Syk Is Required for the Activation of Akt Survival Pathway in B Cells Exposed to Oxidative Stress*

Syk has been demonstrated to play a crucial role in oxidative stress signaling in B cells. Here we report that Syk is required for the activation of the phosphatidylinositol (PI) 3-kinase-Akt survival pathway in B cells exposed to oxidative stress. Phosphorylation and activation of the serine-threonine kinase Akt were markedly increased in B cells treated with H2O2. In Syk-deficient DT40 cells treated with low doses of H2O2 (10–100 μm), Akt activation was considerably reduced. Pretreatment with wortmannin, a PI 3-kinase-specific inhibitor, completely blocked the Syk-dependent Akt activation. Following stimulation by low doses of H2O2, a significant increase in PI 3-kinase activity was found in wild-type but not in Syk-deficient cells. These findings suggest that PI 3-kinase mediates Syk-dependent Akt activation pathway. Furthermore, viability of Syk-deficient cells, after exposure to H2O2, was dramatically decreased and caspase-9 activity was greatly increased compared with that of the wild-type cells. These results suggest that Syk is essential for the Akt survival pathway in B cells and enhances cellular resistance to oxidative stress-induced apoptosis.

Protein-tyrosine kinases (PTKs) 1 play crucial roles in a wide variety of cellular responses including cell proliferation, differentiation, and apoptosis (1). Extracellular stresses such as ionizing radiation, H 2 O 2 treatment, osmotic shock, or genotoxic agents have also been reported to activate various PTKs (2)(3)(4)(5). Syk, which belongs to nonreceptor PTKs and plays a crucial role in B cell receptor-mediated signaling, is also activated by oxidative and osmotic stress (6 -10). Genetic studies using Sykdeficient B cells have revealed that Syk is essential for the increased tyrosine phosphorylation of cellular proteins, Ca 2ϩ release from intracellular stores, and c-Jun N-terminal kinase (JNK) activation following oxidative stress (9,10). Thus, it has been obvious that Syk plays a crucial role in the transduction of oxidative stress signaling in B cells.
It has been demonstrated that oxidative stress induces apoptotic and necrotic cell death in many cell types (11,12). Treatment of cells in vitro with H 2 O 2 causes DNA strand breaks, oxidation of lipids and proteins, activation of poly-(ADP)-ribosylation, and depletion of cellular energy stores (13). On the other hand, oxidative stress can trigger the activation of some signaling pathways that are involved in cell survival including the phosphorylation cascades leading to the activation of mitogen-activated protein kinase, nuclear factor-B (NF-B), and the serine-threonine kinase Akt (10,14,15). Akt becomes activated in a phosphatidylinositol (PI) 3-kinase-dependent manner not only in response to insulin and various growth factors but also in response to extracellular stresses such as H 2 O 2 treatment and heat shock (15)(16)(17)(18)(19). Furthermore, it has been obvious that activation of Akt is necessary for cell survival and the prevention of apoptosis, which occur by phosphorylation of the Bcl-x inhibitor BAD or caspase-9 and by regulation of signaling via transcription factors such as NF-B (20 -22). However, the role of oxidative stress-induced proteintyrosine phosphorylation in the activation of Akt survival pathway is not clear.
In this paper, we report on the role of Syk in the activation of Akt survival pathway in B cells exposed to oxidative stress. We have found that Syk is required for the activation of Akt survival pathway and enhances cellular resistance to cell death in B cells exposed to oxidative stress. Our data provide the first genetic evidence for the mechanism of regulation of Akt by oxidative stress-activated PTKs.

EXPERIMENTAL PROCEDURES
Materials-The RPMI 1640 medium was purchased from ICN Biomedicals. Fetal bovine serum was from Life Technologies, Inc. and Sigma. Protein A-Sepharose CL-4B was from Amersham Pharmacia Biotech AB. Wortmannin was from Sigma. Anti-phosphotyrosine antibody (4G10) was from Upstate Biotechnology, Inc. Anti-Syk antibodies (C20 and N19) were purchased from Santa Cruz Biotechnology, Inc. Anti-Akt and anti-phospho-Akt (Ser-473) antibodies were purchased from New England Biolabs, Inc.
