Acetylation of MEK2 and IκB kinase (IKK) activation loop residues by YopJ inhibits signaling

Abstract

To overcome host defenses, bacterial pathogens of the genus Yersinia inject specific effector proteins into colonized mammalian cells. One such virulence factor, YopJ, inhibits the host inflammatory response and induces apoptosis of immune cells by blocking multiple signaling pathways, including the MAPK and NF-B pathways. In this study, we show that YopJ exerts its deleterious effects by catalyzing the acetylation of two serine residues in the activation loop of the MAP kinase kinase, MEK2. This covalent modification prevents the phosphorylation of these serine residues that is required for activation of MEK2 and downstream signal propagation. We also show that YopJ causes acetylation of a threonine residue in the activation loop of both the ␣ and ␤ subunits of the NF-B pathway kinase, IKK. These results establish a hitherto uncharacterized mode of action for bacterial toxins and suggest the possibility that serine/threonine acetylation may occur even under nonpathogenic conditions and may be a widespread protein modification regulating protein function in eukaryotic cells.inflammation ͉ MEK U nderstanding the mode of action of bacterial toxins has provided insight into the working of mammalian cells especially with regard to signal transduction pathways that impinge upon the activation of the innate immune system (1, 2). Historically, plague has been one of the most devastating diseases to humans, second only to smallpox. The bacillus Yersinia pestis is the causative agent of plague, and two other Yersinia species, Yersinia pseudotuberculosis and Yersinia enterocolitica, cause septicaemic and gastrointestinal disorders (3). These pathogens inject a bouquet of six effector proteins into the mammalian cell cytosol using a type III secretion apparatus (4). These Yersinia outer proteins (Yops) help the pathogen multiply extracellularly in the host by preventing its phagocytosis and by slowing down the onset of the inflammatory response (5). YopE, YopT, and YopO target the Rho family of GTP-binding proteins that control actin cytoskeleton dynamics whereas YopH dephosphorylates focal adhesion proteins, thus inhibiting focal adhesion disassembly. Together, the action of these Yops contributes to the resistance of Yersinia to undergo phagocytosis, a process known to require remodeling of the actin cytoskeleton and of focal adhesions. Suppression of phagocytosis enables Yersinia to evade the macrophage defense network, thereby allowing them to proliferate in Peyer's patches as extracellular microcolonies. The leucine-rich protein, YopM, has been shown to bind to several host cell kinases, resulting in their activation (6). The remaining outer protein, YopJ (also called YopP in Y. enterocolitica) has emerged as an important agent that leads to the reduced host inflammatory response characteristic of Yersinia infections (5). Exposure of macrophages to lipopolysaccharide leads to the activation of NF-B and of several members of the MAPK family that promote the production of proinflammatory cytokines such as TNF-␣. YopJ in...

The Listeria monocytogenes InlC protein interferes with innate immune responses by targeting the IκB kinase subunit IKKα

“…These three bacterial effector proteins are secreted by a type III secretion system (T3SS) and translocated through the Salmonella or Chlamydia vacuolar membrane into the cytosol. Their mode of action differs from that of YopP/YopJ protein of Yersinia, which acetylates IKKα and IKKβ, thereby preventing phosphorylation of IκB (36,37). Enteropathogenic Escherichia coli (EPEC) also has been shown to prevent the phosphorylation of IκB through the use of two T3SS effectors, NleE and NleB, which interact with the TAK1-IKK A B pathway, albeit in an unknown fashion (38).…”

Salmonella Secreted Factor L Deubiquitinase of Salmonella typhimurium Inhibits NF-κB, Suppresses IκBα Ubiquitination and Modulates Innate Immune Responses

“…3, C and D), whereas NEDD8-AMC was hydrolyzed by SENP8, used as positive control. Because YopJ was originally suggested to have DUB activity (16), but subsequently shown to be an acetyltransferase (18,19), we also tested our preparation of SseL catalytic domain but detected no acetyltransferase activity (data not shown). Altogether, these data confirm that SseL has an intrinsic and specific DUB activity, and this activity is dependent on cysteine 285.…”

“…YopJ is homologous to CE cysteine proteases and was reported to hydrolyze ubiquitin and SUMO (a ubiquitinlike protein) (15)(16)(17). YopJ was also shown to act as an acetyltransferase that blocks phosphorylation sites on MAPKK6 and IKK␤ kinases implicated in NF-B activation (18,19). The physiological relevance of YopJ in the pathogenic mechanism of Yersinia has been demonstrated in experiments comparing the lethality of wild-type (WT) and YopJ-deficient bacteria (20).…”

Serine/threonine acetylation of TGFβ-activated kinase (TAK1) by Yersinia pestis YopJ inhibits innate immune signaling

“…In one study, the YopJ homolog, YopP, was shown to inhibit mammalian TAK1; however, no mechanism was identified (11,12). A number of in vitro studies identified the MAP2 kinase family as targets of YopJmediated serine/threonine acetylation (9,10). Through these acetylations, it was argued that YopJ is capable of inhibiting both MAPK (ERK pathway in particular) and NF-κB signaling.…”

“…In this role, YopJ acetylates serines and threonines on various mitogen-activated protein kinase kinases (MAP2Ks). This acetylation blocks the phosphorylation and activation of these kinases, thus neutralizing these signaling pathways (9,10). Interestingly, the Yersinia enterocolitica homolog, YopP, has been shown to inactivate the mammalian MAP3 kinase, hTAK1; however, the mechanism of suppression is not yet established (11,12).…”