Novel Insights into the PKCβ-dependent Regulation of the Oxidoreductase p66Shc*

Dysfunctional mitochondria contribute to the development of many diseases and pathological conditions through the excessive production of reactive oxygen species (ROS), and, where studied, ablation of p66Shc (p66) was beneficial. p66 translocates to the mitochondria and oxidizes cytochrome c to yield H2O2, which in turn initiates cell death. PKCβ-mediated phosphorylation of serine 36 in p66 has been implicated as a key regulatory step preceding mitochondrial translocation, ROS production, and cell death, and PKCβ thus may provide a target for therapeutic intervention. We performed a reassessment of PKCβ regulation of the oxidoreductase activity of p66. Although our experiments did not substantiate Ser36 phosphorylation by PKCβ, they instead provided evidence for Ser139 and Ser213 as PKCβ phosphorylation sites regulating the pro-oxidant and pro-apoptotic function of p66. Mutation of another predicted PKCβ phosphorylation site also located in the phosphotyrosine binding domain, threonine 206, had no phenotype. Intriguingly, p66 with Thr206 and Ser213 mutated to glutamic acid showed a gain-of-function phenotype with significantly increased ROS production and cell death induction. Taken together, these data argue for a complex mechanism of PKCβ-dependent regulation of p66 activation involving Ser139 and a motif surrounding Ser213.

Damage caused by reactive oxygen species contributes to the onset and progression of many diseases and pathological conditions, including diabetes, neurodegeneration, ischemia/reperfusion injury (IRI), 3 stroke, and cardiovascular diseases (1). The use of antioxidants, e.g. for the prevention of IRI, has little benefit (2-7), most likely because of the failure to efficiently and timely target these substances to the site of ROS production and action. ROS produced at the mitochondria are normally the byproduct of the incomplete reduction of O 2 in the electron transport chain (ETC). In particular, complexes I and III are involved in this process (8). Work published over the last years has suggested that mitochondrial processes, including ROS production, are subject to regulation by intracellular signaling (9 -12). Phosphorylation of mitochondrial proteins, including subunits of cytochrome c oxidase (COX, complex IV) by PKA modulated ATP generation and ROS production (12). Similarly, phosphorylation of complex I of the ETC by PKA led to decreased ROS production (13). Lowered mitochondrial ROS levels were observed in cells expressing the survival proteins RAF, AKT, and Bcl-2 (14), whereas p38MAPK has been implicated in causing redox stress (15)(16)(17). PKC⑀ protects cells against stress and regulates the ETC, controlling the processes of respiration and ROS production (18,19). Evidence has also been provided for the cardioprotective effect of directly inhibiting complex I activity (20). Interfering with signaling pathways, which are activated during cellular stress and control mitochondrial function, thus may become an alternative approach for the prevention of oxidative damage.
A direct role in mitochondrial ROS production has been demonstrated for p66 (21). p66 is a redox enzyme that generates H 2 O 2 through the oxidation of cytochrome c (21). p66deficient mice lacked any defects during development or adult life but showed, on average, a 30% prolongation of their life span, which correlated with increased resistance to oxidative stress because of a decreased production of ROS, whereas scavenging systems were not affected (21). p66 ablation in mice was beneficial in many disease settings caused by oxidative stress (22)(23)(24)(25)(26)(27)(28)(29)(30). These observations suggest that p66 may have a key function in the cellular response to stress. Because of the lack of inhibitors of its oxidoreductase function, interfering with its upstream activity pathway holds potential for the development of novel therapeutic approaches for the prevention of clinical conditions associated with excessive production of ROS.
PKC␤ has been proposed as an important regulator of the pro-oxidant and pro-apoptotic function of p66 (31). In partic-ular, the phosphorylation of serine 36 on p66 is required for Pin1 binding and mitochondrial import (31). In this work, we further studied the regulation of p66 by PKC␤. The existence of additional regulatory phosphorylation sites was suggested by the observation of a pronounced effect of PKC␤ inhibition on ROSproductionafterpro-oxidantexposure,whereasSer 36 phosphorylation was not affected. Moreover, the site surrounding Ser 36 conforms to a MAPK phosphorylation motif and may be targeted by p38, JNK, and ERK (32,33). Phosphorylation of p66 on Ser 36 has been reported in response to various stress stimuli (29 -31, 34 -38). We have shown recently that JNK1/2 are involved in the phosphorylation of this site and of p66 prooxidant and pro-death function (39). Here we describe the identification and functional characterization of three novel PKC␤ phosphorylation sites on p66 that are required for mitochondrial ROS production and apoptosis induction in response to oxidative stress. These data, together with our recent demonstration of the p66 Ser 36 kinase activities of JNK1/2 (39), demonstrate that the regulation of p66 activation is more complex than previously anticipated. Our results also support that targeting upstream kinases involved in the activation of p66 during cellular stress offers a novel approach for the prevention of oxidative damage.

