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 H 2 O 2 and TNFα (tumour necrosis factor α). ASK1 induction initiates a signalling cascade leading to apoptosis. After exposure of cells to H 2 O 2 , ASK1 is transiently activated by autophosphorylation at Thr 845 . The protein then associates with PP5 (protein serine/threonine phosphatase 5), which inactivates ASK1 by dephosphorylation of Thr 845 . 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 Thr 845 phosphorylation. The association of endogenous PP2Cϵ and ASK1 was also observed in mouse brain extracts. PP2Cϵ directly dephosphorylated ASK1 at Thr 845 in vitro . In contrast with PP5, PP2Cϵ transiently dissociated from ASK1 within cells upon H 2 O 2 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 H 2 O 2 -induced regulation of ASK1 activity.