A role for peroxiredoxins in H2O2- and MEKK-dependent activation of the p38 signaling pathway

Abstract

The p38 mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in the cellular response to various stresses and its deregulation accompanies pathological conditions such as cancer and chronic inflammation. Hydrogen peroxide (H₂O₂) is a well-established activator of the p38 MAPK signaling pathway. However, the mechanisms of H₂O₂-induced p38 activation are not yet fully understood. In Drosophila cells, we find that H₂O₂-induced activation of p38 depends on the MAPK kinase kinase (MAP3K) Mekk1. In line with the emerging role of peroxiredoxins as H₂O₂ sensors and signal transmitters we observe an H₂O₂-dependent interaction between Mekk1 and the cytosolic peroxiredoxin of Drosophila, Jafrac1. In human cells, MEKK4 (the homologue of Mekk1) and peroxiredoxin-2 (Prx2) interact in a similar manner, suggesting an evolutionarily conserved mechanism. In both organisms, H₂O₂ induces transient disulfide-linked conjugates between the MAP3K and a typical 2-Cys peroxiredoxin. We propose that these conjugates represent the relaying of oxidative equivalents from H₂O₂ to the MAP3K and that the oxidation of Mekk1/MEKK4 leads to the downstream activation of p38 MAPK. Indeed, the depletion of cytosolic 2-Cys peroxiredoxins in human cells diminished H₂O₂-induced activation of p38 MAPK.

Keywords: H(2)O(2) signaling; Hydrogen peroxide; MEKK4; Mitogen activated protein kinase; Peroxiredoxins; Redox relays; p38.

Graphical abstract

Fig. 1

The MAP3K Mekk1 mediates the activation of the p38 signaling pathway in response to H₂O₂ in Drosophila S2R+ cells. (A) S2R+ cells were incubated with increasing concentrations of H₂O₂ and lysed at different time points. Phosphorylation of p38 was visualized by immunoblotting (upper panel). The immunoblot is representative of 3 independent experiments (n = 3). The bar chart (lower panel) presents the mean (+/− SD) ratio of phosphorylated p38 to total p38 from 3 independent experiments. (B) S2R+ cells were treated for 5 days with dsRNAs against the MAP3Ks Mekk1, Ask1 and Tak1, and against an unrelated protein (control). Cells we treated with 500 μM H₂O₂ and p38 phosphorylation was analyzed by immunoblotting at different time points (upper panel). The immunoblot is representative of 3 independent experiments (n = 3). The bar chart (lower panel) presents the mean (+/− SD) ratio of phosphorylated p38 to total p38 from 3 independent experiments. (C) Efficacy of the dsRNAs used to deplete the MAP3Ks was analyzed by qPCR. The bar chart presents the mean (+/− SD) normalized gene expression level from 3 independent experiments (n = 3).

Fig. 2

Drosophila Mekk1 is redox-sensitive and interacts with cytosolic peroxiredoxin Jafrac1 in response to H₂O₂. (A, A′) Tagged versions of Jafrac1 (Jafrac1-SBP) and Mekk1 (Mekk1-myc) were expressed in S2R+ cells. Cells were treated with 500 μM H₂O₂ for 5 min and Jafrac1-SBP was affinity-purified with streptavidin beads. Precipitates (A) and whole cell lysates (WCL) (A′) were analyzed by SDS-PAGE under reducing (R) and non-reducing (NR) conditions followed by immunoblotting (IB). The immunoblots are representative of 3 independent experiments (n = 3). (B, B′) Complementary affinity purification experiment: Reverse-tagged versions of Jafrac1 (Jafrac1-myc) and Mekk1 (Mekk1-SBP) were expressed in S2R+ cells. Cells were treated with 500 μM H₂O₂ for 5 min and Mekk1-SBP was affinity-purified with streptavidin beads. Precipitates (B) and WCL (B′) were analyzed by SDS-PAGE under R and NR conditions followed by IB. e.v.: empty vector, PD: pull down, X: unknown protein. The immunoblots are representative of 3 independent experiments (n = 3).

Fig. 3

Mammalian MEKK4 is redox-sensitive and interacts with cytosolic peroxiredoxin Prx2 in response to H₂O₂. (A, A′) A tagged version of Prx2 (Prx2-SBP) was expressed in HEK293T cells. Cells were treated with H₂O₂ (100 μM) and lysed at indicated time points. Prx2-SBP was affinity-purified using streptavidin beads. Precipitates (A) and whole cell lysates (WCL) (A′) were analyzed by SDS-PAGE under reducing (R) and non-reducing (NR) conditions followed by immunoblotting (IB). The immunoblots are representative of 3 independent experiments (n = 3). (B, B′) Complementary affinity purification experiment: A tagged version of MEKK4 (MEKK4-SBP) was expressed in HEK293T cells. Cells were treated with H₂O₂ (100 μM) and lysed at indicated time points. MEKK4-SBP was affinity-purified using streptavidin beads. Precipitates (B) and WCL (B′) were analyzed by SDS-PAGE under R and NR conditions followed by IB. e.v.: empty vector, PD: pull down, *: endogenous protein, X: unknown protein. The immunoblots are representative of 3 independent experiments (n = 3).

Fig. 4

2-Cys peroxiredoxins participate in the activation of the p38 signaling pathway in response to H₂O₂ and EGF. (A) HAP WT and HAP ΔPrx1+2 cells were incubated with 100 μM H₂O₂ and lysed at indicated time points. Phosphorylation of p38 was analyzed by immunoblotting. The immunoblot is representative of 3 independent experiments (n = 3). The bar chart presents the mean (+/− SD) ratio of phosphorylated p38 to total p38 from 3 independent experiments. (B) HEK293T WT and HEK293T ΔPrx1+2 cells were treated with doxycycline for 4 days to induce scrambled (WT) or Prx1+2 specific (ΔPrx1+2) shRNA expression. Cells were then incubated with 100 μM H₂O₂ and lysed at the indicated time points. Phosphorylation of p38 was analyzed by immunoblotting. The immunoblot is representative of 3 independent experiments (n = 3). The bar chart presents the mean (+/-SD) ratio of phosphorylated p38 to total p38 from 3 independent experiments. (C) HEK293T WT and HEK293T ΔPrx1+2 cells were treated with doxycycline to induce shRNA expression, serum-starved for 16 h, treated with 20 ng/mL EGF and lysed at indicated time points. Phosphorylation of p38 was analyzed by immunoblotting. The immunoblot is representative of 3 independent experiments (n = 3). The bar chart represents the mean (+/− SD) ratio of phosphorylated p38 to total p38 from 3 independent experiments.