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Structural requirements for Yersinia YopJ inhibition of MAP kinase pathways.

Hao YH, Wang Y, Burdette D, Mukherjee S, Keitany G, Goldsmith E, Orth K - PLoS ONE (2008)

Bottom Line: The bacterial effector, YopJ, uses the unique activity of Ser/Thr acetylation to inhibit the activation of the MAPK kinase (MKK) and prevent activation by phosphorylation.Mutations on the hydrophobic face of the conserved G alpha-helix in the kinase domain prevent both binding and acetylation by YopJ.These studies reveal a conserved binding site found on the superfamily of MAPK kinases while providing insight into the molecular interactions required for YopJ inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA.

ABSTRACT
MAPK signaling cascades are evolutionally conserved. The bacterial effector, YopJ, uses the unique activity of Ser/Thr acetylation to inhibit the activation of the MAPK kinase (MKK) and prevent activation by phosphorylation. YopJ is also able to block yeast MAPK signaling pathways using this mechanism. Based on these observations, we performed a genetic screen to isolate mutants in the yeast MKK, Pbs2, that suppress YopJ inhibition. One suppressor contains a mutation in a conserved tyrosine residue and bypasses YopJ inhibition by increasing the basal activity of Pbs2. Mutations on the hydrophobic face of the conserved G alpha-helix in the kinase domain prevent both binding and acetylation by YopJ. Corresponding mutants in human MKKs showed that they are conserved not only structurally, but also functionally. These studies reveal a conserved binding site found on the superfamily of MAPK kinases while providing insight into the molecular interactions required for YopJ inhibition.

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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.
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pone-0001375-g001: 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.

Mentions: We demonstrated that YopJ is able to block the HOG MAPK signaling pathway downstream of the negative regulator Sln1 (Figure 1A) [12]. As shown in Figure 1B, the growth of yeast expressing YopJ is arrested when grown on galactose/sorbitol plates, while strains containing either the empty vector or expressing the catalytically inactive YopJ-C172A mutant (YopJ-C/A) are able to grow on galactose/sorbitol selective media. To confirm that YopJ blocks the HOG pathway in a manner similar to that observed for the mammalian MAPK pathways by preventing activation of MKKs, we generated a yeast strain that contained an activated form of yeast MKKK Ssk2 (Ssk2ΔΝ) under the control of a galactose inducible promoter (Figure 1B) [13]. We observed that growth of the Ssk2ΔΝ strains on galactose was arrested due to constitutive activation of the HOG pathway in the absence of a hyper-osmotic growth environment (Figure 1B) [13]. However, co-expression of YopJ in this cell line rescued the growth arrest phenotype, thereby supporting the hypothesis that YopJ inhibits the HOG pathway downstream of the MKKK (Figure 1A, B).


Structural requirements for Yersinia YopJ inhibition of MAP kinase pathways.

Hao YH, Wang Y, Burdette D, Mukherjee S, Keitany G, Goldsmith E, Orth K - PLoS ONE (2008)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2147050&req=5

pone-0001375-g001: 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.
Mentions: We demonstrated that YopJ is able to block the HOG MAPK signaling pathway downstream of the negative regulator Sln1 (Figure 1A) [12]. As shown in Figure 1B, the growth of yeast expressing YopJ is arrested when grown on galactose/sorbitol plates, while strains containing either the empty vector or expressing the catalytically inactive YopJ-C172A mutant (YopJ-C/A) are able to grow on galactose/sorbitol selective media. To confirm that YopJ blocks the HOG pathway in a manner similar to that observed for the mammalian MAPK pathways by preventing activation of MKKs, we generated a yeast strain that contained an activated form of yeast MKKK Ssk2 (Ssk2ΔΝ) under the control of a galactose inducible promoter (Figure 1B) [13]. We observed that growth of the Ssk2ΔΝ strains on galactose was arrested due to constitutive activation of the HOG pathway in the absence of a hyper-osmotic growth environment (Figure 1B) [13]. However, co-expression of YopJ in this cell line rescued the growth arrest phenotype, thereby supporting the hypothesis that YopJ inhibits the HOG pathway downstream of the MKKK (Figure 1A, B).

Bottom Line: The bacterial effector, YopJ, uses the unique activity of Ser/Thr acetylation to inhibit the activation of the MAPK kinase (MKK) and prevent activation by phosphorylation.Mutations on the hydrophobic face of the conserved G alpha-helix in the kinase domain prevent both binding and acetylation by YopJ.These studies reveal a conserved binding site found on the superfamily of MAPK kinases while providing insight into the molecular interactions required for YopJ inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, USA.

ABSTRACT
MAPK signaling cascades are evolutionally conserved. The bacterial effector, YopJ, uses the unique activity of Ser/Thr acetylation to inhibit the activation of the MAPK kinase (MKK) and prevent activation by phosphorylation. YopJ is also able to block yeast MAPK signaling pathways using this mechanism. Based on these observations, we performed a genetic screen to isolate mutants in the yeast MKK, Pbs2, that suppress YopJ inhibition. One suppressor contains a mutation in a conserved tyrosine residue and bypasses YopJ inhibition by increasing the basal activity of Pbs2. Mutations on the hydrophobic face of the conserved G alpha-helix in the kinase domain prevent both binding and acetylation by YopJ. Corresponding mutants in human MKKs showed that they are conserved not only structurally, but also functionally. These studies reveal a conserved binding site found on the superfamily of MAPK kinases while providing insight into the molecular interactions required for YopJ inhibition.

Show MeSH
Related in: MedlinePlus