Report

Yersinia YopJ Acetylates and Inhibits Kinase Activation by Blocking Phosphorylation

Science  26 May 2006:
Vol. 312, Issue 5777, pp. 1211-1214
DOI: 10.1126/science.1126867

Abstract

Yersinia species use a variety of type III effector proteins to target eukaryotic signaling systems. The effector YopJ inhibits mitogen-activated protein kinase (MAPK) and the nuclear factor κB (NFκB) signaling pathways used in innate immune response by preventing activation of the family of MAPK kinases (MAPKK). We show that YopJ acted as an acetyltransferase, using acetyl–coenzyme A (CoA) to modify the critical serine and threonine residues in the activation loop of MAPKK6 and thereby blocking phosphorylation. The acetylation on MAPKK6 directly competed with phosphorylation, preventing activation of the modified protein. This covalent modification may be used as a general regulatory mechanism in biological signaling.

The bacterial pathogen Yersinia pestis is the causal agent of plague, also known as the Black Death (1). Two related pathogens, Y. pseudotuberculosis and Y. enterocolitica, cause gastroenteritis (2). All three Yersinia species harbor a virulence pathogen that encodes a type III secretion system and secrete effector proteins, referred to as Yops (Yersinia outer proteins) (2). Yops are delivered by this system into a eukaryotic cell to cripple the host defense system (3, 4). The Yersinia species effector protein, YopJ, disrupts signaling essential for eukaryotic cells to elicit an immune response by inhibiting the evolutionarily conserved MAPK and NFκB signaling pathways (2, 3, 5, 6). YopJ contains a catalytic domain that is similar to Clan CE of cysteine proteases, which includes the adenoviral protease (AVP) family and the ubiquitin-like protein protease (Ulp-1) family (7). Mutation of the putative catalytic cysteine residue to an alanine in YopJ (YopJ-C/A) abolishes its ability to inhibit the MAPK and the NFκB signaling pathways (7). YopJ binds MAPK kinases, including MAPKK1, MAPKK3, MAPKK4, MAPKK5, and the related kinase that activates the NFκB pathway, IκB kinase β (IKKβ), and prevents their activation (5). The mechanism by which this binding leads to inactivation of these kinases is unknown.

A cell-free signaling system was developed to recapitulate the inhibition of the MAPK and the NFκB signaling pathways by YopJ (58). Mammalian extracellular signal–regulated kinase (ERK) signaling was activated by addition of recombinant B-Raf to a membrane-free cytosolic lysate (cleared lysate), as demonstrated by the appearance of phosphorylated ERK (Fig. 1A). By contrast, activation of ERK signaling was diminished in cleared lysate isolated from cells transfected with YopJ (Fig. 1A). The catalytic activity in YopJ was required for this inhibition. Addition of B-Raf to cleared lysate isolated from cells expressing mutant YopJ-C/A lead to activation of the ERK pathway (Fig. 1A). For activation of the NFκB pathway, we added a purified active form of recombinant TNF (tumor necrosis factor) receptor–associated factor 6 (TRAF6) (T6RZC) (9). When T6RZC was added to control and YopJ-C/A cleared lysates, the pathway was activated, as indicated by the phosphorylation of IκB (Fig. 1B). However, the addition of T6RZC to YopJ cleared lysate did not result in activation of the NFκB pathway (Fig. 1B). Similarly, when other exogenous stimuli [including NF-κB–inducing kinase (NIK), MAPK kinase kinase 1, and activated Ras-V12 membranes] were added to the lysates, signaling was blocked only in the YopJ lysates. No obvious changes were observed in the molecular weight or the stability of MAPKK1 and MAPKK2 (MAPKK1,2) or IKKβ in the lysates (Fig. 1C). These observations were consistent with previous genetic, microbial, and cellular studies on the activity of YopJ and provided a method for analyzing inhibition of signaling by YopJ in vitro (58).

Fig. 1.

