Implication of Phospholipase D2 in Oxidant-induced Phosphoinositide 3-Kinase Signaling via Pyk2 Activation in PC12 Cells*

The role of phospholipase D (PLD) activation in hydrogen peroxide (H2O2)-induced signal transduction and cellular responses is not completely understood. Here we present evidence that Ca2+-dependent tyrosine kinase, Pyk2, requires PLD activation to mediate survival pathways in rat pheochromocytoma PC12 cells under oxidative stress. The H2O2-induced phosphorylation of two Pyk2 sites (Tyr580, and Tyr881) was suppressed by 1-butanol, an inhibitor of transphosphatidylation by PLD, and also by transfection of catalytically negative mouse PLD2K758R (PLD2KR). Furthermore, we found that PLD2 was associated with Pyk2 and Src, and that activation of PLD2 was required for H2O2-enhanced association of Src with Pyk2 leading to full activation of Pyk2. H2O2-induced phosphorylation of Akt and p70S6K was dependent on phosphatidylinositol 3-kinase (PI3K) activity and was abolished by 1-butanol but not t-butanol. Furthermore, the PI3K/Akt activation in response to H2O2 was reduced by transfection of either PLD2KR or the dominant negative Pyk2DN. This study is the first demonstration that PLD2 activation is implicated in Src-dependent phosphorylation of Pyk2 (Tyr580 and Tyr881) by promoting the complex formation between Pyk2 and activated Src in PC12 cells exposed to H2O2, thereby resulting in activation of the survival signaling pathway PI3K/Akt/p70S6K.

The role of phospholipase D (PLD) activation in hydrogen peroxide (H 2 O 2 )-induced signal transduction and cellular responses is not completely understood. Here we present evidence that Ca 2؉ -dependent tyrosine kinase, Pyk2, requires PLD activation to mediate survival pathways in rat pheochromocytoma PC12 cells under oxidative stress. The H 2 O 2 -induced phosphorylation of two Pyk2 sites (Tyr 580 , and Tyr 881 ) was suppressed by 1-butanol, an inhibitor of transphosphatidylation by PLD, and also by transfection of catalytically negative mouse PLD2K758R (PLD2KR). Furthermore, we found that PLD2 was associated with Pyk2 and Src, and that activation of PLD2 was required for H 2 O 2 -enhanced association of Src with Pyk2 leading to full activation of Pyk2. H 2 O 2induced phosphorylation of Akt and p70S6K was dependent on phosphatidylinositol 3-kinase (PI3K) activity and was abolished by 1-butanol but not t-butanol. Furthermore, the PI3K/Akt activation in response to H 2 O 2 was reduced by transfection of either PLD2KR or the dominant negative Pyk2DN. This study is the first demonstration that PLD2 activation is implicated in Src-dependent phosphorylation of Pyk2 (Tyr 580 and Tyr 881 ) by promoting the complex formation between Pyk2 and activated Src in PC12 cells exposed to H 2 O 2 , thereby resulting in activation of the survival signaling pathway PI3K/Akt/p70S6K.
It is known that the cellular redox state is an important mediator of various signaling systems (1)(2)(3). The major signaling pathways and/or key mediators to influence survival of cells subjected to oxidant injury are the phosphorylation cascades leading to activation of tyrosine phosphorylation of several growth factor receptors and their subsequent downstream signaling events, including Akt and mitogen-activated protein kinases (4 -9). A number of studies have demonstrated that hydrogen peroxide (H 2 O 2 ) stimulates phospholipase D (PLD), 1 which generates phosphatidic acid (PA), a second messenger in various types of cell, such as endothelial cells (10,11), fibroblasts (12,13), PC12 cells (14 -17), vascular smooth muscle cells (18), and leukemia L1210 cells (19). As the mechanisms underlying the oxidant-induced PLD activation, various factors including protein-tyrosine kinase (10,11,13,14), protein kinase C (PKC) (13,16,19), and mitogen-activated protein kinase (15,17) have been proposed. Direct or indirect tyrosine phosphorylation has been thought to be involved in PLD activation in response to H 2 O 2 exposure. In HL60 cells, PLD1 is directly phosphorylated by peroxyvanadate (20). Min et al. (13) also demonstrate that in the presence of vanadate, H 2 O 2 stimulates PLD1 activity and its tyrosine phosphorylation in Swiss-3T3 cells. Our previous studies have suggested that Pyk2 and/or p38 mitogen-activated protein kinase and ERK1/2 were involved in H 2 O 2 -induced PLD activation by using their inhibitors in PC12 cells (14 -15, 17). However, despite many investigations, the biological roles and the exact mechanisms of the oxidant-induced PLD activation remains to be elucidated.
