Glutaredoxin Exerts an Antiapoptotic Effect by Regulating the Redox State of Akt*

Glutaredoxin (GRX) is a small dithiol protein involved in various cellular functions, including the redox regulation of certain enzyme activities. GRX functions via a disulfide exchange reaction by utilizing the active site Cys-Pro-Tyr-Cys. Here we demonstrated that overexpression of GRX protected cells from hydrogen peroxide (H2O2)-induced apoptosis by regulating the redox state of Akt. Akt was transiently phosphorylated, dephosphorylated, and then degraded in cardiac H9c2 cells undergoing H2O2-induced apoptosis. Under stress, Akt underwent disulfide bond formation between Cys-297 and Cys-311 and dephosphorylation in accordance with an increased association with protein phosphatase 2A. Overexpression of GRX protected Akt from H2O2-induced oxidation and suppressed recruitment of protein phosphatase 2A to Akt, resulting in a sustained phosphorylation of Akt and inhibition of apoptosis. This effect was reversed by cadmium, an inhibitor of GRX. Furthermore an in vitro assay revealed that GRX reduced oxidized Akt in concert with glutathione, NADPH, and glutathione-disulfide reductase. Thus, GRX plays an important role in protecting cells from apoptosis by regulating the redox state of Akt.

The redox status of sulfhydryl groups is important to cellular functions such as the synthesis and folding of proteins and regulation of the structure and activity of enzymes, receptors, and transcription factors. To maintain the cellular thiol-disulfide redox status under reducing conditions, living cells possess two major systems, the thioredoxin (TRX) 1 /thioredoxin reductase system and the glutathione (GSH)/glutaredoxin (GRX) system (1).
GRX, also known as thioltransferase, was first discovered as a GSH-dependent hydrogen donor for ribonucleotide reductase in Escherichia coli mutants lacking TRX (2). GRX functions via a disulfide exchange reaction by utilizing the active site Cys-Pro-Tyr-Cys, which specifically and efficiently catalyzes the reduction of protein-S-S-glutathione mixed disulfide (3). Oxidized GRX is selectively recycled to the reduced form by GSH with the formation of glutathione disulfide (GSSG) and regeneration of GSH by coupling with NADPH and GSSG reductase, termed the GSH-regenerating system (4,5). These characteristic interactions of GRX with GSH distinguish it from TRX, which favors intramolecular disulfide substrates and is turned over by NADPH and thioredoxin reductase independent of GSH. Functional overlap or cross-talk between the two systems, however, has been indicated (6,7). GRX also partially shares its function as a redox sensor with TRX (8,9). Although GRX has been shown to play an important role in cytoprotection against oxidative stress (10,11) and apoptosis (12,13), the precise mechanism of the antiapoptotic effect of GRX has not been fully elucidated.
The serine/threonine kinase Akt is a critical component of an intracellular signaling pathway that exerts effects on survival and apoptosis (14). The unphosphorylated form of Akt is virtually inactive, and phosphorylation at Thr-308 and Ser-473 stimulates its activity. Inactivation of Akt also occurs via dephosphorylation of the two phosphorylation sites by protein phosphatase 2A (PP2A) (15,16). Akt activation contributes to the survival of hydrogen peroxide (H 2 O 2 )-treated cells (17). Although H 2 O 2 induces the transient activation of Akt following dephosphorylation and degradation (17)(18)(19), the precise mechanism of H 2 O 2 -induced dephosphorylation of Akt is not well understood. Recently the crystal structure of an inactive Akt2 kinase domain has been deduced. Inactive Akt2 develops a redox-sensitive disulfide bond in its activation loop (20), which suggests that Akt is a redox-regulated protein.
Here we described a novel mechanism for the antiapoptotic effect of GRX via regulation of the redox state of Akt under oxidative stress. An intramolecular disulfide bond formed between Cys-297 and Cys-311 of Akt in cardiac H9c2 cells treated with H 2 O 2 . Overexpression of GRX inhibited oxidation of Akt and protected cells from apoptosis.