Expression of YopJ prevents the activation of mammalian MAPKK (abbreviated in figure labels as MKK) and IKKβ by phosphorylation in vitro. Membrane cleared cell lysates were harvested from human embryonic kidney (HEK) 293 cells transfected with pSFFV empty vector (V), pSFFV-FLAG-YopJ (J), or pSFFV-FLAG-YopJ-C/A (C/A) (10). (A) Lysates were incubated with purified B-Raf for 10 min at 37°C, followed by immunoblotting with antibody to phospho-ERK. (B) V, J, and C/A lysates were incubated with rTRAF6 (9) for 10 min at 37°C, followed by immunoblotting with antibody against phospho-IκB (anti-phospho-IκB). (C) V, J, and C/A lysates were immunoblotted with antibodies to MAPKK1,2 and IKKβ. (D) YopJ coexpressed with rMAPKK6 in bacteria is not phosphorylated by upstream signaling machinery. Purified rMAPKK6, rMAPKK6-J, and rMAPKK6-C/A were incubated with serum-stimulated cleared lysate for 10 min at 37°C, followed by analysis with antibody to phospho-MAPKK6 (anti-phospho-MAPKK6) (lanes 2, 4, and 6). rMAPKK6 was detected by immunoblotting with antibody to Hisx4. Aldolase immunoblotblot was a load control for lysate.

To test whether YopJ acted directly on the MAPKKs and IKKβ, we coexpressed a representative member of this group of kinases, human MAPKK6 (rMAPKK6), with either active YopJ (rMAPKK6-J) or the catalytically inactive form of YopJ (rMAPKK6-C/A) in bacterial cells. We then assessed whether the various rMAPKK6s could be activated in our in vitro signaling assay. Although both rMAPKK6 and rMAPKK6-C/A were robustly phosphorylated when added to cleared lysate, the rMAPKK6-J was not activated by phosphorylation by the upstream signaling machinery (Fig. 1D). Therefore, coexpression of YopJ with MAPKK6 in bacteria produced a kinase that could not be activated by the upstream signaling machinery.

Studies on the YopJ-inactivated rMAPKK6 were undertaken to determine the biochemical nature of the modifications. Although all the rMAPKK6s were indistinguishable by SDS–polyacrylamide gel electrophoresis (PAGE) and gel filtration (fig. S1), mass spectrometry revealed that the total mass of rMAPKK6-J was larger than that of either rMAPKK6 or rMAPKK6-C/A. The majority of YopJ-inactivated rMAPKK6 showed an increase in mass of 126 atomic mass units (amu), whereas smaller populations of rMAPKK6-J exhibited increases in mass of 84 amu or 42 amu (Fig. 2A). We hypothesized that YopJ altered the mass of rMAPKK6 by adding single, double, or triple post-translational modifications equal to a mass of 42 amu.

Fig. 2.

rMAPKK6-J is acetylated on Ser207 and Thr211 residues in its activation loop. (A) Reconstructed molecular mass profiles of rMAPKK6, rMAPKK6-J, and rMAPKK6-C/A. (B and C) Electrospray ionization (ESI) tandem mass spectrometry (MS/MS) spectra of modified tryptic peptide A [mass-to-charge ratio (m/z) of 902.4 (z = 2)] and peptide B [m/z of 825.9 (z = 2)] from rMAPKK6-J. The b and y ions are marked on the MS/MS spectra. The amino acid sequence for each peptide is shown below (18). Acetylated residues are designated with a red circle. Masses that show an increase of 42 amu are marked with an asterisk. Two ions related to acetylated peptide B were detected, m/z of 825.9 (z = 2) and m/z of 833.9 (z = 2). MS/MS data of both ions indicated that peptide B was modified by acetylation on Thr211. The only difference between these two ions is that Met220 is oxidized in m/z of 833.9. Figure 3C shows the MS/MS spectrum of m/z 825.9. A corresponding figure defining the b and y ions is presented in fig. S3. (D) Alignment of the activation loop of the MAPKK superfamily with conserved serine and/or threonine residues that are indicated by asterisks.