Several reports show that the survival signaling via the phosphatidylinositol 3-kinase (PI3K)/Akt pathway is activated by stresses such as heat, hyperosmotic stress, and H 2 O 2 (21)(22)(23). However, little is known regarding the mechanisms for activation of PI3K and Akt caused by stresses. There are reports suggesting that the PLD stimulation participates in the pro-survival signaling at the early stage of the apoptotic process (16,24,25). We have previously shown that the PLD activation induced by sphingosine 1-phosphate (S1P), but not insulin-like growth factor-I, was implicated in the S1P3-mediated PI3K and Akt activation in Chinese hamster ovary cells (26,27). Furthermore, it has been demonstrated that actinomycin D-induced apoptosis was prevented by PLD1 or PLD2 overexpression through activation of the PI3K/Akt signaling pathway, suggesting the notion that PLD plays a role in stimulation of cell survival pathway (28). However, the exact mechanism underlying the activation of the PI3K/Akt pathway via PLD has not been defined. In the present study, we have attempted to gain further insight into the role of PLD activation in response to oxidative stress in PC12 cells and have shown that PLD activation was involved in the PI3K/Akt activation through protein-tyrosine kinases Pyk2 and Src. Thus, these results provided evidence indicating a novel role for PLD2 in the Pyk2/PI3K/Akt pathway that acts as a survival signaling in response to H 2 O 2 .

EXPERIMENTAL PROCEDURES
Materials-[9,10-3 H]Palmitic acid (54.0 Ci/mmol) and [␥-32 P]ATP (3000 Ci/ml) were from PerkinElmer Life Sciences. Rabbit polyclonal * This work was supported in part by Grants-in-aid for Scientific Research (B) 12470042 and (C) 12680633 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.
antibodies against Ser 473 -phosphorlated Akt, Tyr 204 -phosphorylated ERK1/2, Thr 421 /Ser 424 -phosphorylated p70S6K, Tyr 401 -phosphorylated Pyk2, and Tyr 416 -phosphorylated Src, Akt, and ERK1/2 were obtained from Cell Signaling Inc. (Beverly, MA). Rabbit polyclonal antibodies against p70S6K, ␤-actin, mouse monoclonal antibody against Src, and protein A/G Plus-agarose were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse monoclonal anti-Pyk2 and Src were from BD Transduction Laboratories (Lexington, KY). Rabbit polyclonal antibodies against Tyr 881 -and Tyr 580 -phosphorylated Pyk2 were from BioSource International, Inc. (Camarillo, CA). Rabbit polyclonal anti-PLD1 and -PLD2 antibodies were prepared as described previously (28). Anti-rabbit IgG and anti-mouse IgG antibodies conjugated with horseradish peroxidase and the chemiluminescence kit (ECL system) were obtained from Amersham Biosciences. Expression plasmids containing wild-type and catalytically negative mouse PLD2 (K758R) in pCGN were kindly supplied by Dr. Michael A. Frohman (Center for Developmental Genetics and Department of Pharmacology, State University of New York at Stony Brook, Stony Brook, NY). All other reagents were obtained from standard commercial sources.
Cell Culture and Transfections-The PC12 cell line was a generous gift from Dr. Y. Sugimoto (Shirakawa Institute of animal Genetics, Fukushima, Japan). PC12 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum, 5% horse serum, 100 units of penicillin G/ml, and 100 g of streptomycin/ml as described previously (14). The cells were grown to the subconfluent stage at 37°C in a humidified atmosphere containing 5% CO 2 .
For transient transfection PC12 cells were plated at 8 ϫ 10 5 in 100-mm dishes and transfected with plasmid DNA (pCGN-catalytically inactive mouse PLD2 (K758R) or PRK5-dominant negative Pyk2 (Tyr 402 F,Y881F)) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. On the following day the transfected cells were placed in serum-free medium and left for 6 h before treatment with H 2 O 2 in the presence of vanadate.
