N-Ethylmaleimide Inhibits Platelet-derived Growth Factor BB-stimulated Akt Phosphorylation via Activation of Protein Phosphatase 2A*

The redox state plays an important role in gene regulation. Thiols maintain the intracellular redox homeostasis. To understand the role of thiols in redox signaling, we have studied the effect of thiol alkylation on platelet-derived growth factor-BB (PDGF-BB)-induced cell survival events in vascular smooth muscle cells. PDGF-BB stimulated Akt phosphorylation predominantly at Ser-473.N-Ethylmaleimide (NEM), a thiol alkylating agent, blocked PDGF-BB-induced Akt phosphorylation without affecting its upstream phosphatidylinositol 3-kinase (PI3K). On the other hand, LY294002 and wortmannin, specific inhibitors of PI3K, prevented PDGF-BB-induced phosphorylation of Akt and its downstream effector molecules, p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E. NEM also abrogated the phosphorylation of p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E induced by PDGF-BB, suggesting that thiol alkylation interferes with the PI3K/Akt pathway at the level of Akt. In addition, NEM blocked PDGF-BB-induced phosphorylation of BAD and forkhead transcription factor FKHR-L1, and these events correlated with increased apoptosis. NEM alone and in concert with PDGF-BB increased reactive oxygen species (ROS) production and protein phosphatase 2A (PP2A) activity in VSMC. The inhibition of PDGF-BB-induced Akt phosphorylation by NEM was completely reversed by PP2A inhibitors fostriecin and okadaic acid, ceramide synthase inhibitor fumonisin B1, and ROS scavenger N-acetylcysteine (NAC). NAC also attenuated the apoptosis induced by NEM, alone or in combination with PDGF-BB. Together, these findings demonstrate for the first time that PP2A mediates thiol alkylation-dependent redox regulation of Akt and cell survival.

The cellular redox state plays an important role in the regulation of gene expression in prokaryotes and eukaryotes (1)(2)(3)(4). The following observations support this notion: 1) Oxidants regulate the activities of several transcription factors, including activator protein-1, nuclear factor kappa B, and p53 (5-7); 2) Oxidants are capable of activating several early response events, including stimulation of protein tyrosine phosphoryla-tion, activation of mitogen-activated protein kinases and induction of expression of proto-oncogenes (8 -11); 3) Oxidants are produced acutely in response to various agents, including growth factors and cytokines in several cell types (12,13), and a requirement for their production in the mitogenic effects of receptor tyrosine kinase and G protein-coupled receptor agonists has been demonstrated (14,15); and 4) In addition to producing oxidants, cells also possess enzymatic and non-enzymatic mechanisms for their removal (16 -18), and this feature attests to the role of oxidants as second messenger molecules (19). Despite the growing body of information on the role of oxidants in the regulation of gene expression, the mechanisms by which these molecules transmit the extracellular signals from the plasma membrane to the nucleus are less clear. Thiols play a critical role in the reduction/oxidation reactions as well as in the structure and function of several enzymes, transcription factors, and transporters (1,20,21). Most interestingly, cells also possess several enzymatic mechanisms such as thioredoxins and glutaredoxins for regeneration of thiols from their oxidized state, features that orchestrate these molecules as primary targets for oxidant action (22,23). Oxidation of cysteinyl thiols in the active site of protein tyrosine phosphatase 1B has been observed as a mechanism of its reversible inactivation in response to growth factors and oxidants facilitating tyrosine phosphorylation and activation of receptor tyrosine kinases (24,25).