We analyzed tryptic peptides for all three rMAPKK6s (rMAPKK6, rMAPKK6-J, and rMAPKK6-C/A) by using liquid chromatography followed by tandem mass spectrometry (10). After obtaining a complete data set for all the predicted tryptic peptides, we found that rMAPKK6-J, but not rMAPKK6 or rMAPKK6-C/A, contained two tandem peptides [peptide A, MAPKK6 195 to 210 amino acids, and peptide B, MAPKK6 211 to 224 amino acids] modified by acetylation with a consequent increase of 42 amu for each peptide (Fig. 2, B and C). In another partially cleaved tryptic peptide (MAPKK6 195 to 224 amino acids) that contained both peptides A and B, we observed multiple acetylated sites. Peptide A in the rMAPKK6-J protein was modified by acetylation on Ser207 (Fig. 2B), and peptide B was modified by acetylation on Thr211 (Fig. 2C). In the third peptide, it appeared that Lys210 and Ser207 and/or Thr211 were modified by acetylation. Modification of the lysine contributes to the inefficient cleavage of this peptide by trypsin. Residues 195 to 224 map to the end of β strand 9 and the activation loop in MAPKK6, which contains Ser207 and Thr211, the sites that are phosphorylated to activate MAPKK6. Although the serine and threonine residues are conserved throughout the MAPKK superfamily, the lysine residue is not (Fig. 2D). We predict that this residue is modified in a YopJ-dependent manner because of its coincidental location in the activation loop. The observation that YopJ covalently modifies the representative MAPKK, MAPKK6, by acetylation on the same residues that are used for activation of the kinase suggests a mechanism for the inhibition of MAPKKs and IKKβ: namely, acetylation prevents phosphorylation.

YopJ can bind and inhibit MAPKKs and IKKβ but not IKKα (fig. S2) (5), and all of these kinases contain serine and/or threonine residues in their activation loop that must be phosphorylated to activate the kinase (Fig. 2D) (11). rMAPKK6, coexpressed with YopJ and shown to be acetylated at Ser207 and Thr211 (Fig. 2, B and C), was not phosphorylated by upstream signaling machinery (Fig. 1D). These observations support our hypothesis that YopJ functions to modify the MAPKKs without noticeably changing their migration pattern on SDS-PAGE (Fig. 1C and fig. S1).

To determine whether YopJ directly functions as an acetyltransferase, we performed a transferase reaction in the presence of 14C-labeled acetyl–coenzyme A (CoA) (12). rMAPKK6 was modified with the 14C-labeled acetyl moiety only in the presence of recombinant YopJ expressed as a glutathione S-transferase fusion protein (GST-YopJ) and the labeled acetyl donor [14C]acetyl-CoA (Fig. 3A). The 14C label was associated with both rMAPKK6 and GST-YopJ (Fig. 3B). Based on this and analysis of the GST-YopJ protein beads, rMAPKK6 associated with GST-YopJ was the source of the 14C label (Fig. 3C). Thus, YopJ requires both an intact catalytic site and acetyl-CoA to acetylate rMAPKK6. We did not observe any band in reactions that contained only GST-YopJ and [14C]acetyl-CoA, indicating that the charging of YopJ with a [14C]acetyl moiety might be transient, labile, and/or dependent on the presence of a substrate or that the reaction proceeds through direct transfer. Similarly, we have observed that rMAPKK1 was also modified by [14C]acetyl moiety in a YopJ-dependent manner (fig. S4). These experiments show that YopJ acts as an acetyltransferase to modify MAPKKs.

Fig. 3.

In vitro acetylation by YopJ prevents phosphorylation of rMAPKK6 and activation of NFκB pathway. (A) Purified recombinant GST-YopJ or GST-YopJ-C/A was incubated with and without rMAPKK6 in the presence and absence of 14C-labeled acetyl-CoA for 1 hour at 30°C. Samples were separated by SDS-PAGE and analyzed by autoradiography. (B) Purified recombinant GST-YopJ or GST-YopJ-C/A bound to glutathione sepharose beads was incubated with and without 200 pmol of rMAPKK6 in the presence and absence of 35 μM [14C]acetyl-CoA. YopJ beads were washed, and supernatants were trichloroacetic acid (TCA)–precipitated, followed by measurement of the associated radiolabel. (C) Bead samples from (B) were separated by SDS-PAGE, followed by staining with Coomassie blue. (D) Purified recombinant GST-YopJ and GST-YopJ-C/A were incubated with rMAPKK6 in the presence and absence of acetyl-CoA for 1 hour at 30°C, followed by incubation with serum-stimulated cleared lysate for 10 minutes at 37°C and immunoblot analysis with anti-phospho-MAPKK6. (E) Acetyl-CoA (50 μM) was added to cleared lysate (10 mg/ml), followed by addition of recombinant GST-YopJ or GST-YopJ-C/A (100 ng), and incubated for 1 hour at 30°C. Lysates were then incubated with rTRAF6 (9) for 10 min at 37°C, followed by immunoblotting with anti-phospho-IκB, anti-IKKβ, and aldolase.

