YopJ in NF-κB and MAPK signalling
YopJ has been the focus of much research because of its ability to inhibit numerous signal transduction pathways. Recent biochemical analysis has demonstrated that YopJ is a de-ubiquitinating protease (Zhou et al., 2005) capable of targeting multiple distinct ubiquitination-dependent steps in NF-κB activation, such as the TRAF proteins, IKKγ/NEMO and IκBα. In this work, we directly examined the effect of Y. pestis YopJ specifically on discrete TLR-mediated signalling events. The use of HEK293 cell culture allows us to examine the activation of individual TLRs in isolation. This effort revealed that YopJ prevents the production of IL-8 in response to TLR activation, demonstrating that YopJ does indeed effectively target the signalling pathways downstream of these critical innate immune receptors.
To further explore this inhibition, we examined the effect of YopJ on TLR-induced MAPK activation and NF-κB activation, two pathways necessary for the production of IL-8 and other inflammatory cytokines. It is unclear which TLR receptor is most important during Yersinia infection. YopJ-induced apoptosis is mediated by TLR4 signalling but not TLR2 signalling in Yersinia pseudotuberculosis infection. This suggests TLR4 is the critical immune receptor in infection with this bacterium (Zhang and Bliska, 2003), although this situation may not translate directly to Y. pestis as Y. pestis has a modified lipid A structure at 37°C that prevents activation of TLR4 (Montminy et al., 2006). It is possible, however, that inhibition of TLR4 signalling by YopJ is still crucial in very early stages of bubonic infection, where bacteria grown at flea temperature would possess immunostimulatory LPS. Another study reported that LPS stimulation of splenocyte (presumably B-cell) proliferation is inhibited by YopJ (Meijer et al., 2000), but proinflammatory cytokines and NF-κB signalling were not examined in their work. Additionally, TLR2 may be an important target for reducing inflammation, regardless of its role in apoptosis.
We found that YopJ effectively inhibited both p38 MAPK activation and NF-κB activation in response to TLR stimulation, suggesting that the target of YopJ is prior to the branch point (at TAK1) that leads to activation of these distinct responses. In addition, the inhibition of TLR2, TLR3 and TLR4 signalling suggests that the target of YopJ is not a receptor-proximal component of the pathway, as these receptors differ in their requirements for TIR adapters and IRAK kinases during signal transduction. Thus, it is likely that YopJ inhibits the pathway between the receptor complex and the activation of TAK1. YopJ also inhibits NF-κB activation induced by TNFα or IL-1α stimulation. The IL-1 receptor shares its signal transduction machinery with the TLRs. TNFR, on the other hand, recruits a different receptor-proximal complex involving TRADD, FADD, RIP1 and TRAF2 (Silverman and Maniatis, 2001). The first common component of the TNFR and TLR pathways is TAK1, the MAP3K implicated in the activation of both MAPK and NF-κB responses to TLR stimulation. The ability of YopJ to inhibit pathways that activate NF-κB and MAPKs in response to a diversity of stimuli further supports the hypothesis that the earliest signalling activity YopJ could target is the activation of TAK1 by ubiquitination of the various TRAF proteins.
The data examining the ability of YopJ to inhibit NF-κB activation mediated by overexpression of various components of the TLR signalling pathways (Fig. 4) are also consistent with the hypothesis that YopJ targets the activation of TAK1. YopJ inhibited NF-κB activation induced by various TIR-containing adapters and TRAF6 (Fig. 3), suggesting that YopJ does not act upstream of TRAF6. Conversely, NF-κB activation by expression of IKKβ and p65/RelA was insensitive to YopJ (when the protein expression levels of IKKβ and p65/RelA are appropriately controlled, as shown in Fig. 4), suggesting that YopJ functions upstream of these proteins.
Toll-like receptor signalling is thought to induce TRAF6 aggregation resulting in TRAF6 auto-ubiquitination, a required event for activation of TAK1 and downstream signal transduction (Lamothe et al., 2006). It is likely that the role of TRAF6 ubiquitination is to provide an assembly scaffold for downstream components known to bind K63 ubiquitin, such as IKKγ/NEMO and the TAB2/3 proteins (Kanayama et al., 2004; Wu et al., 2006). Expression of TAK1 itself results in no measurable stimulation of NF-κB, but overexpression of the TAK1 accessory protein TAB2, which has been shown to induce TRAF6 ubiquitination and formation of the TRAF6/TAK1/IKK signalling complex (Kanayama et al., 2004; Kishida et al., 2005), leads to NF-κB activation that is strongly inhibited by YopJ (Fig. 3D). Curiously, coexpressing TAK1 with TAB2 activates NF-κB in a YopJ-insensitive manner. TAB2 and TAK1 co-overexpression appears to create conditions that overcome the necessity for TRAF6-regulated assembly of a signalling scaffold, perhaps through the presence of high concentrations of both TAB2 and TAK1. Furthermore, the insensitivity of the TAB2/TAK1 complex when co-overexpressed suggests that the relevant target of YopJ in signal transduction is an upstream component directly involved in the assembly of this activated complex, and not a modification of TAK1 itself. YopP has also been shown to block the activation of TAK1 during IL-1 signalling (without disrupting the interaction of TAK1 with TAB2) (Thiefes et al., 2006), an observation that is entirely consistent with our data and the hypothesis that TRAFs are the target molecules of YopJ and inconsistent with the hypothesis that YopJ is capable of independently inhibiting IKK activity and IκB degradation, as some have proposed (Zhou et al., 2005; Mittal et al., 2006; Mukherjee et al., 2006).
