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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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P1 mutant exhibits a higher basal kinase activity.(A) Pbs2 wild type or P1 mutants expressed in yeast pbs2Δ strain with or without sorbitol stimulation are purified and incubated with MBP and 32P-γ-ATP at 30°C for 30 minutes. The samples are separated by SDS-PAGE, transferred to PVDF membrane and then analyzed by autoradiography. The same membrane is probed with an anti-HA antibody. (B) HEK293 cells are transfected with either Flag-tagged YopJ or YopJ(C172A), HA-tagged ERK and HA-tagged MKK1 wild type or M1 mutant. Cells are lysed and immunoprecipitated using an anti-HA antibody and Protein G beads. Beads are separated using SDS-PAGE and probed with either anti-HA or anti-phospho ERK antibodies.
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pone-0001375-g005: P1 mutant exhibits a higher basal kinase activity.(A) Pbs2 wild type or P1 mutants expressed in yeast pbs2Δ strain with or without sorbitol stimulation are purified and incubated with MBP and 32P-γ-ATP at 30°C for 30 minutes. The samples are separated by SDS-PAGE, transferred to PVDF membrane and then analyzed by autoradiography. The same membrane is probed with an anti-HA antibody. (B) HEK293 cells are transfected with either Flag-tagged YopJ or YopJ(C172A), HA-tagged ERK and HA-tagged MKK1 wild type or M1 mutant. Cells are lysed and immunoprecipitated using an anti-HA antibody and Protein G beads. Beads are separated using SDS-PAGE and probed with either anti-HA or anti-phospho ERK antibodies.

Mentions: Based on growth assays on glucose, the P1 mutant would be expected to exhibit a higher basal kinase activity than the wild type Pbs2. To test this hypothesis, we immunopurified wild type and mutant forms of Pbs2 from yeast that were exposed to normal and hyperosmotic conditions and analyzed them in an in vitro kinase assay. As seen in Figure 5A, the P1 mutant protein, exhibits high basal kinase activity in the absence of stimulus when compared to wild-type Pbs2. As the tyrosine residue that is mutated in P1 (Y423) is conserved in all MKKs, we tested whether the same mutation in mammalian MKK1 (Y130) would alter its basal activity. Mutation of the conserved tyrosine to a histidine in the mammalian MKK1 also resulted in high basal activity (Figure 5B). As expected, MKK1 kinase activity is diminished in the presence of YopJ, because the endogenous kinases are inhibited leaving only the mutant kinases active (Figure 5B). These results support our previous findings that YopJ inhibits the activation of MKKs and that acetylation by YopJ cannot inhibit kinases that are constitutively active (Figure 1) In addition, the P1 mutant might reveal a novel mechanism to activate a kinase.


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)

P1 mutant exhibits a higher basal kinase activity.(A) Pbs2 wild type or P1 mutants expressed in yeast pbs2Δ strain with or without sorbitol stimulation are purified and incubated with MBP and 32P-γ-ATP at 30°C for 30 minutes. The samples are separated by SDS-PAGE, transferred to PVDF membrane and then analyzed by autoradiography. The same membrane is probed with an anti-HA antibody. (B) HEK293 cells are transfected with either Flag-tagged YopJ or YopJ(C172A), HA-tagged ERK and HA-tagged MKK1 wild type or M1 mutant. Cells are lysed and immunoprecipitated using an anti-HA antibody and Protein G beads. Beads are separated using SDS-PAGE and probed with either anti-HA or anti-phospho ERK antibodies.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2147050&req=5

pone-0001375-g005: P1 mutant exhibits a higher basal kinase activity.(A) Pbs2 wild type or P1 mutants expressed in yeast pbs2Δ strain with or without sorbitol stimulation are purified and incubated with MBP and 32P-γ-ATP at 30°C for 30 minutes. The samples are separated by SDS-PAGE, transferred to PVDF membrane and then analyzed by autoradiography. The same membrane is probed with an anti-HA antibody. (B) HEK293 cells are transfected with either Flag-tagged YopJ or YopJ(C172A), HA-tagged ERK and HA-tagged MKK1 wild type or M1 mutant. Cells are lysed and immunoprecipitated using an anti-HA antibody and Protein G beads. Beads are separated using SDS-PAGE and probed with either anti-HA or anti-phospho ERK antibodies.
Mentions: Based on growth assays on glucose, the P1 mutant would be expected to exhibit a higher basal kinase activity than the wild type Pbs2. To test this hypothesis, we immunopurified wild type and mutant forms of Pbs2 from yeast that were exposed to normal and hyperosmotic conditions and analyzed them in an in vitro kinase assay. As seen in Figure 5A, the P1 mutant protein, exhibits high basal kinase activity in the absence of stimulus when compared to wild-type Pbs2. As the tyrosine residue that is mutated in P1 (Y423) is conserved in all MKKs, we tested whether the same mutation in mammalian MKK1 (Y130) would alter its basal activity. Mutation of the conserved tyrosine to a histidine in the mammalian MKK1 also resulted in high basal activity (Figure 5B). As expected, MKK1 kinase activity is diminished in the presence of YopJ, because the endogenous kinases are inhibited leaving only the mutant kinases active (Figure 5B). These results support our previous findings that YopJ inhibits the activation of MKKs and that acetylation by YopJ cannot inhibit kinases that are constitutively active (Figure 1) In addition, the P1 mutant might reveal a novel mechanism to activate a kinase.

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