Tyrosine Phosphorylation Regulates the Proteolytic Activation of Protein Kinase C (cid:1) in Dopaminergic Neuronal Cells* □ S

Oxidative stress is a key apoptotic stimulus in neuronal cell death and has been implicated in the pathogenesis of many neurodegenerative disorders, including Parkinson disease (PD). Recently, we demonstrated that protein kinase C- (cid:1) (PKC (cid:1) ) is an oxidative stress-sensi-tive kinase that can be activated by caspase-3-depend-ent proteolytic cleavage to induce apoptotic cell death in cell culture models of Parkinson disease (Kaul, S., (2003) Eur. J. 18, 1387–1401 A. G., Kitazawa, M., Kanthasamy, A., and Anantharam, V. (2003) Antioxid. Redox. Signal. 5, 609–620). Here we showed that the phosphorylation of a tyrosine residue in PKC (cid:1) can regulate the proteolytic activation of the kinase during oxidative stress, which consequently influences the apoptotic cell death in dopaminergic neuronal cells. Exposure of a mesencephalic dopaminergic neuronal cell line (N27 cells) to H 2 O 2 (0– 300 (cid:2) M ) induced a dose-dependent increase in cytotoxicity, (cid:11) M ) for a period of 4 h, and the cell lysates were used to determine changes in the caspase-3 activity, PKC (cid:1) proteolytic activity, and DNA fragmen- tation. H 2 O 2 (100 (cid:11) M ) induced significant increases in PKC (cid:1) proteolytic cleavage ( A ), caspase-3 activity ( B ), and DNA fragmentation after a 4-h exposure ( C ). Also, comparison of H 2 O 2 -induced PKC (cid:1) cleavage between vector cells and untreated N27 cells showed no difference. All the measured parameters were significantly attenuated in the cells that expressed the PKC (cid:1) Y311F mutant protein. Equal loading of protein was demonstrated by using (cid:3) -actin immunoblotting ( A ). B and C represent n (cid:7) 6 from two to three separate experiments, where ** (cid:7) p (cid:5) 0.001 shows significant differences between untreated and H 2 O 2 treated cells, and # (cid:7) p (cid:5) 0.001 represents significant differences between H 2 O 2 -treated cells and those co-treated with the tyrosine ki- nase inhibitors. Statistical analysis was done using ANOVA and Dunnett’s multiple comparison tests.

PKC␦ is a member of the PKC serine-threonine protein kinase family classified into three groups, namely the classical (␣, ␤, and ␥ activated by DAG and Ca 2ϩ ), the atypical ( and / DAG and Ca 2ϩ -independent), and the novel (␦, ⑀, , and activated by DAG but Ca 2ϩ -independent). PKC␦ activation requires either the phosphorylation of its activation loop residues, leading to enzyme translocation, or the proteolytic cleavage of the kinase to yield catalytically active fragments. In the cellular models of Parkinson disease, we observed a caspase-3 mediated activation of PKC␦ without any evidence of membrane translocation (1). PKC␦ is known to be phosphorylated at tyrosine residues Tyr-52, Tyr-155, Tyr-187, Tyr-311, Tyr-332, and Tyr-565 when activated in response to certain stimuli, particularly to the known oxidative stress-inducing agent hydrogen peroxide (H 2 O 2 ) (19 -21). Src kinase, a member of the nonreceptor protein-tyrosine kinase family, variably modulates PKC␦ activity by increasing tyrosine phosphorylation, depending on the cell type and the insult (22)(23)(24)(25). Other members of the Src family of kinases that influence PKC␦ activity via phosphorylative changes are Fyn and c-Abl kinase (26,27). Furthermore, recent studies have demonstrated that PKC␦, when phosphorylated on the tyrosine residue Tyr-311, exhibits an increased catalytic activity in H 2 O 2 -treated cells (28,29). However, the relationship between PKC␦ tyrosine phosphorylation and its proteolytic cleavage has never been explored, particularly whether PKC␦ tyrosine phosphorylation can regulate its proteolytic activation and proapoptotic function. Here we demonstrate that phosphorylation of the tyrosine residue Tyr-311 in PKC␦ is essential for proteolytic activation, and that inhibition of tyrosine phosphorylation can attenuate oxidative stress-induced apoptotic cell death in dopaminergic neuronal cells.
Cell Culture-The immortalized rat mesencephalic dopaminergic neuronal cell line 1RB3AN 27 , normally referred to as N27 cells, was a kind gift from Dr. Kedar N. Prasad (University of Colorado Health Sciences Center, Denver, CO). N27 cells represent a homogeneous population of tyrosine hydroxylase-positive dopaminergic cells. The cell line is a widely used cell culture model of Parkinson disease (1,2,31,32,34,35). The cells were grown in RPMI 1640 medium containing 10% fetal bovine serum, 2 mM L-glutamine, 50 units of penicillin, and 50 g/ml streptomycin. Cells were maintained in a humidified atmosphere of 5% CO 2 at 37°C as described previously (31,32).