Results
Regulation of p66 Activation by PKC-To define the requirement of PKC␤ for ROS production, we first confirmed that the experimental conditions studied here result in the activation of PKC, monitored by cell membrane translocation of PKC, as shown before (31). 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent PKC activator, was used as a positive control. We observed cell membrane translocation of all isoforms tested (PKC␣, ␤, and ) in the case of pro-oxidant treatment in HEK293 cells (Fig. 1A) as well as in mouse embryonic fibroblasts (MEFs) (data not shown). Next we analyzed PKC␤-deficient immortalized MEFs and measured ROS levels 30 min after treatment with the pro-oxidants H 2 O 2 or t-BHP (40). PKC␤-deficient cells showed decreased mitochondrial ROS production after pro-oxidant treatment, as monitored by Mito-Tracker Red CM-H 2 XRos (or 2Ј,7Ј-dichlorodihydroflourescence (DCF), data not shown) fluorescence, which was comparable with the response observed in cells lacking p66 (Fig. 1B). Expression of PKC␤ in PKC␤ Ϫ/Ϫ MEFs restored basal and prooxidant-induced ROS levels (data not shown). Conditional knockdown of PKC␤ in WT MEF 3T3 cells using siRNA further confirmed its role in stress-induced ROS production (Fig. 1C). siRNA knockdown of PKC␤ was confirmed with quantitative real-time PCR (Fig. 1D). Furthermore, PKC␤ Ϫ/Ϫ cells showed increased resistance to cell death induced by pro-oxidants, as reported for p66 Ϫ/Ϫ cells (41) (Fig. 1E).
Given the importance of Ser 36 phosphorylation for p66 activation, we analyzed PKC␤-deficient and WT MEFs for the presence of this posttranslational modification. We observed in our experiments phosphorylation of p66 on Ser 36 in WT MEFs after 30 min of treatment with H 2 O 2 (Fig. 1F) or t-BHP (data not shown), but we were unable to reduce or prevent Ser 36 phosphorylation by treatment with two different PKC inhibitors, Gö6850 (data not shown) and Gö6976 (Fig. 1F). Inhibition of PKC␤ with Gö6976 was confirmed because it reduced cell membrane translocation of PKC␤ upon TPA treatment (Fig.  1G) as well as after pro-oxidant treatment both in HEK293 cells and in MEFs (data not shown). PKC␤-deficient MEFs showed slightly increased Ser 36 phosphorylation but, like their WT counterpart, responded with further increased phosphorylation to H 2 O 2 treatment (Fig. 1F). However, because PKC␤ inhibition, as well as PKC␤ knockdown and knockout, was very effective in lowering mitochondrial ROS levels in response to pro-oxidant treatment without decreasing Ser 36 phosphorylation, we hypothesized that Ser 36 might not be the critical PKC␤-regulated site on p66.
Ser 139 , Thr 206 , and Ser 213 Are PKC␤-targeted Sites on p66 -A scan for potential PKC phosphorylation sites (42) in p66 predicted three amino acid residues: Ser 139 , Thr 206 , and Ser 213 (43). These predicted phosphorylation sites lie within (Thr 206 and Ser 213 ) or adjacent to (Ser 139 ) the phosphotyrosine binding (PTB) domain of p66 (21). The structure of the PTB domain of Shc1 protein has been resolved by NMR (44, 45) (PDB code 1SHC) and x-ray diffraction (PDB code 4XWX), and visual inspection suggests that these three sites are located on the surface of the protein ( Fig. 2A) and thus freely accessible for the kinases. We confirmed the predicted PKC␤ phosphorylation sites by using in vitro kinase assays with recombinant PKC␤ and peptides harboring any one of these three predicted sites. We could show phosphorylation of peptides containing Ser 139 , Thr 206 , or Ser 213 , but we were not able to detect substantial Ser 36 phosphorylation (Fig. 2B). These results are in agreement with our previous observation where we identified JNK1/2 as p66 Ser 36 kinases (39).
