Redox-sensitive protein phosphatase activity regulates the phosphorylation state of p38 protein kinase in primary astrocyte culture.
Reactive oxygen species (ROS) have been implicated as second messengers that activate protein kinase cascades, although the means by which ROS regulate signal transduction remains unclear. In the present study, we show that interleukin 1beta (IL1beta), H2O2, and sorbitol-induced hyperosmolarity mediate a 5- to 10-fold increase in phosphorylation (activation) of the p38 protein kinase in rat primary glial cells as measured by analyses of Western blots using an antibody directed against the dually phosphorylated (active) p38. Additionally, IL1beta was found to elicit H2O2 synthesis in these cells. Concurrent with p38 phosphorylation, all three stimulation paradigms caused an inhibition of protein phosphatase activity. Phenyl-tert-butyl nitrone (PBN), a nitrone-based free radical trap and N-acetyl-cysteine (NAC), a thiol reducing agent, were examined for their effects on the phosphorylation of p38 as well as phosphatase activity. Pretreatment of cells with either PBN or NAC at 1.0 mM suppressed IL1beta H2O2, and sorbitol-mediated activation of p38 and significantly increased phosphatase activity. These data suggest that ROS, particularly H2O2, are used as second messenger substances that activate p38 in part via the transient inactivation of regulatory protein phosphatases.
Robinson
K A
KA
Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, USA.
Stewart
C A
CA
Pye
Q N
QN
Nguyen
X
X
Kenney
L
L
Salzman
S
S
Floyd
R A
RA
Hensley
K
K
D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D000975:Antioxidants / Q000494:pharmacology; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000378:metabolism*; D003497:Cyclic N-Oxides; D006861:Hydrogen Peroxide / Q000378:metabolism; D007375:Interleukin-1 / Q000494:pharmacology*; D007700:Kinetics; D020928:Mitogen-Activated Protein Kinases; D009589:Nitrogen Oxides / Q000494:pharmacology*; D010084:Oxidation-Reduction; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000502:physiology; D013012:Sorbitol / Q000494:pharmacology; D013113:Spin Labels; D048051:p38 Mitogen-Activated Protein Kinases
D000975:Antioxidants; D003497:Cyclic N-Oxides; D007375:Interleukin-1; D009589:Nitrogen Oxides; D017382:Reactive Oxygen Species; D013113:Spin Labels; C029217:phenyl-N-tert-butylnitrone; D013012:Sorbitol; D006861:Hydrogen Peroxide; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D010749:Phosphoprotein Phosphatases; D000111:Acetylcysteine