EXPERIMENTAL PROCEDURES
Reagents-Anti-mouse GRX antibody was affinity-purified from the serum of a rabbit immunized with a C-terminal 16-mer peptide of mouse GRX (mouse GRX-(91-106)). Anti-PP2A scaffolding A subunit (PR65) antibody was obtained from Santa Cruz Biotechnology. Anti-Akt, anti-phospho(Ser-473)-Akt, anti-phospho(Thr-308)-Akt, anti-Akt5G3, and anti-Akt1G1 antibodies were from Cell Signaling Technology. Anti-PP2A catalytic C subunit (PP2Ac) antibody was from BD Transduction Laboratories. Anti-Myc tag antibody, Akt1 cDNA Allelic Pack, and purified recombinant Akt protein (Akt/inactive and Akt/ active) were from Upstate Biotechnology. c-Myc monoclonal antibody-* This work was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Cell Culture-H9c2 cells, a clonal line derived from embryonic rat heart, were obtained from American Type Culture Collection (CRL-1446). H9c2 cells and gene-transfected cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum in a humidified atmosphere of 95% air and 5% CO 2 at 37°C.
Vector Construction-Full-length mouse GRX cDNA subcloned into pBluescript SK(ϩ) was obtained as described previously (21). The mouse GRX open reading frame was amplified by PCR techniques. As the 5Ј-primer oligonucleotides, 5Ј-CGGGGATCCATGGCTCAGGAGTT-TGTGAACTGC-3Ј, which annealed to the 5Ј-end of GRX cDNA and introduced a BamHI site, and as the 3Ј-primer oligonucleotides, 5Ј-CTCGAATTCTTATAACTGCAGAGCTCCAATCTG-3Ј complementary to the 3Ј terminus of the GRX cDNA and inserting an EcoRI site, were used. The amplified DNA fragment was digested with BamHI and EcoRI and then cloned into the pCMV-tag2B expression vector (Stratagene). GRX cDNA accompanied at the 5Ј-end with the FLAG sequence (5ЈFLAG-GRX) was digested with NotI and EcoRV and then cloned into NotI/EcoRV-cut pTRE2-Hyg (BD Biosciences Clontech) and termed pTRE2Hyg-GRX. 5ЈFLAG-GRX was also digested with EcoRI and XhoI and then cloned into EcoRI-XhoI-cut pGEX-6p-1 (Amersham Biosciences) and termed pGEX-GRX. The nucleotide sequences were confirmed by sequencing with an ALFexpress II system (Amersham Biosciences).
Site-directed Mutagenesis-The QuikChange XL site-directed mutagenesis kit (Stratagene) was used to make point mutations of cAkt cDNA. The following are the various primers, which were used for converting two cysteine residues (Cys-297 and Cys-311) to serine in cAkt cDNA to create mutants: sense primer oligonucleotide (5Ј-GACT-TCGGGCTGTCCAAGGAGGGGATC-3Ј) and antisense primer oligonucleotide (5Ј-GATCCCCTCCTTGGACAGCCCGAAGTC-3Ј) for C297S; sense primer oligonucleotide (5Ј-GGGGCCAGGTACTCCGGCGTTCC-GGAGAATGTCTTCATAGTGGC-3Ј) and antisense primer oligonucleotide (5Ј-GCCACTATGAAGACATTCTCCGGAACGCCGGAGTACCTG-GCCCC-3Ј) for C311S. A double mutant (C297S/C311S) was constructed using the cAkt-C297S single mutant as a DNA template and primers for C311S. These experiments were performed according to the manufacturer's protocol. The nucleotide sequences were confirmed by sequencing with an ALFexpress II system.
Gene Transfection and Selection of Cells-Gene transfection was performed using LipofectAMINE Plus reagent (Invitrogen) according to the manufacturer's protocol. A Tet-On gene expression system (BD Biosciences Clontech) was used to establish the cell line overexpressing GRX. First, H9c2 cells were transfected with the pTet-on regulation vector. Stable transfectants were screened by culturing with 500 g/ml G418. The cloned G418-resistant cells were then transfected with pTRE2hyg or pTRE2Hyg-GRX using the same procedure as for pTet-on. Stable transfectants were screened with 100 g/ml hygromycin B. The cloned G418-resistant and hygromycin B-resistant cells were screened for expression of GRX. After screening, cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum containing 75 g/ml G418 and 75 g/ml hygromycin B.