Oxidant stress has been implicated in the pathogenesis of a variety of diseases, including atherosclerosis and cancer (26,27). Depletion of cellular thiols causes oxidant stress (26). In view of the above information, we hypothesize that the cellular thiol redox state plays a determinant role in agonist-induced cell survival/apoptotic signals from the plasma membrane to the nucleus leading to induction of expression of target genes enabling the cellular response. Assuming such an important role for cellular thiols in the signal transduction pathways, one would expect that blockade of these inorganic sulfur groups should affect the signal transduction events that are dependent on oxidation/reduction of these molecules either positively or negatively. The PI3K 1 /Akt pathway plays an important role in cell survival and growth in response to a variety of agents, including cytokines, growth factors, and hormones (28 -33). To understand the role of thiol-sensitive redox mechanisms in the regulation of cell survival and growth, we have studied the effect of thiol alkylation on PDGF-BB-induced activation of the PI3K/Akt pathway in VSMC. Here we report that: 1) NEM, a thiol-alkylating agent, blocks PDGF-BB-induced Akt phosphorylation without affecting its upstream PI3K; 2) NEM also blocks p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E phosphorylation induced by PDGF-BB, suggesting that thiol alkylation interferes with the PI3K/Akt pathway at the level of Akt; 3) NEM attenuates PDGF-BB-induced phosphorylation of BAD and FKHR-L1, and these events correlate with activation of caspase-3 and induction of apoptosis; 4) The blockade of PDGF-BB-induced Akt phosphorylation by NEM was completely reversed by fostriecin, okadaic acid, and fumonisin B1, specific inhibitors of PP2A and ceramide synthase, respectively, suggesting a role for these enzymes in thiol alkylationdependent inhibition of PDGF-BB-stimulated Akt phosphorylation; 5) NEM alone and in concert with PDGF-BB increased ROS production and PP2A activity in VSMC; 6) NAC, an ROS scavenger, reversed the inhibition of PDGF-BB-stimulated Akt phosphorylation by NEM; and 7) NAC also reduced the apoptosis induced by NEM alone or in combination with PDGF-BB. Together, these findings demonstrate for the first time that PP2A mediates thiol-sensitive redox regulation of Akt and cell survival.  MN). Anti-phospho-Akt (9271), anti-phospho-4E-BP1 (9451), anti-phospho-eIF4E (9741), anti-phospho-p70S6K (9205), anti-phospho-ribosomal protein S6 (2211), and anti-phospho-BAD (9295) rabbit polyclonal antibodies were obtained from Cell Signaling Technology (Beverly, MA). Anti-phospho-FKHR-L1 (06-953), anti-FKHR-L1 (06-951), and anti-PI3K (06-195) rabbit polyclonal antibodies and an Akt Immunoprecipitation Kinase Assay Kit  and Ser/ Thr Phosphatase Assay Kit (17-127) were from Upstate Biotechnology Inc. (Lake Placid, NY). Anti-Akt (SC-5298) and anti-caspase-3 (SC-7148) antibodies were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-PP2A catalytic ␣ antibodies were from Transduction Laboratories (San Diego, CA). A cell death detection enzymelinked immunosorbent assay kit was obtained from Roche Molecular Biochemicals (Indianapolis, IN). [␥-32 P]ATP (3000 Ci/mmol) was obtained from PerkinElmer Life Sciences (Boston, MA).

Reagents
Cell Culture-VSMC were isolated from the thoracic aortae of 200-to 300-g male Sprague-Dawley rats by enzymatic dissociation as described earlier (34). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 units/ml penicillin, and 100 g/ml streptomycin. Cultures were maintained at 37°C in a humidified 95% air and 5% CO 2 atmosphere. Cells were growth-arrested by incubating in DMEM containing 0.1% FBS for 72 h and used to perform the experiments unless otherwise stated.
Cell Death Assay-This assay is based on the quantitative sandwichenzyme immunoassay using mouse monoclonal antibodies directed against DNA and histones. The assay was performed according to the manufacturer's protocol (Roche Molecular Biochemicals). Cells were seeded in a 24-well culture plate at a density of 8 ϫ 10 3 cells/well in 2 ml of DMEM supplemented with 10% FBS and grown in a humidified incubator (95% air-5% CO 2 ) at 37°C. At about 80% confluence, cells were growth-arrested by incubating in DMEM with 0.1% FBS for 72 h. Growth-arrested cells were then treated with and without PDGF-BB (20 ng/ml) in the presence and absence of NEM (20 M) for 6 h. After the treatments, cell extracts were prepared, and histone-associated DNA fragments (mono-and oligonucleosomes) in the cytosolic fraction of the cell lysates were measured spectrophotometrically at 405 nm in a Spectra Max 190 microtiter plate reader.
Akt Assay-After appropriate treatments, VSMC were lysed and assayed for Akt activity using its immunoprecipitation kinase assay kit following the supplier's protocol (Upstate Biotechnology Inc.).
