Plodia interpunctella is a highly prevalent food pest in human dwellings, and has been shown to contain a number of allergens. So far, only one of these, the arginine kinase (Plo i 1) has been identified.The Indianmeal moth The aim of this study was to identify further allergens and characterise these in comparison to Plo i 1.P. interpunctella was screened with the serum of a patient with indoor allergy and IgE to moths, and thioredoxin was identified as an IgE-binding protein. Recombinant thioredoxin was generated in E. coli, and tested together with Plo i 1 and whole moth extracts in IgE immunoblots against a large panel of indoor allergic patients' sera. BALB/c mice were immunised with recombinant thioredoxin and Plo i 1, and antibody production, mediator release from RBL cells, T-cell proliferation and cytokine production were measured.A cDNA library from whole adult P. interpunctella thioredoxin, compared to 25% reacting with recombinant Plo i 1. In immunised BALB/c mice, the recombinant allergens both induced classical Th2-biased immune responses such as induction IgE and IgG1 antibodies, upregulation of IL-5 and IL-4 and basophil degranulation.For the first time a thioredoxin from an animal species was identified as allergen. About 8% of the sera from patients with IgE against moth extracts reacted with recombinant P. interpunctella allergen.Thioredoxin from moths like Plo i 1 acts like a classical Type I allergen as do the thioredoxins from wheat or corn. This clearly supports the pan-allergen nature of thioredoxin. The designation Plo i 2 is suggested for the new  Arthropods represent more than three quarters of all animal species, and get into contact with humans in multiple ways. Arthropod antigens can cause Type I allergies with various manifestations, such as the acute and life-threatening insect venom allergies Tineola bisselliella in an infested home Galleria mellonella in a company producing fish bait Although moths like cockroaches are typical and widespread household pests, there is much less research on Type I allergy against moth proteins. Historically, that moths cause inhalant allergies had been reported as early as 1928 by Vaughan Plodia interpunctella is a common household and stored product pest. Its larvae feed on dry foodstuffs such as grains, nuts, dried fruit, or chocolate. This pest had been suspected early to be a possible source of allergens in mills P. interpunctella allergen to be identified on the molecular level (Plo i 1) was an arginine kinase e.g. from shrimp The Indianmeal moth P. interpunctella whole larval extracts were positive in 51% when probed with sera from indoor allergic patients P. interpunctella revealed a further allergen, a thioredoxin.IgE immunoblots of Malassezia spp. Thioredoxins are ubiquitous redox-active proteins found in eukaryotes and prokaryotes. They activate proteins by reduction of cysteine disulphide bonds In this study, the allergenic potential of thioredoxin as a new arthropod allergen was examined in comparison to Plo i 1 by IgE immunoblotting and in a mouse immunisation model.The study was approved by the ethics committee of the Medical University of Vienna and the Vienna General Hospital (application EK Nr. 322/2008). The anonymised sera to be tested in immunoblots  are displayed in detail in P. interpunctella was screened with the serum from an indoor-allergic patient with IgE reactivity to Indianmeal moths.The diagnosis of Type I allergy was based on case history and symptoms , skin prick testing as well as CAP-RAST  testing using a panel of extracts from indoor  and outdoor  allergen sources. The cDNA library from adult P. interpunctella larvae were left to hide for metamorphosis in a roll of cardboard with vertical holes. When the moths emerged, they were immediately frozen in liquid nitrogen. Four hundred moths (around 3.2 g) were homogenised in 30 ml of TRIzol reagent , and about 10 mg of total RNA were extracted. Poly(A)+ RNA was prepared using the Poly(A)Ttract system  with a yield of 4.9 µg from 4 mg of total RNA. The cDNA library prepared with the Uni-ZAP system  6 clones.5) from the primary library were plated on Escherichia coli XL1-Blue (Stratagene). Synthesis of recombinant proteins was induced by adding nitrocellulose filters  soaked in 10 mM isopropylthio β-D-galactoside (IPTG). The filters were blocked, and probed with a 1/10 dilution of a serum which contained IgE against several moth proteins. IgE bound to recombinant moth proteins was detected by 125I-labeled anti-IgE antibodies (Pharmacia) followed by autoradiography. Further rounds of screening were then performed to obtain single phage clones.λZAP phages (2.8×10in vivo excision The cDNA-containing plasmids were obtained from the isolated phages by C ATA TGT CGA TCC ACA TCA AAG ACG TTG AGG AC-3′5′-GGG ACT TC and ExTrxR CT CGA GTT AAT GAT GGT GAT GAT GAT GCT TGA GTT TCA AAA TGG TGG ACC G-3′5′-TCC G were used for PCR to amplify the P. interpunctella thioredoxin coding sequence from the cDNA template (clone 1). The primers contain NdeI and XhoI restriction sites (bold) for cloning into the expression plasmid pET-17b  plus a hexahistidine-encoding sequence (underlined) for recombinant protein purification.The oligodeoxynucleotide primers ExTrxF E. coli BL21-CodonPlus (DE3) (Stratagene). The cultures were grown to an OD600 of 0.75, recombinant protein production was induced by addition of 1 mM IPTG and shaking overnight at 18°C. Harvesting of cells, lysis and purification of recombinant thioredoxin via metal chelate affinity chromatography under native conditions were performed according to the supplier's protocol . Recombinant P. interpunctella arginine kinase (Plo i 1) was produced as described The recombinant pET-17b plasmid was transformed into Ephestia kuehniella), a species closely related to P. interpunctella but considerably larger, were obtained by grinding of 150 moths with liquid nitrogen in a mortar. Fifteen ml of 1× reducing gel loading buffer were added, samples were denatured for 10 min at 95°C, and debris was removed by centrifugation. The whole extracts or, respectively, the purified recombinant allergens (arginine kinase (Plo i 1) and moth thioredoxin) were separated on preparative 12.5% SDS - polyacrylamide gels with an approximate protein concentration of 20 µg/cm (extracts) or 10 µg/cm (of each purified recombinant protein) as