Thioltransferase Activity Assay-Thioltransferase activity was assayed as described previously (5). In brief, cell lysate or purified mouse GRX was mixed with a reaction buffer consisting of 137 mM Tris-HCl buffer (pH 8.0), 0.5 mM GSH, 1.2 units of GSSG reductase, 2.5 mM Cys-SO 3 , 0.35 mM NADPH, and 1.5 mM EDTA (pH 8.0). The reaction proceeded at 30°C, and thioltransferase activity was measured spectrophotometrically at 340 nm. The net enzymatic reaction rate was obtained by subtraction of the non-enzymatic reaction rate from the total rate.
Apoptosis Assay-Apoptosis was detected by flow cytometry with the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) method using an ApopTag Plus fluorescein in situ apoptosis detection kit (Intergen) as described previously (16).
Immunoblot Analysis-Cultured cells were harvested and lysed for 20 min at 4°C in lysis buffer as described previously (16). The supernatants obtained by centrifugation of the lysates at 8000 ϫ g for 15 min were used in subsequent experiments. Protein concentrations were determined using a BCA assay kit (Pierce). Protein samples were electrophoresed on 10, 12.5, or 15% SDS-polyacrylamide gels under reducing conditions with the exception of thiol-modified protein samples. The proteins in the gels were transferred onto a nitrocellulose membrane. The membranes were blocked in Tris-buffered saline (10 mM Tris-HCl (pH 7.5) and 0.15 M NaCl; TBS) containing 0.05% (v/v) Tween 20 (TBST) and 5% (w/v) nonfat dry milk and then reacted with primary antibodies in TBST containing 3% (w/v) bovine serum albumin overnight with constant agitation at 4°C. After several washes with TBST, the membranes were incubated with horseradish peroxidase-conjugated anti-IgG antibodies. Proteins in the membranes were then visualized using the enhanced chemiluminescence (ECL) detection kit (Amersham Biosciences) according to the manufacturer's instructions.
Akt Activity Assay-Akt activity was assayed using an Akt assay kit (Cell Signaling Technology) according to the manufacturer's protocol with GSK3␣/␤ fusion protein (GSK3␣/␤) as a substrate. Phosphorylation of GSK3␣/␤ was assessed by immunoblot analysis using specific antibody.
Protein Phosphatase Assay-PP2A activity was assayed spectrophotometrically using the Ser/Thr phosphatase assay kit 1 (Upstate Biotechnology) according to the manufacturer's protocol. The phosphopeptide RKpTIRR (where pT is phosphothreonine) and p-nitrophenyl phosphate were used as phosphatase substrates.
Cell Viability Assay-The viability of cultured cells was evaluated using MTT as described previously (22). The cells (5ϫ10 3 ) were placed in 100 l of medium/well in 96-well plates and cultured overnight. After treatment with or without H 2 O 2 for a period of time, 10 l of 0.5% MTT solution was added, and the cells were incubated for 4 h. The reaction was stopped by adding 100 l of lysis buffer (20% SDS, 50% N,Ndimethylformamide (pH 4.7)), and then cell viability was evaluated by measuring the absorbance at 570 nm using a microplate reader.
Lactate Dehydrogenase (LDH) Release Assay-The activity of LDH released into the medium was measured with an MTX-LDH kit (Kyokuto Pharmaceutical Industrial Co., Ltd., Tokyo, Japan) according to the manufacturer's instructions. The activity of the cytoplasmic enzyme released was shown as a percentage of LDH activity in the medium over the total enzyme activity. Total enzyme activity was determined by measuring the LDH activity in the lysate of cells treated with 0.2% Tween 20, which caused complete cell death.