Treatment Paradigm-H 2 O 2 (0 -300 M) or MPP ϩ (300 M) was added to the cells for the duration of the experiment. The cells were removed from the flask by using a cell scraper and centrifuged at 200 ϫ g for 5 min, washed with PBS twice, and homogenized as described previously (1). Cell lysates, collected by spinning down the cell fragments at 20,000 ϫ g for 45 min at 4°C, were used for immunoprecipitation studies to determine caspase-3 enzyme activity, DNA fragmentation, and PKC␦ cleavage. Untreated cells were grown in the complete medium and used as control samples. For real time fluorescence imaging, the cells were grown in 24-well plates and viewed in the culture wells.
Cytotoxicity Assays-Cell death was determined after exposing the N27 cells to H 2 O 2 (100 M) using the Sytox® green cytotoxicity assay and the LIVE/DEAD® viability/cytotoxicity kit (Molecular Probes, Eugene, OR). The Sytox® green cytotoxicity assay is based on the principle that Sytox® green cannot enter cells with intact membranes (live cells) but permeates cells with compromised plasma membranes and intercalates with DNA to produce green fluorescence (36,37). Briefly, N27 cells were grown in 24-well cell culture plates at equal densities and treated with H 2 O 2 (0 -300 M) and 1 M Sytox® green fluorescent dye for a period of 4 h. The Sytox® green assay allows dead cells to be viewed directly under the fluorescence microscope as well as quantitatively measured with a fluorescence microplate reader (excitation 485 nm; emission 538 nm) (SpectraMax Gemini XS model, Molecular Devices, Sunnyvale, CA). The LIVE/DEAD® kit consists of a combination of two dyes: SYTO 10 (green fluorescence), a highly cell permeable cell dye which stains all cells, and an ethidium homodimer (DEAD Red; red fluorescence), a membrane impermeant dye that only binds to nucleic acids in cells with compromised cell membranes. Fluorescent images were taken after exposure to H 2 O 2 with a NIKON TE2000 microscope, and pictures were captured with a SPOT digital camera.
In Situ Fluorometric Analysis of Caspase Activity-FITC-VAD-FMK, a cell-permeable fluorescent probe that binds to active caspase-3, was used as an in situ marker for caspase activity. The entire procedure was performed according to Promega's CaspACE® kit, as described previously (1). Fluorescent images were captured using a SPOT digital camera (Diagnostic Instruments, Sterling Heights, MI).
Immunoprecipitation and Kinase Assay-Immunoprecipitation studies were conducted to determine the phosphorylative changes in the PKC␦ protein obtained from H 2 O 2 -treated N27 cells. Briefly, cells were washed once with 1ϫ PBS and resuspended in 500 l of PKC lysis buffer (25 mM HEPES (pH 7.5), 20 mM ␤-glycerophosphate, 0.1 mM sodium orthovanadate, 0.1% Triton X-100, 0.3 M NaCl, 1.5 mM MgCl 2 , 0.2 mM EDTA, 0.5 mM dithiothreitol, 10 mM NaF, and 4 g/ml each of aprotinin and leupeptin) (1). The cell suspension was kept on ice for 30 min and then centrifuged at 13,000 ϫ g for 5 min. The resultant supernatant was collected as the cytosolic fraction. Cytosolic protein (ϳ200 g total) was immunoprecipitated overnight at 4°C using 20 -40 l of anti-phosphotyrosine (4G10)-agarose conjugated antibody. The Sepharose-bound antigen-antibody complexes were washed three times with buffer. Samples were then mixed with 2ϫ SDS-PAGE loading buffer, boiled for 5 min, and then separated on 10 -12% SDS-PAGE. For the kinase assay, the immunoprecipitation was done by using a polyclonal PKC␦ rabbit antibody and protein-Sepharose A and washed three times with kinase buffer (40 mM Tris (pH 7.4), 20 mM MgCl 2 , 20 M ATP, 2.5 mM CaCl 2 ). The reaction was started by adding 20 l of buffer containing 0.4 mg of histone and 5 Ci of [␥-32 P]ATP (4,500 Ci/mM). After incubation for 10 min at 30°C, SDS loading buffer (2ϫ) was added to the samples to terminate the reaction. The reaction products were separated on SDS-PAGE (12.5%), and the H1-phosphorylated bands were detected using a Personal Molecular Imager (FX model, Bio-Rad Labs) and quantified with Quantity One 4.2.0 software.
DNA Fragmentation Assay-DNA fragmentation was measured using a recently developed Cell Death Detection ELISA Plus assay kit, a fast, highly sensitive, and reliable assay for the detection of early apoptotic death (32,39). Briefly, after treatment with 100 M H 2 O 2 , the cells were spun down at 200 ϫ g for 5 min and washed once with PBS. Cells were then lysed in 450 l of lysis buffer (provided with the kit) and spun down again at 5,000 rpm for 10 min to collect the supernatant, which was used to measure DNA fragmentation as per the manufacturer's protocol. Readings were taken in a Spectramax multiwell plate reader at 405 nm, with 490 nm as a reference reading.
