Supporting Figures

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Fig. 4. Stimulation of MEK and Erk phosphorylation by various extracellular stimuli. HeLa cells that had been serum-starved for 16 h were stimulated with either EGF (20 ng/ml), TPA (200 ng/ml), or isoproterenol (10 mM) at 37°C, and the activation (by phosphorylation) of MEK and Erk was detected by the use of phospho-specific antibodies. Isoproterenol was chosen as the stimulus for the experiments reported in this article because the rapid activation and decay of the signals allowed us to collect a complete time course.

Fig. 5. YopJ acetylates MEK2 on Thr-13. Tryptic digests of MEK2 purified from YopJ-coexpressing cells (+) and control cells (-) were subjected to LC-MS/MS. A mass difference of 42 amu (corresponding to the mass of one acetyl group) was detected in the peptides corresponding to aa5-aa39. Collision-induced fragmentation of these peptides is depicted here. Both peptides display the same y-ion series (up to y13, although data only up to y6 is shown here) and identical m/z ratios for the b-series ion, b8. The difference between the b9 (and subsequent) ions in the two preparations indicates that the threonine residue at position 13 in MEK2 is O-acetylated by YopJ.

Fig. 6. YopJ protects IkB from degradation in IKKb -overexpressing cells by modifying IKK. HEK293 cells were cotransfected with IKKa and either YopJ-wt or YopJ-C172A (Left). Similarly, they were cotransfected with IKKb and either YopJ-wt or YopJ-C172A (Right). Twenty-four hours after transfection, cells were serum-starved for 16 h and then stimulated with 20 ng/ml TNF-a. At the indicated time points, cells were harvested and processed for Western blotting. The boxed regions in the Upper panel of anti-IkB blots show that the resting amount of IkB is much lower in cells overexpressing IKKb and inactive YopJ compared with those expressing IKKb and active YopJ. This result is owing to the fact that (even in the absence of TNF-a stimulation) overexpressed IKKb autoactivates and phosphorylates IkB, leading to its ubiquitination and degradation by the proteasome. This results in the reduced resting levels of IkB seen in YopJ-C172A coexpressing cells. Coexpressed WT YopJ, however, modifies IKKb , inhibiting its ability to phosphorylate IkB, thus resulting in higher resting levels of IkB. This interpretation is reinforced by the phosphoIkB blot that shows elevated levels of high molecular weight polyubiquitinated phospho IkB in the C172A coexpressing cells. Also, the phosphoIkB blot shows that, upon TNF-a treatment, the kinetics of appearance of phosphoIkB is reduced in C172A coexpressing cells (red box), reflecting the lowered amount of IkB present in the cells at the time of TNF-a stimulation. TNF-a treatment results in the complete degradation of IkB, presumably due to phosphorylation by the small percentage of unmodified IKKb that would be present in YopJ-wt coexpressing cells. Similar effects are not seen with IKKa overexpressing cells because, even though IKKa autoactivates (see phosphoIKKa blot), its preferred target is not IkB. The Lower three panels show that both IKKa and IKKb are modified by WT YopJ such that their activation is inhibited, but total protein amounts are unaffected.

Fig. 7. YopJ acetylates IKKa on Thr-179. Flag-IKKa was prepared by immunoprecipitation from cells coexpressing either YopJ-wt or the inactive mutant YopJ-C172A. Peptides resulting from chymotrypsin treatment of the preparations were resolved by nanoscale liquid chromatography and analyzed by mass spectrometry. From such an LC-MS/MS experiment, a doubly charged ion at m/z 735.3 corresponding to the amino acid sequence A¹⁶⁹KDVDQGSLC*TSF¹⁸¹ (C* is carbamidomethylcysteine), with an additional mass of 42 amu (one acetyl group), was detected. This ion was selected and subjected to collision-induced fragmentation. Comparison of the fragmentation data from this modified peptide (Upper) with that from the unmodified doubly charged ion at m/z 714.3 (Lower) shows that they have the same b-ion series (b2 to -10) and y1 and y2 ions. The difference of 42 amu in y4 ions indicates that the acetylation site is on threonine 179 of IKKa.