Immunoprecipitation and Immunoblotting-Stimulated cells (1 ϫ 10 7 cells/ml) were lysed in ice-cold lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 100 M Na 3 VO 4 , 2 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, 10 g/ml aprotinin). Lysates were clarified by centrifugation at 12,000 ϫ g for 10 min at 4°C. The supernatants were incubated sequentially (1 h for each incu-* This work was supported by Grants-in-Aid for Scientific Research (B), Scientific Research on Priority Areas (A) from the Ministry of Education, Science, Sports and Culture of 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.
PI 3-Kinase Assay-PI 3-kinase assay was performed by the method of Aagaard-Tillery et al. (25). Briefly, 20 l of phosphatidylinositol 4,5-diphosphate at 1 mg/ml were added to immunoprecipitates obtained from cell lysates using anti-phosphotyrosine antibody.  1) were added, and samples were vortexed and centrifuged at 15,000 ϫ g for 5 s at 4°C. Aqueous phase was discarded, and 160 l of methanol, 1 M HCl (1:1) was added to the organic phase; samples were vortexed and centrifuged; and the organic phase was extracted again. Following evaporation under a stream of nitrogen, lipid residues were dissolved in 30 l of chloroform/methanol (2:1). Phosphoinositides were separated by thin layer chromatography on Silica Gel 60 plates in running solvent (n-propanol, 2 M acetic acid (65:35)). Radiolabeled phosphoinositides were detected by autoradiography.
Cell Viability-Wild-type DT40 and mutant cells (5 ϫ 10 5 cells/ml) were stimulated using the indicated concentrations of H 2 O 2 . After 22 h, cell viability was determined by the trypan blue dye exclusion method.
DNA Fragmentation Analysis-Wild-type and Syk-deficient DT40 cells (5 ϫ 10 5 cells/ml) were stimulated with the indicated concentration of H 2 O 2 for 22 h. 5 ϫ 10 6 cells were lysed in 0.5 ml of lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM EDTA, 200 mM NaCl, 0.4% Triton X-100, and 0.1 mg/ml protein K) for 20 min at room temperature followed by a 30-min incubation with 0.1 mg/ml RNase A at 50°C. DNA fragmentation was analyzed using a 2.5% agarose gel in the presence of 0.5 g/ml ethidium bromide.
Detection of Caspase-9 Activity-After stimulation by H 2 O 2 , cell lysates were tested for protease activity by the addition of a caspase-9specific peptide that is conjugated to the color reporter molecule pnitroanilide (LEHD-pNA substrate) using the Caspase-9 Colorimetric Assay (R&D Systems Inc.) according to the manufacturer's instructions.

Akt Is Activated in a Syk-dependent Manner by Treatment with Low Doses of H 2 O 2 -We have previously reported that
Syk is activated by oxidative stress and plays a crucial role in Ca 2ϩ release from intracellular stores and JNK activation in B cells, suggesting that Syk is a key molecule in the transduction of oxidative stress signals (9,10). It has been reported that cells exhibit resistance to oxidative stress-induced apoptosis via the activation of Akt survival pathway (19). In this study, we have examined the role of Syk in the activation of Akt survival pathway in DT40 B cells exposed to oxidative stress. Wild-type or Syk-deficient cells were treated with various concentrations of H 2 O 2 , and the whole cell lysates were subjected to immunoblotting analysis using anti-phospho-Akt antibody, which recognizes phosphorylated Ser-473 of Akt, an active form of Akt. In wild-type cells, Akt was rapidly phosphorylated and reached maximum phosphorylation at 10 min after stimulation with 100 M H 2 O 2 , whereas phosphorylation of Akt in Syk-deficient cells was almost completely abolished (Fig. 1A). In contrast, when cells were exposed to high doses of H 2 O 2 (1-5 mM), the levels of Akt phosphorylation were about the same in both the wild-type and the Syk-deficient cells (Fig. 1B). Although phosphorylation of Akt on Ser-473 correlates with maximal activation of Akt (26), in order to confirm Akt activation, we investigated the enzymatic activity of Akt by in vitro kinase assay using histone 2B as a substrate. Consistent with the phospho- A, wild-type (WT) or Syk-deficient (Syk Ϫ ) DT40 cells were treated with 100 M H 2 O 2 for the indicated times, and whole cell lysates were subjected to immunoblotting (IB) analysis using antiphospho-Akt or anti-Akt antibodies. B, wild-type and Syk-deficient cells were treated with the indicated doses of H 2 O 2 , and whole cell lysates were subjected to immunoblotting analysis. C, anti-Akt immunoprecipitates (IP) were subjected to in vitro kinase assay (IVK) using histone 2B as a substrate. The results shown are from one representative experiment that was replicated four times. rylation of Akt, the enzymatic activation of Akt by stimulation with 100 M H 2 O 2 was also abolished in Syk-deficient cells (Fig.  1C). These data showed that Akt activation in B cells stimulated with low doses of H 2 O 2 was dependent on Syk.
