Pellino 3b Negatively Regulates Interleukin-1-induced TAK1-dependent NFκB Activation

Abstract

IL-1 receptor-associated kinase (IRAK) is phosphorylated, ubiquitinated, and degraded upon interleukin-1 (IL-1) stimulation. In this study, we showed that IRAK can be ubiquitinated through both Lys-48-and Lys-63-linked polyubiquitin chains upon IL-1 induction. Pellino 3b is the RING-like motif ubiquitin protein ligase that promotes the Lys-63-linked polyubiquitination on IRAK. Pellino 3b-mediated Lys-63-linked IRAK polyubiquitination competed with Lys-48-linked IRAK polyubiquitination for the same ubiquitination site, Lys-134 of IRAK, thereby blocking IL-1-induced IRAK degradation. Importantly, the negative impact of Pellino 3b on IL-1-induced IRAK degradation correlated with the inhibitory effect of Pellino 3b on the IL-1-induced TAK1-dependent pathway, suggesting that a positive role of IRAK degradation in IL-1 induced TAK1 activation. Taken together, our results suggest that Pellino 3b acts as a negative regulator for IL-1 signaling by regulating IRAK degradation through its ubiquitin protein ligase activity.Interleukin-1 (IL-1), 3 a major pro-inflammatory cytokine, has a wide range of biological activities in inflammation. Genetic and biochemical studies revealed that IL-1R mediates a very complex pathway, involving a cascade of kinases organized by multiple adapter molecules into signaling complexes, leading to activation of the transcription factor NFB. Based on studies by our group and others, we postulated a model for the IL-1 pathway (1-7). Upon IL-1 stimulation, the IL-1 receptor recruits adapter molecule MyD88 (8) and mediates the formation of complex I (IL-1R-MyD88-IRAK4-IRAK-TRAF6), where IRAK4 (IL-1 receptor-associated kinase 4 (9)) is activated, leading to hyperphosphorylation of IRAK (10), which creates an interface for its interaction with adapter Pellino 1 (11). The receptor proximal components are then released from the receptor to form an intermediate complex, followed by formation of complex II (IRAK-TRAF6-TAK1-TAB2-TAB3), leading to phosphorylation of TAK1 (transforming growth factor ␤-activated kinase, a MAP3K) and TAB2 (TAK1-binding protein 2) and TAB3 on the membrane (1, 2-7). Although the membraneassociated modified IRAK is ubiquitinated and degraded, complex III (TRAF6-TAK1-TAB2-TAB3) is then dissociated from complex II and translocated from the membrane to the cytosol, where TAK1 is activated, followed by the activation of IB kinase (IKK) and NFB (7).Chen and co-workers (14, 15) showed that protein ubiquitination plays an important role in TRAF6-mediated TAK1 and IKK activation. The ubiquitin pathway generally involves three types of enzymes, ubiquitin-activating enzyme (E1 or Uba), ubiquitin-conjugating enzyme (E2 or Ubc), and ubiquitin protein ligase (E3 or Ubr) (16). The E3 ubiquitin protein ligases play a key role in recognition and selection of proteins targeted for ubiquitination. Many RING finger proteins have been shown to act as E3s, either by themselves or as part of a multisubunit E3 protein complex (17). TRAF6, a RING domain protein, has been shown to function as a ubiquit...

The Pellino family: IRAK E3 ligases with emerging roles in innate immune signalling

“…This was supported by a study showing that coproduction of Pellino1 or Pellino2, but not Pellino3, with IRAK leads to IRAK polyubiquitylation [43]. Interestingly, two independent groups have shown that all three Pellino proteins, including both splice variants of Pellino3, can induce intense polyubiquitylation of co-expressed IRAK1 [33,42]. In vitro ubiquitylation assays have shown that recombinant and endogenous forms of Pellino1-3 possess E3 ligase activity and, thus, Pellino proteins are likely to directly catalyse polyubiquitylation of IRAK1 [33,42,45].…”

“…By contrast, detailed studies using mass spectrometry show that Pellino1 can combine with UbcH3 to catalyse formation of K48-linked polyubiquitin chains, whereas, in combination with UbcH4, UbcH5a or UbcH5b, it promotes formation of K48-and K11-linked polyubiquitin chains [45]. However, cellular studies to date indicate that the co-expression of Pellino proteins with IRAK1 induces only K63-linked polyubiquitylation of IRAK1 [33,45]. It is possible that Pellino proteins might be subject to regulatory processes in vivo that facilitate their interaction with different E2 enzymes and, hence, confer the ability to promote the formation of polyubiquitin chains that are linked by other lysine residues.…”

“…Given this model, there is currently intense interest in identifying the E3 ubiquitin ligase(s) that catalyses polyubiquitylation of IRAK1. It has been proposed [29] that there is, at least, a partial involvement of TRAF6 in the polyubiquitylation of IRAK1, but this has been opposed by two other groups [16,33]. Although the role of TRAF6 as the E3 ligase for IRAK1 remains ambiguous, the existence of a non-TRAF6 E3 ligase(s) that is capable of catalysing K63-linked polyubiquitylation of IRAK1 is widely agreed upon.…”

“…Furthermore, a yeast two-hybrid screen using IRAK4 as bait independently identified Pellino2 as a protein-binding partner [40]. Under physiological conditions, the Pellino-IRAK interactions are likely to be dependent on prior TLR or IL-1R stimulation because endogenous IRAK-Pellino associations are not observed in resting cells but manifest in response to stimulation by IL-1 [33,[35][36][37]44] and LPS [44].…”

“…Interestingly, two independent groups have shown that all three Pellino proteins, including both splice variants of Pellino3, can induce intense polyubiquitylation of co-expressed IRAK1 [33,42]. In vitro ubiquitylation assays have shown that recombinant and endogenous forms of Pellino1-3 possess E3 ligase activity and, thus, Pellino proteins are likely to directly catalyse polyubiquitylation of IRAK1 [33,42,45]. Intriguingly, in an in vitro setting, the Pellino proteins are capable of catalysing the formation of K63-and K48-linked polyubiquitin chains.…”