To demonstrate that the modification on rMAPKK6 by YopJ prevents activation via phosphorylation, we used our in vitro signaling system. Pretreatment of rMAPKK6 in the presence of both YopJ and acetyl-CoA diminished the ability of the upstream signaling machinery to activate rMAPKK6 by phosphorylation (Fig. 3D). Hence, the acetylation of a MAPKK by YopJ prevents phosphorylation and activation of this kinase.

YopJ protein is delivered into the cytoplasm of a host cell by a type III secretion system, where it inhibits the activation of the MAPKKs and IKKβ (5). Previously, adding recombinant YopJ to lysates did not show an inhibitory affect on signaling pathways. However, by using an acetyl-CoA–supplemented cleared lysate, we observed that addition of GST-YopJ but not GST-YopJ-C/A resulted in the inhibition of the NFκB signaling pathway in vitro (Fig. 3E). Thus, as observed during infection, when delivered to a lysate, YopJ uses acety-CoA to target and inactivate MAPKKs and IKKβ.

The mechanism of YopJ inhibition is elegant in its simplicity. On the basis of current studies on a representative kinase, MAPKK6, we propose that YopJ blocks signaling of the MAPKK and NFκB pathways by binding and acetylating critical residues in the activation loop of MAPKKs and IKKβ, respectively, thereby preventing these residues from being phosphorylated. Analysis of the predicted secondary structure of YopJ demonstrated similarities with the protease AVP (13). Because of the similarities between AVP and its distant relative, Ulp1, it was proposed that YopJ might act as an Ulp1-like protease or a general hydrolase (7). Inconsistent with this earlier hypothesis is the observation that YopJ selectively targets MAPKKs and IKKβ without any obvious changes in their migration on SDS-PAGE (26, 8) (Fig. 1C and fig. S1). However, because YopJ shares similarities with a family of cysteine proteases, this provides mechanistic insight into the chemistry of YopJ catalysis (7). A likely first step in the reaction is that YopJ is acetylated on Cys172 by formation of a thioester bond, and in the second step of the reaction this bond is attacked, not by a water molecule, but by a hydroxyl moiety on a serine or threonine residue of MAPKK, resulting in the formation of an acetylated amino acid. The substrate specificity determined by a bacterial effector protein, YopJ, and its interaction with target host proteins, MAPKKs and IKKβ, illustrates a common mechanism used by many bacterial effectors to ensure that their potent activity does not harm the bacterial host (3).

We find that YopJ-dependent acetylation occurs on the critical serine or threonine residues, thereby directly competing with the posttranslational modification, phosphorylation. Although the possibility exists that this is a unique modification developed by pathogenic bacteria to affect signaling in eukaryotic cells, a major characteristic of bacterial effector proteins is that they usurp or mimic a eukaryotic activity and refine this activity to produce an extremely efficient mechanism to combat eukaryotic signaling. Therefore, a more appealing hypothesis is that the modification of phosphorylatable residues by acetylation is a commonly used eukaryotic mechanism that simply has not been detected previously. Our findings support the provocative hypothesis that modification of amino acids other than lysine by acetylation is used to regulate eukaryotic cellular machineries. Enzymes that acetylate lysines have been studied for many years, including the eukaryotic and bacterial N-acetyltransferases that use acetyl-CoA and a catalytic triad, which appears similar to papain-like cysteine proteases (Cys-Glu/Asp-His) (1416). Immunoblotting and the interpretation of tandem mass spectrometry data by MASCOT are commonly used for the identification of lysine acetylation (17). However, these assays do not detect acetylation of serines and threonines. In view of the current finding, a more careful manual analysis of liquid chromatography followed by tandem mass spectrometry data may be required to determine whether an amino acid other than lysine is modified by acetylation. The characterization of a bacterial effector as a Ser or Thr acetyl transferase presents a previously unknown paradigm to be considered for other biological signaling pathways.

Supporting Online Material

www.sciencemag.org/cgi/content/full/312/5777/1211/DC1

Materials and Methods

Figs. S1 to S4

References and Notes

References and Notes

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