Regulation of protein phosphatase 2A by hydrogen peroxide and glutathionylation.
The regulation of protein phosphatase 2A (PP2A) and protein threonine phosphorylation by H(2)O(2) was determined in Caco-2 cell monolayer. Incubation with H(2)O(2) (20 microM) resulted in threonine phosphorylation of a cluster of proteins at the molecular mass range of 170-250 kDa. PKC activity and plasma membrane localization of several isoforms of PKC were not affected by H(2)O(2). However, H(2)O(2) reduced 80-85% of okadaic acid-sensitive protein phosphatase activity. Immunocomplex protein phosphatase assay demonstrated that H(2)O(2) 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 H(2)O(2)-treated Caco-2 cell monolayer. These results indicate that PP2A activity can be regulated by glutathionylation, and that H(2)O(2) inhibits PP2A in Caco-2 cells, which may involve glutathionylation of PP2A.
Rao
R K
RK
Department of Physiology, University of Tennessee Health Sciences Center, Memphis, TN 38163, USA. rkrao@physiol1.utmem.edu
Clayton
L W
LW
D002462:Cell Membrane / Q000201:enzymology; D005978:Glutathione / Q000494:pharmacology*; D019803:Glutathione Disulfide / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D054648:Protein Phosphatase 2; D014407:Tumor Cells, Cultured
D007527:Isoenzymes; D010769:Phosphothreonine; D006861:Hydrogen Peroxide; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D005978:Glutathione; D019803:Glutathione Disulfide