In addition, IKKγ/NEMO has also been shown to be a target of TRAF6-mediated ubiquitination (Lamothe et al., 2006; Sun et al., 2004). Targeting of IKKγ by YopJ, as recently suggested (Haase et al., 2005), provides an additional mechanistic explanation for the inhibition of NF-κB activation, however, it is not possible to determine if the effect of YopJ on IKKγ ubiquitination is direct or a consequence of TRAF6 inactivation. Another article has shown that IKKβ is mono-ubiquitinated after stimulation by strong overexpression of the T-cell oncogene Tax, and that this ubiquitination is blocked by YopJ (Carter et al., 2003). However, this study did not address the target of YopJ in this atypical constitutive Tax-induced NF-κB activation. Tax itself is ubiquitinated (but not degraded) and this conjugation is associated with its activation of NF-κB (Harhaj et al., 2007); it is possible that Tax itself or some other upstream component is the target of YopJ in this system.
To validate TRAF6 as a target of YopJ, we examined the ubiquitination of FLAG-tagged TRAF6 when YopJ was coexpressed. We observed a reduction in TRAF6 ubiquitination in the presence of YopJ (Fig. 6A and B), an observation that has recently been independently confirmed by two other investigators (Haase et al., 2005; Zhou et al., 2005). Because K63 ubiquitination of TRAF6 (and presumably TRAF3) is essential for signalling (Wang et al., 2001; Lamothe et al., 2006), removal of ubiquitin from these molecules by YopJ is likely to make an important contribution to the interference of signal transduction by YopJ. Indeed, when TRAF6 is conjugated with a mutant of ubiquitin that does not contain K63, the resulting ubiquitination is not sensitive to YopJ (Fig. 6C), but conjugation with a mutant of ubiquitin that does not contain K48 (but has K63) is strongly inhibited by the presence of YopJ (Fig. 6C). This observation is in contrast to that of Zhou et al. (2005), who suggested that YopJ inhibits both K63 and K48 ubiquitination. The reason for this difference is unclear; however, their work lacked two controls that are important for full interpretation of this experiment. Specifically, they did not present data with the mutant YopJ in their experiments with ubiquitin variants, nor did they demonstrate that the level of HA-ubiquitin expression was unaffected during coexpression of YopJ. We have found that at least one of these controls is critical to rule out expression artefacts, as coexpression of other proteins (regardless of activity) can often alter the amount of HA-ubiquitin that is made, making the empty vector control insufficient.
YopJ in IRF3-mediated signalling
The interferon response is a primary mechanism by which mammalian cells respond to infection by viral pathogens. TLRs 3, 4, 7/8 and 9, along with intracellular receptors such as RIG-I and Mda5, activate type I interferons through the transcription factors IRF1, 3, 5 and/or 7 (Schoenemeyer et al., 2005; Schroder and Bowie, 2005; Negishi et al., 2006). Surprisingly, we found that YopJ is capable of blocking the activation of an interferon-sensitive reporter element (ISRE) reporter by the TLR3 ligand poly [I:C]. The YopJ-mediated inhibition of TLR3–induced interferon signalling has important implications for this signalling cascade. YopJ has de-ubiquitinating activity, implicating a ubiquitin-dependent signalling event in the activation of IRF3 by TLR3. The suspected targets of YopJ-mediated de-ubiquitination in TLR- and TNFR-induced NF-κB signalling (TRAF2/6 activation of TAK1) do not play a role in TLR3-mediated IRF activation, however, two recent reports demonstrate that the putative RING-finger containing E3 ubiquitin ligase TRAF3 is critical for this pathway (Hacker et al., 2005; Oganesyan et al., 2005). Current understanding of the TLR3-induced interferon pathway posits that the signal is transduced via TRAF3 from the TIR-adapter TRIF to the MKK/IKK superfamily member TANK-binding kinase (TBK) 1 (Fitzgerald et al., 2003; Sato et al., 2003; Hemmi et al., 2004; Hacker et al., 2005; Oganesyan et al., 2005), which then activates IRF3 by phosphorylation. Consistent with the lack of a MAP3K in this pathway, the activation loop of TBK1 contains a phosphomimetic glutamic acid at residue 168, in place of serine 177 in the IKKβ‘activation loop’ which is phosphorylated by TAK1 during NF-κB signalling (Wang et al., 2001; Kishore et al., 2002). YopJ potently inhibits this pathway, as shown, even though it may not require a MAP3K for activation.
