doi: 10.1093/emboj/cdg513.
Redox regulation of PI 3-kinase signalling via inactivation of PTEN
Affiliations
- PMID: 14532122
- PMCID: PMC213768
- DOI: 10.1093/emboj/cdg513
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Redox regulation of PI 3-kinase signalling via inactivation of PTEN
EMBO J.
.
Abstract
The tumour suppressor PTEN is a PtdIns(3,4,5)P(3) phosphatase that regulates many cellular processes through direct antagonism of PI 3-kinase signalling. Here we show that oxidative stress activates PI 3-kinase-dependent signalling via the inactivation of PTEN. We use two assay systems to show that cellular PTEN phosphatase activity is inhibited by oxidative stress induced by 1 mM hydrogen peroxide. PTEN inactivation by oxidative stress also causes an increase in cellular PtdIns(3,4,5)P(3) levels and activation of the downstream PtdIns(3,4,5)P(3) target, PKB/Akt, that does not occur in cells lacking PTEN. We then show that endogenous oxidant production in RAW264.7 macrophages inactivates a fraction of the cellular PTEN, and that this is associated with an oxidant-dependent activation of downstream signalling. These results show that oxidants, including those produced by cells, can activate downstream signalling via the inactivation of PTEN. This demonstrates a novel mechanism of regulation of the activity of this important tumour suppressor and the signalling pathways it regulates. These results may have significant implications for the many cellular processes in which PtdIns(3,4,5)P(3) and oxidants are produced concurrently.
Figures
Fig. 1. PTEN, but not SHIP-2, is highly sensitive to oxidative inactivation in vitro. Recombinant PTEN (A) and SHIP-2 (B) were separately purified by affinity chromatography and immunoprecipitation respectively. Enzymes were then washed in ambient conditions lacking additional reducing or oxidizing agents, and then assayed in the presence of either 5 mM DTT, no additional reducing or oxidizing agents, or of different concentrations of H2O2. Data in (A) show d.p.m. released labelled phosphate, and in (B) show production of labelled PtdIns(3,4)P2 determined by densitometry. Presented data points represent the mean of assays performed in duplicate and the range of these duplicates. The measured activity using boiled enzyme is presented as a negative control.
Fig. 2. Cellular PTEN is inactivated by oxidative stress. (A) The indirect alkylation method used to analyse the effects of oxidative stress on PTEN activity is shown. It is described in detail in the text and Materials and methods. Iodoacetic acid is abbreviated to IAA and immunoprecipitation to IP. The method causes PTEN that is oxidized and inactive at the moment of lysis to be recovered as activity. Reduced active cellular enzyme is alkylated and this activity is lost. This method is used in panel B. (B) For indirect redox analysis of PTEN, Swiss 3T3 cells were treated for 10 min with the indicated concentrations of H2O2 before being lysed in buffer containing the alkylating agent IAA. PTEN was immunoprecipitated and IAA washed away before PTEN was assayed in reducing conditions. Control cells were also lysed in buffer lacking alkylating agent, but with 5 mM DTT reducing agent and immunoprecipitated, washed and assayed in reducing conditions. A fraction of the first sample from each condition was analysed by western blotting (WB) using a different antibody raised against PTEN. (C) Direct analysis of PTEN activity. Swiss 3T3 cells were treated with or without 1 mM H2O2 for 10 min. Cells were then lysed, and endogenous PTEN was then immunoprecipitated and assayed, all in anaerobic conditions in the absence of additional reducing or oxidizing agents. As a positive control 5 µg of alkaline phosphatase (AP) was used. A fraction of each PTEN IP sample was analysed by western blotting using a different antibody raised against PTEN. Data is presented as mean activity (d.p.m. released phosphate) from duplicate samples ± the range of these duplicates, with the exception of the PTEN IPs from (C) which are derived from triplicate samples.
Fig. 2. Cellular PTEN is inactivated by oxidative stress. (A) The indirect alkylation method used to analyse the effects of oxidative stress on PTEN activity is shown. It is described in detail in the text and Materials and methods. Iodoacetic acid is abbreviated to IAA and immunoprecipitation to IP. The method causes PTEN that is oxidized and inactive at the moment of lysis to be recovered as activity. Reduced active cellular enzyme is alkylated and this activity is lost. This method is used in panel B. (B) For indirect redox analysis of PTEN, Swiss 3T3 cells were treated for 10 min with the indicated concentrations of H2O2 before being lysed in buffer containing the alkylating agent IAA. PTEN was immunoprecipitated and IAA washed away before PTEN was assayed in reducing conditions. Control cells were also lysed in buffer lacking alkylating agent, but with 5 mM DTT reducing agent and immunoprecipitated, washed and assayed in reducing conditions. A fraction of the first sample from each condition was analysed by western blotting (WB) using a different antibody raised against PTEN. (C) Direct analysis of PTEN activity. Swiss 3T3 cells were treated with or without 1 mM H2O2 for 10 min. Cells were then lysed, and endogenous PTEN was then immunoprecipitated and assayed, all in anaerobic conditions in the absence of additional reducing or oxidizing agents. As a positive control 5 µg of alkaline phosphatase (AP) was used. A fraction of each PTEN IP sample was analysed by western blotting using a different antibody raised against PTEN. Data is presented as mean activity (d.p.m. released phosphate) from duplicate samples ± the range of these duplicates, with the exception of the PTEN IPs from (C) which are derived from triplicate samples.