Next we confirmed these three sites in an in vitro kinase assay by using full-length recombinant p66 protein. The protein was incubated with JNK1 or PKC␤ kinase alone or in combination under kinase assay conditions. The reaction products were separated by SDS-PAGE, which was further immunoblotted to detect p66 Ser 36 (Fig. 3A) or stained with Coomassie. GST-Shc was excised from the gel and digested either with trypsin or Lys C to yield peptides containing Ser 139 and Thr 206 /Ser 213 , respectively, detectable by MS. As shown in Fig. 3B, incubation with PKC␤ resulted in a time-dependent increase in the number of p66 Ser 139 -phosphorylated peptides alone or in combination with JNK but not with JNK alone. p66 Ser 36 phosphorylation was observed with JNK alone or in combination with PKC␤ but not with PKC␤ alone (Fig. 3A). However, Thr 206 -and Ser 213phosphorylated peptides were only detected when both kinases were used together (Fig. 3, B-D). The percentage of Thr 206 / Ser 213 -containing phosphopeptides detected was significantly lower than that of Ser 139 -phosphorylated peptides.
In our cellular assays, increased p66 Ser 36 phosphorylation upon pro-oxidant treatment was not affected by the PKC inhibitor Gö6976 (Fig. 4A), which efficiently decreased Ser 139 phosphorylation that is detected by MS (Fig. 4, B and C). However, in contrast to the in vitro experiments (Fig. 3, B-D), no Thr 206 / Ser 213 phosphorylation was detected.
The Role of Ser 139 in PKC␤-mediated Activation-Ser 139 in p66, equivalent to Ser 29 in p52Shc, has already been reported to be phosphorylated by the PKC␣, ⑀, and ␦ isoforms and is essential for the interaction of ShcA with the protein tyrosine phos-phatase protein tyrosine phosphatase PEST (PTP-PEST) (43). However, a role in pro-oxidant signaling by PKC␤/p66 has never been analyzed. To address the function of Ser 139 in p66 activation/activity, we reconstituted p66-deficient cells with either the S36A or S139A mutant of p66 or with WT p66 and measured ROS production following H 2 O 2 treatment. Equal expression of p66 and its mutants was confirmed by immunoblotting (Fig. 5A). As shown in Fig. 5, B and C, the alanine exchange caused significantly reduced ROS production upon stress. We were also able to show that reduced ROS production in p66S139A reconstituted MEFs correlated with decreased cell death (Fig. 5D).
Thr 206 and Ser 213 in p66 Are Critical for the Control of ROS Production and Cell Death by PKC␤-The physiological relevance of the two PKC␤ phosphorylation sites was evaluated following stable expression of mutant p66 proteins in p66 Ϫ/Ϫ MEFs. Equal protein expression was confirmed by immunoblotting (Fig. 6A). Two different phenotypes were observed (Fig. 6B). Although mutating Thr 206 did not affect the ability of p66 to produce ROS, maintaining the integrity of Ser 213 was essential for the pro-oxidant function of p66 both under basal growth conditions and in response to H 2 O 2 treatment (Fig. 6B). Additional mutations tested included the individual exchange of these residues to glutamic acid. However, these mutants did FIGURE 1. Regulation of p66 redox activity by PKC. A, HEK293 cells were transfected with pECFP-PKC␣, ␤, or , and fluorescence images were acquired before and after treatment with TPA (1 g) or an oxidative challenge (H 2 O 2 1 mM). Before stress application, all PKC isoforms showed a predominantly cytosolic distribution as described previously (31). Upon TPA stimulation or H 2 O 2 treatment, distinct membrane fluorescence was detected (n Ն 3). All images were acquired using a ϫ63 oil immersion objective, and for better visualization, the images were adjusted, and representative cells are shown. not behave differently from the alanine mutants for all parameters studied here (data not shown). Exchanging Thr 206 and Ser 213 to alanine resulted in a protein that did not differ from S213A (Fig. 6B). In contrast, replacing both residues with glutamic acid created a protein that was hyperactive with regard to ROS production (Fig. 6B). This, to our knowledge, is the first demonstration of a constitutively active form of p66. To assure that mutant p66 specifically affected mitochondrial ROS production, we treated cells with low concentrations of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Such mild uncoupling of mitochondria is known to decrease mitochondrial ROS production (14). As shown in Fig. 6C, this treatment significantly reduced p66-dependent ROS production.