PI3K Assay-PI3K activity was measured as described previously (34). Briefly, after appropriate treatments, cells were lysed in 1 ml of lysis buffer (20 mM HEPES, pH 7.4, 2 mM EGTA, 50 mM ␤-glycerophosphate, 1 mM dithiothreitol, 1 mM Na 3 VO 4 , 1% Triton X-100, 10% glycerol, 2 M leupeptin, 10 units/ml aprotinin, and 400 M PMSF) for 20 min on ice. The cell lysates were cleared by centrifugation at 12,000 rpm for 15 min at 4°C. The protein content of the supernatants was determined using a Micro BCA TM protein assay reagent kit (Pierce, Rockford, IL). Five-hundred micrograms of protein from control and each treatment was immunoprecipitated with 3 g of anti-PI3K antibodies for 2 h at 4°C, followed by incubation with 40 l of 50% (w/v) protein A-Sepharose beads for an additional hour. The immunoprecipitates were washed three times with lysis buffer, three times with wash buffer, and three times with TNE buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 1 mM EDTA, and 10 M Na 3 VO 4 ). The kinase activity was measured by resuspending the immunoprecipitates in 30 l of TNE buffer and incubating with 10 l of 2 mg/ml phosphatidylinositol, 10 l of 100 mM MgCl 2 , 2 l of 100 mM ATP, and 20 Ci of [␥-32 P]ATP for 10 min at 22°C. The reaction was terminated by addition of 20 l of 5 N HCl and 200 l of chloroform:methanol (1:1) mix. The aqueous and organic phases were separated by centrifugation at 2000 rpm for 10 min. The organic phase containing the phosphoinositol phosphates was spotted onto a Silica Gel 60A TLC plate coated with 1% potassium oxalate and separated in a solvent system consisting of chloroform:methanol:water: ammonium hydroxide (90:70:14.6:5.4, v/v). The TLC plate was exposed to X-Omat AR x-ray film for 4 -6 h at Ϫ80°C and developed.
PP2A Assay-PP2A activity was measured using a kit following the supplier's instructions (Upstate Biotechnology Inc.). After appropriate treatments, VSMC were lysed in 1 ml of lysis buffer consisting of 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 2 M leupeptin, 100 units/ml aprotinin and 400 M PMSF. Cell lysates consisting of 500 g of protein from control and each treatment were immunoprecipitated with 3 g of PP2A catalytic alpha subunit (PP2Ac␣) antibodies overnight at 4°C, at which time 40 l of 50% (w/v) protein G-Sepharose CL-4B beads was added and incubation continued for another 2 h. The immunoprecipitates were washed three times with lysis buffer and resuspended in 25 l of assay buffer (50 mM Tris-HCl, pH 7.0, and 0.1 mM CaCl 2 ). The reaction was initiated by the addition of 5 l of 1 g/l phosphopeptide substrate (200 mM) (KRpTIRR) and incubating at 37°C for 10 min. The reaction was then terminated by the addition of 100 l of Malachite Green solution. The reaction mixture was spun down, and the absorbance of the supernatant was measured at 620 nm in a Spectra Max 190 microtiter plate reader (Molecular Devices Inc., Sunnyvale, CA). Phosphatase activity was calculated using a phosphate standard curve.
Reactive Oxygen Species Detection-After appropriate treatments, cells were rinsed twice with DMEM and incubated for 10 min with 1 mg/ml DCFDA in DMEM. DCF fluorescence produced by ROS was measured on an arbitrary gray scale with a Nikon Eclipse TE 300 fluorescence microscope following a previously published procedure (15).
Western Blot Analysis-After appropriate treatments, VSMC were rinsed with cold phosphate-buffered saline and frozen immediately in liquid nitrogen. Cells were lysed by thawing in 250 l of lysis buffer (phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 g/ml PMSF, 100 g/ml aprotinin, 1 g/ml leupeptin, and 1 mM sodium orthovanadate) and scraped into 1.5-ml Eppendorf tubes. After standing on ice for 20 min, the cell lysates were cleared by centrifugation at 12,000 rpm for 20 min at 4°C. Cell lysates containing equal amounts of protein were resolved by electrophoresis on 0.1% SDS and 10% polyacrylamide gels. The proteins were transferred electrophoretically onto a nitrocellulose membrane (Hybond, Amersham Biosciences, Piscataway, NJ). After blocking in 10 mM Tris-HCl buffer, pH 8.0, containing 150 mM NaCl, 0.1% Tween 20, and 5% (w/v) nonfat dry milk, the membrane was treated with appropriate primary antibodies followed by incubation with horseradish peroxidaseconjugated secondary antibodies. The antigen-antibody complexes were detected using a chemiluminescence reagent kit (Amersham Biosciences).