estimated by Coomassie blue-stained test gels. Proteins were blotted onto nitrocellulose membranes (Schleicher & Schuell), and 5 mm strips were cut after the transfer. The strips were blocked 2×5 min and 1×30 min at room temperature with buffer G  Tween 20, 0.5% (w/v) bovine serum albumin, 0.05% (w/v) NaN3, pH 7.5), and incubated with a 1/10 dilution of patients' sera in buffer G overnight at 4°C. After washing 2×5 min and 1×30 min in buffer G, bound IgE was detected by overnight incubation at room temperature with 125I-labeled anti-IgE antibodies , washing as above, and autoradiography at −80°C.Whole extracts from imagines of the Mediterranean flour moth . Control mice were sham-treated with PBS. Blood samples were taken before treatment (day 0), and 7 days after the last immunisation (day 49) by tail bleeding. At sacrifice cell suspensions from spleens were prepared as described Female BALB/c mice  were purchased from the Research Institute for Laboratory Animal Breeding . Sensitisation was performed by four subcutaneous (s.c.) injections  of 1 µg, 5 µg or 25 µg of Plo i 1 (arginine kinase) or Plo i 2 (thioredoxin) adsorbed to aluminium hydroxide (Al(OH)Microtiter plates  were coated with 5 µg/ml of the recombinant allergens Plo i 1 or Plo i 2, respectively, and incubated with sera. Serum samples were diluted 1/500 for IgG2a, 1/1000 for IgG1 and 1/10 for IgE. Rat anti-mouse IgG2a, IgG1 and IgE antibodies  were used, followed by peroxidase-conjugated mouse anti-rat IgG antibodies  Sera were incubated with RBL-2H3 cells  at dilutions of 1/10. IgE-dependent degranulation of RBL cells was induced by adding 0.03 µg of each allergen (Plo i 1 or Plo i 2) diluted in 100 µl Tyrode's buffer. Supernatants were analysed for ß-hexosaminidase activity as described previously 5 cells/well) after stimulation with the recombinant allergens Plo i 1 or, respectively, Plo i 2 (2 µg/well) was measured as described previously i.e. the stimulation index [SI], was calculated.Proliferation of splenocytes (2×106 cells/well) incubated for 48 hours with Plo i 1 or Plo i 2 (10 µg/well) Levels of IL-4, IL-5 and IFN-γ were measured in spleen cell cultures .As described above, a λZAP cDNA library from P. interpunctella thioredoxin (Pi) aligned with selected other thioredoxins. The most closely related sequence with 83% amino acid identity was from the domestic silkworm Bombyx mori (Bm). The thioredoxin from the more distantly related arthropod Litopenaeus vannamei (Lv) and human thioredoxin (Hs) were 58% and 46% identical, respectively, to P. interpunctella thioredoxin. The allergenic fungal thioredoxin (2J23_A) Mala s 13 from Malassezia sympodialis (Ms) and wheat (Triticum aestivum) thioredoxin Tri a 25 (Ta) (CAB96931) were 46% and 41% identical. All the sequences contained the active site consensus WCGPC with the two redox-active cysteines.The BlastP search clearly identified the protein as thioredoxin. We obtained only two other IgE-positive cDNAs from the same screen not coding for thioredoxin. The delicate extraction of RNA from adult moth material, a possible advantage of λZAP phage expressing thioredoxin leading to a preponderance in the composition of the plated library, or a peculiarity of the patient's serum used for screening may contribute to this finding. We did not investigate this matter in more detail and focused on the characterisation of thioredoxin as a novel allergen from moths.P. interpunctella were separated together under denaturing and reducing conditions in SDS-PAGE, blotted onto nitrocellulose, and strips were probed with the sera that had shown positive reactivity with the whole moth extracts. The signals from the recombinant allergens arginine kinase Plo i 1 and thioredoxin are displayed in Recombinant thioredoxin (11.7 kDa) and arginine kinase, Plo i 1, (39.9 kDa) P. interpunctella thioredoxin, six in the various indoor allergic groups and one in the seafood allergic group, but none in mite negative group. This corresponds to 8% of all moth extract positive patients, 5% of all allergic patients of this study. Again the strongest signals were observed in patients with concomitant mite allergy or concomitant mite and dander allergy, and in the seafood allergic patients. Due to this small but significant number of sera with IgE to moth thioredoxin, we proposed to name this allergen Plo i 2, and it is now available in the IUIS allergen nomenclature database as Plo i 2.0101.Counting only the clearly positive signals, seven patients' sera had IgE towards P. interpunctella thioredoxin better than the E. kuehniella homolog. To investigate this cross-reactivity, we performed an inhibition experiment as done previously P. interpunctella thioredoxin overnight at 4°C, and then used to probe strips of blotted E. kuehniella antigens as described above  and arginine kinase (Plo i 1) we sensitised mice four times subcutaneously with three different concentrations of the recombinant allergens . Treatment with thioredoxin induced strong Th2 responses with increased IgG1 levels for all three concentrations, whereas treatment with 5 µg and 25 µg showed significantly increased antibody levels in comparison to sham-treated mice. IgE antibody production in thioredoxin-treated mice was significantly upregulated after sensitisation with the lowest concentration of 1 µg and with the highest concentration of 25 µg allergen, respectively, compared to controls. The typical Th1-associated IgG2a was only enhanced in mice, treated with 1 µg and 25 µg thioredoxin .Arginine kinase-specific IgG1 was induced with all three concentrations, but only treatment with 25 µg showed a significantly increased antibody production, compared to controls. Whereas arginine kinase-specific IgE was significantly induced with a concentration of 5 µg as well as 25 µg, in comparison to sham-treated mice, IgG2a production was significantly increased in 25 µg- arginine kinase-treated mice .For thioredoxin, IgE-dependent basophil degranulation was induced with all three concentrations, significantly increased only in 1 µg-sensitised group in comparison to controls. Similarly, immunisation with 1 µg and 5 µg arginine kinase induced significantly high IgE-depended basophil degranulation compared to controls .Both arginine kinase and thioredoxin induced strong cellular responses with high IL-5 and IL-4 levels in restimulated spleen cell