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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.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.

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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Interaction between YopJ and Pbs2 or MKK1 mutants.(A) Yeast two-hybrid assay of the interaction between YopJ or YopJ(C172A) mutant and Pbs2 wild type or Pbs2 mutants from the screening. (B) Yeast two-hybrid assay of the interaction between YopJ or YopJ(C172A) mutant and corresponding MKK1 mutants.
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pone-0001375-g003: Interaction between YopJ and Pbs2 or MKK1 mutants.(A) Yeast two-hybrid assay of the interaction between YopJ or YopJ(C172A) mutant and Pbs2 wild type or Pbs2 mutants from the screening. (B) Yeast two-hybrid assay of the interaction between YopJ or YopJ(C172A) mutant and corresponding MKK1 mutants.

Mentions: From previous studies we have observed that YopJ inhibits only the kinases to which it binds (all MKKs and IKKβ) but not kinases it does not bind (IKKα, Raf, p38, ERK1/2, Jnk) [4]. Therefore, we predicted that one mechanism to suppress YopJ inhibition would be to prevent binding of YopJ to its target kinase. Using the yeast two-hybrid assay, we analyzed whether the suppressor mutants could bind to YopJ and observed that one of these mutants, P4, was unable to interact with YopJ (Figure 3A). Because the remaining suppressor mutants (P1, P5) were able to bind to YopJ, we propose that binding of YopJ to Pbs2 may be necessary but not sufficient for YopJ's inhibition. As expected, analogous mutations made in mammalian MKK1, resulted a similar profile of binding to YopJ as assessed by yeast two-hybrid (Figure 3B), reinforcing the proposal that YopJ inhibits the MKKs by a common mechanism [4], [10].


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)

Interaction between YopJ and Pbs2 or MKK1 mutants.(A) Yeast two-hybrid assay of the interaction between YopJ or YopJ(C172A) mutant and Pbs2 wild type or Pbs2 mutants from the screening. (B) Yeast two-hybrid assay of the interaction between YopJ or YopJ(C172A) mutant and corresponding MKK1 mutants.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0001375-g003: Interaction between YopJ and Pbs2 or MKK1 mutants.(A) Yeast two-hybrid assay of the interaction between YopJ or YopJ(C172A) mutant and Pbs2 wild type or Pbs2 mutants from the screening. (B) Yeast two-hybrid assay of the interaction between YopJ or YopJ(C172A) mutant and corresponding MKK1 mutants.
Mentions: From previous studies we have observed that YopJ inhibits only the kinases to which it binds (all MKKs and IKKβ) but not kinases it does not bind (IKKα, Raf, p38, ERK1/2, Jnk) [4]. Therefore, we predicted that one mechanism to suppress YopJ inhibition would be to prevent binding of YopJ to its target kinase. Using the yeast two-hybrid assay, we analyzed whether the suppressor mutants could bind to YopJ and observed that one of these mutants, P4, was unable to interact with YopJ (Figure 3A). Because the remaining suppressor mutants (P1, P5) were able to bind to YopJ, we propose that binding of YopJ to Pbs2 may be necessary but not sufficient for YopJ's inhibition. As expected, analogous mutations made in mammalian MKK1, resulted a similar profile of binding to YopJ as assessed by yeast two-hybrid (Figure 3B), reinforcing the proposal that YopJ inhibits the MKKs by a common mechanism [4], [10].

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.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.

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