Structural Requirements for Yersinia YopJ Inhibition of MAP Kinase Pathways
Figure 1
YopJ binds, acetylates and inhibits activation of Pbs2.
(A) Schematic representation of the canonical MAPK pathway aligned with the yeast HOG MAP kinase pathway. (B) Yeast pbs2Δ strain is cotransformed with pRS416-GPD1-Pbs2 wild type, S514D or T518D mutants and pRS413-Gal1 vector, YopJ or YopJ(C172A) mutant. pRS415-Gal1-Ssk2ΔN mutant is also transformed to the pRS416-GPD1-Pbs2 wild type strains (the third panel). Strains are plated on media containing 2% glucose or 2% raffinose, 1% galactose with or without 1 M sorbitol and incubated at 30°C. (C) Yeast two-hybrid assay of the interaction between YopJ or YopJ(C172A) mutant and Pbs2 wild type, Pbs2 dead mutant (AA) or Pbs2 constitutively active mutant (DD). (D) Recombinant YopJ or YopJ(C172A) are incubated with or without purified Pbs2 kinase domain (Pbs2KD(WT)) or Pbs2 (S514A, T518A) mutant (AA) kinase domain (Pbs2KD(AA)) in the presence or absence of 14C-labeled acetyl-CoA at 30°C for 1 hour. Samples are separated by SDS-PAGE and analyzed by autoradiography.