Syk-dependent Akt Phosphorylation following Oxidative Stress Requires Syk Kinase Activity-We have previously reported that Syk was phosphorylated and activated by treatment with high doses of H 2 O 2 (1-10 mM) (9, 10). In this paper, we show that tyrosine phosphorylation of Syk was also increased by treatment with 100 M H 2 O 2 ( Fig. 2A). To further examine the role of Syk kinase activity and its autophosphorylation site in Akt activation, we analyzed the Akt phosphorylation induced by 100 M H 2 O 2 using Syk-deficient cells expressing kinase-negative Syk (K395R; here designated KD) or Syk having mutations in the autophosphorylation site (Y518F/Y519F, here designated Autop Ϫ ). As shown in Fig. 2B, in cells expressing KD or Autop Ϫ as well as in Syk-deficient cells, Akt phosphorylation was significantly reduced. These findings suggest that both the kinase activity and the autophosphorylation sites of Syk were required for Akt phosphorylation.
Syk-dependent Akt Phosphorylation following Oxidative Stress via PI 3-Kinase-PI 3-kinase is believed to be an upstream activator of Akt in growth factor-activated survival pathway (27). Recently, it has been reported that Akt is activated by oxidative stress in fibroblasts in a PI 3-kinase-dependent or -independent manner (17,18). Hence, the involvement of PI 3-kinase in oxidative stress-induced Akt activation remains controversial. Therefore, we have studied the involvement of PI 3-kinase in Syk-dependent Akt phosphorylation using B cells. Pretreatment of cells with wortmannin (100 nM, 30 min), a specific PI 3-kinase inhibitor, completely blocked Akt phosphorylation in B cells subsequently treated with 100 M H 2 O 2 (Fig.  3A). Furthermore, PI 3-kinase activity in wild-type cells is significantly increased in a H 2 O 2 dose-dependent manner, whereas Syk-deficient cells demonstrate little increase of PI 3-kinase activity following stimulation with low doses of H 2 O 2 .
These findings indicate that in B cells stimulated with low doses of H 2 O 2 Syk activates PI 3-kinase, thereby inducing Akt activation.
Syk Enhances Cellular Resistance to H 2 O 2 -induced Apoptosis-Akt has been shown to be a general mediator of growth factor-induced survival and to suppress cell death induced by a variety of stimuli (16). We have examined whether Syk was involved in suppressing oxidative stress-induced cell death. Wild-type and various mutant cells were treated with low doses of H 2 O 2 for 22 h, and cell viability was determined by the trypan blue dye exclusion method. About 30% of wild-type and Syk revertant cells (Syk/Syk Ϫ ) died after 22 h of treatment with 100 M H 2 O 2 , whereas more than 80% of Syk-deficient and kinase-inactive Syk (KD)-expressing cells died under similar conditions (Fig. 4A). It was obvious that the viability of Syk-deficient cells declined dramatically after exposure to H 2 O 2 . Furthermore, DNA ladder formation was detected in B cells treated with low doses of H 2 O 2 . As shown in Fig. 4B, a deficiency of Syk produced a significantly enhanced, dose-dependent apoptotic response based on DNA fragmentation. Recently, it has been reported that Akt phosphorylates caspase-9 and inhibits its protease activity (21). We therefore studied caspase-9 activity in B cells treated with 50 M H 2 O 2 for 12 h. It was notable that following stimulation with H 2 O 2 , caspase-9 activity in Syk-deficient cells had increased more strongly than that in wild-type cells (Fig. 5). These results suggest that Syk plays a role in the protection of B cells from oxidative stressinduced apoptosis. DISCUSSION It has been well established that oxidative stress induces the activation of various PTKs, subsequently leading to an increase in the cytoplasmic free calcium and the activation of mitogenactivated protein kinases (2, 5, 8 -10). We have previously demonstrated that in B cells exposed to oxidative stress, Syk plays a crucial role in signal transduction, including Ca 2ϩ release from intracellular pools and