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. 2006 Dec 5;103(49):18574-9.
doi: 10.1073/pnas.0608995103. Epub 2006 Nov 20.

Acetylation of MEK2 and I kappa B kinase (IKK) activation loop residues by YopJ inhibits signaling

Rohit Mittal  1 , Sew-Yeu Peak-Chew, Harvey T McMahon
Affiliations

Acetylation of MEK2 and I kappa B kinase (IKK) activation loop residues by YopJ inhibits signaling

Rohit Mittal et al. Proc Natl Acad Sci U S A. .

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-kappaB 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 alpha and beta subunits of the NF-kappaB 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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
MEK modification by YopJ. (a) The three-component MAPK cascade is an important signaling module that is shared by many signal transduction pathways; the MAPK pathway investigated in this study focuses on the MAP kinase kinases MEK1/2 that, in turn, activate the MAP kinases Erk1/2; this and similar pathways mediate the immediate host response to infection by Yersinia species. (b) Immunoblots from HeLa cells transfected with either YopJ-wt or the C172A mutant. Transfected cells were treated with 10 μM ISO at 37°C for the indicated times. (Upper) In YopJ-expressing cells, there is no signal corresponding to phospho-MEK, indicating that MEK activation is blocked by YopJ; in cells transfected with the C172A mutant of YopJ MEK, activation is clearly seen with a return to baseline levels by 30 min. (Lower) Loss of immunoreactivity with the CST9122 antibody against MEK1/2 in YopJ-wt expressing cells. (c) The block of MEK activation depends on the dosage of transfected YopJ. Indicated amounts (in ng) of plasmid pSFV-YopJ were transfected into a 35 mm dish of HeLa cells. Twenty-four hours after transfection, cells were serum-starved for 6 h and then treated with 10 μM ISO for 5 min at 37°C. Total lysates were examined by western analysis for phospho MEK1/2 and for total MEK1/2 (using the CST9122 antibody). (d) The loss of MEK signal is specific for the MEK1/2 (CST9122) antibody. Lysates from HeLa cells expressing either WT YopJ or the C172A mutant were immunoblotted by using the indicated antibodies. Whereas the MEK1/2 (CST9122) antibody discriminates between WT and C172A lanes, the other antibodies do not. This observation suggests that expression of active YopJ modifies MEK1/2 in a manner that masks the epitope recognized by the MEK1/2 (CST9122) antibody.
Fig. 2.
Fig. 2.
YopJ modifies MEK2 by acetylation. (a) Before mass spectrometric analysis, myc-MEK2-(His)6 prepared from YopJ-coexpressing cells (+) and control cells (−) was blotted against different antibodies to ensure it was modified. (b and c) Mass spectrometric analysis of a peptide from MEK2 spanning amino acid residues 210–231. The fragmentation of the modified peptide is shown in b and for the unmodified peptide in c. It can be seen that, whereas Cys-211 is carbamidomethylated (CAM) in each case, Ser-222 and Ser-226 are O-acetylated (OAc) only in YopJ-coexpressing cells. (d) GST-MEK2 (0.5 μg) is acetylated in vitro by YopJ-wt in the presence of [14C]acetyl coenzymeA. Increasing amounts of YopJ lead to higher acetyl transfer. (e) Time course of acetylation of GST-MEK (0.5 μg) by YopJ (80 ng). Progressively greater acetyl transfer is seen over time. The inactive C172A mutant of YopJ does not show any activity in these assays.
Fig. 3.
Fig. 3.
YopJ modifies IKKα and IKKβ. (a) Various proinflammatory stimuli activate the NF-κB-signaling pathway by activating the IκB kinase (IKK) complex. IKK activation results in the phosphorylation and subsequent ubiquitin-mediated degradation of IκB, the inhibitor or NF-κB. The removal of IκB results in NF-κB translocation to the nucleus where it can regulate gene expression. (b) HEK293 cells transfected with YopJ-wt or YopJ-C172A were stimulated with TNF-α. IκB degradation is slowed down (Top) by the expression of YopJ-wt (but not the C172A inactive mutant of YopJ). This effect is due to reduced phosphorylation of IκB in these cells (Middle) caused by inhibition of activation of IKK (Bottom) caused by WT YopJ. (c and d) Overexpression of IKKα (c) or IKKβ (d) results in their activation in cells expressing inactive YopJ-C172A but this activation is blocked by WT YopJ (Upper). Total levels of IKKα and IKKβ are not affected by expression of YopJ (Lower).
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