Measurement of PLD Activity-PLD activity was assessed by measuring the formation of [ 3 H]phosphatidylbutanol by transphosphatidylation. PC12 cells were subcultured in 35-mm-diameter tissue culture dishes at 5 ϫ 10 5 cells/dish and grown for 3 days. The subconfluent cells were labeled for 3 h with [ 3 H]palmitic acid (3 Ci/dish), washed twice with serum-free Dulbecco's modified Eagle's medium, and preincubated for 30 min at 37°C in the presence or absence of the indicated inhibitors and 100 M Na 2 VO 4 , and 1-butanol was added at a final concentration of 0.3% (v/v) and further incubated for 10 min. Cells were then treated with 0.5 mM H 2 O 2 for 30 min at 37°C. The reactions were stopped by removal of the assay medium followed by the immediate addition of 1 ml of an ice-cold phosphate-buffered saline/methanol (2:5, v/v) mixture to the culture dishes. After extraction of cellular lipids, [ 3 H]phosphati-dylbutanol was separated by TLC on silica gel LK6D plates using a solvent system of an upper phase of ethyl acetate/2,2,4-trimethylpentane/acetic acid/water (13:2:3:10, by volume) as previously described (14). The amount of [ 3 H]phosphatidylbutanol formed was expressed as the percentage of the total radioactivity recovered from the TLC plate. Phosphoinositide 3-Kinase Assay-PI3K activity was assayed as described previously (27). The immunoprecipitation was performed using anti-phosphotyrosine (PY-20) and p110␤ PI3K antibodies, and PI3K activity in the immunoprecipitates was assayed using PI as substrate.
Immunoprecipitation Analysis-For immunoprecipitation, 300 g of protein of cell lysate was diluted with 300 l of the buffer containing 1% Nonidet P-40, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 20 mM HEPES, 3 mM MgCl 2 , 1 mM phenylmethylsulfonyl fluoride, 20 g/ml leupeptin, 20 mM ␤-glycerophosphate, 1 mM sodium orthovanadate, and 1 mM sodium fluoride, pH 7.4, and 2 g of the appropriate antibody (anti-PLD2, anti-Pyk2, or anti-Src) at 4°C for 2 h followed by incubation with protein A/G-agarose 4B overnight. Agarose beads were collected by centrifugation, washed 3 times with the same buffer, resuspended in SDS sample buffer, and then boiled for 10 min. Proteins were separated as described for Western blot analysis.

RESULTS AND DISCUSSION
Phosphorylation of Src and Pyk2 in H 2 O 2 -or EGF-stimulated PC12 Cells-Our previous studies have demonstrated that H 2 O 2 -induced PLD activation in PC12 cells was inhibited by protein-tyrosine kinase inhibitors, such as PP2, genistein, and herbimycin, but enhanced in the presence of vanadate, a protein-tyrosine phosphatase inhibitor (14,15,17). These observations lead us to assume that tyrosine phosphorylation of PLD2 expressed in this cell may regulate its activity. However, PLD2 tyrosine phosphorylation by H 2 O 2 was marginal, and direct phosphorylation of the enzyme was unlikely to stimulate the activity. Other studies also report that tyrosine phosphorylation of PLD enzyme is not involved in the regulation of its activity (29,30). The depletion of exogenous Ca 2ϩ by EGTA resulted in a complete abolishment of H 2 O 2 -mediated PLD activation in PC12 cells (14,15). It was then considered that Src-type protein-tyrosine kinase and a Ca 2ϩ -dependent proline-rich cytoplasmic protein-tyrosine kinase (Pyk2), which is highly active in PC12 cells, should be involved in H 2 O 2 -mediated up-regulation of PLD activity in PC12 cells.
It has been reported that autophosphorylation of Pyk2 (Tyr 402 ) leads to Src activation, which causes further phosphorylation of two other tyrosine residues (Tyr 580 , Tyr 881 ) in the C terminus of Pyk2, resulting in the full activation of Pyk2 (31)(32)(33). We examined phosphorylation of Src and Pyk2 (Tyr 402 , Tyr 580 , and Tyr 881 ) after H 2 O 2 treatment of PC12 cells. For site-specific phosphorylation of Pyk2, the antibodies were used that specifically recognize phospho-Pyk2 (Tyr 402 , autophosphorylation and Src binding site; Tyr 580 , regulatory kinase activation loop; Tyr 881 , Grb2-SH2 binding site) in PC12 cells. As shown in Fig. 1, in the presence of vanadate, H 2 O 2 induced a rapid, remarkable tyrosine phosphorylation of Src and three Pyk2 sites (Tyr 402 , Tyr 580 , and Tyr 881 ). Similar levels of Pyk2 phosphorylation were observed in three tyrosine phosphorylation sites. On the other hand, upon stimulation with EGF, tyrosine phosphorylation of Src and these three Pyk2 sites was much weaker. The PLD activation in response to H 2 O 2 was much higher than EGF stimulation in PC12 cells (data not shown), suggesting involvement of PLD activation in phosphorylation of Pyk2 and Src.
