Selective Inhibition of Type III Secretion Activated Signaling by the Salmonella Effector AvrA
Figure 3
The ERK signaling pathway is required for AvrA phosphorylation.
A Effect of inhibitiors of MAP kinase pathways on AvrA phosphorylation. Cultured Henle cells were incubated with 10 µM each (alone or in combination) of the ERK (UO126), JNK (SP600125), or p38 (SB203580) MAP kinase pathways 30 min prior to S. typhimurium infection and were left throughout the experiment. Cells were infected with a S. Typhimurium strain expressing C-terminally triple FLAG tagged AvrA and at the indicated times after infection, the presence of AvrA and activated ERK1/2 in the translocated protein fractions were probed by western immunoblot with an anti FLAG or anti phospho-p44/42 MAPK antibodies respectively. The middle panel shows a longer exposure of the gel to visualize the dually phosphorylated form of AvrA. B S. Typhimurium ΔavrA strain expressing wild type AvrA or its catalytic mutant AvrAC172S activates ERK to similar levels. Henle-407 cells infected with the indicated S. Typhimurium strains for the indicated times and cell lysates were probed by western immunoblot with an anti FLAG or anti phospho-p44/42 MAPK antibodies respectively. The middle panel shows a longer exposure of the gel to visualize the dually phosphorylated form of AvrA. C AvrA interacts with ERK2 but not MEK1 while YopJ interacts with MEK1 but not ERK2 in a yeast two-hybrid assay. AvrAC172S or YopJC172S were expressed in yeast as baits along with the ERK pathway components as the targets as indicated in the diagram. See Materials and Methods for details.