Determination of Redox States-The redox states of proteins were assessed by modifying free thiol with AMS (23). Briefly, after incubation with or without H 2 O 2 , cell lysates or proteins were treated with trichloroacetic acid at a final concentration of 7.5% to denature and precipitate the proteins as well as to avoid any subsequent redox reactions. The protein precipitates were collected by centrifugation at 12,000 ϫ g for 10 min at 4°C, washed with acetone twice, and dissolved in a buffer containing 50 mM Tris-HCl (pH 7.4), 1% SDS, and 15 mM AMS. Proteins were then separated by 10% SDS-PAGE without using any reducing agents and blotted to a nitrocellulose membrane. Proteins in the membranes were then visualized by immunoblotting as described above.
Protein Purification-FLAG-tagged mouse GRX was purified with a GST gene fusion system (Amersham Biosciences) according to the manufacturer's protocol. In brief, competent E. coli strain BL-21(DE3) cells were transformed with pGEX-GRX, and expression was induced by adding 1 mM isopropyl-1-thio-␤-D-galactopyranoside for 3 h at 37°C. GST-fused GRX (GST-GRX) was affinity purified from cell lysates using glutathione-Sepharose 4B, and digested with PreScission protease. The cleaved GST was removed with glutathione-Sepharose 4B.
Peroxide Quantification-Peroxide was quantified using the PeroXOquant quantitative peroxide assay (Pierce) according to the manufacturer's instructions. In brief, H 2 O 2 was incubated in buffer containing components of the GSH/GRX system as indicated in Fig.7E at room temperature for 30 min. After a 1:10 dilution of each sample was made, 10 volumes of working reagent was added to 1 volume of diluted sample and mixed well. After incubation at room temperature for 15-20 min, the purple product composed of Fe 3ϩ -xylenol orange complex was detected spectrophotometrically at 570 nm.

Establishment and Characterization of H9c2 Cells
Overexpressing the GRX Gene-To investigate the functional effect of the overexpression of GRX on the intracellular redox state, we constructed a FLAG-tagged GRX gene expression vector and transfected rat cardiac H9c2 cells with it. The Tet-On gene expression system was utilized to obtain H9c2 cells stably overexpressing GRX. After the two-step screening of G418resistant and hygromycin B-resistant transfectants, the expression level of GRX was characterized immunologically. We obtained three clones (H9c2-GRX22, H9c2-GRX30, and H9c2-GRX49) that overexpressed GRX without doxycycline, so-called leaky expression (Fig. 1A). Although the anti-mouse GRX antibody was useful in detecting rat brain GRX immunohistochemically (24), the expression of GRX in parental and mocktransfected H9c2 cells (H9c2-Vector) was immunologically undetectable. We used no doxycycline to induce further expression of GRX in any experiments. These clones have more thioltransferase activity than parental and H9c2-Vector cells (Fig. 1B).
Overexpression of GRX Protects H9c2 Cells from H 2 O 2 -induced Apoptosis-A lower concentration (Ϫ400 M) of H 2 O 2 induces apoptosis or early mitochondrial dysfunction followed by a loss of plasma membrane integrity in H9c2 cells (25,26). To examine the functional role of overexpressed GRX in protecting H9c2 cells against oxidative stress, mock-transfected and GRX gene-transfected H9c2 cells were treated with H 2 O 2 . As shown in Fig. 2A, the MTT assay revealed that 100 M H 2 O 2 decreased the viability of H9c2-Vector cells in a time-dependent manner but not that of GRX gene-transfected cells. In the LDH release assay, loss of plasma membrane integrity was observed in H9c2-Vector cells treated with H 2 O 2 but not in H9c2-GRX49 cells (Fig. 2B). A TUNEL assay was carried out to clarify whether apoptosis contributed to the cell damage seen in H9c2-Vector cells treated with H 2 O 2 . An increase in fluorescence intensity derived from DNA strand breaks was detected in H9c2-Vector cells treated with H 2 O 2 but not in H9c2-GRX49 cells (Fig. 2C). H9c2-GRX22 and H9c2-GRX30 cells showed results similar to H9c2-GRX49 cells in the LDH release assay and TUNEL assay (data not shown).