Measurement of ROS Generation-The ROS generation in N27 cells was measured using the fluorescence probe dihydroethidine, as described previously (40). Briefly, N27 cells were plated in 24-well plates at a density of 2 ϫ 10 6 cells per well for a period of 24 h prior to treatment. After 24 h, the RPMI culture medium was removed from the wells and replaced with clear HBSS medium supplemented with 2 mM CaCl 2 . 1 M dihydroethidine (final concentration) was added to the HBSS for a period of 15 min. The cells were then treated with either H 2 O 2 (100 M) alone or along with the tyrosine kinase inhibitors genistein (25 M) and TSKI (5 M) for a period of 1 h. After the 1-h incubation period, fluorescence was measured by using a microplate reader (Beckman and Coulter). The data were quantified using SpectraMax spectrophotometer analysis software.
Transient Transfections-cDNA encoding PKC␦ catalytic fragment (PKC␦-CF) from the pEGFPN1 vector was subcloned into the lentiviral expression vector plenti6/V5-D-TOPO (herein referred to as plenti/PKC␦-CF) by PCR using standard cloning procedures. PKC␦ Y311F encoded in pcDNA3 vector encodes a protein in which tyrosine residue at position 311 is mutated to phenylalanine. The expression of PKC␦-CF in mammalian cells can be monitored by using an antibody directed against V5 epitope. Transfections of PKC␦-CF and PKC␦ Y311F mutants were done by using an AMAXA® Nucleofector TM kit for cell lines (AMAXA GmbH, Germany). Plasmid pCDNA3.1 was used as vector control. Briefly, N27 cells were grown in T-175 flasks at a density of 3 ϫ 10 6 per ml and harvested for the transfection procedure. The Nucleofector TM solution V was primed by adding a supplement solution (provided by manufacturer) and plasmid DNA. The cells were then resuspended in this DNA-containing Nucleofector solution at an optimal density of 3 million cells per 100 l of solution. Electroporation was carried out with AMAXA® Nucleofector TM transfector instrument as per the manufacturer's protocol. The procedure was repeated for each subsequent sample. 100 l of cell suspension containing 5 g of pmax GFP DNA (provided with the kit) was used to determine the transfection efficiency. The transfected cells were then transferred to T-75 flasks or 6-well plates as desired and allowed to grow for a 24-h period before being used for the treatment paradigm. Transfection efficiency was determined to be Ͼ75% as determined by GFP expression.
Statistical Analysis-Data were analyzed with Prism 3.0 software (GraphPad Software, San Diego, CA). Bonferroni's and Dunnett's multiple comparison testing were used in order to delineate significant differences between the MPP ϩ -treated groups and the control (untreated) and the inhibitor-treated samples. Differences with p Ͻ 0.05, p Ͻ 0.01, and p Ͻ 0.001 were considered to be statistically significant and are indicated by asterisks in the figures.

RESULTS
Low Dose H 2 O 2 Exposure Induces Dose-dependent Cell Death in Dopaminergic Neuronal Cells-H 2 O 2 is an oxidative insult commonly used to investigate oxidative stress-induced apoptotic signaling in many cell types including dopaminergic neuronal cells (41,42). To determine the effects of low dose oxidative stress on the dopaminergic system, we treated mesencephalic dopaminergic clonal cells (N27) with low doses of H 2 O 2 (0 -300 M) for a period of 4 h, and cytotoxicity was assayed at 1-h intervals over the entire treatment period. We also measured the cytotoxicity both qualitatively and quantitatively by using another fluorescence dye Sytox® green, which stains only dead/dying cells. Cellular apoptosis is often marked by the final precipitating events of chromatin breakdown and DNA fragmentation, which are considered hallmarks of programmed cell death. Therefore, we determined the extent of DNA fragmentation following H 2 O 2 treatment in N27 cells using a DNA ELISA technique. H 2 O 2 (100 M) induced time-dependent increases in DNA fragmentation of 109, 154, and 392% at 1, 2, and 4 h, respectively, as compared with the untreated cells (Fig. 2B). Together, these data clearly indicate that N27 dopaminergic cells are highly sensitive to low dose oxidative stress and can undergo activation of caspase-3 and subsequent DNA fragmentation.