Involvement of PLD Activation in Tyrosine Phosphorylation of Src and Pyk2 in PC12 Cells Exposed to H 2 O 2 -We examined involvement of Src and PLD in site-specific tyrosine phosphorylation of Pyk2. As depicted in Fig. 2A, the blockade of Src kinase by PP2 prevented phosphorylation of Pyk2 (Tyr 580 and Tyr 881 ) but not autophosphorylation at Tyr 402 , which is the binding site for the SH2 domain of Src (31). In fact, the dominant negative Pyk2 (Y402F,Y881F; Pyk2DN) prevented H 2 O 2 -induced Src phosphorylation in PC12 cells (data not shown). Therefore, activation of Src via Pyk2-dependent mechanism was likely to occur in PC12 cells exposed to H 2 O 2 . To examine the involvement of PLD in the Pyk2 and Src tyrosine phosphorylation, we used 1-butanol, which inhibits PA generation by PLD activation and t-butanol, its inactive analogue. As shown in Fig. 2, A and C, 1-butanol, but not t-butanol, caused reduction of phosphorylation of Pyk2 (Tyr 580 and Tyr 881 ) but not Pyk2 (Tyr 402 ) and Src tyrosine phosphorylation. In contrast, there were no inhibitory effects of 1-butanol on EGF-induced phosphorylation of Pyk2 and Src (Fig. 2B). These results suggest that PLD activation is involved in phosphorylation at Tyr 580 and Tyr 881 but not Tyr 402 of Pyk2 and Src phosphorylation.
To ascertain the involvement of PLD activation in Pyk2 (Tyr 580 and Tyr 881 ) phosphorylation induced by H 2 O 2 , we have transfected catalytically inactive PLD2-cDNA (K758R; PLD2KR) into PC12 cells and examined phosphorylation of Pyk2 (Tyr 580 and Tyr 881 ). Overexpression of PLD2KR significantly reduced the H 2 O 2 -induced phosphatidylbutanol formation as compared with that of the vector control (Fig. 3A). In PLD2KRoverexpressed PC12 cells, H 2 O 2 -induced Pyk2 (Tyr 580 and Tyr 881 ) phosphorylation was much reduced (Fig. 3, B and C), whereas phosphorylation of Pyk2 (Tyr 402 ) and Src was not af-fected. These results provided confirmative support for data from the experiments with 1-butanol (Fig. 2, A and C), indicating that PLD2 was implicated in phosphorylation of Pyk2 (Tyr 580 and Tyr 881 ) via Src activation in PC12 cells exposed to H 2 O 2 .
Association of PLD2 with Pyk2 and Src in Control and PLD2KR-overexpressed PC12 Cells-It has been known that Pyk2 has both kinase and scaffolding functions and that in many cell systems, Pyk2 is associated with Src and other SH2containing proteins including PI3K (32,33). In PC12 cells the requirement for both Pyk2 and Src in H 2 O 2 -induced PLD activation suggests that these kinases would be associated with PLD2. Using a co-immunoprecipitation approach, we found that PLD2 and Pyk2 were co-immunoprecipitated in PC12 cells stimulated with H 2 O 2 or EGF, indicating that PLD2 and Pyk2 are physically associated in PC12 cells (Fig. 4, A-D). Furthermore, to confirm the role of PLD2 activity in association with Pyk2, we examined the Pyk2 association in PLD2KR-overexpressed PC12 cells. As shown in Fig. 4E, the PLD2KR overexpression attenuated both basal and H 2 O 2 -induced association of Pyk2 with PLD2. This suggests that the PLD2 association with Pyk2 requires its catalytic site.