Statistics-All the experiments were repeated at least three times with similar results. Data for Akt, PI3K, PP2A, ROS, and apoptosis are presented as mean Ϯ S.D. The treatment effects were analyzed by Student's t test. p values Ͻ 0.05 were considered to be statistically significant. In the case of Western blot analysis, one representative set of data is shown.

RESULTS
The PI3K/Akt pathway plays an important role in cell survival and growth in response to a variety of agents, including cytokines, growth factors, and hormones (28 -33). To understand the role of thiols in redox-signaling events related to cell survival/apoptosis, we have studied the effect of thiol alkylation on activation of Akt by PDGF-BB in VSMC. NEM has been used extensively as a specific thiol-alkylating agent (36,37). To determine thiol alkylation by NEM, growth-arrested VSMC were treated with and without 20 M NEM for 30 min, and free thiols were measured using 5,5Ј-dithiobis(2-dinitrobenzoic acid) reagent (35). NEM (20 M) alkylated 60% of the available thiols in VSMC (control, 522 Ϯ 11 nmol/mg of protein versus NEM treatment, 213 Ϯ 4 nmol/mg of protein). At higher concentrations, NEM was found to be toxic to VSMC, and, therefore, it was used at 20 M concentration throughout this study. Growth-arrested VSMC were treated with and without PDGF-BB (20 ng/ml) in the presence and absence of NEM (20 M) for the indicated times, and cell extracts were prepared. Equal amounts of protein from control and each treatment were analyzed by Western blotting for Akt Ser-473 and Thr-308 phosphorylation using its phospho-specific antibodies. PDGF-BB stimulated Akt phosphorylation both on Ser-473 and Thr-308 residues in a time-dependent manner (Fig. 1A). Increases in PDGF-BB-stimulated Akt Ser-473 phosphorylation occurred at 5 min (5-fold) and peaked by 30 min (7-fold), and these levels sustained thereafter for at least 2 h. Maximal increases in PDGF-BB-stimulated Akt-308 phosphorylation were observed at 5 min (3-fold), and these levels decreased thereafter. Furthermore, PDGF-BB-induced phosphorylation of Akt was found to be severalfold higher on Ser-473 than Thr-308. NEM significantly (80%) inhibited PDGF-BB-stimulated Akt Ser-473 and Thr-308 phosphorylation. Because PDGF-BB-induced Akt phosphorylation on Ser-473 was severalfold higher than Thr-308, all the subsequent experiments were focused on Akt Ser-473 phosphorylation. To test whether the observed changes in Akt phosphorylation levels correlate with its activity, growth-arrested VSMC were treated with and without PDGF-BB (20 ng/ml) in the presence and absence of NEM (20 M) for the indicated times, and cell extracts were prepared. Equal amounts of protein from control and each treatment were assayed for Akt activity using a kit (Upstate Biotechnology Inc.). As shown in Fig. 1B, PDGF-BB induced Akt activity by 2-to 3-fold as compared with control, and it was significantly suppressed by NEM. PI3K is upstream to, and mediates Akt phosphorylation and activation in response to a variety of agonists, including growth factors and cytokines (31)(32)(33). Therefore, to understand the mechanism by which NEM inhibits PDGF-BB-stimulated Akt phosphorylation, we studied the effect of thiol alkylation on PI3K activity. PDGF-BB stimulated PI3K activity in a time-dependent manner with a 5-fold increase at 2 h (Fig. 2). Thiol alkylation by NEM alone caused an increase in PI3K activity, and it had an additive effect on PDGF-BB-induced activation of this kinase (Fig. 2).