Involvement of Akt Signaling Pathway in the Protective Effect of GRX against H 2 O 2 -induced Apoptosis-
The importance of the Akt signaling pathway in protecting cardiomyocytes from apoptosis has been reported (27). We investigated the phosphorylation of Akt immunologically in the cells treated with 100 M H 2 O 2 . In H9c2-Vector cells, Akt activity increased to a maximum 10 -30 min after the addition of H 2 O 2 and then returned to basal levels by 60 min. After 120 min of treatment, Akt underwent degradation. On the other hand, a sustained phosphorylation of Akt for at least 240 min was observed without degradation in H9c2-GRX49 cells treated with H 2 O 2 (Fig. 3,  A and B).
To clarify the significance of the sustained activation of the Akt signaling pathway in protecting H9c2 cells from apoptosis under oxidative stress, H9c2 cells transfected with myr-Akt1-pUSEamp(ϩ), which expresses an N-terminal myristoylated constitutively active Akt (myrAkt), were treated with 100 M H 2 O 2 for 2 h, and apoptosis was evaluated by TUNEL assay. Overexpression of myrAkt enhanced intracellular Akt kinase activity (Fig. 3, C, D, and E). Treatment with 100 M H 2 O 2 induced apoptosis in pUSEamp(ϩ) vector-transfected cells (H9c2-Vector2) but not in cells overexpressing myrAkt (H9c2-myrAkt) (Fig. 3F).  Cells were treated with 100 M H 2 O 2 for the period indicated. Phosphorylation of Akt was detected by immunoblot analysis using specific antibodies as described under "Experimental Procedures." B, the band intensity was estimated densitometrically, and the phosphorylation rates are expressed as the relative intensity of phosphorylated Akt to total Akt (pAkt/Akt). C, pUSEamp(ϩ) or myr-Akt1-pUSEamp(ϩ) was introduced into H9c2 cells as described under "Experimental Procedures." After 48 h, cells were harvested, and overexpression of myrAkt in H9c2-myrAkt cells was detected by immunoblot analysis using specific antibodies as described under "Experimental Procedures." Anti-Akt antibody detected both myrAkt and endogenous Akt in H9c2-myrAkt. D, after 48 h of transfection of pUSEamp(ϩ) or myr-Akt1-pUSEamp(ϩ) as described above, Akt was immunoprecipitated (IP) from cell lysates with anti-Akt monoclonal antibody-conjugated and c-Myc monoclonal antibody-conjugated agarose beads. Akt activity was measured by phosphorylation of GSK3␣/␤ as described under "Experimental Procedures." E, the band intensity was estimated densitometrically. F, apoptosis was evaluated as in Cadmium Diminished the Protective Effect of GRX on H 2 O 2induced Apoptosis-Cadmium is an inhibitor of GRX (12). Treatment with 200 M CdCl 2 reduced cellular thioltransferase activity by over 60% in H9c2-GRX49 cells (Fig. 6A). The LDH release assay (Fig. 6B) and MTT assay (data not shown) re-vealed that treatment with 100 M H 2 O 2 in the presence of 200 M CdCl 2 caused cell damage to H9c2-GRX49 cells similar to that seen with 100 M H 2 O 2 alone in H9c2-Vector cells (Fig. 2,  A and B). Pretreatment with 200 M CdCl 2 did not enhance the cytotoxic effect of H 2 O 2 in H9c2-Vector cells (compare Fig. 6B to Fig. 2B). Treatment with H 2 O 2 in the presence of CdCl 2 induced apoptosis in H9c2-GRX49 cells, but CdCl 2 alone did not (Fig. 6C). Cadmium also blocked the sustained activation of Akt observed in H9c2-GRX49 cells treated with H 2 O 2 alone (Fig. 6, D and E). Furthermore treatment with H 2 O 2 in the presence of CdCl 2 induced Akt oxidation in a time-dependent manner (Fig. 6F) and enhanced interaction between Akt and PP2A in H9c2-GRX49 cells (Fig. 6G). These results strongly support that GRX plays an important role in regulating the redox state of Akt under oxidative stress in vivo.