Oxidative Stress Induces Caspase-3-mediated Proteolytic Cleavage of PKC␦-We showed previously (1, 32) that apoptotic cell death is mediated by caspase-3-dependent proteolytic activation in Parkinson disease models. To determine whether oxidative stress induces the proteolytic activation of PKC␦ in dopaminergic neuronal cells, we examined the effect of H 2 O 2 on PKC␦ cleavage in N27 cells. N27 cells were exposed to H 2 O 2 (100 M) and assayed for cytotoxicity using the Live-dead® cytotoxicity assay, which demonstrates dead cells with a bright red stain and the live cells with a green fluorescence (A), and Sytox® green stain, which stains nuclear DNA of dead cells with compromised plasma membranes only (B). The two panels demonstrate phase contrast images (left) and fluorescence micrographs (right) to show the extent of Sytox® green staining of cells in the field of view. C, quantitative cytotoxicity analysis indicated a dosedependent increase in N27 dopaminergic cell death when exposed to increasing concentrations of H 2 O 2 (0 -300 M). Data represent mean Ϯ S.E. of n ϭ 6 from two separate experiments. **, p Ͻ 0.01 as indicated by one-way ANOVA analysis using Dunnett's multiple comparison tests. Nonlinear curve regression analysis yielded an EC 50  50 M of the caspase-3 specific inhibitor Z-DEVD-FMK (Fig.  3B). Together, these results indicate that oxidative stress induces capase-3-dependent proteolytic cleavage of PKC␦ in dopaminergic cells.
Oxidative Stress-induced Tyrosine Phosphorylation Regulates Proteolytic Cleavage of PKC␦-To determine whether PKC␦ tyrosine phosphorylation modulates its proteolytic cleavage and kinase activity, we tested the effects of tyrosine kinase inhibitors on PKC␦ activity in N27 dopaminergic cells following H 2 O 2 treatment. Both the broad spectrum Src kinase inhibitor genistein (25 M) and the specific p60 Src peptide inhibitor TSKI (5 M) inhibited the H 2 O 2 -induced PKC␦ proteolytic cleavage (Fig. 4A). The inhibitory effect of TSKI was complete, as compared with the partial block induced by genistein treatment. To examine whether the observed effects of genistein and TSKI were due to inhibition of PKC␦ tyrosine phosphorylation, we measured the level of PKC␦ tyrosine phosphorylation in cells treated with H 2 O 2 in the presence or absence of genistein and TSKI. Tyrosine-phosphorylated proteins were first immunoprecipitated using phosphotyrosine antibody conjugated with agarose beads. The immunoprecipitates were then separated on SDS-PAGE and immunoblotted with PKC␦ to determine changes in tyrosine phosphorylation. As shown in Fig. 4B, PKC␦ cleavage products were tyrosine-phosphorylated in N27 cells when treated with H 2 O 2 for a period of 4 h. This phosphorylation was, however, significantly inhibited by co-treatment with both genistein and TSKI. These results indicate that tyrosine kinase inhibitors can indeed attenuate tyrosine phosphorylation of PKC␦ and its proteolytic cleavage during oxidative insult in dopaminergic neuronal cells.
Tyrosine Phosphorylation of PKC␦ by Oxidative Stress Is Essential for Its Kinase Activity-Next, we determined whether the inhibition of H 2 O 2 -induced PKC␦ proteolytic cleavage by TSKI and genistein affects its kinase activity. N27 cells were treated with H 2 O 2 (100 M) with or without co-treatment with the Src tyrosine kinase inhibitors genistein (25 M) and TSKI (5 M) as well as the caspase-3 inhibitor Z-DEVD-FMK (50 M) for 4 h. PKC␦ was subsequently immunoprecipitated from each sample, and kinase activity was measured by using the 32 Plabeled histone phosphorylation assay in the absence of lipid activators. The data indicate that H 2 O 2 induced a significant increase in the PKC␦ kinase activity that was significantly attenuated by co-treatment with genistein, the caspase-3 inhibitor Z-DEVD-FMK (Fig. 5A), and the specific p60 Src kinase inhibitor TSKI (Fig. 5B). Collectively, these results demonstrate that tyrosine kinase inhibitors effectively block the Values with * ϭ p Ͻ 0.05 and ** ϭ p Ͻ 0.01 depict significant differences between control and treatment groups at each given time point. One-way ANOVA analysis and Dunnett's multiple comparison tests were performed to obtain significance values. Caspase activity in N27 cells exposed to H 2 O 2 (100 M) was further confirmed by using an in situ fluorescence substrate, Z-VAD-FITC co-incubated 10 min before the end of the treatment period. The cells were then fixed and viewed under a FITC filter to determine an increase in green fluorescence, which indicated an increase in caspase activity (inset). FU, fluorescence units. B, cell lysates from the cells treated with H 2 O 2 for 1, 2, and 4 h were used to determine DNA fragmentation and cellular apoptosis using a DNA-ELISA kit. Cells treated with H 2 O 2 (100 M) showed a time-dependent increase in DNA fragmentation. The data represent n ϭ 6 from three separate experiments where ** ϭ p Ͻ 0.01 denotes significant differences between untreated cells and the treatment groups at each time point. The significant values were obtained using one-way ANOVA and Dunnett's multiple comparison tests. AU, absorbance units. PKC␦ tyrosine phosphorylation and proteolytic activation of the kinase.