Akt is downstream to and mediates several of the PI3K-dependent events, including phosphorylation of p70S6K, 4E-BP1, BAD, and FKHR family of transcriptional factors (31, 38 -40), although other mechanisms that are independent of PI3K and Akt have also been reported, at least, in the phosphorylation of p70S6K and 4E-BP1 (41)(42)(43). Because thiol alkylation had no effect on PDGF-BB-stimulated PI3K activity, we were interested to learn the consequences of Akt inactivation by thiol alkylation on PDGF-BB-induced phosphorylation of p70S6K and 4E-BP1 and their downstream effector molecules ribosomal protein S6 and eIF4E. To gain information on this aspect, we first studied the role of PI3K on PDGF-BB-induced phosphorylation of Akt. LY294002 (25 M) and wortmannin (1 M), two structurally different and potent inhibitors of PI3K, blocked both basal and PDGF-BB-induced phosphorylation of Akt (Fig. 3, top panel). LY294002 also inhibited PDGF-BBinduced phosphorylation of p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E (Fig. 3, lower panel). These results suggest that PDGF-BB-induced phosphorylation of Akt, p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E is PI3K-dependent. We now tested the effect of thiol alkylation on PDGF-BB-induced phosphorylation of the above molecules. NEM completely inhibited PDGF-BB-induced phosphorylation of p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E (Fig. 4).
Akt promotes cell survival via phosphorylating and inactivating pro-apoptotic molecules such as BAD and FKHR-L1 (31, 38 -40). Upon phosphorylation BAD dissociates from Bcl-2, an anti-apoptotic protein, which in turn, prevents the release of cytochrome c from the mitochondria to the cytoplasm (44). The release of cytochrome c from the mitochondria to the cytoplasm is required for activation of caspase-9, an initial event in the execution of apoptosis (44 -46). In the case of FKHR-L1, it is a FIG. 1. Thiol alkylation inhibits PDGF-BB-stimulated phosphorylation and activity of Akt. Growth-arrested VSMC were treated with and without PDGF-BB (20 ng/ml) in the presence and absence of NEM (20 M) for the indicated times and cell extracts were prepared. A, 40 g of protein from control and each treatment was analyzed by Western blotting for Akt using its Ser-473 and Thr-308 phospho-specific antibodies. B, 500 g of protein from control and each treatment was immunoprecipitated with 3 g of anti-Akt antibodies, and the kinase activity in the immunocomplexes was measured using a kit following the supplier's instructions (Upstate Biotechnology Inc.). *, p Ͻ 0.01 versus control; **, p Ͻ 0.01 versus PDGF-BB treatment. member of the forkhead family of transcriptional factors and plays a role in the regulation of cell cycle arrest and apoptosis via induction of expression of p27 kip1 and retinoblastoma-like p130 protein (38 -40). Because thiol alkylation prevented PDGF-BB-stimulated Akt phosphorylation, we also wanted to examine the effect of thiol alkylation on PDGF-BB-induced phosphorylation of its immediate substrate molecules BAD and FKHR-L1. Cell extracts of VSMC that were treated with and without PDGF-BB (20 ng/ml) in the presence and absence of NEM (20 M) for the indicated times were analyzed by Western blotting for phosphorylation of BAD and FKHR-L1 using their phospho-specific antibodies. PDGF-BB stimulated BAD Ser-136 and FKHR-L1 Ser-253 phosphorylation in VSMC (Fig. 5). Maximal increases in PDGF-BB-stimulated phosphorylation of BAD and FKHR-L1 occurred at 30 min (2-to 3-fold), and these increases were sustained thereafter for at least 2 h. NEM completely inhibited PDGF-BB-stimulated phosphorylation of BAD and FKHR-L1. To test whether inhibition of phosphorylation of BAD and FKHR-L1 by thiol alkylation correlate with activation of caspase cascade, growth-arrested VSMC were treated with and without PDGF-BB (20 ng/ml) in the presence and absence of NEM (20 M) for 2 h, and cell extracts were prepared and analyzed by Western blotting for caspase-3 using an antibody that recognizes both of its pro-and active forms. NEM, while alone causing a modest increase of 1.8-fold in the conversion of pro-caspase-3 into active form, in combination with PDGF-BB it increased active caspase-3 production by 3-fold (Fig. 6A). No active caspase-3 levels were detected in control or PDGF-BB-treated cells.