Redox Regulation of Akt by the GSH/GRX System in Vitro-To clarify whether the GSH/GRX system directly regulates the redox state of Akt, we examined whether GRX could protect Akt from disulfide bond formation under oxidative stress in vitro. First we purified mouse GRX using the GST gene fusion system (Fig. 7A). Purified GRX expressed thioltransferase activity (Fig. 7B). Then we examined the effect of purified GRX on the redox state of recombinant Akt under oxidative stress with H 2 O 2 . Inactive Akt existed as a fully oxidized form. On the other hand, active Akt existed as both reduced and partially oxidized forms (Fig. 7C). After reduction with 100 mM DTT for 1 h on ice following its removal by gel filtration, active Akt was incubated with or without 1 mM H 2 O 2 under various conditions (Fig. 7D). H 2 O 2 directly oxidized Akt independent of GSH (lane 2). GRX protected Akt from oxidation in a dose-dependent manner in the presence of the GSHregenerating system (GSH/GSSG and NADPH/GSSG reductase, lanes 10 and 12-15). Unexpectedly GRX oxidized active Akt in GSH/GSSG buffer without H 2 O 2 (lane 5). Yeast GRX1 and GRX2 possess glutathione peroxidase activity (28). The TRX/thioredoxin reductase and GSH/GRX systems are efficient electron donors to human plasma glutathione peroxidase, which exists where GSH levels are low (29). We also measured the peroxide scavenging activity of mouse GRX in vitro (Fig.  7E). GRX alone had the ability to reduce peroxide (lane 9) and worked more efficiently in the presence of the GSH/GSSG buffer (lane 10) or GSH-regenerating system (lanes [11][12][13]. Under the same conditions as in Fig. 7D, lane 15, GRX scavenged ϳ60% of 1 mM H 2 O 2 . However, 250 M H 2 O 2 was enough to fully oxidize Akt with the GSH-regenerating system (data not shown). Taken together, mouse GRX regulates the redox state of Akt in concert with the GSH-regenerating system independent of its peroxide scavenging activity under oxidative stress.
Recombinant active Akt was reduced by incubation with DTT or oxidized by incubation with GRX in the GSH/GSSG buffer. The activity of the active form of Akt was not influenced by further reduction or oxidation (Fig. 8). DISCUSSION We have shown that when overexpressed GRX regulated the redox state of Akt, resulting in the protection of H9c2 cells against apoptosis under oxidative stress, and that the GSH/ GRX system protected Akt from H 2 O 2 -induced disulfide bond formation in vitro. Akt existed predominantly in the reduced form in the cells not under oxidative stress. Akt developed a disulfide bond between Cys-297 and Cys-311 following treatment with H 2 O 2 , accompanying an increased association with PP2A. This is one possible mechanism for the transient activation of Akt following dephosphorylation under oxidative stress. Overexpression of GRX prevented Akt from developing a disulfide bond and associating with PP2A under oxidative stress. The importance of the redox regulation of protein functions through the formation of an intramolecular disulfide bond has been demonstrated from E. coli to mammals, and both GRX and TRX are involved in this mechanism. In E. coli, the transcription factor OxyR is activated through intramolecular disulfide bond formation and is inactivated by enzymatic reduction with GRX1. TRX is also capable of reducing OxyR in vitro (30). OxyR is sensitive to oxidation and activates the expression of antioxidant genes in response to H 2 O 2 . The gene encoding GRX1 is regulated by OxyR (31), thus providing a mechanism for autoregulation. Likewise RsrA, an anti-factor in Streptomyces coelicolor, is regulated by redox change, and TRX reduces oxidized RsrA (32). RsrA-TRX is also suggested to create feedback homeostasis loops for its own expression. Another transcription factor and a molecular chaperone have also been shown to be activated by intramolecular disulfide bond formation (33,34). Yap1, a functional homologue of the bacte-rial OxyR, regulates hydroperoxide homeostasis in Saccharomyces cerevisiae. Although Yap1 is activated by oxidation, it is not directly oxidized. Glutathione peroxidase 3 was identified as a second component of the pathway, serving as a sensor and transducer of the hydroperoxide signal sent to Yap1 (35). TRX turns off the pathway by reducing both sensor and regulator (35,36). In mammalian HeLa and NIH3T3 cells, the tumor suppressor PTEN undergoes reversible intramolecular disulfide bond formation and inactivation by H 2 O 2 (37), which is also one of the mechanisms of transient activation of Akt following dephosphorylation under oxidative stress. Although the best candidate for the intracellular reducing agent of oxidized PTEN was considered to be TRX, the role of the GSH/GRX system was not fully elucidated.