cultures, while IFN-γ was induced only by arginine kinase. Th2 cytokine production in splenocytes of thioredoxin-treated mice showed significantly induced IL-5 levels in 1 µg-sensitised groups, compared to sham-treated mice. Thioredoxin-specific IL-4 was increased in all treated groups, in comparison to controls. Thioredoxin did not induce IFN-γ . ArgininThioredoxin restimulated splenocytes showed an increased proliferation response (SI: 4) for all 3 treatment groups. Immunisation with 25 µg thioredoxin provoked a significantly induced proliferation compared to sham-treated mice.Arginine kinase-sensitised mice showed cellular responses only in the group with 1 µg treatment concentration, not with 5 µg or 25 µg, in comparison to controls .The IgE immunoblot experiments probing indoor allergic patients' sera with extracts from adult moths confirmed previous results on larval extracts in vitro reactivity is weaker than in mite positive patients. As some of these individuals may bind to cross-reactive carbohydrate determinants (CCDs) in the moth extracts, we do not know at this time how much mite-independent moth allergy could exist. Certainly, the allergens cross-reactive between moth and mite may contribute to this enhanced IgE reactivity. An additional phenomenon is that the mite and animal dander allergic patients together with the seafood allergic patients show the highest levels of IgE binding to moths. It appears as if these polysensitised patients show a stronger atopic predisposition and higher IgE levels. So far we do not know what is the contribution of moth allergens in the sensitisation of these patients.The complex and only partially resolved immunoblot band patterns show that there are numerous moth IgE antigens. Although many patients without mite allergy have IgE against moth components, the P. interpunctella cDNA library produced a number of immunopositive clones mostly encoding thioredoxin. Testing the moth positive sera against recombinant thioredoxin gave a positive reaction in 5% of all examined allergic patients, 8% of moth positive patients, whereas recombinant Plo i 1 tested positive in 14% of all sera, 25% of moth positive sera. The data show that thioredoxin (now named Plo i 2) is a minor allergen, however, the majority of those with positive reactivity showed a high level of thioredoxin-specific IgE. The prevalence of thioredoxin reactivity was highest in the group of indoor allergic patients reacting with mites and animal dander.IgE screening of a Recombinant thioredoxin like arginine kinase induced a Th2-biased immune response with significantly enhanced IgE and IgG1 antibody levels at the humoral site as well as a marked upregulation of IL-5 and IL-4 cytokines on the cellular site. Moreover, significantly enhanced IgE-mediated basophil degranulation with even 1 µg allergen confirmed the clinical relevant Th2-biased immune response of thioredoxin as well.Thioredoxin is a truly ubiquitous protein with pleiotropic activities found both in eukaryotes and prokaryotes. It had been identified as an allergen in wheat and maize Finally, we have noted that under certain conditions thioredoxins may also diminish the allergic response


Saccharomyces cerevisiae. Sensitivity to trichothecenes increased when functional mitochondria were required for growth, and trichothecenes inhibited mitochondrial translation at concentrations, which did not inhibit total translation. In organello translation in isolated mitochondria was inhibited by type A and B trichothecenes, demonstrating that these toxins have a direct effect on mitochondrial translation. In intact yeast cells trichothecenes showed dose-dependent inhibition of mitochondrial membrane potential and reactive oxygen species, but only at doses higher than those affecting mitochondrial translation. These results demonstrate that inhibition of mitochondrial translation is a primary target of trichothecenes and is not secondary to the disruption of mitochondrial membranes.  Fusarium head blight (FHB) reduces crop yield and results in contamination of grains with trichothecene mycotoxins. We previously showed that mitochondria play a critical role in the toxicity of a type B trichothecene. Here, we investigated the direct effects of type A and type B trichothecenes on mitochondrial translation and membrane integrity in  Fusarium graminearum, which causes head blight or scab of wheat and barley, resulting in yield reduction and contamination of grains with trichothecene mycotoxins -Met  was added. The reaction was stopped after 20 min by washing and resuspending cells in 500 µL (20 mM) cold methionine and 75 µL Rodel Mix . An equal volume of 50% TCA (trichloroacetic acid) was added to the mix and filtered through 2.4 cm glass fiber filters . Filters were then washed, once each, with 5% TCA and 95% ethanol and scintillation counts per minute (CPM) measured. CPM readings were finally normalized to OD600 to indicate total translation.Cells were grown in synthetic methionine dropout (SD-Met) media  containing MSG as the nitrogen source and 2% raffinose, which prevents glucose repression and enhances respiration. Cultures grown to an OD600.The mitochondrial translation assay was done as outlined in 600. All centrifugation steps, unless otherwise noted, were carried out for 5 min at 2500 g. The pellet was washed with H2O and incubated in freshly prepared TD buffer  for 5 min with gentle shaking. The pellet was then resuspended in SP buffer  to which zymolyase was added for spheroplast formation. The spheroplasts were carefully collected and resuspended in 2X SHP buffer  to which equal volume of ice-cold H2O containing 1 mM PMSF was added. The resulting suspension was then carefully homogenized with a glass homogenizer. The homogenate was then centrifuged twice at 4 °C and the supernatant collected from each spin was combined and centrifuged for further 10 min at 12,000 g at 4 °C. The resulting pellet was resuspended in 1X SH buffer  for the in organello translation assay after protein quantification by the Bradford assay [Mitochondria were isolated from yeast as described previously . Brieflyrd assay .In organello translation, using isolated yeast mitochondria, was done as described previously [eviously  with somNeurospora crassa, as described previously [-DA) for ROS generation and MitoTracker Red CMXRos for mitochondrial membrane