Redox-sensitive protein phosphatase activity regulates the phosphorylationstate of p38 protein kinase in primary astrocyte culture

Reactive oxygen species (ROS) have been implicated as second messengers tha t activate protein kinase cascades, although the means by which ROS regulat e signal transduction remains unclear. In the present study we show that in terleukin 1 beta (IL1 beta), H2O2, and sorbitol-induced hyperosmolarity med iate a 5- to10-fold increase in phosphorylation (activation) of the p38 pro tein kinase in rat primary glial cells as measured by analyses of Western b lots using an antibody directed against the dually phosphorylated (active) p38. Additionally, IL1 beta was found to elicit H2O2 synthesis in these cel ls. Concurrent with p38 phosphorylation, all three stimulation paradigms ca used an inhibition of protein phosphatase activity. Phenyl-tert-butyl nitro ne (PBN), a nitrone-based free radical trap and N-acetyl-cysteine (NAC), a thiol reducing agent, were examined for their effects on the phosphorylatio n of p38 as well as phosphatase activity, Pretreatment of cells with either PEN or NAC at 1.0 mM suppressed IL1 beta H2O2, and sorbitol-mediated activ ation of p38 and significantly increased phosphatase activity. These data s uggest that ROS, particularly H2O2, are used as second messenger substances that activate p38 in part via the transient inactivation of regulatory pro tein phosphatases. (C) 1999 Wiley-Liss, Inc.