Next we tested whether the ROS-deficient phenotype of cells expressing p66 mutated in Ser 213 also resulted in increased resistance to apoptosis in response to different stress stimuli. 24-h treatment of p66 Ϫ/Ϫ MEFs expressing p66S213A with t-BHP resulted in about 80% vital cells (Annexin V-and propidium iodide-negative) similar to p66-deficient cells, in contrast to WT MEFs showing a viability of about 50% (Fig. 7A). We could observe the same resistance of cells expressing p66 mutated in Ser 213 after treatment with hydrogen peroxide (H 2 O 2 ), although less pronounced (data not shown). As observed for ROS production, mutation of Thr 206 had no effect on the ability of the protein to induce cell death (Fig. 7A), whereas expression of p66T206E/S213E, as expected from its ROS phenotype, greatly enhanced cell death. To assure that apoptosis in response to t-BHP was caused by excessive ROS production, we treated cells with the anti-oxidant N-acetylcysteine (NAC). Pretreatment of cells with NAC rescued them from death after pro-oxidant treatment (Fig. 7B).

Discussion
Excessive production or inefficient detoxification of ROS is critically involved in the initiation and progression of many pathological conditions ranging from cancer, stroke, and neurodegenerative diseases to IRI. Although the role of ROS in their development is clearly established, the use of antioxidants for the prevention of oxidative damage failed to provide significant long-term benefits (6, 46 -48). Dysfunctional mitochondria cause a shortage in energy provision, enhance ROS production, and perturb mitochondrial Ca 2ϩ levels. ROS damage nucleic acids, lipids, or proteins, alter the function of signaling proteins, and eventually cause cell death (49) but also lead to the activation of the inflammasome (50) or autophagy (51). p66 meets the criteria for a stress-regulated ROS-producing enzyme involved in death of cells leading to the loss or func- tional impairment of organs: its activation in the cytosol is triggered by cellular stress (including IR) and controlled by signaling proteins in a complex way that is not fully understood; p66 directly causes mitochondrial ROS production and cell damage; the presence of survival signals and normoxic conditions precludes p66 activation; the absence of p66 does not affect physiological ROS signaling, as evidenced by the normal development and postnatal life of p66-deficient mice (21); and a protective effect of p66 ablation was observed against IRI (27), experimental diabetic glomerulopathy (25), vascular cell apoptosis and atherogenesis induced by a high-fat diet (23), cardiomyocyte apoptosis in an experimental model of streptozotocin-induced diabetes (52), and cerebral cortex hypoxia (53).
Although no inhibitors of p66 oxidoreductase activity are currently known, interfering with its activation may lead to novel therapeutic approaches to prevent or ameliorate the conditions described above.
