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

ASK I (apoptosis signal-regulating kinase 1), a MKKK (mitogen-activated protein kinase kinase kinase), is activated in response to cytotoxic stresses, such as H2O2 and TNF alpha (tumour necrosis factor a). ASK1 induction initiates a signalling cascade leading to apoptosis. After exposure of cells to H2O2, 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 PP2C epsilon (protein phosphatase 2C epsilon), a member of PP2C epsilon family, in the regulation of ASK1 signalling. Following expression in HEK-293 cells (human embryonic kidney cells), wild-type PP2C epsilon inhibited ASK1-induced activation of an AP-1 (activator protein 1) reporter gene. Conversely, a dominant-negative PP2C epsilon mutant enhanced AP-1 activity. Exogenous PP2C epsilon associated with exogenous ASK1 in HEK-293 cells under non-stressed conditions, inactivating ASK1 by decreasing Thr(845) phosphorylation. The association of endogenous PP2C epsilon and ASK1 was also observed in mouse brain extracts. PP2C epsilon directly dephosphorylated ASK1 at Thr(845). in vitro. In contrast with PP5, PP2C epsilon transiently dissociated from ASK1 within cells upon H2O2 treatment. These results suggest that PP2C epsilon maintains ASK1 in an inactive state by dephosphorylation in quiescent cells, supporting the possibility that PP2C epsilon and PP5 play different roles in H2O2-induced regulation of ASK1 activity.