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. 2004 Nov 23;101(47):16419-24.
doi: 10.1073/pnas.0407396101. Epub 2004 Nov 8.

Reversible oxidation and inactivation of the tumor suppressor PTEN in cells stimulated with peptide growth factors

Jaeyul Kwon  1 , Seung-Rock Lee, Kap-Seok Yang, Younghee Ahn, Yeun Ju Kim, Earl R Stadtman, Sue Goo Rhee
Affiliations

Reversible oxidation and inactivation of the tumor suppressor PTEN in cells stimulated with peptide growth factors

Jaeyul Kwon et al. Proc Natl Acad Sci U S A. .

Abstract

Stimulation of cells with various peptide growth factors induces the production of phosphatidylinositol 3,4,5-trisphosphate (PIP3) through activation of phosphatidylinositol 3-kinase. The action of this enzyme is reversed by that of the tumor suppressor PTEN. With the use of cells overexpressing NADPH oxidase 1 or peroxiredoxin II, we have now shown that H2O2 produced in response to stimulation of cells with epidermal growth factor or platelet-derived growth factor potentiates PIP3 generation and activation of the protein kinase Akt induced by these growth factors. We also show that a small fraction of PTEN molecules is transiently inactivated as a result of oxidation of the essential cysteine residue of this phosphatase in various cell types stimulated with epidermal growth factor, platelet-derived growth factor, or insulin. These results suggest that the activation of phosphatidylinositol 3-kinase by growth factors might not be sufficient to induce the accumulation of PIP3 because of the opposing activity of PTEN and that the concomitant local inactivation of PTEN by H2O2 might be needed to increase the concentration of PIP3 sufficiently to trigger downstream signaling events. Furthermore, together with previous observations, our data indicate that peroxiredoxin likely participates in PIP3 signaling by modulating the local concentration of H2O2.

