Point of
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Yersinia Inhibits Host Signaling by Acetylating MAPK Kinases James B. Bliska* Department of Molecular Genetics and Microbiology, Center for Infectious Diseases, SUNY Stony Brook, Stony Brook, New York 11794-5222
T
ype III secretion systems (TTSSs) are specialized protein export pathways present in several Gram-negative bacteria that are pathogenic for animals or plants (1). The Yersinia spp. Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica are causative agents of plague or enteric infections in humans. These pathogens use a TTSS to counteract development of innate and adaptive immune responses during infection (2). The Yersinia TTSS delivers into host cells a set of six effectors known as Yops (2). One of these effectors, YopJ (known as YopP in Y. enterocolitica) has been the subject of intense study. YopJ functions biologically to induce apoptosis in macrophages (3, 4) and to inhibit cytokine production in infected host cells (5). However, until recently, the molecular function of YopJ had remained mysterious. In a recent issue of Science, Mukherjee et al. (6) report that YopJ has acetyltransferase (ATF) activity. This unexpected finding may have important implications for understanding how signaling pathways are normally regulated in eukaryotic cells. Previously, it had been established that induction of apoptosis and inhibition of cytokine production by YopJ was a consequence of its ability to deactivate or inhibit the mitogen-activated protein kinase (MAPK) and nuclear factor B (NFB) signaling pathways (5, 7, 8). Subsequently, Orth et al. (9) showed that YopJ binds to members of the MAPK kinase (MKK) superfamily and blocks their ability to be activated by phoswww.acschemicalbiology.org
phorylation (9). The MKKs are activated by phosphorylation of two serine or threonine residues within an activation loop. An additional clue to the function of YopJ came from the discovery that YopJ is predicted to share secondary structure similarity with the CE clan of cysteine proteases, which includes the ubiquitin (Ub)-like protease family (10). These enzymes contain a triad of residues (His, Asp/Glu, and Cys) required for catalysis, and mutational analysis of YopJ indicated that these residues were also required for biological function (10). YopJ activity was associated with decreased global levels of protein modification by the Ub-like protein small Ub-related modifier (SUMO) (10). It was thus proposed that YopJ removed Ub-like modifications from signaling proteins to inactivate MAPK and NFB response pathways (10). In line with this idea, it was demonstrated that YopJ activity prevented ubiquitination of I, an MKK family member that is integral to the NFB pathway (11). Furthermore, Zhou et al. (12) found evidence for a broad deubiquitinylating activity in YopJ and reported in vitro activity on an artificial Ub substrate. YopJ activity was associated with decreased deubiquitinylating of several proteins within the NFB signaling pathway, including tumor necrosis factor receptor-associated factor-6 (TRAF6) and inhibitor of NFB␣. Given the prevailing belief that YopJ acts as a protease on Ub-like substrates, it was quite surprising when Mukharjee et al. (6) published evidence that YopJ inactivates
A B S T R A C T Pathogenic Yersinia spp. secrete the effector YopJ (YopP) into host cells to counteract cytokine production and to induce programmed cell death (apoptosis). YopJ achieves these aims by inactivating mitogen-activated protein kinase (MAPK) and nuclear factor B signaling pathways. YopJ was shown to bind to members of the MAPK kinase (MKK) family and was predicted to have protease activity toward ubiquitin (Ub)like proteins. In a recent report, YopJ was demonstrated to inactivate MKKs via acetylation of critical serine or threonine residues. The ramifications of these exciting results are discussed in the context of other studies implicating YopJ as a Ub-like protease.
*Corresponding author,
[email protected].
Published online July 21, 2006 10.1021/cb600261k CCC: $33.50 © 2006 by American Chemical Society
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Figure 1. Model of MAPK signaling pathway and inactivation by YopJ ATF activity. a) Shown is a simplified pathway of MAPK signaling through sequential MKKK, MKK, and MAPK modules. MKK is activated by phosphorylation (P) of serine or threonine residues in an activation loop. b) Acetylation of MKK by YopJ. YopJ binds through protein–protein interactions to MKK and, using acetyl-CoA as a cofactor, acetylates serine and threonine residues in an activation loop. c) Mechanism of MKK inactivation by acetylation. Acetylation of Serine and Threonine residues in an activation loop of MKK prevents phosphorylation by upstream MKKK. As a result, the pathway is blocked (red X).
