TNFR1 signaling kinetics: spatiotemporal control of three phases of IKK activation by posttranslational modification

Abstract

TNFα is a pleotropic cytokine that plays a central role in the inflammatory response by activating the NF-κB signaling pathway, and is targeted in a range of chronic inflammatory diseases, underscoring the therapeutic importance of understanding its underlying molecular mechanisms. Although K63-linked ubiquitination of RIP1 by TRAF2/5 and cIAP1/2 was thought to serve as a scaffold to activate the NF-κB pathway, the recent accumulation of conflicting results has challenged the necessity of these proteins in NF-κB activation. In addition, several serine/threonine kinases have been implicated in TNFα-induced IKK activation; however, the targeted disruption of these kinases had no effect on transient IKK activation. The recent discovery of RIP1-dependent and -independent activation of the early and delayed phases of IKK and TRAF2 phosphorylation-dependent activation of the prolonged phase of IKK offers a reconciliatory model for the interpretation of contradictory results in the field. Notably, the TNFα-induced inflammatory response is not exclusively controlled by the NF-κB pathway but is subject to regulatory crosstalk between NF-κB and other context-dependent pathways. Thus further elucidation of these spatiotemporally-coordinated signaling mechanisms has the potential to provide novel molecular targets and therapeutic strategies for NF-κB intervention.

Fig. 1

TNFα-mediated transient IKK activation pathways. (A) Signaling pathways to full IKK activation. Upon ligation, TNFR1 recruits TRADD, RIP1, TRAF2, and cIAP1/2. TRAF2 and cIAP1/2, in conjunction with UbcH5, catalyze RIP1 ubiquitination through linear (M1-pUb) and K63 linkages (K63-pUb), and also undergo autoubiquitination through K63-linkage. K63-pUb recruits the TAK1-TAB1-TAB2 complex, and M1-pUb recruits the IKKα-IKKβ-NEMO complex, leading to TAK1-mediated activation of the early phase of IKK. Thereafter, K63-pUb recruits the HOIL-1L–HOIP complex, which in turn recruits and catalyzes linear ubiquitination of NEMO, leading to more IKK recruitment and full activation of IKK. Such full IKK activation is required for the efficient expression of NF-κB target genes. (B) RIP1-independent pathway to delayed IKK activation. RIP1 protein is not detectable in embryonic hepatocytes. As a result, upon TNFR1 ligation, receptor-recruited TRAF2 and cIAP1/2 undergo increased autoubiquitination through K63 and K48 linkages. The K63-pUb chains then recruit the TAK1-TAB1-TAB2 and HOIP–HOIL-1 complexes, leading to activation of the delayed phase of IKK. Such delayed IKK activation is sufficient for induction of some NF-κB target genes (e.g., IκBα and IP-10).

Fig. 2

TRAF2 phosphorylation regulates the prolonged phase of IKK activation. (A) Schematic illustration of the TRAF2 protein domains, phosphorylation sites, consensus C and H residues in the RING domain. (B) Upon TNFR1 ligation, the membrane bound complex-I activates the early and delayed phases of IKK through RIP1-dependent and -independent pathways. Thereafter, TRAF2 is phosphorylated at Ser-11 and Ser-55, which triggers the translocation of the TRADD/RIP1/TRAF2/cIAP1 complex from TNFR1 to the cytoplasm to form complex-IIA. This leads to activation of the prolonged phases of IKK in a TRAF2 phosphorylation-dependent manner, which is also required for the efficient expression of some NF-κB target genes.

Fig.3

Crosstalk in TNFR1 signaling. Ligation of TNFR1 leads to activation of the pro-inflammatory NF-κB and JNK, and pro-apoptotic caspase-8 and ROS pathways. However, in normal cells, NF-κB target gene products suppress pro-apoptotic pathways. For instance, cIAP1/2 and cFLIP inhibit caspase-8 activation, XIAP and GADD45β suppress prolonged JNK activation, and FHC and Mn-SOD inhibit ROS accumulation. Therefore, unless NF-κB pathway is blocked, TNFα is insufficient to induce cell death.