To find whether the increases in active caspase-3 levels result in increased VSMC apoptosis, growth-arrested VSMC were treated with and without PDGF-BB (20 ng/ml) in the presence and absence of NEM (20 M) for 6 h, and apoptosis was measured by determining the cytoplasmic levels of his- tone-associated DNA fragments. As expected, growth-arrested VSMC exhibited a mild basal apoptotic activity, and this was reversed in response to treatment with PDGF-BB (Fig. 6B). NEM alone and in the presence of PDGF-BB induced VSMC apoptosis by 3-and 5-fold, respectively. Earlier studies have reported that PP2A plays a role in apoptosis via dephosphorylation and inactivation of Bcl-2 and CREB (47)(48)(49). In addition, ceramide-induced apoptosis was reported to be dependent on activation of PP2A (50,51). To understand the molecular mechanism by which thiol alkylation induces apoptosis, the roles of PP2A and ceramide synthase were studied. Growth-arrested VSMC that were exposed or unexposed to NEM (20 M) were treated with and without PDGF-BB (20 ng/ml) in the presence and absence of fostriecin (1 M) or okadaic acid (1 M), two structurally different and potent inhibitors of PP2A (52,53), or fumonisin B1 (25 M), a potent inhibitor of ceramide synthase (54), for 30 min, and cell extracts were prepared. Equal amounts of protein from each condition were analyzed by Western blotting for Akt using its phospho-specific antibodies. Both PP2A and ceramide synthase inhibitors completely reversed the thiol alkylation-induced inhibition of PDGF-BB-stimulated Akt Ser-473 phosphorylation (Fig. 7). To find whether PP2A associates with Akt, co-immunoprecipitation experiments were performed. Equal amounts of protein from VSMC that were treated with PDGF-BB (20 ng/ml) in the presence and absence of NEM 20 (M) or left untreated were immunoprecipitated with 3 g of anti-PP2Ac␣ antibodies, and the resulting immunocomplexes were analyzed by Western blotting for Akt using its specific antibodies. Western blot analysis of anti-PP2Ac␣ antibody immunocomplexes with anti-Akt antibodies detected a protein with a molecular mass of 70 kDa (Fig. 8A). Con-

FIG. 6. Thiol alkylation alone and in concert with PDGF-BB induces the conversion of pro-caspase-3 into active caspase-3. A,
growth-arrested VSMC were treated with and without PDGF-BB (20 ng/ml) in the presence and absence of NEM (20 M) for 2 h, and cell extracts were prepared. 40 g of protein from control, and each treatment was analyzed by Western blotting for pro-and active caspase-3 using an antibody that recognizes both forms. B, growth-arrested VSMC were treated with and without PDGF-BB (20 ng/ml) in the presence and absence of NEM (20 M) for 6 h, and apoptosis was measured by determining the cytoplasmic levels of histone-associated DNA fragments. *, p Ͻ 0.01 versus control.  8. PP2A exists as a complex with Akt and thiol alkylation increases its activity in response to PDGF-BB. Growth-arrested VSMC were treated with and without PDGF-BB (20 ng/ml) in the presence and absence of NEM (20 M) for 30 min and cell extracts were prepared. A, 500 g of protein from control and each treatment was immunoprecipitated with anti-Akt or anti-PP2Ac␣ antibodies, and the resulting immunocomplexes were subjected to Western blot analysis using the indicated antibodies. B, 600 g of protein from control and each treatment was immunoprecipitated with anti-PP2Ac␣ antibodies, and PP2A activity was measured in the immunocomplexes using a phosphopeptide (KRpTIRR) as a substrate. *, p Ͻ 0.05 versus control.
versely, Western blot analysis of anti-Akt antibody immunocomplexes with anti-PP2Ac␣ antibodies detected a protein with a molecular mass of 36 kDa (Fig. 8A). Western blot analysis of the immunoprecipitates of non-immune serum with anti-Akt or anti-PP2Ac␣ antibodies did not detect either 70-or 36-kDa proteins (data not shown). These results suggest that PP2A exists as a complex with Akt. To obtain additional evidence for the role of PP2A in thiol alkylation-induced inhibition of PDGF-BB-stimulated Akt phosphorylation, the effect of NEM on PP2A activity was determined. Growth-arrested VSMC were treated with and without PDGF-BB (20 ng/ml) in the presence and absence of NEM (20 M) for 30 min, and cell extracts prepared. Equal amounts of protein from each condition were immunoprecipitated with anti-PP2Ac␣ antibodies, and PP2A activity was measured in the immunocomplexes using a phosphopeptide (KRpTIRR) as a substrate. NEM increased PP2A activity 1.7-fold, alone and in concert with PDGF-BB (Fig. 8B).