TSKI Does Not Promote PKC␦ Degradation-To examine whether TSKI blocks H 2 O 2 -induced proteolytic cleavage of PKC␦ by preventing tyrosine phosphorylation of PKC␦ or by promoting the degradation of the cleaved PKC␦ fragment, we transiently expressed the 41-kDa PKC␦ catalytic fragment with a V5 tag in N27 cells and then exposed them to 5 M TSKI for 4 h. Western blot analysis with an antibody directed against the V5 tag revealed that the 41-kDa PKC␦ catalytic fragment was not degraded in TSKI-treated cells (Fig. 6). Densitometric analysis revealed no significant differences in the 41-kDa PKC␦ catalytic fragment expression between control and TSKItreated N27 cells. This result suggests that the PKC␦ catalytic fragment does not undergo degradation by TSKI treatment, but rather TSKI blocks PKC␦ tyrosine phosphorylation and subsequent proteolytic cleavage.
Tyrosine Phosphorylation of PKC␦ at Position 311 Is Essential for Oxidative Stress-mediated Cellular Apoptosis in N27 Dopaminergic Cells-We further investigated whether Tyr-311, which is in close proximity to the PKC␦ cleavage site, regulates the proteolytic activation and proapoptotic function of PKC␦. As shown in Fig. 7A, N27 cells treated with H 2 O 2 (100 M) showed a significant increase in PKC␦ Tyr-311 phosphorylation. Src tyrosine kinase inhibitors genistein and TSKI significantly attenuated H 2 O 2 -induced PKC␦ Tyr-311 phosphorylation (Fig. 7A). We also examined whether inhibition of PKC␦ Tyr-311 phosphorylation by genistein and TSKI alters the H 2 O 2 -induced apoptosis in dopaminergic cells. TSKI and genistein co-treatment significantly attenuated H 2 O 2 -induced caspase-3 activity (Fig. 7B) and DNA fragmentation (Fig. 7C)   shown previously (1) that proteolytic activation of PKC␦ contributes to the Parkinsonian toxin MPP ϩ -induced apoptotic cell death in dopaminergic cells. To confirm the results obtained with the generic oxidant H 2 O 2 , we tested the effects of the p60 Src peptide inhibitor TSKI on PKC␦ proteolytic cleavage and apoptotic cell death in N27 dopaminergic cells following a 24-h treatment with 300 M MPP ϩ . TSKI (5 M) co-treatment inhibited the MPP ϩ -induced PKC␦ proteolytic cleavage (Fig. 8A) and DNA fragmentation (Fig. 8B). TSKI almost completely inhibited both PKC␦ cleavage and DNA fragmentation. These results indicate that PKC␦ tyrosine phosphorylation also regulates dopaminergic cell death induced by the Parkinsonian toxin MPP ϩ .

Neuroprotective Effect of Src Kinase Inhibitors Genistein and TSKI against H 2 O 2 -induced Dopaminergic Cell Death-We
further determined whether inhibition of tyrosine phosphorylation by genistein and TSKI had any effect on the oxidative stress-induced cellular toxicity in dopaminergic neuronal cells. Genistein (25 M) and TSKI (5 M) were co-treated with H 2 O 2 (100 M) for 4 h, and then cytotoxicity was measured by the Sytox® green cytotoxicity assay. Both inhibitors significantly inhibited the cell death induced by H 2 O 2 in N27 cells. Fig. 9A is representative of the fluorescent imaging of H 2 O 2 -treated N27 cells (top panel) compared with those treated with the Src tyrosine kinase inhibitors genistein (middle panel) or TSKI (bottom panel). Quantification of Sytox® green fluorescence revealed that H 2 O 2 (100 M) induced an increase in cell death of 438% over the untreated cells, whereas the cells co-treated with tyrosine kinase inhibitors were almost completely protected against cell death (Fig. 9B). We also tested the effect of c-Abl protein-tyrosine kinase inhibition on H 2 O 2 -induced dopaminergic cell death. For this purpose we used a specific c-Abl protein kinase inhibitor STI571 (Gleevac-Novartis). Co-treatment with STI571 at various concentrations (10 -300 nM) did not afford any neuroprotection in N27 cells exposed to H 2 O 2 (100 M) over a period of 4 h, suggesting that c-Abl does not mediate PKC␦ phosphorylation in dopaminergic neuronal cells (supplemental Fig. 1). Taken together, these data indicate that inhibition of PKC␦ tyrosine phosphorylation by a select group of pharmacological inhibitors can confer neuroprotection in dopaminergic neuronal cells exposed to oxidative stress.
