Oxidative Stress Impairs the Stimulatory Effect of S100 Proteins on Protein Phosphatase 5 Activity.
Oxidative stress is the consequence of an imbalance between the production of harmful reactive oxygen species and the cellular antioxidant system for neutralization, and it activates multiple intracellular signaling pathways, including apoptosis signal-regulating kinase 1 (ASK1). Protein phosphatase 5 (PP5) is a serine/threonine phosphatase involved in oxidative stress responses. Previously, we reported that S100 proteins activate PP5 in a calcium-dependent manner. S100 proteins belong to a family of small EF-hand calcium-binding proteins involved in many processes such as cell proliferation, differentiation, apoptosis, and inflammation. Therefore, we investigated the effects of oxidative stress on S100 proteins, their interaction with PP5, and PP5 enzyme activity. Recombinant S100A2 was easily air-oxidized or Cu-oxidized, and oxidized S100A2 formed cross-linked dimers and higher molecular-mass complexes. The binding of oxidized S100A2 to PP5 was reduced, resulting in decreased PP5 activation in vitro. Oxidation also impaired S100A1, S100A6, S100B, and S100P to activate PP5, although the low dose of oxidized S100 proteins still activated PP5. Hydrogen peroxide (H2O2) induced S100A2 oxidation in human keratinocytes (HaCaT) and human hepatocellular carcinoma (Huh-7) cells. Furthermore, H2O2 reduced the binding of S100A2 to PP5 and decreased PP5 activation in HaCaT and Huh-7 cells. Importantly, even the low dose of S100A2 achieved by knocking down increased dephosphorylation of ASK1 and reduced caspase 3/7 activity in Huh-7 cells treated with H2O2. These results indicate that oxidative stress impairs the ability of S100 proteins to bind and activate PP5, which in turn modulates the ASK1-mediated signaling cascades involved in apoptosis.
Department of Cell Physiology, Faculty of Medicine, Kagawa University.
Fuminori
F
Yamaguchi
Mitsumasa
M
Tsuchiya
Seiko
S
Shimamoto
Tomohito
T
Fujimoto
Hiroshi
H
Tokumitsu
Masaaki
M
Tokuda
Ryoji
R
Kobayashi
D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects; D055785:Gene Knockdown Techniques; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D048848:MAP Kinase Kinase Kinase 5 / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism*; D010084:Oxidation-Reduction / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D055503:Protein Multimerization / Q000187:drug effects; D009418:S100 Proteins / Q000378:metabolism*
D009687:Nuclear Proteins; D009418:S100 Proteins; D006861:Hydrogen Peroxide; D048848:MAP Kinase Kinase Kinase 5; C482858:MAP3K5 protein, human; D010749:Phosphoprotein Phosphatases; C089641:protein phosphatase 5; D020169:Caspases