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Figures

Fig. 1.
Fig. 1.
Effects of Nox1 overexpression on the production of PIP3 and on PI 3-kinase activity in PDGF-stimulated cells. (A) NIH 3T3 cells either stably expressing Nox1 or stably transfected with the empty pEF-PAC vector (control) were labeled with [32P]Pi and stimulated for the indicated times with 30 ng/ml PDGF. The amount of [32P]PIP3 in the cells was then determined and expressed as a percentage of total radioactivity associated with the phospholipid fraction. Data are means of values obtained from two separate experiments. (B) Control and Nox1-overexpressing NIH 3T3 cells were stimulated with PDGF as in A, and cell lysates were processed for assay of PI 3-kinase activity. Data are expressed in arbitrary units and are from an experiment that was performed twice with similar results.
Fig. 2.
Fig. 2.
Effects of Prx II on PIP3 accumulation and Akt phosphorylation in EGF-stimulated HeLa cells. (A) HeLa cells stably expressing Prx II-WT or Prx II-DN or those transfected with the empty pCR3.1 vector (control) were labeled with [32P]Pi and stimulated for the indicated times with 25 ng/ml EGF. The amount of [32P]PIP3 in the cells was then determined and is expressed as a percentage of the total radioactivity associated with the phospholipid fraction. Data are representative of three independent experiments. (B) HeLa cells were transfected for 48 h with pCR3.1 encoding Prx II-WT (PrxII) or with the empty vector, and cell lysates were subsequently subjected to immunoblot analysis with Abs to Prx II and to β-actin (loading control). The transfected cells were also stimulated with 2 ng/ml EGF for the indicated times, after which cell lysates were subjected to immunoblot analysis with Abs specific for Akt phosphorylated on Ser-473 (pAkt); the filter was reprobed with Abs to β-actin. Data are representative of three independent experiments.
Fig. 3.
Fig. 3.
Detection of oxidized PTEN on the basis of an electrophoretic mobility shift in cells treated with exogenous H2O2 or with growth factors. (A) HeLa cells were incubated for the indicated times with 0.2 mM H2O2, after which cell lysates were subjected to alkylation with NEM and fractionated by nonreducing SDS/PAGE as described (11). Reduced (Red) and oxidized (Oxi) forms of PTEN were detected by immunoblot analysis with rabbit Abs to PTEN. (B) HeLa cells were incubated for the indicated times with 100 ng/ml EGF, after which cell lysates were subjected to alkylation with NEM followed by immunoprecipitation with a mAb (A2B1) to PTEN. The resulting precipitates were subjected to nonreducing SDS/PAGE and immunoblot analysis with rabbit Abs to PTEN as in A. (C) NIH 3T3 cells were transfected for 48 h with a mixture of Effectene (Qiagen) and a pCGN-derived vector encoding human PTEN tagged with HA at its NH2 terminus. The cells were subsequently stimulated for the indicated times with 25 ng/ml PDGF or for 5 min with 5 mM H2O2, after which cell lysates were analyzed as in B with the exception that the PTEN immunoprecipitates were either left untreated (Left) or treated with 1 mM DTT (Right) before immunoblot analysis with Abs to HA. The blots were exposed to x-ray film for 5 min (Upper) or 10 s (Lower).
Fig. 4.
Fig. 4.
Detection of oxidized PTEN by biotinylation in cells treated with exogenous H2O2 or with growth factors. NIH 3T3 cells were incubated either for the indicated times with 1 mM H2O2 (A) or for 5 min with the indicated concentrations of H2O2 (B); HeLa cells were incubated for the indicated times with 100 ng/ml EGF (C); NIH 3T3 cells were incubated for the indicated times with 25 ng/ml PDGF (D); and HEK293 cells were incubated for the indicated times with 0.5 μg/ml insulin (E). Cell lysates were then prepared and subjected to labeling with biotin-conjugated maleimide as described in Materials and Methods. A portion (10%) of each biotinylated sample was subjected to immunoblot analysis with rabbit Abs to PTEN as a control (Lower), whereas biotinylated proteins in the remaining fraction were precipitated with avidin-conjugated agarose and then subjected to immunoblot analysis with Abs to PTEN to detect biotinylated PTEN (BM-PTEN) (Upper).
Fig. 5.
Fig. 5.
Detection by phosphatase assay of PTEN oxidized in NIH 3T3 cells stimulated with PDGF. Cells were incubated for 0, 5, or 10 min with 30 ng/ml PDGF or for 5 min with 5 mM H2O2, lysed in the presence of iodoacetic acid, and divided into three equal portions. PTEN was immunoprecipitated from each portion with a mAb to PTEN and then assayed for phosphatase activity under reducing conditions as described (15). Data are expressed as cpm of radioactivity released as Pi from the [32P]PIP3 substrate and are means ± SEM of the triplicate samples.
Fig. 6.
Fig. 6.
Model for the production, signaling role, and removal of H2O2 in growth factor-stimulated cells. Stimulation of cells with a growth factor induces the activation of PI 3-kinase (PI3K), which catalyzes the conversion of PI(4,5)P2 to PIP3. PIP3 activates the NADPH oxidase (NOX) complex, resulting in the production of H2O2. The H2O2 so generated likely mediates inactivation of cytosolic Prx molecules located nearby through a two-step oxidation of the active site Cys–SH to Cys–SO2H. The inactivation of Prx in turn promotes local accumulation of H2O2. The results of the present study suggest that the accumulated H2O2 molecules inactivate PTEN by oxidizing the catalytic cysteine residue. This inactivation of PTEN increases the abundance of PIP3 sufficiently to trigger downstream signaling events. The H2O2 signal is likely terminated by the reactivation of sulfinylated Prx and the consequent removal of H2O2. As the local concentration of H2O2 decreases, oxidized PTEN is reactivated by thioredoxin (Trx), which in turn receives reducing equivalents from NADPH by means of thioredoxin reductase (TrxR).
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References

    1. Rhee, S. G., Bae, Y. S., Lee, S.-R. & Kwon, J. (2000) Sci. STKE 2000, pe1. - PubMed
    1. Lambeth, J. D. (2004) Nat. Rev. Immunol. 4, 181–189. - PubMed
    1. Sundaresan, M., Yu, Z. X., Ferrans, V. J., Irani, K. & Finkel, T. (1995) Science 270, 296–299. - PubMed
    1. Bae, Y. S., Kang, S. W., Seo, M. S., Baines, I. C., Tekle, E., Chock, P. B. & Rhee, S. G. (1997) J. Biol. Chem. 272, 217–221. - PubMed
    1. Burdon, R. H. (1995) Free Radical Biol. Med. 18, 775–794. - PubMed

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