Based on the data presented here, we propose that once activated, TRAF3 functions as an E3-ligase required for activation of the IKK-like kinase TBK1. Based on analogy to TRAF2/6, it is likely that TRAF3 ubiquitinates itself and/or other proteins [perhaps the NEMO analogue NAP1 (Sasai et al., 2006)] to serve as a scaffold for further signalling complex assembly. In Fig. 7, we show that TRAF3 is ubiquitinated when it is expressed in HEK293 cells, and that YopJ removes this modification (Fig. 7A and B). As with TRAF6, YopJ only affects modification of TRAF3 by the activating K63 polymer of ubiquitin, not through lysine 48 (Fig. 7C). This further supports the hypothesis that the inhibitory mechanism of YopJ is the proteolytic cleavage of K63-polymerized ubiquitin from TRAF proteins. Based on these results, we would also predict that TLR4-mediated interferon induction, which also proceeds through the TIR adapter TRIF, will be blocked by YopJ. However, TLR4-mediated IRF3 activation is difficult to observe in the HEK293 model system used in this study and was therefore not addressed.
Detection of intracellular viral RNA by RIG-I or Mda5 induces IRF3 through a pathway distinct from the TLR3 signalling pathway. These receptors activate IRF3 through the adapter MAVS (also known as IPS-1, VISA or Cardif) (Kawai et al., 2005; Meylan et al., 2005; Seth et al., 2005; Xu et al., 2005). Through a mechanism that is still poorly understood and may involve RIP-1 and FADD (Balachandran et al., 2004), MAVS activates TBK1 which can phosphorylate and activate IRF3 (Fitzgerald et al., 2003). This pathway may also use another IKK-related kinase, IKKε, for the activation of IRF3. In Fig. 5 we show that YopJ can inhibit TLR-mediated, but not RIG-I-mediated, ISRE activation. It remains to be determined how MAVS stimulates TBK1 or IKKε and subsequent IRF3 activation. Although TRAF3 is believed to function in this pathway (Saha et al., 2006), the lack of YopJ inhibition suggests that K63 ubiquitination is not involved in this signal transduction. Alternatively, it is possible that the involvement of IKKε, which is not important for TLR3 responses but serves as an alternative mechanism of IRF activation in the RIG-I pathway (Hemmi et al., 2004; McWhirter et al., 2004; Perry et al., 2004), somehow renders the RIG-I pathway refractory to YopJ.
Although the implications of IFN inhibition in Yersinia pathogenesis are unclear, recent evidence suggests that type I interferons are involved in the response to bacterial as well as viral infections (Fehr et al., 1997; O'Connell et al., 2005; Opitz et al., 2006; Stetson and Medzhitov, 2006; Henry et al., 2007). It is possible that Y. pestis gains an advantage from the inhibition of IRF3 activation. In contrast, cytosolic intracellular bacteria such as Francisella and Listeria stimulate an IFN response that potentiates inflammasome-mediated apoptosis, an effect which at least in Listeria contributes to microbial pathogenesis (O'Connell et al., 2004; Henry et al., 2007). It is also possible that IRF inhibition is simply an ‘off-target’ effect caused by the similarity of TLR-mediated IRF signalling to other TLR-induced pathways, in particular the recently discovered requirement for a TRAF family member, TRAF3, in TLR3-induced IRF activation.
Two recent articles make the surprising claim that YopJ is a bacterial acetyltransferase that modifies and inactivates MKK6 and IKKα/β by blocking the sites of activating phosphorylation with acetyl group attachment (Mittal et al., 2006; Mukherjee et al., 2006). We do not see any evidence in support of this hypothesis when YopJ is expressed in mammalian cells; activation by IKKβ expression is not inhibited by YopJ unless there is an artefactual decrease in protein expression level (Figs 3 and 4). Furthermore, this hypothesis does not account for our observation that YopJ inhibition is circumvented by coexpression of TAB2 and TAK1. In addition, this proposed mechanism is unlikely to explain how YopJ inhibits TLR-mediated IRF3 activation, yet has no effect on RIG-I induced activation. The data presented here are most consistent with the proposed de-ubiquitinating activity of YopJ and of TRAFs as the critical targets, however, it remains possible that YopJ has both protease and acetyltransferase activities.