Redox regulation of ceramide production by cytokines has been reported previously (54). To understand the mechanism by which NEM activates PP2A and thereby suppresses PDGF-BB-stimulated Akt phosphorylation, we tested its effect on ROS production. Growth-arrested VSMC were treated with and without PDGF-BB (20 ng/ml) in the presence and absence of NEM (20 M) for 30 min and ROS production was measured by DCF fluorescence (15). As shown in Fig. 9, NEM alone and in combination with PDGF-BB increased ROS production by 2and 5-fold, respectively, as compared with control. PDGF-BB also increased ROS production at levels those are lower than the levels produced by NEM alone or in combination with PDGF-BB. In addition, although the effect of PDGF-BB on ROS production was found to be acute, the effects of NEM and NEM and PDGF-BB on ROS production were found sustained (data not shown). To determine the role of ROS in NEM-induced inhibition of PDGF-BB-stimulated Akt phosphorylation, we studied the effect of NAC, an ROS scavenger. Growth-arrested VSMC that were exposed or unexposed to NAC (20 mM) were treated with and without PDGF-BB (20 ng/ml) in the presence and absence of NEM (20 M) for 30 min, and Akt phosphorylation was measured. NAC completely reversed NEM-induced inhibition of PDGF-BB-stimulated phosphorylation of Akt (Fig.  10A). NAC also completely suppressed the apoptosis induced by NEM alone or in combination with PDGF-BB (Fig. 10B). NAC alone had no effect either on Akt phosphorylation or apoptosis. DISCUSSION The important findings of the present study are as follows: 1) Thiol alkylation inhibited PDGF-BB-stimulated phosphorylation of Akt and its downstream effector molecules, p70S6K, 4E-BP1, BAD, and FKHR-L1; 2) Decreased p70S6K and 4E-BP1 phosphorylation also led to a decrease in the phosphorylation state of their effector molecules ribosomal protein S6 and eIF4E, respectively; 3) Decreased PDGF-BB-stimulated BAD and FKHR-L1 phosphorylation by thiol alkylation correlated with increased active caspase-3 production and apoptosis; 4) The inhibition of PDGF-BB-stimulated Akt phosphorylation by thiol alkylation exhibited a requirement for activation of PP2A and ceramide synthase; 5) PP2A was found to be associated with Akt and thiol alkylation alone and in concert with PDGF-BB increased PP2A activity; and 6) Thiol alkylation increased ROS production by PDGF-BB, and NAC, an ROS scavenger, reversed NEM-induced inhibition of PDGF-BBstimulated Akt phosphorylation. A large number of studies have demonstrated that PI3K-dependent Akt activation plays a critical role in cell survival (28,29). One of the several

FIG. 10. NAC reverses thiol alkylation-induced inhibition of PDGF-BB-stimulated Akt phosphorylation and apoptosis.
Growth-arrested VSMC that were exposed or unexposed to NAC (20 mM) were treated with and without PDGF-BB (20 ng/ml) in the presence and absence of NEM (20 M) for 30 min, and cell extracts were prepared. A, 40 g of protein from control and each treatment was analyzed by Western blotting for Akt using its Ser-473 phospho-specific antibodies. B, growth-arrested VSMC that were exposed or unexposed to NAC (20 mM) were treated with and without PDGF-BB (20 ng/ml) in the presence and absence of NEM (20 M) for 6 h, and apoptosis was measured by determining the cytoplasmic levels of histone-associated DNA fragments. *, p Ͻ 0.01 versus control; **, p Ͻ 0.01 versus NEM ϩ PDGF-BB or NEM treatment. NAC was added to cells 2 h prior to the addition of NEM. mechanisms by which Akt enhances the cell survival activity is the phosphorylation of BAD, a pro-apoptotic protein, thereby causing it to dissociate from Bcl-2, an anti-apoptotic protein, which in turn, perhaps via inhibiting the activity of voltage-dependent anion channels present in the outer mitochondrial membranes, prevents the release of cytochrome c from the mitochondria to the cytoplasm (30, 44 -46). In the cytoplasm, cytochrome c activates caspase-9, which, in turn, induces the conversion of pro-caspase-3 into active caspase-3 (44,45). It has been reported that ceramides induce apoptosis as well as mediate cytokine-induced apoptosis (55,56). Ceramides have also been shown to activate PP2A (50,51), and a role for PP2A in apoptosis has been demonstrated (47). Importantly, redox regulation of ceramide production and voltage-dependent anion channel activity has also been reported (55,57). The