potential according to manufacturer’s protocol. Stained cells were then examined with the Olympus BX41 microscope. All images were captured and analyzed using the MetaMorph® Microscopy Automation & Image Analysis software . Mitochondrial morphology was examined using an epifluorescence microscope (Olympus BX41). BY4743 cells were transformed with pVT100U-mtGFP, which contains the green fluorescent protein (GFP) targeted to the mitochondria, with the presequence from the subunit 9 of the F0-ATPase of eviously . Trichot® . For each sample 25-50,000 events were recorded. Channel gating and histogram plots were made using the CFlow Plus Analysis software . Changes in MitoTracker Red and DCFH-DA fluorescence were detected using the FL1 and FL3 channel respectively.Trichothecene-treated and untreated cells, following staining with the appropriate dyes, were analyzed using the Accuri C6 Flow CytometerData from the growth and translation assays were analyzed and the graphs were plotted using Microsoft Excel. 0 strains, devoid of functional mitochondria can only grow in media containing a fermentable sugar, such as dextrose. A rho0 strain derived from the wild type strain by ethidium bromide treatment was less sensitive to each trichothecene tested  inhibited mitochondrial translation to a greater extent in wild type yeast than in strains selected for trichothecene resistance, implicating mitochondrial translation as a site of action . We invein organello assay, in which mitochondria isolated from wild type yeast (W303) [in organello translation relative to mitochondrial translation in intact cells is likely due to the short duration (10 min) of treatment of the isolated mitochondria relative to the longer (6 h) treatment of intact cells. These results demonstrate that trichothecenes directly inhibit mitochondrial translation and provide evidence that inhibition of mitochondrial translation is not a secondary effect of the inhibition of cytosolic translation. To determine if trichothecenes have a direct effect on mitochondrial translation, we used an t (W303)  were tret (W303) . As showWe previously showed that Tcin treatment led to the fragmentation of the tubular mitochondrial network in yeast, and Tcin-mediated cell death was partially rescued by mutants that regulate mitochondrial fusion and maintenance of the tubular network of mitochondria . To deteM) [M, we analyzed yeast cells stained with MitoTracker Red using flow cytometry. Wild type cells stained with MitoTracker Red exhibited an active mitochondrial membrane potential observed as an increase in MitoTracker Red staining  . To detestaining A. In con control A. Treatmchondria    showed vstaining B. ROS lestaining D, in agrstaining A and theining . These results indicate that the effects of trichothecenes on ΔΨM and ROS generation are dose and time-dependent. Although mitochondrial membrane integrity was not compromised at 6 h post treatment with the low doses of trichothecenes, mitochondrial translation was significantly inhibited, suggesting that the inhibition of mitochondrial translation is not a consequence of the damage to the mitochondrial membrane integrity. Moreover, ΔΨM and ROS production were inhibited and mitochondrial morphology was altered after treatment of yeast with the low doses of trichothecenes for 18 h  and B (Tcin) trichothecenes on mitochondrial translation and membrane integrity. Wild type yeast treated with trichothecenes showed an increase in growth sensitivity when functional mitochondria were required for growth . Yeast cin organello translation. Translation was inhibited in isolated yeast mitochondria after treatment with increasing concentrations of type A and type B trichothecenes (To determine if trichothecenes affect mitochondrial translation, we examined inhibition of total and mitochondrial translation after exposure of yeast cells to increasing doses of trichothecenes. When yeast cells were treated with low doses of trichothecenes, total translation was not inhibited. However, mitochondrial translation was inhibited A. Dose-dthecenes E. These The inhibition of mitochondrial translation could be due to the damage to the mitochondrial membranes, which might render mitochondria nonfunctional. To address this possibility, we investigated the effects of type A and B trichothecenes on mitochondrial morphology. The tubular mitochondrial network, characteristic of actively respiring cells, was severely fragmented 6 h after treatment of yeast with the high doses of trichothecenes A, but noM and ROS levels, two biomarkers for mitochondrial integrity. T-2 toxin was previously reported to promote severe damage to the DNA via generation of ROS in both yeast and mammals [To further investigate the effects of trichothecenes on mitochondrial membranes, we measured ΔΨ mammals ,19,32. M mammals ,34,35,36 mammals ,38. A de mammals ,40,41. M (M (Unlike actively respiring cells, which exhibited an active ΔΨM A and lowM B, cells M (M C. A corrM (M D. The frM (M A could tM (Mitochondrial translation was inhibited when yeast cells were treated with low doses of trichothecenes for 6 h A when thM A. These M and ROS levels in cells treated with the low doses of trichothecenes, which did not inhibit total translation. ΔΨM increased in cells treated with low doses of Tcin at 6 h after treatment (M (M observed at 18 h could be due to an increase in ROS that occurred earlier. ROS levels may begin to drop as the mitochondrial membrane becomes depolarized by the trichothecenes. In a previous study, Chaudhari et al. [xrefTo determine if mitochondrial membranes were affected due to inhibition of cytosolic translation, we examined ΔΨreatment A. Moderareatment B were altment (M C and ROStment (M D decreas al. xref observed al. xref. The fraFusarium mycotoxins, such as enniatins [Recent studies have implicated mitochondria as a target of other toxins. A type I RIP, saporin-6 was shown to specifically cleave the human mitochondrial DNA D-loop . Mitochonniatins , and fumnniatins . Our resThe present work shows that type A and type B trichothecenes inhibit mitochondrial translation independent of their effects on cytosolic translation and mitochondrial membrane integrity. Inhibition of mitochondrial translation represents a novel mode of action for trichothecene mycotoxins and a potential target for developing protection strategies.