Phosphorylation of Ser 36 has been shown to be critical for the activation of p66 (31). However, in our experiments, we observed greatly reduced ROS production in the presence of PKC inhibitors or following PKC ablation without significant inhibition of Ser 36 phosphorylation (Figs. 1 and 4A). These findings confirm the PKC␤ dependence of the pro-oxidant function of p66 but strongly argue for the existence of an additional kinase responsible for Ser 36 phosphorylation. The sequence surrounding Ser 36 predicts phosphorylation by pro- line-directed kinases (54), and MAPKs have been implicated previously in the phosphorylation of Ser 36 (33,55,56). Recent work by us found Ser 36 phosphorylation by JNK1/2, which was required for ROS production and cell death induction (39). Inspection of the amino acid sequence of p66 suggested three PKC phosphorylation sites with high probability: Ser 139 , Thr 206 , and Ser 213 . All of these three sites were phosphorylated when short peptides served as substrates for recombinant PKC␤ (Fig. 2B), whereas a peptide containing Ser 36 only displayed background phosphorylation. Similarly, using p66 phosphorylated in vitro or immunoprecipitated from HEK293, we detected phosphorylation of Ser 139 but not of Ser 36 (Figs. 3 and  4). Phosphorylated Ser 36 was readily detected in in vitro kinase assays with recombinant JNK1 (39) (Fig. 3A). To verify phosphorylation of Thr 206 and/or Ser 213 in cellular p66, a variety of MS experiments were performed, including collision-induced dissociation, higher-energy collisional dissociation, electron transfer dissociation (ETD), wide-band activation, and threestage mass spectrometry (MS3). Moreover, immobilized metal affinity chromatography (IMAC) and TiO 2 enrichment of phosphopeptides and digestion of proteins with two different enzymes (trypsin and lysozyme C) was carried out. In no case was phosphorylation of Thr 206 and Ser 213 found, which lets us speculate that the amount of p66 phosphorylated on Thr 206 and Ser 213 is below the detection limit. This assumption is corroborated by the demonstration that, in the in vitro kinase assay, we Cells lysates were analyzed for p66 Ser 36 phosphorylation by Western blotting or immunoprecipitated for mass spectrometry. B and C, proteins were digested on beads with trypsin and analyzed by nano-LC coupled via an electrospray ionization interface to a Velos mass spectrometer. The amounts of phosphorylated and non-phosphorylated peptide were calculated by the peak heights of the extracted ion chromatograms provided. A summary graph of more than three individual biological experiments and mass spectrometry analyses is provided. Statistical significance was determined by using ANOVA (*, p Ͻ 0.05; **, p Ͻ 0.01).
were able to detect only a small quantity of Thr(P) 206 /Ser(P) 213 peptide (1.1%) compared with peptides phosphorylated on Ser 139 (18%). If we predict the same ratio for our cellular assays, where, on average, we found 1.6% of the peptides to be phosphorylated on Ser 139 , detection of Thr(P) 206 /Ser(P) 213 peptide will be impossible with the approach used currently.
Characterization of Ser 139 confirmed its importance in the production of ROS and cell death induction following pro-oxidant treatment (Fig. 5). Although the mutation of Thr 206 to alanine did not affect p66 function, exchange of serine in position 213 to alanine generated a mutant protein that was no longer able to respond with enhanced ROS production and cell death induction under stress (Figs. 6 and 7). However, a more complex regulation that may also involve Thr 206 has been suggested by the demonstration that the acidic exchange of both residues rendered p66 constitutively active in terms of basal as well as inducible activity (Figs. 6 and 7). This, to our knowledge, is the first mutant of p66 displaying this property. We currently do not know which aspect in the regulation of p66 activation is affected by these mutations or whether, possibly, the intrinsic oxidoreductase activity of p66 was affected. Based on our data, we propose a model for p66 phosphorylation activation where JNK1/2 are required for the phosphorylation of Ser 36 , which may result in Pin1 binding and mitochondrial import, as reported previously for PKC␤ (21). Subsequent PKC␤ phosphorylation of Ser 139 , Thr 206 , and Ser 213 is required for full activation. Which steps in the previously postulated sequence of p66 activation (57) are thereby regulated is currently unclear. The WW domain of the peptidyl-prolyl cis/trans-isomerase (PPIase) Pin1 binds phosphorylated Ser/Thr-Pro (Ser(P)/ Thr-Pro) motifs, and the PPIase domain then catalyzes cis/ trans-isomerization of such proline-containing peptides (58). Sequence inspection of the candidate phosphorylation sites (Fig. 2B) indicates that only Ser 36 conforms to a possible Pin1 binding side. Nevertheless, we have started to address a possible role of the PKC␤ phosphorylation sites identified here in Pin1 binding and mitochondrial import of p66. We can confirm the expected reduction in Pin1 binding for p66 mutated in S36A but failed to detect altered binding for the p66 Ser 139 or p66T206A/S213A mutants, suggesting that these novel sites are not directly involved in Pin1 binding and, thus, Pin1-dependent mitochondrial translocation of p66 (data not shown). However, the whole situation may be much more complex because Pin1-mediated prolyl isomerization is a critical step in JNK activation (59). Also, evidence has been obtained that JNK1/2 bind to p66 following H 2 O 2 treatment (39) and that JNK1/2 can translocate to the mitochondria under stress via binding to the outer mitochondrial membrane protein Sab, resulting in ROS production and cell death (60 -62). It remains to be shown whether this can provide an alternative route for the mitochondrial translocation of p66.
