The regulation of protein phosphatase 2A (PP2A) and protein threonine phosphorylation by H2O2 was determined in Caco-2 cell monolayer. Incubation with H2O2 (20 muM) resulted in threonine phosphorylation of a cluster of proteins at the molecular mass range of 170-250kDa. PKC activity and plasma membrane localization of several isoforms of PKC were not affected by H2O2. However, H2O2 reduced 80-85% of okadaic acid-sensitive protein phosphatase activity. Immunocomplex protein phosphatase assay demonstrated that H2O2 reduced the activity of PP2A, but not that of PP2C or PP1. Oxidized glutathione inhibited PP2A activity in plasma membranes prepared from Caco-2 cells and the phosphatase activity of an isolated PP2A. PP2A activity was also inhibited by N-ethylmaleimide, iodoacetamide, and p-chloromercuribenzoate. Inhibition of PP2A by oxidized glutathione was reversed by reduced glutathione. Glutathione also restored the PP2A activity in plasma membranes isolated from H2O2-treated Caco-2 cell monolayer. These results indicate that PP2A activity can be regulated by glutathionylation, and that H2O2 inhibits PP2A in Caco-2 cells, which may involve glutathionylation of PP2A. (C) 2002 Elsevier Science (USA). All rights reserved.