Regulation of apoptosis signal-regulating kinase 1 by protein phosphatase 2Cϵ

ASK1 (apoptosis signal-regulating kinase 1), a MKKK (mitogen-activated protein kinase kinase kinase), is activated in response to cytotoxic stresses, such as H₂O₂ and TNFα (tumour necrosis factor α). ASK1 induction initiates a signalling cascade leading to apoptosis. After exposure of cells to H₂O₂, ASK1 is transiently activated by autophosphorylation at Thr⁸⁴⁵. The protein then associates with PP5 (protein serine/threonine phosphatase 5), which inactivates ASK1 by dephosphorylation of Thr⁸⁴⁵. Although this feedback regulation mechanism has been elucidated, it remains unclear how ASK1 is maintained in the dephosphorylated state under non-stressed conditions. In the present study, we have examined the possible role of PP2Cϵ (protein phosphatase 2Cϵ), a member of PP2C family, in the regulation of ASK1 signalling. Following expression in HEK-293 cells (human embryonic kidney cells), wild-type PP2Cϵ inhibited ASK1-induced activation of an AP-1 (activator protein 1) reporter gene. Conversely, a dominant-negative PP2Cϵ mutant enhanced AP-1 activity. Exogenous PP2Cϵ associated with exogenous ASK1 in HEK-293 cells under non-stressed conditions, inactivating ASK1 by decreasing Thr⁸⁴⁵ phosphorylation. The association of endogenous PP2Cϵ and ASK1 was also observed in mouse brain extracts. PP2Cϵ directly dephosphorylated ASK1 at Thr⁸⁴⁵in vitro. In contrast with PP5, PP2Cϵ transiently dissociated from ASK1 within cells upon H₂O₂ treatment. These results suggest that PP2Cϵ maintains ASK1 in an inactive state by dephosphorylation in quiescent cells, supporting the possibility that PP2Cϵ and PP5 play different roles in H₂O₂-induced regulation of ASK1 activity.