We thus cannot exclude that these novel PKC␤ phosphorylation sites are somehow involved in mitochondrial import. However, one fact that complicates the analysis of mitochon- drial translocation is the observation we consistently made in various cell systems that mitochondrial p66 is present in substantial quantities already in unstimulated cells (here we also did not observe a difference in the Ala and Glu mutants studied so far) and that the mitochondrial translocation following stimulation is not immediately obvious, as e.g. in the case of JNK1/2 (data not shown). It may therefore very well be that it is the import of de novo phosphorylated protein, which may be a minor fraction, that is critical for the mitochondrial function of p66.
In summary, our experiments provide evidence for a complex mechanism regulating the activation of p66 under stress. Given the importance of p66 in the development of various disease conditions, targeting the key kinases (PKC␤ and JNK1/2) involved in triggering the pro-oxidant and pro-apoptotic function of p66 may become a realistic therapeutic option.
Site-directed Mutagenesis-Primers for mutagenesis of p66 were obtained from Eurofins Genomics (Ebersberg, Germany). Site-directed mutagenesis was performed using the Quik-Change II site-directed mutagenesis kit from Stratagene (La Jolla, CA). Amino acid exchange was confirmed by DNA sequencing at the Department of Genetic Epidemiology at the Medical University Innsbruck or at Microsynth AG (Switzerland).
Coomassie Staining-Following electrophoresis, the gel was soaked in fixing buffer consisting of 40% (v/v) ethanol and 10% (v/v) acetic acid for 1 h. After fixation, the gel was washed for 1 h with deionized water and incubated for 2 h in Coomassie stain (Sigma) with continuous agitation. The gel was destained with deionized water.
MS Analyses-After the in vitro kinase assay, protein bands were excised from Coomassie-stained gel and digested with trypsin from porcine pancreas (Sigma-Aldrich) or Lys C (Sigma-Aldrich) as described previously (65). Tryptic digests were analyzed using an UltiMate 3000 nano-HPLC system (Thermo Scientific, Bremen, Germany) coupled to a Q Exactive Plus or Q Exactive HF mass spectrometer (Thermo Scientific) equipped with a Nanospray Flex ionization source. Settings different to Q Exactive Plus are bracketed. The peptides were separated on a homemade fused-silica microcapillary column (75 m inner diameter ϫ 280 m outer diameter ϫ 10 cm length) packed with 3-m reverse-phase C18 material (Reprosil). Solvents for HPLC were 0.1% (v/v) formic acid (solvent A) and 0.1% (v/v) formic acid in 85% (v/v) acetonitrile (solvent B). The gradient profile was as follows: 0 -2 min, 4% B; 2-55 min, 4 -50% B; 55-60 min, 50 -100% B, and 60 -65 min, 100% B. The flow rate was 250 nl/min. The Q Exacitve Plus mass spectrometer was operating in data-dependent mode selecting the top 20 most abundant isotope patterns with a charge Ͼ1 from the survey scan with an isolation window of 1.6 m/z. Survey full-scan MS spectra were acquired from 300 -1750 m/z at a resolution of 60,000 with a maximum injection time of 120 ms and automatic gain control (AGC) target 1e6. The selected isotope patterns were fragmented by higher-energy collisional dissociation with normalized collision energy of 28 at a resolution of 15,000 with a maximum injection time of 120 ms and automatic gain control target 5e5.
Data analysis was performed using Proteome Discoverer 1.4.1.14 (Thermo Scientific) with the search engine Sequest. The raw files were searched against a database (545 entries) containing the most common contaminants and p66. Precursor and fragment mass tolerance was set to 10 ppm and 0.02 Da, respectively, and up to two missed cleavages were allowed. Carbamidomethylation of cysteine, oxidation of methionine, and phosphorylation of serine, threonine, and tyrosine were set as variable modifications. The peak area of the phosphorylated and non-phosphorylated peptides was calculated by summing up the peak areas or the corresponding precursor ions.