MKKs by acetylation. A prototypical MKK, MKK6, was acetylated on Ser207 and Thr211 when co-expressed with YopJ in Escherichia coli. Ser207 and Thr211 are the residues that are normally phosphorylated to activate MKK6. The authors also show that purified YopJ acetylates purified MKK6 or MEK1, another MKK, in a reaction that requires acetyl-CoA. Finally, in a cell-free signaling system, acetylation of MKK6 by YopJ was associated with decreased phosphorylation of this kinase by upstream activators. Together, these data strongly support the idea that YopJ acetylates activation loop serine or threonine residues in MKKs; this prevents phosphorylation of these residues and activation by upstream kinases (Figure 1). The fact that YopJ is specifically targeted to MKKs through protein–protein interactions (9) is an especially attractive feature of the model (6). These recent findings raise the interesting question of whether YopJ is both an ATF and a deubiquitinylating enzyme (Figure 2). In this context, it is important to note that the latter activity has been revealed under conditions in which YopJ and its putative substrates are overexpressed together in cell lines. For example, experiments in which proteins such as TRAF6 and IB␣ were shown to be deubiquitinylated in the presence of YopJ utilized transfection vectors to overproduce YopJ and an epitope-tagged form of Ub in cell lines (12). It now appears that such experiments can give misleading results because YopJ can inhibit protein expression from certain commonly used 350
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transfection vectors. In fact, Orth et al. (10) observed that the expression of unconjugated epitope-tagged SUMO was decreased in the presence of YopJ; this could explain the global decrease in SUMOylated proteins observed under these conditions. Other observations also argue against the possibility that the deubiquitinylating activity linked to YopJ in cell lines is biologically important. For example, IB␣ could be ruled out as an important target of YopJ because earlier experiments indicated that YopJ acts at, or above, the level of IKK to block the NFB pathway (13, 14). It is possible that under certain conditions YopJ does exhibit protease activity toward Ub. This could explain why Zhou et al. (12) observed cleavage of a Ub substrate by YopJ in an in vitro assay. In this case, the substrate was Ub-conjugated at its C-terminus to 7-amino-4-methylcoumarin, which is a sensitive substrate for Ub hydrolases. In this context, it is interesting that Mukherjee et al. (6) observed acetylation of Lys210 of MKK6 by YopJ in E. coli. If YopJ encountered this residue with an attached Ub, might it first remove the Ub before the acetyl group is added? There is another observation in the field that is difficult to reconcile with the idea that YopJ specifically targets MKKs and inactivates these enzymes by acetylation. Haase et al. (15) have obtained evidence that YopP inhibits the NFB pathway upstream of IKK, at the level of the TA1 (Figure 2). Although Mukherjee et al. (6) showed in a cell-free signaling system that YopJ could BLISKA
block phosphorylation of IKK, it has not been demonstrated that YopJ directly acetylates IKK. Could YopJ acetylate TAK1 instead of IKK? Alternatively, could acetylation of IKK by YopJ somehow affect the activation of TAK1? These important questions must be resolved in the future. The YopJ proteins encoded by Y. pestis and Y. pseudotuberculosis are highly conserved at the primary sequence level (99–100% identical over 288 residues). Greater sequence divergence occurs between the YopJ and YopP proteins (⬃94% identity). Interestingly, the YopP proteins from two different serogroups of Y. enterocolitica (O:8 and O:9) also show sequence divergence (94% identity) with 17 amino acid differences between the proteins. Because all YopJ/YopP proteins contain the same catalytic triad (His, Glu, and Cys), investigators in the field generally consider them interchangeable. However, evidence already exists that sequence differences in YopP proteins can have important biological consequences. Ruckdeschel et al. (16) have shown that YopP proteins with an Arg at position 143 (serogroup O:8) have greater activity toward inhibiting the NFB pathway compared with YopP proteins with a serine at this position (serogroup O:9). In future experiments designed to investigate whether these proteins function only as ATFs, or as ATFs and deubiquitinylating enzymes, researchers must keep in mind that sequence differences among the YopJ/ YopP proteins could impact the outcome. Mukharjee et al. (6) use their knowledge of the chemistry of cysteine protease reactions to suggest a model for the acetylation reaction catalyzed by YopJ. In this model, a thioester bond is formed between the essential Cys172 of YopJ and the acetyl group derived from acetyl-CoA. Subsequently, the thioester bond is attacked by a hydroxyl group on a serine or threonine residue within the activation loop of an MKK member. A well-studied family of arylamine N-ATFs contains a catalytic triad of Cys, His, www.acschemicalbiology.org
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Figure 2. Model of MAPK and NF, signaling pathways and mechanisms of inactivation by YopJ. Shown is a simplified pathway of MAPK signaling through sequential steps involving TRAF6, TAK1, MKK6, and MAPK. A parallel pathway involving TRAF6, TAK1, IKK, and IB␣ and resulting in activation of NFB is shown. MKK6 and IKK are activated by phosphorylation (P) of serine or threonine residues in their activation loops. Also shown are proposed steps at which YopJ inactivates these pathways. Acting as an ATF (6), YopJ binds specifically to MKK6 and IKK. YopJ then acetylates regulatory serine or threonine residues in each activation loop, and the result is molecules that cannot be phosphorylated by TAK1. Acting as a broad-specificity deubiquitinase (12), YopJ removes Ub from proteins, including TRAF6 and IB␣. Also shown is the possibility that YopJ inactivates TAK1 by an unknown mechanism (15).
and Asp residues and may utilize a similar mechanism (17). These enzymes are responsible for acetylation of several arylamine compounds, including the antimycobacterial drug isoniazid. Histone ATFs modify protein function by acetylation of His residues, and this activity plays a key role in transcriptional regulation in eukaryotes (18). However, YopJ appears to be the first enzyme identified that acetylates serine and threonine residues on proteins. Mukharjee et al. (6) raise the provocative idea that YopJ mimics a previously unidentified class of eukaryotic enzymes that regulate signaling via acetylation of Ser or Thr residues. If true, this would once again show how the study of bacterial virulence factors leads to important new insights into the biology of the host cell. Acknowledgment: I thank Yue Zhang for helpful suggestions to improve this article. Work in my laboratory on the biology of Yop proteins is sup-
www.acschemicalbiology.org
ported by a grant from the National Institutes of Health (R01 AI 433890).
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