Tyrosine Kinase Inhibitors Genistein and TSKI Do Not Attenuate H 2 O 2 -induced Oxidative Stress in Dopaminergic Neurons-Recent studies attributed the protective effect of the soy isoflavone-derived tyrosine kinase inhibitor genistein to the attenuation of oxidative stress, especially in human cortical neuronal cells (43). Therefore, we next determined whether the tyrosine kinase inhibitors used in our studies attenuated cell death via tyrosine kinase inhibition or from their antioxidant properties. Treatment with genistein (25 M) and TSKI (5 M) did not alter the H 2 O 2 (100 M)-induced oxidative stress or the basal level of ROS generation (supplemental Fig. 2). However, a higher concentration of genistein (50 M) significantly attenuated H 2 O 2 -induced ROS generation (data not shown). This clearly indicates that the neuroprotective effects of the tyrosine kinase inhibitors at the doses selected result from inhibition of tyrosine kinase activity rather than antioxidant effects.
Overexpression of PKC␦ Y311F Mutant Attenuates Proteolytic Cleavage of PKC␦-We used the PKC␦ Y311F mutant to further confirm the role of PKC␦-Tyr-311 phosphorylation in the proteolytic activation and proapoptotic function of the kinase. Tyrosine has been mutated to phenylalanine at position 311 in PKC␦ Y311F , which confers insensitivity to phosphorylation at that amino acid residue. As shown in Fig. 10A, exposure to 100 M H 2 O 2 induced proteolytic cleavage in both normal N27 cells and pcDNA3.1 vector expressing N27 cells, whereas overexpression of PKC␦ Y311F significantly attenuated 100 M H 2 O 2induced PKC␦ proteolytic cleavage, indicating that Tyr-311 is required for the proteolytic cleavage. Also, no cleavage was observed in untreated pcDNA3.1 and PKC␦ Y311F -expressing N27 cells (Fig. 10A). Densitometric analysis of PKC␦ expression in vector-and PKC␦ Y311F -transfected cells showed a slight (Ͻ7%) but not statistically significant decrease in the PKC␦ expression level as compared with the untransfected N27 cells (data not shown). Additionally, we examined whether PKC␦ Y311F -transfected cells are resistant to oxidative stressinduced apoptosis. As shown in Fig. 10, B and C, PKC␦ Y311Fexpressing cells showed significantly reduced H 2 O 2 (100 M)induced caspase-3 activity and DNA fragmentation compared with vector cells. Together, the data clearly indicate that PKC␦ tyrosine phosphorylation at Tyr-311 can effectively regulate proteolytic activation and proapoptotic function of the oxidative stress-sensitive kinase in dopaminergic cells. DISCUSSION The present study reveals that oxidative stress has a profound effect on PKC␦, an important member of a novel PKC isoform family, in dopaminergic neuronal cells. The key findings of this study are as follows: (i) H 2 O 2 induces caspase-3-dependent proteolytic activation of PKC␦ in dopaminergic neuronal cells; (ii) phosphorylation of the Tyr-311 residue in PKC␦ is essential for proteolytic cleavage and activation of the kinase; and (iii) Src tyrosine kinase inhibitors prevent oxidative stressinduced PKC␦ proteolytic activation and apoptotic cell death in dopaminergic neuronal cells. These findings are highly significant because biochemical mechanisms in the neurodegenerative process of PD are not clearly understood despite the numerous implications of oxidative damage in the disease pathogenesis (13,14). The results also suggest that PKC␦ may be an attractive target for development of neuroprotective strategies against oxidative damage in PD.
H 2 O 2 can induce its toxic response by activating the apoptotic cascade in both neuronal and non-neuronal cells (41,44,45). N27 cells were transfected with PKC␦-CF plasmid construct that contains the V5 fusion protein, as described under "Experimental Procedures." Following a 24-h post-transfection, cells were exposed to 5 M TSKI for 4 h, and cell lysates were evaluated for PKC␦-CF protein expression using an antibody directed against the V5 epitope. TSKI treatment did not alter the expression of PKC␦-CF. Densitometric analysis of the 41-kDa PKC␦ catalytic fragment is represented below the Western blot image. Data represents Ϯ S.E. of n ϭ 3.
lease and capsase-9 activation (51). Activated caspase-3 recognizes a specific sequence, DXXD, on protein substrates to induce proteolytic cleavage of these substrates (52). Some of the known substrates of activated caspase-3 are poly(ADP-ribose) polymerase, DNA-PK, topoisomerases, and lamin B1 (53). These proteins can be either activated (54) or inactivated (55) upon proteolysis by caspase-3, leading to apoptosis by either the induction of DNA damage or the impairment of DNA repair.
