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Stimulation through the interleukin-1 receptor (IL-1R) and some Toll-like receptors (TLRs) induces ubiquitination of TRAF6 and IRAK-1, signaling components required for NF-kappaB and mitogen-activated protein kinase activation. Here we show that although TRAF6 and IRAK-1 acquired Lys63 (K63)-linked polyubiquitin chains upon IL-1 stimulation, only ubiquitinated IRAK-1 bound NEMO, the regulatory subunit of IkappaB kinase (IKK). The sites of IRAK-1 ubiquitination were mapped to Lys134 and Lys180, and arginine substitution of these residues impaired IL-1R/TLR-mediated IRAK-1 ubiquitination, NEMO binding, and NF-kappaB activation. K63-linked ubiquitination of IRAK-1 required enzymatically active TRAF6, indicating that it is the physiologically relevant E3. Thus, K63-linked polyubiquitination of proximal signaling proteins is a common mechanism used by diverse innate immune receptors for recruiting IKK and activating NF-kappaB.

TRAF2 and ASK1 play essential roles in tumor necrosis factor alpha (TNF-alpha)-induced mitogen-activated protein kinase signaling. Stimulation through TNF receptor 2 (TNFR2) leads to TRAF2 ubiquitination and subsequent proteasomal degradation. Here we show that TNFR2 signaling also leads to selective ASK1 ubiquitination and degradation in proteasomes. c-IAP1 was identified as the ubiquitin protein ligase for ASK1 ubiquitination, and studies with primary B cells from c-IAP1 knock-out animals revealed that c-IAP1 is required for TNFR2-induced TRAF2 and ASK1 degradation. Moreover, in the absence of c-IAP1 TNFR2-mediated p38 and JNK activation was prolonged. Thus, the ubiquitin protein ligase activity of c-IAP1 is responsible for regulating the duration of TNF signaling in primary cells expressing TNFR2.

The transcription factor NF-kappaB is sequestered in the cytoplasm in a complex with IkappaB. Almost all NF-kappaB activation pathways converge on IkappaB kinase (IKK), which phosphorylates IkappaB resulting in Lys 48-linked polyubiquitination of IkappaB and its degradation. This allows migration of NF-kappaB to the nucleus where it regulates gene expression. IKK has two catalytic subunits, IKKalpha and IKKbeta, and a regulatory subunit, IKKgamma or NEMO. NEMO is essential for NF-kappaB activation, and NEMO dysfunction in humans is the cause of incontinentia pigmenti and hypohidrotic ectodermal dysplasia and immunodeficiency (HED-ID). The recruitment of IKK to occupied cytokine receptors, and its subsequent activation, are dependent on the attachment of Lys 63-linked polyubiquitin chains to signalling intermediates such as receptor-interacting protein (RIP). Here, we show that NEMO binds to Lys 63- but not Lys 48-linked polyubiquitin, and that single point mutations in NEMO that prevent binding to Lys 63-linked polyubiquitin also abrogates the binding of NEMO to RIP in tumour necrosis factor (TNF)-alpha-stimulated cells, the recruitment of IKK to TNF receptor (TNF-R) 1, and the activation of IKK and NF-kappaB. RIP is also destabilized in the absence of NEMO binding and undergoes proteasomal degradation in TNF-alpha-treated cells. These results provide a mechanism for NEMO's critical role in IKK activation, and a key to understanding the link between cytokine-receptor proximal signalling and IKK and NF-kappaB activation.

Inhibitor of apoptosis proteins (IAPs) c-IAP1 and c-IAP2 were identified as part of the tumor necrosis factor receptor 2 (TNFR2) signaling complex and have been implicated as intermediaries in tumor necrosis factor alpha signaling. Like all RING domain-containing IAPs, c-IAP1 and c-IAP2 have ubiquitin protein ligase (E3) activity. To explore the function of c-IAP1 in a physiologic setting, c-IAP1-deficient mice were generated by homologous gene recombination. These animals are viable and have no obvious sensitization to proapoptotic stimuli. Cells from c-IAP1(-/-) mice do, however, express markedly elevated levels of c-IAP2 protein in the absence of increased c-IAP2 mRNA. In contrast to reports implicating c-IAPs in the activation of NF-kappaB, resting and cytokine-induced NF-kappaB activation was not impaired in c-IAP1-deficient cells. Transient transfection studies with wild-type and E3-defective c-IAP1 revealed that c-IAP2 is a direct target for c-IAP1-mediated ubiquitination and subsequent degradation, which are potentiated by the adaptor function of TRAF2. Thus, the c-IAPs represent a pair of TNFR-associated ubiquitin protein ligases in which one regulates the expression of the other by a posttranscriptional and E3-dependent mechanism.

NEMO is the regulatory subunit of the IκB kinase (IKK) in NF-κB activation and its CC2-LZ region interacts with Lys63 (K63)-linked polyubiquitin to recruit IKK to receptor signaling complexes. In vitro, CC2-LZ also interacts with tandem diubiquitin. Here we report the crystal structure of CC2-LZ with two dimeric coiled coils representing CC2 and LZ, respectively. Surprisingly, mutagenesis and nuclear magnetic resonance experiments reveal that the binding sites for diubiquitins at LZ are composites of both chains and that each ubiquitin in diubiquitins interacts with symmetrical NEMO asymmetrically. For tandem diubiquitin, the first ubiquitin uses the conserved hydrophobic patch and the C-terminal tail while the second ubiquitin uses an adjacent surface patch. For K63-linked diubiquitin, the proximal ubiquitin uses its conserved hydrophobic patch while the distal ubiquitin mostly employs the C-terminal arm including the K63-linkage residue. These studies uncover the energetics and the common U-shaped geometry for mutual recognition of NEMO and diubiquitins.

