Ubiquitination of RIP Is Required for Tumor Necrosis Factor α-induced NF-κB Activation

Abstract

Stimulation of cells with tumor necrosis factor (TNF␣) triggers a recruitment of various signaling molecules, such as RIP, to the TNF␣ receptor 1 complex, leading to activation of NF-B. Previous studies indicate that RIP plays an essential role for TNF␣-induced NF-B activation, but the molecular mechanism by which RIP mediates TNF␣ signals to activate NF-B is not fully defined. Earlier studies suggest that RIP undergoes a ligand-dependent ubiquitination. However, it remains to be determined whether the ubiquitination of RIP is required for TNF␣-induced NF-B activation. In this study, we have identified Lys 377 of RIP as the functional ubiquitination site, because mutating this residue to arginine completely abolished RIP-mediated NF-B activation. The K377R mutation of RIP cannot undergo ligand-dependent ubiquitination and fails to recruit its downstream signaling components into the TNF␣ receptor 1 complex. Together, our studies provide the first genetic evidence that the ubiquitination of RIP is required for TNF␣-induced NF-B activation.NF-B is a homo-or heterodimeric transcription factor that controls the expression of various genes involved in inflammatory, apoptotic, and immune responses. In resting cells, the activity of NF-B is controlled through its cytoplasmic sequestration by a family of inhibitors, IB (Inhibitor of NF-B) (1). In response to extracellular stimuli, IB proteins are phosphorylated by the IB kinase (IKK) 2 complex, then ubiquitinated, and rapidly degraded, which leads to the nuclear localization and activation of NF-B (2). One of the most potent NF-B activators is the proinflammatory cytokine, tumor necrosis factor (TNF)␣. TNF␣ functions through the TNF receptors, mainly TNFR1, on the cell surface. The binding of TNF␣ to TNFR1 causes trimerization of the receptor and recruitment of the adaptor protein TRADD (TNF receptor-associated death domain) (3). TRADD further recruits TARF2 (TNF receptor-associated factor 2) (4), FADD (Fas-associated death domain) (4), and RIP (receptor-interacting protein) (5, 6).RIP is a serine/threonine kinase that plays an essential role in TNF␣-induced NF-B activation (6, 7). It contains an N-terminal kinase domain, an intermediate domain, and a C-terminal death domain. Earlier studies indicate that the RIP kinase domain is dispensable for the activation of NF-B (6, 8), whereas its death domain is required for the association with the upstream signaling component TRADD (5). The intermediate domain is required for RIP-mediated NF-B activation (6) and plays an important role for interacting with its downstream signaling components such as NEMO, the regulatory subunit of the IKK complex, and other molecules (9). Therefore, RIP likely functions as an adaptor molecule to mediate the TNF␣ signaling cascade. Although genetic studies have demonstrated that TAK1 (tumor growth factor ␤-activated kinase 1) and MEKK3 are involved in mediating TNF␣-induced NF-B activation downstream of RIP (10 -15), the mechanism by which RIP activates these downstream kinases is not fully def...

Evidence that TNF-TNFR1-TRADD-TRAF2-RIP-TAK1-IKK pathway mediates constitutive NF-κB activation and proliferation in human head and neck squamous cell carcinoma

“…Whether RIP has any role in the constitutive activation of NF-kB in HNSCC was also examined. We found that DN-RIP (K337R mutant) that lacks K63 polyubiquitylation site (Li et al, 2006) inhibited the constitutive activation of NF-kB in a dose-dependent manner in HN5 cells (Figure 4c). TNF-induced activation of NF-kB SEAP was also inhibited by DN-RIP and DN-TAK1 (Figure 4d).…”

“…Ubiquitinated RIP then interacts with transforming growth factor-b-activated kinase 1 (TAK1) through TAK1-binding protein (TAB1) to activate IKK complex (Li et al, 2006). Therefore, we tested the role of RIP and TAK1 in the constitutive activation of NF-kB in HNSCC cells.…”

NF-κB and the immune response

“…Signaling downstream of RIP1 requires TAK1 for the activation of IKK Shim et al, 2005). Whether TAK1 directly activates IKK or this process proceeds through an intermediary such as MEKK3 is not yet clear (Takaesu et al, 2003;Li et al, 2006). However, it does not appear that TAK1 is involved in the activation of the non-canonical NF-kB pathway .…”

RIP kinases: key decision makers in cell death and innate immunity

“…[38][39][40] RIP1 can also mediate TNF-induced activation of the mitogen-activated protein kinases (MAPKs) ERK, p38 and JNK and, interestingly, although the kinase activity of RIP1 is dispensable for activating NFkB, p38 and JNK, it is required to stimulate ERK activity. 41 Although ubiquitinated RIP1 serves to promote downstream activation of NFkB and gene expression that drives inflammation and protects cells from death by apoptosis, 42 NFkB can also induce the deubiquitinating enzymes CYLD Figure 1 The RIP kinase family. The domain structures of members of the RIP kinase family are indicated.…”