Total serum immunoglobulin (IgE) test is usually performed to aid in the diagnosis of allergic diseases, but its reference values may vary among people of different ethnic backgrounds.To establish reference values of total IgE in Asian children and to assess their significance in the diagnosis of atopy and allergic diseases.1321 Asian children aged 5-18 years in the Prediction of Allergies in Taiwanese CHildren (PATCH) study, a population-based cohort, were evaluated for total and specific IgE by ImmunoCAP and Phadiatop Infant, respectively.1/FVC, were associated with higher total IgE levels in univariate analyses. Multivariate analysis revealed that atopy was the single most important determinant explaining 66.1% of the variability of total IgE levels in this population. The area under the receiver-operator characteristic (ROC) curve of total IgE for diagnosing atopy, asthma, rhinitis, and eczema were 0.92, 0.72, 0.70, and 0.70, respectively. The sensitivity, specificity, and positive and negative predictive values of total IgE at the optimal cutoff of 77.7 kU/L on the ROC curve for diagnosing atopy were 82.3%, 87.1%, 89.5%, and 78.6%, respectively. The corresponding values using the upper 95% CI of total IgE (164.3 kU/L) in non-atopic children were 61.2%, 95.0%, 94.3%, and 64.6%, respectively; whereas a customary cutoff (100 kU/L) provided accuracy between that of the aforementioned two cutoffs. Total IgE at the cutoff of 77.7 kU/L provided modest sensitivity and specificity (49.0%-78.3%) for diagnosing allergic diseases, but had high negative predictive values (84.2%-97.9%).Male, atopy, allergic diseases, recent symptoms of upper respiratory infection, and lower FEVTotal serum IgE discriminates Asian children with and without atopy independent of allergic symptoms, with an optimal cutoff of 77.7 kU/L. The study confirms the insufficient diagnostic accuracy of total IgE alone to detect allergic diseases, but low total IgE levels may help exclude allergic diseases. Serum immunoglobulin E (IgE) is the immunoglobulin mediating allergic sensitization to various allergens. Several studies have shown a quantitative relationship between serum IgE and atopy or various allergic diseases . IgE levPrevious studies have demonstrated ethnic differences in total IgE levels ,10. ThusThis study aimed to establish reference values of total IgE in a large population-based sample of Asian children, and to assess the significance of these values in the diagnosis of atopy  and allergic diseases.The study was a part of the Prediction of Allergies in Taiwanese CHildren (PATCH) study, a population-based prospective cohort study that was launched in 2007 to investigate the epidemiology and predictive factors of allergies and asthma in children ,16. Brie2) whereas body surface area (BSA) was defined as the square root of product of weight (kg) times height (cm) divided by 3600. This study was approved by the Institutional Review Board of Chang Gung Memorial Hospital and the parents of each subject provided written informed consent.Weights and heights were measured according to standard protocols. Body mass index (BMI) was defined as weight (kg) divided by the height squared . Specific IgE was determined by a commercial assay for IgE  against the most common inhalant and food allergens  . Univariate analyses were performed using simple linear regression and unpaired t-tests to assess associations between log-transformed total IgE and the following variables: age, anthropometric measurements, pulmonary function variables, sex, allergic sensitization, symptoms of upper respiratory infection (URI) in the past two weeks, passive smoking, preterm birth. Variables with a The characteristics of the 1,321 subjects  are shown in r = 0.551, P < 0.001; 1/FVC ratio . Asthma, rhinitis, and eczema were all associated with higher total IgE levels .  Boys had significant higher total IgE levels than the girls  and thus none of the candidate variables were excluded from the multivariate analysis. In the current study, the values of tolerance were all larger than 0.1 (ranging from 0.885 to 0.968) and the VIF values were all smaller than 2 (ranging from 1.033 to 1.130). Thus, multicollinearity was not considered a problem in this analysis. Only atopy was significantly and independently associated with total IgE levels in a multiple linear regression model which explained 66.1% of total variation in total IgE levels in this large population cohort of children  for these three allergic diseases.We also performed sex stratified ROC analyses, which yielded very similar cutoff levels for boys and girls. Specifically, the optimal cutoff levels of total IgE on the ROC curves for diagnosing atopy were 79.7 kU/L and 77.7 kU/L for boys and girls, respectively. This large population-based study defines the reference value of total IgE levels in Asian children, which is independent of age and sex. Total IgE level discriminates Asian children with and without atopy independent of allergic symptoms, with an optimal cutoff of 77.7 kU/L. In contrast, this study confirms the insufficient diagnostic accuracy of total IgE levels alone to detect allergic diseases. The findings provide important information on interpreting total IgE levels in Asian children.There has been considerable interest and debate regarding the validity of serum total IgE test as a diagnostic tool for atopy and allergic diseases. The current study assesses three sets of potential reference values of total IgE levels. The majority of reference values of total IgE levels published to date refer to the upper 95% CI of total IgE in non-atopic or non-allergic subjects ,11,12,13This study demonstrates the insufficient diagnostic accuracy of total IgE levels to detect allergic diseases regardless of which cutoff value is being used, indicating that total IgE is linked more to atopy than directly to symptoms. These results are in accordance with previous studies ,11,12,13This population-based study identifies that atopy is the single most important determinant of total IgE levels in Asian children, which explains 66.1% of total variation in total IgE levels in this population. Moreover, the clear dose-response correlation between total IgE levels and allergic sensitization in terms of Phadiatop Infant titers suggests that the relationship between IgE levels and atopy is not only qualitative but also quantitative. The reference value of total IgE levels established in this study is independent of age, sex and smoking. These results ensure that a simple set of reference value can be applied to children in the same age range. A number of previous studies, mostly conducted in adults, suggest the role of age, sex, or smoking on affecting total IgE levels, while other studies disagree ,4,11,21.The representative sampling of children in the community, large sample size, incorporation of objective markers of atopy all add strength to the results of this study. In addition, the population-based design in this study allows us to determine an optimal cutoff on the ROC curve, bringing the advantage that the ROC curve is independent of disease prevalence . Apart fIn conclusion, this large population-based study provides important practical information regarding the interpretation of total IgE levels in Asian children. Total serum IgE test discriminates Asian children with and without atopy independent of allergic symptoms, with an optimal cutoff of 77.7 kU/L. The study confirms the insufficient diagnostic accuracy of total IgE levels alone to detect allergic diseases. Another important message for physicians is that low serum total IgE levels in children may help exclude the diagnosis of allergic diseases.