Immunoprecipitated proteins were digested on beads with trypsin and analyzed using an UltiMate 3000 nano-HPLC system coupled via an electrospray ionization interface to an LTQ Velos mass spectrometer (Thermo Scientific). HPLC conditions were the same as with the in vitro kinase assay, except the particle size of the column material, which was 5-m reversephase C18. The LTQ Velos mass spectrometer was operating in data-dependent mode selecting the top four most abundant isotope patterns with a charge Ͼ1 from the survey scan with an isolation window of 2.0 m/z. Survey full-scan MS spectra were acquired from 300 -1800 m/z at an enhanced scan rate. Twostage mass spectrometry (MS2) and MS3 (neutral loss from MS2) scans were fragmented by collision-induced dissociation with a normalized collision energy of 35. The settings for data analysis were the same as with the in vitro kinase assay, except for fragment mass tolerance, which was set to 0.8 Da. The amount of phosphorylated and non-phosphorylated peptide was calculated by the peak heights of the extracted ion chromatograms ROS Measurements-Mitochondrial ROS were imaged by fluorescence microscopy after staining the cells with 100 nM MitoTracker Red CM-H 2 XROS from Molecular Probes (Invitrogen) in serum-free DMEM. For this purpose, 30,000 -120,000 cells/chamber were seeded in 8-well chambered Lab-Tek coverglasses (Nalge Nunc, Rochester, NY). Staining was performed for 30 min at 37°C. Images were taken using an Olympus IX-70 inverted microscope with an Olympus ϫ40 water (numerical aperture, 0.8) and an Olympus U-RFL-T mercury vapor lamp. Image acquisition was performed with a Kappa ACC1 camera and Kappa ImageBase software (Oakland, CA). For MitoTracker Red CM-H 2 XROS, a 568-nm filter was used with a light expose of 1.6 s. Gray values were measured using Scion Image software (Sigma-Aldrich, St. Louis, MO) for Windows. For every experimental condition, gray values from 100 cells were averaged. Alternatively, cellular ROS levels were determined by staining with DCF-diacetate (DCF-DA) (Sigma). Cells were loaded with 7 M DCF-DA and incubated for 15 min at 37°C before FACS measurement. Analysis was done using CellQuest software for FACScalibur (BD Biosciences).
siRNA, RNA Isolation, and Real-time PCR-ON-TARGET Plus SMARTpool mouse PRKCB1 siRNA was purchased from Dharmacon (Lafayette, CO) and transfected using Lipofectamine 2000 (Invitrogen) according to the protocol of the manufacturer. RNA isolation was performed using Qiagen RNeasy mini kit (Qiagen, Hilden, Germany), and cDNA synthesis was done using the Fermentas RevertAid first strand cDNA synthesis kit (St. Leon-Rot, Germany). PKC␤ expression was determined by real-time PCR using RT 2 real-time SYBR Greenfluorescein PCR master mix (Bio-Rad) according to the protocol provided on an iQ5 multicolor real-time PCR detection system from Bio-Rad. Using 100 nM PKC␤ siRNA, on average 86% knockdown was achieved and was used for further experiments. The RT 2 qPCR Primer Assay for Mouse PRKCB1 was ordered from Qiagen. The expression was normalized to the housekeeping gene RSP-29. Data analysis was performed using the 2 Ϫ⌬⌬Ct method (66). Melting curve analysis was done for quality control.
Immunoprecipitation-HEK293 cells were transfected with 2 g of pBABEpuro p66 (68) using Lipofectamine (Invitrogen). 48 -72 h after transfection, 300,000 or 600,000 cells were lysed in 1 ml of Nonidet P-40 lysis buffer supplemented with a protease inhibitor mixture set from Calbiochem (Merck). 40 l of the lysate was kept as lysate control, mixed with 8 l of 6ϫ Laemmli buffer, and incubated for 5 min at 95°C. Preclearing was performed with 40 l of protein G-agarose beads from Roche, incubation for 1 h at 4°C, and shaking at 300 rpm. Immunoprecipitation was done for 4 h with 40 l of protein G-agarose (Roche) and 2.0 g of Shc antibody. Cell Death Assays-Apoptosis was determined using FACS after staining with Annexin V-FITC (Enzo Lifesciences, Farmingdale, NY) and propidium iodide (Carl Roth, Karlsruhe, Germany) or by counting stained (dead) and unstained (living) cells with a Neubauer counting chamber after incubation with trypan blue (Sigma-Aldrich). A detailed protocol has been described previously (39).