Fig. 2. Cellular PTEN is inactivated by oxidative stress. (A) The indirect alkylation method used to analyse the effects of oxidative stress on PTEN activity is shown. It is described in detail in the text and Materials and methods. Iodoacetic acid is abbreviated to IAA and immunoprecipitation to IP. The method causes PTEN that is oxidized and inactive at the moment of lysis to be recovered as activity. Reduced active cellular enzyme is alkylated and this activity is lost. This method is used in panel B. (B) For indirect redox analysis of PTEN, Swiss 3T3 cells were treated for 10 min with the indicated concentrations of H2O2 before being lysed in buffer containing the alkylating agent IAA. PTEN was immunoprecipitated and IAA washed away before PTEN was assayed in reducing conditions. Control cells were also lysed in buffer lacking alkylating agent, but with 5 mM DTT reducing agent and immunoprecipitated, washed and assayed in reducing conditions. A fraction of the first sample from each condition was analysed by western blotting (WB) using a different antibody raised against PTEN. (C) Direct analysis of PTEN activity. Swiss 3T3 cells were treated with or without 1 mM H2O2 for 10 min. Cells were then lysed, and endogenous PTEN was then immunoprecipitated and assayed, all in anaerobic conditions in the absence of additional reducing or oxidizing agents. As a positive control 5 µg of alkaline phosphatase (AP) was used. A fraction of each PTEN IP sample was analysed by western blotting using a different antibody raised against PTEN. Data is presented as mean activity (d.p.m. released phosphate) from duplicate samples ± the range of these duplicates, with the exception of the PTEN IPs from (C) which are derived from triplicate samples.
Fig. 3. Oxidative stress increases cellular PtdIns(3,4,5)P3 levels only in cells expressing wild-type PTEN. Cellular levels of PtdIns(3,4,5)P3 (A and B) and PtdIns(3,4)P2 (C) were analysed in U87MG cells ± PTEN activity, stimulated with PDGF and/or H2O2. PTEN-null U87MG cells were labelled with [3H]inositol for 48 h in inositol-free culture medium. For the last 24 h of this incubation, cells were either left uninfected or infected with virus expressing wild-type GFP–PTEN (A and C), or infected with viruses expressing phosphatase-dead GFP–PTEN C124S or wild-type GFP–PTEN (B). Some cells were then stimulated as shown with 1 mM H2O2 or 50 ng/ml PDGF for 10 min before lysis. Cellular phosphoinositides were then purified, deacylated and analysed by HPLC. Data are presented as the mean of duplicate samples plus the range of these duplicates, with data for (A) and (C) being derived from the one experiment. These experiments were performed three times with similar results. The percentage of label incorporated into each lipid in unstimulated uninfected or C124S-infected cells corresponds to 1641, 1317 and 1462 d.p.m. above background in (A), (B) and (C), respectively.
Fig. 3. Oxidative stress increases cellular PtdIns(3,4,5)P3 levels only in cells expressing wild-type PTEN. Cellular levels of PtdIns(3,4,5)P3 (A and B) and PtdIns(3,4)P2 (C) were analysed in U87MG cells ± PTEN activity, stimulated with PDGF and/or H2O2. PTEN-null U87MG cells were labelled with [3H]inositol for 48 h in inositol-free culture medium. For the last 24 h of this incubation, cells were either left uninfected or infected with virus expressing wild-type GFP–PTEN (A and C), or infected with viruses expressing phosphatase-dead GFP–PTEN C124S or wild-type GFP–PTEN (B). Some cells were then stimulated as shown with 1 mM H2O2 or 50 ng/ml PDGF for 10 min before lysis. Cellular phosphoinositides were then purified, deacylated and analysed by HPLC. Data are presented as the mean of duplicate samples plus the range of these duplicates, with data for (A) and (C) being derived from the one experiment. These experiments were performed three times with similar results. The percentage of label incorporated into each lipid in unstimulated uninfected or C124S-infected cells corresponds to 1641, 1317 and 1462 d.p.m. above background in (A), (B) and (C), respectively.