Summary Background The proinflammatory cytokine tumor necrosis factor-α (TNF-α) elicits cellular responses by signaling through a receptor complex that includes the essential adaptor molecule RIP. One important consequence of signaling is activation of the transcription factor NF-κB, and failure to downregulate TNF-induced NF-κB transcriptional activity results in chronic inflammation and death. Internalization of the receptor complex plays an important regulatory role in TNF signaling. Results We report that CARP-2, a RING domain-containing ubiquitin protein ligase (E3), is a negative regulator of TNF-induced NF-κB activation. By virtue of its phospholipid-binding FYVE domain, CARP-2 localized to endocytic vesicles, where it interacted with internalized TNF-receptor complex, resulting in RIP ubiquitination and degradation. Knockdown of CARP-2 stabilized TNFR1-associated polyubiquitinated RIP levels after TNF simulation and enhanced activation of NF-κB. Conclusions CARP-2 acts at the level of endocytic vesicles to limit the intensity of TNF-induced NF-κB activation by the regulated elimination of a necessary signaling component within the receptor complex.

The transcription factor NF-kappaB is sequestered in the cytoplasm in a complex with IkappaB. Almost all NF-kappaB activation pathways converge on IkappaB kinase (IKK), which phosphorylates IkappaB resulting in Lys 48-linked polyubiquitination of IkappaB and its degradation. This allows migration of NF-kappaB to the nucleus where it regulates gene expression. IKK has two catalytic subunits, IKKalpha and IKKbeta, and a regulatory subunit, IKKgamma or NEMO. NEMO is essential for NF-kappaB activation, and NEMO dysfunction in humans is the cause of incontinentia pigmenti and hypohidrotic ectodermal dysplasia and immunodeficiency (HED-ID). The recruitment of IKK to occupied cytokine receptors, and its subsequent activation, are dependent on the attachment of Lys 63-linked polyubiquitin chains to signalling intermediates such as receptor-interacting protein (RIP). Here, we show that NEMO binds to Lys 63- but not Lys 48-linked polyubiquitin, and that single point mutations in NEMO that prevent binding to Lys 63-linked polyubiquitin also abrogates the binding of NEMO to RIP in tumour necrosis factor (TNF)-alpha-stimulated cells, the recruitment of IKK to TNF receptor (TNF-R) 1, and the activation of IKK and NF-kappaB. RIP is also destabilized in the absence of NEMO binding and undergoes proteasomal degradation in TNF-alpha-treated cells. These results provide a mechanism for NEMO's critical role in IKK activation, and a key to understanding the link between cytokine-receptor proximal signalling and IKK and NF-kappaB activation.

Signaling through tumor necrosis factor receptor 2 (TNF-R2) results in ubiquitination of TRAF2 by the E3 c-IAP1. In this report, we confirm that TRAF2 translocates to a Triton X-100 (TX)-insoluble compartment upon TNF-R2 engagement. Moreover, TRAF2 ubiquitination occurs in this compartment, from which TRAF2 is degraded in a proteasome-dependent manner. Confocal microscopy demonstrated that the TX-insoluble compartment is perinuclear and co-localizes with endoplasmic reticulum (ER) markers. The ER transmembrane Ubc6 bound to c-IAP1 and served as a cognate E2 for c-IAP1's E3 activity in vitro. Furthermore, Ubc6 co-localized with translocated TRAF2/c-IAP1 in the ER-associated compartment in vivo, and a catalytically inactive Ubc6 mutant inhibited TNF-alpha-induced, TNF-R2-dependent TRAF2 degradation. These results indicate that upon TNF-R2 signaling, translocation of TRAF2 and c-IAP1 to an ER-associated, Ubc6-containing perinuclear compartment is required for the ubiquitination of TRAF2 by c-IAP1. Therefore, the ER plays a key role in the TNF-R-mediated signal transduction cascade by acting as a site of assembly for E2/E3/substrate complexes.

Cellular Inhibitors of Apoptosis 1 and 2 (c-IAP1 and c-IAP2) are ubiquitin protein ligases (E3s) that constitutively ubiquitinate and induce proteasomal-mediated degradation of NF-κB Inducing Kinase (NIK) and repress non-canonical NF-κB activation. Mice expressing an E3-inactive c-IAP2 mutant (c-IAP2(H570A)) have constitutive activation of non-canonical NF-κB, resulting in B cell hyperplasia and T cell costimulation-independence. If, and if so to what extent, c-IAP1 and c-IAP2 are redundant in NF-κB regulation in these mice is not known. Here we have generated mice expressing a mutant c-IAP1 that lacks E3 activity (c-IAP1(H582A)). These mice were phenotypically normal and did not have constitutive NF-κB activation in B cells or MEFs. siRNA-mediated knockdown of c-IAP2 showed that accumulated c-IAP2, resulting from lack of c-IAP1-dependent degradation, compensated for absent c-IAP1 E3 activity. Surprisingly, c-IAP1(H582A) T cells had a lower p100/p52 ratio than wild type T cells, and in the absence of costimulation proliferated to a degree intermediate between wild type and c-IAP2(H570A) T cells. Therefore, although c-IAP1 and c-IAP2 both can repress constitutive NF-κB activation, the relative importance of each varies according to cell type.