PKC␦ is a member of the novel PKC family that can be activated in response to numerous cellular stimuli by various mechanisms. Some of the activation pathways include membrane translocation (25,56), tyrosine phosphorylation (19,57), and proteolytic cleavage by an activated enzyme (3,39,58). Recent studies have shown that H 2 O 2 can activate PKC␦ during apoptosis by either membrane translocation (59) or tyrosine phosphorylation (29,60,61) but not through proteolytic cleavage-mediated activation. H 2 O 2 treatment has been shown to induce PKC␦ translocation to the plasma membrane in CaCo2 cells (59) and to the mitochondrial membrane in NIH3T3 cells (62). However, H 2 O 2 can also induce a remarkable increase in PKC␦ kinase activity and tyrosine phosphorylation in cardiomyocytes without any evidence of membrane translocation (28,60). Although oxidative stress-induced PKC␦ activation has been well documented, none of these studies have shown proteolytic cleavage of the kinase as a possible mode of activity, especially in H 2 O 2 -induced apoptosis. In dopaminergic neuronal cells, we report here for the first time that H 2 O 2 treatment induces the proteolytic cleavage of PKC␦ protein into its catalytically active fragments, which leads to a persistent increase in its kinase activity and apoptosis ( Fig. 3 and Fig. 5, A and B). Furthermore, the H 2 O 2 -induced PKC␦ cleavage was completely abolished by the caspase-3 inhibitor Z-DEVD-FMK (Fig. 3B), indicating that the activation of this kinase was dependent on caspase-3 enzyme activity. PKC␦ cleavage induced by caspase-3 activation has been well documented in UV radiation-treated keratinocytes (63), etoposide-treated salivary acinar cells (39,58,63), and in methylcyclopentadienyl managanese tricarbonyl and dieldrin-treated PC12 cells (64). Recently, caspase-3 independent cleavage of PKC␦ in a colon cancer cell line resulted in a kinase-inactive catalytic fragment of PKC␦ (65). Furthermore, Konishi et al. (29) also showed that tyrosine phosphorylation of PKC␦ increases its kinase activity and promotes H 2 O 2 -induced apoptosis in COS cells without any proteolytic cleavage. These findings may be explained by the reported identification of a caspase-3-insensitive PKC␦ isoform, PKC␦-II, in certain cell types such as mouse thymocytes (66), which does not undergo proteolytic activation. We have observed a high expression of PKC␦-I isoform in N27 dopaminergic neuronal cells (2). Hence, it appears that different modes of PKC␦ activation are highly dependent on type of stimulus, cell line, and the expression characteristics of PKC␦.
Tyrosine phosphorylation of PKC␦ has been reported to either increase or decrease the kinase activity during different types of cellular stimulation. The majority of the literature suggests that oxidative stress induces an increase in PKC␦ kinase activity. Some of the nonreceptor tyrosine kinases that have been known to phosphorylate PKC␦ during H 2 O 2 treatment are Src (67), Lck (68), and Syk (69). The tyrosine residues on PKC␦ that can be phosphorylated during oxidative stress  include Tyr-52 (20) and Tyr-187 (70) on the N terminus of the protein, Tyr-512 and Tyr-523 (26, 71) on the C terminus, and Tyr-311 at the intermediate hinge region (22). Konishi et al. (29) have demonstrated that H 2 O 2 treatment induces the phosphorylation of PKC␦ at various tyrosine residues including Ty-311, Tyr-332, and Tyr-512 in COS cells, but phosphorylation at Tyr-311 is critical for initiation of PKC␦ catalytic activity. PKC␦ tyrosine phosphorylation has also been shown to regulate prooxidant etoposide-induced apoptotic cell death in C-6 glial cells (72). Our results demonstrate that phosphorylation of PKC␦ at Tyr-311 is essential for its caspase-3-dependent proteolytic activation. Because Tyr-311 is in close proximity to the caspase-3 cleavage site, DIPD, in PKC␦, it is likely that phosphorylation of this residue may cause conformational change in the kinase structure to expose its cleavage site to caspase-3. The complete inhibition of H 2 O 2 -induced PKC␦ cleavage, kinase activity, and tyrosine phosphorylation by the p60 Src peptide inhibitor TSKI in the present study suggests that p60 Src may be an upstream kinase responsible for the Tyr-311 phosphorylation in dopaminergic cells. In this study, we also show that TSKI treatment does not induce degradation of the cleaved PKC␦ catalytic fragment as determined by the exogenous expression of the PKC␦ catalytic fragment. Re-cently, Src kinase has been shown to phosphorylate PKC␦ on the residue Tyr-311 during H 2 O 2 treatment in primary keratinocytic cells (19,25), but these studies did not characterize the relationship between tyrosine phosphorylation and the proteolytic cleavage of PKC␦. The important functional consequence of inhibition of PKC␦ Tyr-311 phosphorylation by TSKI and the PKC␦ T311F mutant is the attenuation of H 2 O 2 and MPP ϩ -induced DNA fragmentation in dopaminergic neuronal cells. Together this study clearly demonstrates that PKC␦ tyrosine phosphorylation regulates dopaminergic cell death induced by the generic oxidant H 2 O 2 as well as by the Parkinsonian toxin MPP ϩ . Therefore, this study demonstrates that the PKC␦ Tyr-311 phosphorylation site might be a potential target for development of neuroprotective agents against oxidative damage in Parkinson disease. Tyrosine kinase inhibitors derived from soy FIG. 8. Effect of TSKI on MPP ؉ -induced proteolytic cleavage of PKC␦ and apoptotic cell death. Briefly, N27 cells were exposed to MPP ϩ (300 M) in the presence or absence of TSKI (5 M) for 24 h. After treatment, cell lysates were assessed for PKC␦ cleavage by Western blot (A) and DNA fragmentation (B). The data in B represent Ϯ S.E. of n ϭ 6 from two separate experiments. **, p Ͻ 0.001 demonstrates significant differences between the untreated cells and treatment with MPP ϩ ; ##, p Ͻ 0.001 represents significant differences between the MPP ϩtreated cells and those co-treated with TSKI. Significance was determined using ANOVA and Bonferroni's multiple comparison. AU, absorbance units.