Next, we examined the association of PLD2 and Src. The cell lysates of PC12 cells stimulated with H 2 O 2 or EGF were immunoprecipitated with anti-Src antibody. As shown in Fig. 4F, PLD2 was co-immunoprecipitated with Src, suggesting association of PLD2 with Src as observed in other cell types (29,34). It was also shown that the association of PLD2 with Src was increased 2-fold by stimulation of H 2 O 2 but not EGF. Furthermore, this association was markedly reduced by enforced expression of PLD2KR in PC12 cells exposed to H 2 O 2 compared with the vector control (Fig. 4G, upper panel). It has previously been shown that Src interacts with Pyk2 upon stimulation of PC12 cells with various agonists, leading to phosphorylation of Pyk2 (31)(32)(33). We have then checked involvement of the PLD activation induced by H 2 O 2 in the association of Src and Pyk2. As shown in Fig. 4G, lower panel, stimulation with H 2 O 2 induced association of Src with Pyk2 in empty vector-transfected cells, but this association was greatly suppressed in PLD2KR-expressed cells. It is, thus, of great interest to note that PLD2 activation is required for complex formation between Pyk2 and activated Src. Further studies are required to define the mechanism by which PLD activation regulates its association with Src and the Pyk2/Src interaction, although intracellularly generated PA via PLD2 activation would be considered to participate in these interactions. Taken together, it was indicated that the association of PLD2 with Pyk2 and with Src is independent of and dependent on PLD2 activation, respectively, and also that the Pyk2/Src interaction is enhanced by PLD2 activation. (16,24,25). However, the precise mechanism of the role of PLD2 activation remains to be elucidated. It has been reported that oxidative stress by reactive oxygen species (ROS) induces the activation of mitogenic or survival signaling, including serine/threonine protein kinase Akt and ERK1/2 (6,8,9). In PC12 cells, both Akt and Erk1/2 were phosphorylated by H 2 O 2 in a time-dependent manner, although their temporal profiles were distinct from those observed in EGF stimulation (Fig. 5A). Phosphorylation of Akt and ERK1/2 by H 2 O 2 showed slow increases peaking at 10 and 20 min, respectively, whereas EGF induced rapid increases in their phosphorylation.

Involvement of PLD2 Activation in Phosphorylation of Akt and ERK1/2 in H 2 O 2 -treated PC12 Cells-Previous studies have suggested that the H 2 O 2 -induced PLD2 activation in PC12 cells is involved in the anti-apoptotic process
In PC12 cells, bradykinin, and carbachol have been shown to transactivate EGF receptor leading to ERK1/2 activation (35,36). Furthermore, Pyk2 is implicated in calcium ionophore-and phorbol ester-stimulated ERK1/2 phosphorylation via a direct interaction with Src (32,37) or via EGF receptor transactivation (38). To know which pathway mediates H 2 O 2 -induced Akt and ERK activation, we examined the effect of a specific inhibitor of the EGF receptor-tyrosine kinase, AG1478, on phosphorylation of these kinases in H 2 O 2 -treated PC12 cells. As shown in Fig. 5B, treatment of PC12 cells with AG1478 reduced phosphorylation of ERK1/2 in response to H 2 O 2 by ϳ80%, whereas it completely blocked EGF-induced ERK1/2 phosphorylation. On the other hand AG1478 had no significant effect on the H 2 O 2 -induced Akt phosphorylation under the condition where EGF-induced Akt phosphorylation was completely abolished. These results suggest that H 2 O 2 -induced ERK1/2 activation was largely dependent on EGF receptor transactivation, but Akt activation was not.
To examine the upstream events of H 2 O 2 -induced Akt activation, PC12 cells were pretreated with various inhibitors, including genistein, PI3K inhibitor (LY294002), and calcium chelator (EGTA) and then exposed to H 2 O 2 . H 2 O 2 -induced Akt phosphorylation was reduced by LY294002 and genistein in a dose-dependent manner and was diminished by EGTA (Fig. 6,  A and B), suggesting that Akt phosphorylation in response to H 2 O 2 was mediated by PI3K and calcium-dependent tyrosine kinase, Pyk2. Next, we examined involvement of PLD activation in Akt and ERK1/2 activation after H 2 O 2 treatment. As depicted in Fig. 6C, the pretreatment of PC12 cells with 1-butanol abolished H 2 O 2 -induced phosphorylation of Akt but not ERK1/2. However, the same treatment did not affect EGFinduced phosphorylation of either Akt or ERK1/2. It is, thus, interesting to note that PLD activation participates in H 2 O 2induced Akt activation but not in ERK1/2 phosphorylation. In contrast, EGF-mediated phosphorylation of Akt and ERK1/2 was independent of PLD activation.