Radiation-induced normal brain injury is associated with acute and/or chronic inflammatory responses, and has been a major concern in radiotherapy. Recent studies suggest that microglial activation is a potential contributor to chronic inflammatory responses following irradiation; however, the molecular mechanism underlying the response of microglia to radiation is poorly understood. c-Jun, a component of AP-1 transcription factors, potentially regulates neural cell death and neuroinflammation. We observed a rapid increase in phosphorylation of N-terminal c-Jun (on serine 63 and 73) and MAPK kinases ERK1/2, but not JNKs, in irradiated murine microglial BV2 cells. Radiation-induced c-Jun phosphorylation is dependent on the canonical MEK-ERK signaling pathway and required for both ERK1 and ERK2 function. ERK1/2 directly interact with c-Jun in vitro and in cells; meanwhile, the JNK binding domain on c-Jun is not required for its interaction with ERK kinases. Radiation-induced reactive oxygen species (ROS) potentially contribute to c-Jun phosphorylation through activating the ERK pathway. Radiation stimulates c-Jun transcriptional activity and upregulates c-Jun-regulated proinflammatory genes, such as tumor necrosis factor-α, interleukin-1β, and cyclooxygenase-2. Pharmacologic blockade of the ERK signaling pathway interferes with c-Jun activity and inhibits radiation-stimulated expression of c-Jun target genes. Overall, our study reveals that the MEK-ERK1/2 signaling pathway, but not the JNK pathway, contributes to the c-Jun-dependent microglial inflammatory response following irradiation. Radiation therapy is an important modality of treatment for brain tumors and cancers that have metastasized to the brain. However, the side effects of radiation therapy, such as acute and chronic brain injury, are a major concern and limit its clinical application Activation of transcription factors, such as AP-1 proteins, is one of the cellular responses to ionizing radiation c-Jun can be phosphorylated by many different types of kinases, especially mitogen-activated protein kinases (MAPKs) JNKs, ERK1/2 (p44 and p42), and p38 MAPKs represent the well characterized MAPK subfamilies. Various AP-1 proteins are regulatory targets of these MAPKs. JNKs have been recognized as the predominant c-Jun kinases targeting Ser63 and Ser73 in the c-Jun N-terminus; whereas ERK1/2 and p38 are critical for c-fos phosphorylation In this study, we used BV2 murine microglial cells as a model to explore the molecular signaling pathway required for c-Jun activation in response to ionizing radiation. We discovered that the canonical MAPK MEK-ERK1/2 signaling cascade, but not JNK pathway, directly mediated c-Jun N-terminal phosphorylation. Blockade of MEK-ERK1/2 signaling pathway compromised c-Jun activity, leading to a decreased expression of several proinflammatory genes regulated by c-Jun.BV2 cells were irradiated with a single dose of 10 Gy γ-ray and cell lysates were collected for Western blot analyses. Radiation resulted in a prominent increase in c-Jun phosphorylation on Ser63 and Ser73 , the criERK1 and ERK2 expression vectors were cotransfected into BV2 cells in increasing amounts to overexpress these two proteins. Forty-eight hours after transfection, c-Jun phosphorylation on Ser63 showed a dose-dependent increase, indicating the capability of ERK1/2 to stimulate c-Jun phosphorylation . After preincubating BV2 cells with apocynin, we observed a marked reduction of radiation-induced ROS in these cells (Cellular oxidative stress is one of primary consequences caused by irradiation. We and others have observed increased generation of reactive oxygen species (ROS) following radiation in various cells, including microglia reatment . Conversreatment , revealise cells . Importase cells , suggest32P labeled AP-1 probes. As shown in To validate the importance of c-Jun phosphorylation on Ser63 and Ser73 in BV2 cells, we conducted a Gal4/UAS Gaussia luciferase assay To further substantiate the role of ERK signaling pathway on c-Jun transcriptional activity, we studied several well-characterized c-Jun target genes, including four proinflammatory genes  and cyclin D1In summary, we proposed a working model  based onOwing to its functional significance, c-Jun activation is subtly regulated by various post-transcriptional modifications in cells, such as phosphorylation, which have profound impacts on c-Jun function. c-Jun is phosphorylated on multiple sites in different cellular processes In the mammalian kinome, MAPKs are predominantly responsible for c-Jun phosphorylation on Ser63 and Ser73. JNKs were initially identified and are generally recognized as the preferential enzymes for c-Jun N-terminal phosphorylation, especially on Ser63 and Ser73. JNK kinases bind to a small portion of the c-Jun N-terminus and consequently exert their kinase function on c-Jun Our study indicated that the ERK1/2, but not the JNKs, were rapidly phosphorylated in microglial cells after irradiation. We provided multiple lines of evidence using overexpression, pharmacological inhibitor, and gene knockdown approaches to prove that both ERK1 and ERK2 are indispensable for radiation-induced c-Jun phosphorylation on Ser63 and Ser73. In accordance with this novel discovery, ERK1 and ERK2 directly interact with c-Jun in vitro, and their phosphorylated forms are endogenously associated with c-Jun in cells. Interestingly, ERK2 was more efficient than ERK1 in binding to c-Jun in vitro, although they are highly homologous to each other. Meanwhile, the phosphorylated form of ERK2 is more abundant than that of ERK1 in BV2 cells, as indicated by Western blots. However, it seems that these differences do not make ERK2 more important in mediating c-Jun phosphorylation, since individual knockdown of these two kinases was insufficient in blocking radiation-induced c-Jun phosphorylation in our study, suggesting that ERK1 and ERK2 are functionally compensating each other in regulating c-Jun. Despite functional resemblance between ERK1/2 and JNKs, discrepancies exist between these two subtypes of MAPKs in their binding pattern with c-Jun. The N-terminal c-Jun contains the JBD region that is indispensable for JNK-mediated c-Jun phosphorylation. However, the c-Jun mutant (aa1-62) containing JBD region showed a weaker binding ability to ERK1 and ERK2, compared to other c-Jun truncation mutants. Our mapping study could not further