FIG. 9. H 2 O 2 -induced dopaminergic neuronal cell death is attenuated by co-treatment with Src tyrosine kinase inhibitors.
N27 cells cultured in 24-well plates were exposed to H 2 O 2 (100 M) for a period of 4 h with or without the addition of Src tyrosine kinase inhibitors genistein (25 M), TSKI (5 M), and the c-Abl kinase inhibitor STI571 (0 -300 nM). Cytotoxicity assay was performed using the Sytox® green nucleic acid dye. A, the left panels represent the phase contrast imaging of cells, and the right panels represents the fluorescent images, as seen under a FITC filter. Cells exposed to H 2 O 2 showed numerous green fluorescent cells as compared with those treated with the tyrosine kinase inhibitors. This indicated a markedly reduced rate of cell death in N27 cells treated with the tyrosine kinase inhibitors and exposed to H 2 O 2 . B, Sytox® green fluorescence in cells treated with H 2 O 2 was also quantified using a Beckman microplate reader. The data represent n ϭ 6 from two separate experiments. #, p Ͻ 0.01 represents significant differences between control and H 2 O 2 -treated groups; **, p Ͻ 0.001 represents significant differences between the H 2 O 2 -treated group and the cells treated with tyrosine kinase inhibitors. Significant values were determined by one-way ANOVA and Dunnett's multiple comparison tests. isoflavones like genistein have been shown to be protective in primary cortical neuronal cultures against oxidative stress by scavenging reactive oxygen species (43). It should be noted that the aforementioned study used a higher dose of genistein at 50 -100 M to demonstrate its antioxidant effect. Furthermore, another study demonstrated that genistein did not attenuate oxidative stress at lower doses (20 -40 M) but exhibited antioxidant properties only at higher concentrations (50 -100 M) (73). In our study we did not observe any attenuation of H 2 O 2induced oxidative stress by either genistein or TSKI (Fig. 8) at the doses used in the tyrosine kinase inhibitors experiments. Thus, the observed neuroprotective effect of genistein and TSKI in our studies was, in all probability, due to the inhibition of tyrosine kinases and not due to antioxidant action.
Oxidative stress has been described as integral to the pathogenesis of dopaminergic degeneration in PD, but the cellular mechanisms of the selective neurodegenerative processes still remain to be established (14,33,74). Our results demonstrate, for the first time, that tyrosine phosphorylation of PKC␦ can regulate the proteolytic activation of the oxidative stress-sensitive kinase PKC␦ in a cell culture model of PD and that modulation of tyrosine phosphorylation can offer neuroprotection against oxidative damage in dopaminergic neuronal cells. FIG. 10. Overexpression of the PKC␦ Y311F mutant in N27 cells attenuates H 2 O 2 -induced cellular apoptosis. N27 cells were transfected to express the Y311F mutant form of PKC␦ using the AMAXA® nucleofection kit as described under "Experimental Procedures." N27 cells expressing empty vector were used as vector control. N27 cells expressing vector or PKC␦ Y311F were then treated with H 2 O 2 (100 M) for a period of 4 h, and the cell lysates were used to determine changes in the caspase-3 activity, PKC␦ proteolytic activity, and DNA fragmentation. H 2 O 2 (100 M) induced significant increases in PKC␦ proteolytic cleavage (A), caspase-3 activity (B), and DNA fragmentation after a 4-h exposure (C). Also, comparison of H 2 O 2 -induced PKC␦ cleavage between vector cells and untreated N27 cells showed no difference. All the measured parameters were significantly attenuated in the cells that expressed the PKC␦ Y311F mutant protein. Equal loading of protein was demonstrated by using ␤-actin immunoblotting (A). B and C represent n ϭ 6 from two to three separate experiments, where ** ϭ p Ͻ 0.001 shows significant differences between untreated and H 2 O 2treated cells, and # ϭ p Ͻ 0.001 represents significant differences between H 2 O 2 -treated cells and those co-treated with the tyrosine kinase inhibitors. Statistical analysis was done using ANOVA and Dunnett's multiple comparison tests.