Transcription of GRX is regulated by an oxidative stressinduced transcription factor from E. coli to mammals. Recently the human GRX gene has been reported to be regulated by the transcription factor AP-1 under oxidative stress in lens epithelial cells (38). GRX protects cerebellar granule neurons from dopamine-induced cell death by dual activation of the Rasphosphatidylinositol 3-kinase-Akt and c-Jun N-terminal kinase pathways (13), indicating the existence of cross-talk between the two pathways. Furthermore GSK3, a substrate of Akt, negatively regulates the transcription factor AP-1 (39,40). These findings imply the existence of an Akt-GRX autoregulation loop like OxyR-GRX1 or RsrA-TRX.
In  A, measurement of kinase activity of reduced or oxidized active Akt. Recombinant active Akt was incubated with 100 mM DTT on ice for 1 h or with 1 mM GSH, 0.05 mM GSSG, and 40 g of mouse GRX at room temperature for 30 min to form reduced (Red) or oxidized (Ox) active Akt, respectively. Akt activity was measured by phosphorylation of GSK3␣/␤ as described under "Experimental Procedures." B, the band intensity was estimated densitometrically, and the Akt kinase activity is expressed as the relative intensity of phosphorylated GSK3␣/␤/ immunoprecipitated Akt. The data represent three independent experiments. IP, immunoprecipitation; IB, immunoblot; pGSK3␣/␤, phospho-GSK3␣/␤. Cys-297 forms a redox-sensitive disulfide bond with Cys-311 (20). Akt belongs to the so-called AGC superfamily of serine/ threonine kinases. The cysteine residues identical to Cys-297 and Cys-311 of Akt are conserved in most members of the AGC superfamily, such as p70-S6K, serum-and glucocorticoid-inducible kinase, protein kinase C-related kinase 2, and protein kinase C. Several studies have reported that the AGC superfamily underwent oxidative regulation (41). Although the precise mechanisms of redox regulation of these kinases have not been elucidated, disulfide bond formation in the conserved cysteine residues would provide a possible explanation.
The effect of thiol alkylation on platelet-derived growth factor-BB-induced cell survival events has been studied (42). Platelet-derived growth factor-BB-induced Akt phosphorylation was found to be blocked by N-ethylmaleimide at the level of Akt, which supports our findings that Akt undergoes redox regulation. N-Ethylmaleimide alone or in concert with plateletderived growth factor-BB increased PP2A activity, which depended on ceramide production following reactive oxygen species generation. In our study, no increase of PP2A activity was observed (Fig. 5A), but interaction between Akt and PP2A increased coinciding with Akt oxidation and dephosphorylation (Fig. 5B) in H9c2-Vector cells treated with H 2 O 2 . The redox state of Akt itself, however, did not affect the Akt activity in vitro (Fig. 8). These results imply the relevance of PP2A in dephosphorylation of Akt after transient phosphorylation under oxidative stress.
PP2A consists of three subunits (43). The core enzyme is a dimer, consisting of a catalytic C subunit (PP2Ac) and a scaffolding A subunit (PR65). A third regulatory B subunit can be associated with this core structure. The B subunit constitutes four different families, which can bind to the AC dimer to form a wide variety of heterotrimeric complexes. B subunits determine the substrate specificity, subcellular localization, and catalytic activity of the core enzyme. Although glutathionylation is one of the mechanisms of regulation of PP2A activity (44), PP2Ac and PR65 did not undergo redox regulation under oxidative stress (Fig. 5A). The regulatory B subunit specific for Akt, although unidentified, might be redox-regulated and be another key regulator of Akt dephosphorylation under oxidative stress.
In summary, GRX plays an important role in protecting cells from H 2 O 2 -induced apoptosis by regulating the redox state of Akt through the GSH-regenerating system. The identification of other redox-sensitive proteins regulated by GRX is crucial for understanding further the antiapoptotic functions of GRX.