define a specific ERK-binding domain on c-Jun, since it appears that multiple regions of c-Jun are likely involved in its interaction with ERKs.ERK 1 and ERK2 are autophosphorylable on both threonine and tyrosine residues and the autophosphorylation correlates with their activation ROS generation is one of the earliest cellular responses after ionizing radiation We found that radiation did not increase expression and activation of RelA/p65, an important component of NF-kB signaling pathway, in the irradiated BV2 cells, and the MEK inhibitor had no effect on RelA/p65 . HoweverERK inhibitor , U0126 and SP600125 were purchased from Calbiochem . The antioxidant N-acetyl-L-cysteine (NAC) was purchased from Sigma-Aldrich Corporation . The antibodies for JNK (#9258), phosphorylated JNK , cyclin D1 (#2926) and phosphorylated c-Jun  were purchased from Cell Signaling Technology . Antibodies for c-Jun (sc-45), phosphorylated c-Jun , ERK1 (sc-94), phosphorylated ERK1/2 , phosphorylated MEK (sc-7995R), COX-2 (sc-1745) and HA (sc-7392) were purchased from Santa Cruz Biotechnology . MEK antibody (RB-1662) was purchased from Thermo Scientific .2. Cells were transiently transfected with varied amounts of plasmids using Fugene HD (Roche Applied Science) according to the manufacturer's instruction. Forty-eight hours post transfection, the cells were subjected to further analysis. BV2 cells were used under passage 30. In the kinase inhibitor assay, the inhibitors were supplemented to culture medium 1 h before exposure to irradiation. For radiation treatment, cells were starved with serum-free DMEM medium for 24 h prior to irradiation. Cells were irradiated with a 137Cs irradiator  at a dose rate of 3.7 Gy/min. All irradiations were performed at room temperature; control cells were received sham irradiation. After irradiation, the culture dishes were returned to the incubator.Murine microglial BV2 cells K97M) In this study, murine cDNAs of c-Jun, ERK1, ERK2, and MEK1 were cloned into appropriate expression vectors, and the shRNA constructs were designed to target murine mRNA sequences. The expression plasmids pCMV-SPORTS6/ERK1 and pCMV-SPORTS6/ERK2 were obtained from Open Biosystems . The coding regions of c-Jun and MEK1 were amplified with RT-PCR using cDNA from BV2 cells and subcloned into different vectors. The kinase-dead MEK1  to concentrate viral particles. To infect BV2 cells, the medium was added with concentrated lentivirus and polybrene (8 mg/ml) and incubated for 6 h before replacing with normal medium.AGGTCTGAAGGTAGGAAGG), mIL-1b (F: ATCCGTGTCTTCCTAAAGTATG/R: CCTGAGCGACCTGTCTTG), mCOX-2 (F: TGTCTTCCAGCCCATTGAAC/R: CAGCGTTTCTCGTAGTAAAGTG), mIL-6 (F: GCTGGAGTCACAGAAGGAG/R: GAGAACAACATAAGTCAGATACC), and GAPDH (F: GCCTTCCGTGTTCCTACC/R: CTTCACCACCTTCTTGATGTC). Total RNA was prepared by Trizol  method, and further purified with the RNeasy Mini kit  after DNase I treatment. Reverse transcription was performed with MMLV  following the manufacturer's instructions. Real-time PCR was performed at ABI7000 using FastStart universal SYBR green master mix (Rox) . The samples were analyzed in quadruplicate and repeated in three independent experiments. The comparative Ct method was used to calculate relative levels of gene expression based on normalization to GAPDH.The primers used for real-time PCR were: mTNFa (F: CACCACCATCAAGGACTC/R: 5′-CGCTTGATGAGTCAGCCGGAA-3′ and 3′-GCGAACTACTCAGTCGGCCTT-5′), which was labeled with γ-32P-ATP  using T4 polynucleotide kinase . The labeled probe was cleaned with a Sephadex G-25 column  to remove the free γ-32P-ATP and radioactivity was quantified using the LS6500 liquid scintillation counter . The EMSA procedure was performed by using the Gel-Shift Core Assay Kit  with slight modifications. Briefly, 10 μg of nuclear protein was incubated with labeled AP-1 probe (∼150 cpm/reaction) at 37°C for 1 h. The binding products were then resolved on a 5% native polyacrylamide gel (0.5× TBE). The gel was subsequently vacuum-dried  for 2 h at 85°C before autoradiography.Nuclear protein was extracted from treated and untreated BV2 cells following a procedure described previously t test was used to evaluate significance of differences between two groups. A P value of <0.05 was considered statistically significant.All experimental data were collected, unless otherwise indicated, from 3 independent experiments, and presented as mean ± SD. Statistical analysis was performed with SigmaPlot 10.0  and Student's Figure S1Protein interaction between c-Jun and ERK1/2 in cells. A lentivirual vector pSL2 was used to construct overexpression vectors for HA-ERK1, HA-ERK2, and Flag-c-Jun. BV2 cells were infected with the lentiviruses to express the indicated proteins. Forty-eight hours after infection, cell lysates were subjected to immunoprecipitation assay with the anti-Flag antibody  and control antibody, and then the HA monoclonal antibody  was used to detect the precipitated HA-ERK1 or HA-ERK2 in a Western blot assay. To show binding specificity, the cell lysates expressing HA-ERK1 or HA-ERK2 alone were included in the co-immunoprecipitation assay. The expression of Flag-c-Jun, HA-ERK1, HA-ERK2, and GAPDH (loading control) in the co-immunoprecipitation lysates is shown in the lower panel, respectively.(PDF)Click here for additional data file.Figure S2The relative levels of p-c-Jun and p-ERK1/2 in Western blots normalized against the levels of their total proteins. Quantification was performed with the Quantity One 1-D Analysis software . (A) and (B) Quantification results of p-c-Jun and p-ERK1/2 in U0126 inhibitor assay shown in p<0.05; **p<0.01.(PDF)Click here for additional data file.Figure S3Ser63 and 73 are critical for c-Jun activity in BV2 cells. (A) Schematic diagram for 4XUAS reporter and Gal4-c-Jun fusion constructs. 4XUAS was amplified from 4XUAS-TK-Luc p<0.05.(PDF)Click here for additional data file.Figure S4Model for radiation-induced c-Jun phosphorylation and its role in neuroinflammation.(PDF)Click here for additional data file.Figure S5Radiation does not induce NFkB activation in BV2 cells. (A) RelA/p65 level in the irradiated BV2 cells. (B) Levels of nuclear form of RelA/p65 in irradiated BV2 cells with or without MEK inhibitor U0126 treatment. Histone H3 and Ponceau S staining represent loading controls for the nuclear extracts.(PDF)Click here for additional data file.Table S1Primers for cloning.(DOC)Click here for additional data file.