other mechanism by which Akt promotes cell survival is the phosphorylation and inactivation of forkhead family of transcriptional factors such as FKHR-L1 leading to down-regulation of expression of cell cycle arrest molecules p27 kip1 and retinoblastoma-like p130 protein (38 -40). Our results show that thiol alkylation suppresses PDGF-BB-induced phosphorylation of Akt downstream to PI3K via involving PP2A and ceramide synthase leading to dephosphorylation and activation of proapoptotic molecules BAD and FKHR-L1. Because thiol alkylation increased ROS production by PDGF-BB, and NAC, an ROS scavenger, prevented NEM-induced inhibition of PDGF-BBstimulated Akt phosphorylation and apoptosis, it is likely that ROS via generation of ceramides activate PP2A. Activated PP2A via dephosphorylating suppresses PDGF-BB-stimulated phosphorylation of Akt. The facts that PP2A exists as a complex with Akt and the ability of the inhibitors of ceramide synthase (fumonisin B1), PP2A (fostriecin and okadaic acid), and ROS scavenger (NAC) to reverse the thiol alkylation-induced inhibition of PDGF-BB-stimulated Akt phosphorylation strongly support such a mechanism. It was also reported that PP2A associates with, dephosphorylates, and inactivates Akt in response to integrin ␣ 2 ␤ 1 in cells adherent to collagen (58). Thiol alkylation by NEM, although having no effect on PDGF-BB-induced PI3K activity, inhibited the PDGF-BB-stimulated phosphorylation of Akt as well as BAD, FKHR-L1, p70S6K, ribosomal protein S6, 4E-BP1, and eIF4E suggests that thiolsensitive redox mechanisms exert a second level of regulation of cell survival events independent of PI3K and dependent on Akt. Because thiol alkylation inhibited PDGF-BB-stimulated Akt phosphorylation via producing ROS and activating ceramide synthase and PP2A, these enzymes appear to be critical in redox regulation of Akt and cell survival.
A large number of studies have shown that PI3K/PDK/Akt/ mTOR pathway plays a major role in agonist-induced phosphorylation of 4E-BP1 and p70S6K (42, 59 -61). 4E-BP1 binds to eIF4E and represses translation (59). Upon phosphorylation it dissociates from, and facilitates the phosphorylation and activation of eIF4E by its upstream kinases, resulting in an enhancement in its translation activity (41)(42)(43)59). In the case of p70S6K, it phosphorylates and activates ribosomal protein S6, an event that is important in the initiation of translation. PDGF-BB-induced phosphorylation of 4E-BP1, eIF4E, p70S6K, and ribosomal protein S6, to a major extent, is dependent on PI3K in VSMC, because LY294002, a specific inhibitor of this enzyme, completely suppressed these effects. Similarly, PDGF-BB-induced Akt phosphorylation is dependent on PI3K activity. It was reported that peptide inhibitors of eIF4E activity induces apoptosis (62). Based on these observations, a role for a decreased translational activity could not be ruled out in thiol alkylation-induced apoptosis in PDGF-BBtreated VSMC. In addition, earlier studies have reported that CREB plays a role in cell survival and PP2A dephosphorylates and inactivates it (48,49). PP2A was also reported to dephosphorylate Bcl-2 and cause apoptosis in other cell types (50). In view of these findings, it is possible that thiol alkylationinduced activation of PP2A affects both the apoptotic and translation activities by simultaneous dephosphorylation of the molecules of these pathways, including Akt and CREB. In any case, the present study for the first time demonstrates that PP2A mediates thiol-sensitive redox regulation of Akt and apoptosis.
Acknowledgments-We are thankful to Drs. Christopher M. Waters and R. K. Rao for allowing us to use their Nikon Eclipse TE 300 fluorescence microscope and Spectra Max 190 microtiter plate reader, respectively.
Addendum-While this report was being submitted for publication, a study from other laboratories reported that overexpression of calreticulin induces apoptosis in rat cardiac myoblast H9c2 cells via dephosphorylation of Akt (63). This study further showed that the decrease in Akt phosphorylation correlates with an increase in PP2A activity, and this event is independent of PI3K. Thus, the above study and ours have independently reached similar conclusions on the role of PP2A in the dephosphorylation of Akt leading to apoptosis in two different cell types in response to two different cellular stress conditions.