As described above, H 2 O 2 -induced Akt activation was likely to be dependent on PI3K by using LY294002 (Fig. 6, A and B). To ascertain this notion, we examined PI3K activation by H 2 O 2 exposure. H 2 O 2 caused marked stimulation of PI3K activity, but this activation was suppressed by 1-butanol (Fig. 7A), suggesting involvement of PLD in PI3K activation. However, PI3K activation by EGF stimulation was not prevented by 1-butanol (data not shown). It was also observed that p110 ␤-type, but not p110␣-type, was attributed to the H 2 O 2 -induced PI3K activation (Fig. 7B). Moreover, to assess whether PLD activity is involved in H 2 O 2 -induced PI3K activation, PC12 cells were transfected with PLD2KR-cDNA. Fig. 7B showed that overexpression of PLD2KR attenuated activation of PI3K␤. The activation of PI3K␤ by H 2 O 2 was also repressed in Pyk2DN-overexpressed cells (Fig. 7, B and D). Accordingly, as expected, Akt phosphorylation induced by H 2 O 2 was reduced by overexpression of either PLDKR or Pyk2DN (Fig. 7, C and  D). Taken together, it was indicated that PLD2 activity was implicated in the PI3K/Akt signaling pathway mediated through Pyk2/Src in PC12 cells exposed to H 2 O 2 .
It has recently been reported that PLD activation is involved in the proliferation signaling pathway through mTOR/p70S6 kinase (p70S6K), where mTOR is activated by direct interaction with PA generated by PLD activation (39). In PC12 cells p70S6K was markedly phosphorylated by stimulation with H 2 O 2 (Fig. 8). However, the H 2 O 2 -induced phosphorylation of p70S6K was completely abolished by either LY294002 or 1-butanol. In contrast, p70S6K phosphorylation induced by EGF was not affected by these inhibitors. These results suggested that PI3K-dependent p70S6K activation induced by H 2 O 2 was regulated by the upstream PLD activation, which is required for full activation of Pyk2.
In conclusion, we have shown for the first time that PLD2 is associated with Pyk2 and Src in PC12 cells where the Pyk2 acts as a scaffold protein. As indicated in Fig. 9, we could demonstrate a possible implication of PA generated by H 2 O 2 -induced PLD2 activation in Src-dependent phosphorylation of Pyk2 (Tyr 580 and Tyr 881 ). Furthermore, we showed that PLD2 activation by H 2 O 2 was most likely to participate in stimulation of the PI3K/Akt/p70S6K pathway via Pyk2/Src activation involved in regulation of cell survival of PC12 cells. On the other hand, the major pathway of H 2 O 2 -induced ERK activation, which is mediated by transactivation of EGF receptor, was independent of PLD2 activation in PC12 cells. FIG. 9. A proposed model involving of PLD2 in the survival signaling pathway via Pyk2 and Src activation in PC12 cells exposed to H 2 O 2 . PLD2 is associated with cytosolic proline-rich, Ca 2ϩdependent Pyk2, which acts as a scaffold protein and also with Src in PC12 cells. Upon stimulation with H 2 O 2 , intracellular Ca 2ϩ is increased by influx, resulting in autophosphorylation of Tyr 402 of Pyk2 and then Src phosphorylation. The Src activation induces PLD2 activation to generate PA, which would promote association of PLD2 with Src and the Pyk2/Src interaction followed by Pyk2 full activation with phosphorylation at Tyr 580 and Tyr 881 . The tyrosine phosphorylation of PI3K␤ by H 2 O 2 would be mediated via phosphorylation of Pyk2 (Tyr 881 ). Thus, this hypothetical model suggests that in PC12 cells exposed to H 2 O 2 , the survival signaling is mediated by the Pyk2(Tyr 402 )/Src/PLD2(PA)/ Pyk2(Tyr 580,881 )/PI3K/Akt/p70S6K pathway. H 2 O 2 -induced ERK activation is mainly mediated by transactivation of EGF receptor (EGFR).