Fig. 3. Oxidative stress increases cellular PtdIns(3,4,5)P3 levels only in cells expressing wild-type PTEN. Cellular levels of PtdIns(3,4,5)P3 (A and B) and PtdIns(3,4)P2 (C) were analysed in U87MG cells ± PTEN activity, stimulated with PDGF and/or H2O2. PTEN-null U87MG cells were labelled with [3H]inositol for 48 h in inositol-free culture medium. For the last 24 h of this incubation, cells were either left uninfected or infected with virus expressing wild-type GFP–PTEN (A and C), or infected with viruses expressing phosphatase-dead GFP–PTEN C124S or wild-type GFP–PTEN (B). Some cells were then stimulated as shown with 1 mM H2O2 or 50 ng/ml PDGF for 10 min before lysis. Cellular phosphoinositides were then purified, deacylated and analysed by HPLC. Data are presented as the mean of duplicate samples plus the range of these duplicates, with data for (A) and (C) being derived from the one experiment. These experiments were performed three times with similar results. The percentage of label incorporated into each lipid in unstimulated uninfected or C124S-infected cells corresponds to 1641, 1317 and 1462 d.p.m. above background in (A), (B) and (C), respectively.
Fig. 4. Oxidative stress activates cellular PKB only in cells expressing PTEN. PTEN-null U87MG cells growing at low cell density were either left uninfected or infected with viruses encoding wild-type GFP–PTEN for 24 h. Cells were then stimulated with 1 mM H2O2 or 50 ng/ml PDGF for 10 min before cell lysis and determination of the activity (A) and phosphorylation (B) of PKB/Akt. (A) After lysis, cellular PKB was immunoprecipitated and assayed in vitro. Data is presented as the mean + SD d.p.m. of labelled phosphate incorporated into peptide substrate from triplicate samples. (B) After lysis, the phosphorylation of PKB was analysed by western blotting using antibodies specific for total PKB and for phosphoserine-473 PKB. These experiments were performed three times with similar results.
Fig. 5. Inactivation of cellular PTEN by stimulation of RAW264.7 macrophages. RAW264.7 macrophages were either grown in normal medium (A), or primed with 100 ng/ml interferon γ for 24 h (B), before some cells were stimulated with 100 ng/ml LPS and 1 µM PMA for the indicated times. Cellular PTEN was then immunoprecipitated in the presence of the alkylating agent IAA (as Figure 2A and B), and the fraction of cellular PTEN protected from alkylation by oxidation was determined. Data in (A) are presented as the mean of five samples ± the SEM. This experiment was performed three times with similar results. Data in (B) are presented as the mean of six samples ± SEM. This experiment was performed on three occasions with similar results. In (A) and (B), the mean fully reduced control PTEN activity were 3362 and 5515 d.p.m. respectively. (C) RAW264.7 macrophages were also stimulated for 10 min with 100 ng/ml LPS alone or 100 ng/ml LPS and 1 µM PMA, and subsequently cells were lysed and PTEN immunoprecipitated and assayed all in reducing conditions in the absence of alkylating agents. (D) The oxidant dependence of the protection of PTEN from alkylation in stimulated macrophages (as panel A) was investigated. The stimulated production of ROS in RAW macrophages was antagonized by a 5 min pre-treatment with either 10 µM DPI or 10 mM NAC before stimulation with 100 ng/ml LPS and 1 µM PMA for 10 min. Cells were then lysed and the protection of PTEN from alkylation was investigated as above (Figures 2 and 5A and B). Data are presented as the mean of three samples ± SEM. (E) PKB phosphorylation in lysates used for the investigation of the oxidation of cellular PTEN (see panels A and B) was analysed in duplicate samples by Western blotting using antibodies specific for phosphoserine-473 PKB and total PKB. The presence of the alkylating agent IAA in the lysis buffer did not appear to interfere with this analysis. (F) The oxidation of PTEN was analysed using a biotinlyated alkylating agent as described in Materials and methods. RAW264.7 macrophages were treated with or without either 1 mM H2O2 or with 100 ng/ml LPS and 1 µM PMA. Cells were lysed in the presence of alkylating agent and proteins sequentially reduced and alkyated with a second biotinylated alkylating agent. PTEN was then immunoprecipitated and analysed either by western blotting with different antibodies against the protein, or with streptavidin–HRP.
