Biochemical and Biophysical Research Communications
Identification and H2O2 sensitivity of the major constitutive MAPK phosphatase from rat brain
Section snippets
Materials and methods
Materials. Rat brains were purchased from Pel-Freez Biologicals (Tucson, AZ). Phosphopeptides (>95% purity) and Biomol Green phosphate reagent were purchased from Biomol Research Laboratories (Plymouth Meeting, PA). Anti-protein phosphatase 2A catalytic subunit antibody (product # P8484), protein phosphatase inhibitors, glutathione disulfide, and protease inhibitor cocktail were purchased from Sigma Chemical (St. Louis, MO). PVDF membranes were purchased from NOVEX (San Diego, CA). The Bio-Gel
The MAPK phosphatase is enriched in the microsomal and soluble fractions
The constitutively expressed MAPK phosphatase was operationally defined as the phosphatase activity in subcellular fractions from rat brain directed towards a low (75 μM) concentration of a 13-amino acid dual-phosphorylated peptide (D-H-T-G-F-L-pT-E-pY-V-A-T-R) corresponding to the activation motif of ERK [18] as substrate. MAPK phosphatase activity was detected in all of the particulate fractions (P1, P2, and microsomal) and the soluble fraction (Fig. 1A). The specific MAPK phosphatase activity
Discussion
Two major findings are reported here. First, the phosphatase activity directed towards a dual-phosphorylated MAPK peptide was attributable largely or entirely to PP2A in both microsomal and soluble fractions from rat brain cerebral cortex. Second, the defined MAPK phosphatase activity of PP2A was inhibited via an apparent reversible thiol oxidation by hydrogen peroxide and glutathione disulfide, both of which are central mediators of cellular oxidant stress.
The identification of the measured
Acknowledgements
This work was supported by NIH Grant AG022357 from the National Institute on Aging and in part from an Internal Research Award from the University of Scranton.
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