)(16)(17)(18).…”

Linear Polyubiquitination: A Crucial Regulator of NF-κB Activation

“…We thus showed that linear di-ubiquitin conjugated to NEMO is recognized by another NEMO in trans, which triggers dimerization of the IKK complex and subsequent trans-autophosphorylation of IKK2 (Fig. 4 ) [ 17 ].…”

“…4 ) and conjugates linear ubiquitin chains onto the proteins. Conjugation of di-ubiquitin (dimer) of linear linkage to NEMO appears enough to activate the IKK complex [ 17 ]. Activation of the IKK complex is mediated by the phosphorylation of IKK2 [ 6 ].…”

“…In the case of LUBAC-mediated canonical NF-κB activation, NEMO, an integral subunit of the IKK complex, is the substrate of LUBAC, and linear polyubiquitination of NEMO is shown to be involved in the activation of IKK that leads to canonical NF-κB activation [ 6 ]. Recent analyses revealed the molecular mechanism of linear polyubiquitination of NEMO-mediated IKK activation [ 17 ]. Upon stimulation with agents such as TNF-α, LUBAC recognizes NEMO using the Npl4-type zinc fi nger 1 (NZF1) domain of HOIP (Fig.…”