Redox regulation of PTEN by S-nitrosothiols.
PTEN (phosphatase with sequence homology to tensin) is a phosphatidylinositol 3,4,5-trisphosphate phosphatase that regulates many cellular processes. Activity of the enzyme is dependent on the redox state of the active site cysteine such that oxidation by H2O2 leads to inhibition. Because S-nitrosothiols are known to modify enzymes containing reactive cysteines, we hypothesized that S-nitrosothiols would oxidize PTEN and inhibit its phosphatase activity. In the present study, we show that S-nitrosocysteine (CSNO), S-nitrosoglutathione (GSNO), and S-nitroso-N-acetylpenicillamine (SNAP) reversibly oxidized recombinant PTEN. In addition, CSNO led to concentration- and time-dependent oxidation of endogenous cellular PTEN. However, in contrast, GSNO and SNAP were effective only when coincubated with cysteine, suggesting that these nitrosothiols must react with cysteine to form CSNO, which can be transferred across cell membranes. Oxidation of cellular PTEN resulted from thiol modification and led to reversible inhibition of phosphatase activity. Although oxidation of PTEN by H2O2 led to formation of an intramolecular disulfide, oxidation of PTEN by CSNO seemed to lead to formation of a mixed disulfide. Glutathionylation of cellular proteins by incubating cells with diamide or incubating cellular extracts with GSSG oxidized PTEN in a manner similar to that of CSNO. Overall, these data demonstrate for the first time that S-nitrosothiols oxidatively modify PTEN, leading to reversible inhibition of its phosphatase activity, and suggest that the oxidized species is a mixed disulfide.
Yu
Chang-Xi
CX
Department of Pharmacology and Cancer Biology, Box 3813, C 138B Lev. Sci. Res. Center, Duke University Medical Center, Durham, NC 27710, USA.
Li
Sheng
S
Whorton
A Richard
AR
D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D004220:Disulfides / Q000378:metabolism; D006801:Humans; D010084:Oxidation-Reduction; D051059:PTEN Phosphohydrolase; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D026403:S-Nitrosothiols / Q000494:pharmacology*; D025521:Tumor Suppressor Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*
D017931:DNA Primers; D004220:Disulfides; D011994:Recombinant Proteins; D026403:S-Nitrosothiols; D025521:Tumor Suppressor Proteins; D010744:Phosphoric Monoester Hydrolases; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human