Regulation of apoptosis signal-regulating kinase 1 by protein phosphatase 2Cepsilon.
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 TNFalpha (tumour necrosis factor alpha). 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 PP2Cepsilon (protein phosphatase 2Cepsilon), a member of PP2C family, in the regulation of ASK1 signalling. Following expression in HEK-293 cells (human embryonic kidney cells), wild-type PP2Cepsilon inhibited ASK1-induced activation of an AP-1 (activator protein 1) reporter gene. Conversely, a dominant-negative PP2Cepsilon mutant enhanced AP-1 activity. Exogenous PP2Cepsilon associated with exogenous ASK1 in HEK-293 cells under non-stressed conditions, inactivating ASK1 by decreasing Thr845 phosphorylation. The association of endogenous PP2Cepsilon and ASK1 was also observed in mouse brain extracts. PP2Cepsilon directly dephosphorylated ASK1 at Thr845 in vitro. In contrast with PP5, PP2Cepsilon transiently dissociated from ASK1 within cells upon H2O2 treatment. These results suggest that PP2Cepsilon maintains ASK1 in an inactive state by dephosphorylation in quiescent cells, supporting the possibility that PP2Cepsilon and PP5 play different roles in H2O2-induced regulation of ASK1 activity.
Saito
Jun-ichi
J
Department of Biochemistry, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryomachi, Aoba-ku, Sendai 980-8575, Japan.
Toriumi
Shinnosuke
S
Awano
Kenjiro
K
Ichijo
Hidenori
H
Sasaki
Keiichi
K
Kobayashi
Takayasu
T
Tamura
Shinri
S
D002460:Cell Line; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide; D048848:MAP Kinase Kinase Kinase 5 / Q000235:genetics / Q000378:metabolism*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D000071636:Protein Phosphatase 2C
D006023:Glycoproteins; C021240:tissue-factor-pathway inhibitor 2; D006861:Hydrogen Peroxide; D048848:MAP Kinase Kinase Kinase 5; D020929:Mitogen-Activated Protein Kinase Kinases; D010749:Phosphoprotein Phosphatases; D000071636:Protein Phosphatase 2C