Fig. 5. Inactivation of cellular PTEN by stimulation of RAW264.7 macrophages. RAW264.7 macrophages were either grown in normal medium (A), or primed with 100 ng/ml interferon γ for 24 h (B), before some cells were stimulated with 100 ng/ml LPS and 1 µM PMA for the indicated times. Cellular PTEN was then immunoprecipitated in the presence of the alkylating agent IAA (as Figure 2A and B), and the fraction of cellular PTEN protected from alkylation by oxidation was determined. Data in (A) are presented as the mean of five samples ± the SEM. This experiment was performed three times with similar results. Data in (B) are presented as the mean of six samples ± SEM. This experiment was performed on three occasions with similar results. In (A) and (B), the mean fully reduced control PTEN activity were 3362 and 5515 d.p.m. respectively. (C) RAW264.7 macrophages were also stimulated for 10 min with 100 ng/ml LPS alone or 100 ng/ml LPS and 1 µM PMA, and subsequently cells were lysed and PTEN immunoprecipitated and assayed all in reducing conditions in the absence of alkylating agents. (D) The oxidant dependence of the protection of PTEN from alkylation in stimulated macrophages (as panel A) was investigated. The stimulated production of ROS in RAW macrophages was antagonized by a 5 min pre-treatment with either 10 µM DPI or 10 mM NAC before stimulation with 100 ng/ml LPS and 1 µM PMA for 10 min. Cells were then lysed and the protection of PTEN from alkylation was investigated as above (Figures 2 and 5A and B). Data are presented as the mean of three samples ± SEM. (E) PKB phosphorylation in lysates used for the investigation of the oxidation of cellular PTEN (see panels A and B) was analysed in duplicate samples by Western blotting using antibodies specific for phosphoserine-473 PKB and total PKB. The presence of the alkylating agent IAA in the lysis buffer did not appear to interfere with this analysis. (F) The oxidation of PTEN was analysed using a biotinlyated alkylating agent as described in Materials and methods. RAW264.7 macrophages were treated with or without either 1 mM H2O2 or with 100 ng/ml LPS and 1 µM PMA. Cells were lysed in the presence of alkylating agent and proteins sequentially reduced and alkyated with a second biotinylated alkylating agent. PTEN was then immunoprecipitated and analysed either by western blotting with different antibodies against the protein, or with streptavidin–HRP.
Fig. 5. Inactivation of cellular PTEN by stimulation of RAW264.7 macrophages. RAW264.7 macrophages were either grown in normal medium (A), or primed with 100 ng/ml interferon γ for 24 h (B), before some cells were stimulated with 100 ng/ml LPS and 1 µM PMA for the indicated times. Cellular PTEN was then immunoprecipitated in the presence of the alkylating agent IAA (as Figure 2A and B), and the fraction of cellular PTEN protected from alkylation by oxidation was determined. Data in (A) are presented as the mean of five samples ± the SEM. This experiment was performed three times with similar results. Data in (B) are presented as the mean of six samples ± SEM. This experiment was performed on three occasions with similar results. In (A) and (B), the mean fully reduced control PTEN activity were 3362 and 5515 d.p.m. respectively. (C) RAW264.7 macrophages were also stimulated for 10 min with 100 ng/ml LPS alone or 100 ng/ml LPS and 1 µM PMA, and subsequently cells were lysed and PTEN immunoprecipitated and assayed all in reducing conditions in the absence of alkylating agents. (D) The oxidant dependence of the protection of PTEN from alkylation in stimulated macrophages (as panel A) was investigated. The stimulated production of ROS in RAW macrophages was antagonized by a 5 min pre-treatment with either 10 µM DPI or 10 mM NAC before stimulation with 100 ng/ml LPS and 1 µM PMA for 10 min. Cells were then lysed and the protection of PTEN from alkylation was investigated as above (Figures 2 and 5A and B). Data are presented as the mean of three samples ± SEM. (E) PKB phosphorylation in lysates used for the investigation of the oxidation of cellular PTEN (see panels A and B) was analysed in duplicate samples by Western blotting using antibodies specific for phosphoserine-473 PKB and total PKB. The presence of the alkylating agent IAA in the lysis buffer did not appear to interfere with this analysis. (F) The oxidation of PTEN was analysed using a biotinlyated alkylating agent as described in Materials and methods. RAW264.7 macrophages were treated with or without either 1 mM H2O2 or with 100 ng/ml LPS and 1 µM PMA. Cells were lysed in the presence of alkylating agent and proteins sequentially reduced and alkyated with a second biotinylated alkylating agent. PTEN was then immunoprecipitated and analysed either by western blotting with different antibodies against the protein, or with streptavidin–HRP.
Fig. 6. Stimulation of RAW264.7 macrophages induces an oxidant-dependent activation of PKB. RAW264.7 macrophages were left untreated (–) or pre-treated for 5 min with 10 mM NAC (N) or DPI (D). Cells were then left untreated (control) or stimulated with 100 ng/ml LPS, 1 µM PMA, 10 µM insulin or 1 mM H2O2, each for 10 min. Cells were lysed and the activity state of cellular PKB was analysed using an in vitro kinase assay of immunoprecipitated PKB (A), or PKB phosphorylation analysed using western blotting with antibodies specific for phosphoserine-473 PKB, total PKB and PTEN (B). Data in (A) are presented as the mean activity ± SEM from a minimum of three samples. These experiments were performed three times with similar results.
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