Identification and H2O2 sensitivity of the major constitutive MAPK phosphatase from rat brain

https://doi.org/10.1016/j.bbrc.2004.01.096Get rights and content

Abstract

The present study examined in subcellular fractions from rat brain the nature and sensitivity to hydrogen peroxide of constitutively expressed mitogen-activated protein kinase (MAPK) phosphatase activity. MAPK phosphatase activity was defined as the activity directed towards a dual-phosphorylated (pT/pY) peptide corresponding to the activation domain of the extracellular-regulated kinase (ERK) subtype of the MAPKs. The use of phosphatase inhibitors and biochemical analyses demonstrate that the MAPK phosphatase activity, which was highest in the microsomal membrane and soluble fractions, was attributable mainly, if not entirely, to protein phosphatase 2A (PP2A). Moreover, hydrogen peroxide (in the absence and presence of reduced glutathione) and glutathione disulfide inhibited the MAPK phosphatase activity by a dithiothreitol-reversible mechanism. These results provide direct support for mounting evidence that PP2A is a major regulator of MAPK phosphorylation in brain and suggest that inhibition of PP2A activity via reversible oxidation of a cysteine thiol(s) may underlie at least in part the activation of MAPKs occurring in response to hydrogen peroxide and oxidative stress.

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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