the activation of JNK (9,10). In this study, we have found that Syk also induces PI 3-kinase activation followed by Akt activation in B cells following oxidative stress. It has been reported that Syk is upstream of PI 3-kinase in platelet activation and B cell receptor signaling (28,29). We have previously demonstrated that Syk associates with PI 3-kinase and enhances its activity following thrombin stimulation in platelets (28). However, we could not demonstrate the association of Syk with PI 3-kinase in B cells exposed to oxidative stress, although PI 3-kinase activation was dependent on Syk. In B cell receptor signaling, Syk phosphorylates Cbl at the PI 3-kinase binding site, whereby Cbl binds PI 3-kinase, leading to activation of PI 3-kinase (29). In oxidative stress signaling, as well as in B cell receptor signaling, some molecules such as Cbl may mediate Syk-dependent PI 3-kinase activation. Further investigation is warranted to clarify the molecular mechanism of Syk-dependent PI 3-kinase activation following oxidative stress.
In this work, Syk-dependent Akt activation has been observed after treatment with low doses of H 2 O 2 (10 -100 M) but not with high doses of H 2 O 2 (1-5 mM). The various cell lines exhibited different susceptibilities to H 2 O 2 -induced cell death, and B cells were relatively sensitive to H 2 O 2 treatment (11)(12)(13). We have observed that DNA ladder formation, which is characteristic of apoptosis, was detectable in B cells treated with low doses of H 2 O 2 (Fig. 4B) but not in B cells treated with high doses of H 2 O 2 (data not shown). Thus, high doses of H 2 O 2 induce cell necrosis, whereas cell death induced by low doses of H 2 O 2 seems to reflect cellular apoptosis in B cells. One prominent feature of H 2 O 2 in cell apoptosis is to activate mitochondrial permeability transition pore and release the mitochondrial protein cytochrome c into the cytoplasm (30). In the cytosol, cytochrome c in combination with Apaf-1 activates caspase-9, which leads to the activation of the caspase cascade and DNA fragmentation (31). We have demonstrated that the caspase-9 activity in Syk-deficient cells after H 2 O 2 stimulation was higher than in wild-type cells. These observations suggest that Syk is involved in the regulation of the caspase-9 activity in oxidative stress-induced B cell apoptosis. It has been reported that Akt phosphorylates caspase-9 and inhibits its protease activity (21), so we suggest that Akt, which is activated in a Syk-dependent manner following oxidative stress, inhibits caspase-9 activity. However, we could not determine whether caspase-9 was the substrate for Akt in B cells following oxidative stress. There is another possibility, that Syk modulates caspase-9 activation mediated by cytochrome c. Further studies are required to clarify the anti-apoptotic function of Syk in oxidative stress.
We have previously reported that Syk is required for JNK activation in B cells stimulated with high doses of H 2 O 2 (0.5-1 mM) (10). It has also been shown that JNK is activated by stressful stimuli, and a high level of JNK activity is correlated in many instances with the induction of apoptosis (32). However, in B cells stimulated with a low dose of H 2 O 2 (100 M), JNK is hardly activated (10). Therefore, we suspect that JNK has little if any role in B cell apoptosis induced by low doses of H 2 O 2 .
In summary, our findings provide another physiological role for Syk in oxidative stress signaling. Syk induces the activation of the PI 3-kinase-Akt survival pathway following oxidative stress, thereby enhancing cell survival against oxidative stress. FIG. 5. Caspase-9 activity in wild-type and Syk-deficient cells exposed to H 2 O 2 . Cells were treated with 50 M H 2 O 2 for 12 h at 37°C, and cell lysates were assayed for caspase-9 activity as described under "Experimental Procedures." The results are expressed as -fold increase in caspase-9 activity of apoptotic cells over that of noninduced cells.