HIV-1 transcription is activated by the viral Tat protein that recruits host positive transcription elongation factor-b (P-TEFb) containing CDK9/cyclin T1 to the HIV-1 promoter. P-TEFb in the cells exists as a lower molecular weight CDK9/cyclin T1 dimer and a high molecular weight complex of 7SK RNA, CDK9/cyclin T1, HEXIM1 dimer and several additional proteins. Our previous studies implicated CDK2 in HIV-1 transcription regulation. We also found that inhibition of CDK2 by iron chelators leads to the inhibition of CDK9 activity, suggesting a functional link between CDK2 and CDK9. Here, we investigate whether CDK2 phosphorylates CDK9 and regulates its activity.90SPYNR94) that is consensus CDK2 phosphorylation site. CDK9 was phosphorylated on Ser90 by CDK2 in vitro. In cultured cells, CDK9 phosphorylation was reduced when Ser90 was mutated to an Ala. Phosphorylation of CDK9 on Ser90 was also detected with phospho-specific antibodies and it was reduced after the knockdown of CDK2. CDK9 expression decreased in the large complex for the CDK9-S90A mutant and was correlated with a reduced activity and an inhibition of HIV-1 transcription. In contrast, the CDK9-S90D mutant showed a slight decrease in CDK9 expression in both the large and small complexes but induced Tat-dependent HIV-1 transcription. Molecular modeling showed that Ser 90 of CDK9 is located on a flexible loop exposed to solvent, suggesting its availability for phosphorylation.The siRNA-mediated knockdown of CDK2 inhibited CDK9 kinase activity and reduced CDK9 phosphorylation. Stable shRNA-mediated CDK2 knockdown inhibited HIV-1 transcription, but also increased the overall level of 7SK RNA. CDK9 contains a motif (Our data indicate that CDK2 phosphorylates CDK9 on Ser 90 and thereby contributes to HIV-1 transcription. The phosphorylation of Ser90 by CDK2 represents a novel mechanism of HIV-1 regulated transcription and provides a new strategy for activation of latent HIV-1 provirus. Eradication of latent HIV-1 provirus is especially challenging as integrated HIV-1 is not affected by the existing anti-HIV-1 drugs until its transcription is activated . While lnscripts . The HIVnscripts . Nucleosnscripts . Knockdonscripts . P-TEFb nscripts ,8. The hnscripts -11 and tnscripts ,13. HEXInscripts . The hignscripts . The impnscripts . HIV-1 Tnscripts . Tat wasnscripts ,19. P-TEnscripts . P-TEFb nscripts ,22. The nscripts . CDK2 wa[in vitro and in vo[in vivo. Inhibito[in vivo, inhibito[in vivo. Roscovio[in vivo, suggesto[in vivo,27. Furto[in vivo,29. CDK9o[in vivo,31 and to[in vivo,31. Autoo[in vivo and the o[in vivo has alsoo[in vivo.Here, we investigated the phosphorylation of CDK9 by CDK2, using a siRNA and shRNA-mediated knockdown approach. We identified Ser 90 of CDK9 as a novel and key CDK2 phosphorylation site, and analyzed its effect on the distribution of P-TEFb between large and small molecular weight complexes and the ability of CDK9 to activate HIV-1 transcription. We also modeled Ser90 in the CDK9 structure.Previously, we utilized siRNA-mediated CDK2 knockdown (KD) to demonstrate the importance of CDK2 for HIV-1 transcription . Here, w32P) orthophosphate to allow CDK9 phosphorylation. CDK9 was precipitated with anti-Flag antibodies from the cellular lysates, and CDK9 phosphorylation was analyzed by Phosphor Imaging. CDK9 phosphorylation was reduced in the cells transfected with CDK2-targeted siRNA in comparison to the control siRNA  showed about 40% reduction of CDK2 mRNA . Only 293T cells expressing the shRNA that targeted the nef. For normalization, MTT assay was used. Luciferase activity was significantly reduced in 293T-CDK2 KD cells compared to 293T cells . We also generated HeLa CDK2 KD cell line, which showed reduced CDK2 protein expression PX(K/R) sequence . Analysiin vitro. Flag-tagged WT CDK9 and CDK9 S90A mutants were expressed in 293T cells, immunoprecipitated with anti-FLAG antibodies and subjected to phosphorylation with recombinant CDK2/cyclin E. Phosphorylation of the CDK9 S90A mutant was reduced  orthophosphate. CDK9 was immunoprecipitated with anti-FLAG antibodies and resolved on SDS-PAGE. CDK9 phosphorylation was analyzed with the Phosphor Imager. Both the S90A and S90D substitutions reduced CDK9 phosphorylation . In the absence of Tat, there was a small inhibitory effect of CDK9 S90A (about 20%), but no effect of CDK9 S90D on basal HIV-1 transcription Figure C.versus Leu81-Thr87 and Ser98-Asp104 in original PDB), while residues Ser90-Cys95 form flexible loop. Local energy minimization of side chains was performed for each of these conformations and the structure with the lowest energy was selected . Phosphorylation of peptides containing Thr29, Ser175 or Ser90 showed that only Ser90 was phosphorylated by CDK2. These results were confirmed with the CDK9 S90A mutant, which was less phosphorylated by recombinant CDK2 in vitro and in cultured cells. Previously, CDK9 was shown to be phosphorylated on at least ten sites that included Thr186 [A CDK2 consensus phosphorylation site adheres to the sequence (S/T)PX(K/R) , and is d Thr186 ,31, Thr2d Thr186  and the d Thr186 . Thus thd Thr186 . We alsod Thr186 . The modEarlier studies showed that CDK2 activity is required for the entry into S-phase ,43 and tTaken together, our study has identified Ser90 as a novel phosphorylation site of CDK9. The phosphorylation of Ser90 by CDK2 represents a novel mechanism of HIV-1 regulated transcription and provides a new strategy for activation of latent HIV-1 provirus.2 fragment was purchased from GE Healthcare . All other inorganic reagents were purchased from Fisher Scientific  or Sigma . Radioactive materials were purchased from Perkin-Elmer . ARC -pyrimidine-5-Carboxamide) [293T cells were purchased from ATCC . Histone H1 was purchased from Upstate Cell Signaling Solutions . Anti-FLAG monoclonal antibodies, protein G and protein A agarose were purchased from Sigma . Recombinant CDK2/cyclin E and CDK9/cyclin T1 were purchased from ProQinase . Antibodies against CDK9, CDK2 and cyclin T1 were purchased from Santa Cruz Biotechnology . Anti-CDK9 phospho-Thr186 polyclonal antibodies were a kind gift from Dr. Qiang Zhou . Preparation of CDK9 Ser 90 phospho-specific antibodies is described below. Horseradish peroxidase (HRP)-conjugated F(ab)oxamide)  was a gi-E-  was obtained from the NIH AIDS Research and Reference Reagent Program. The Tat expression plasmid was a gift from Dr. Ben Berkhout (University of Amsterdam). WT HIV-1 LTR (−105 to +77) followed by the luciferase reporter gene (KB SP WT) was kindly provided by Dr. Manuel López-Cabrera .The HIV-1 genomic vector, pNL4-3.Luc.R293T cells were seeded in 6 well plates to achieve 50% confluence at the day of transfection. The cells were transfected with indicated plasmids using Lipofectamine and Plus reagents (Life Technologies) following manufacturer’s protocol. The efficiency of transfection was verified using a plasmid encoding green fluorescent protein. The cells were cultured for 48 hrs post-transfection and then analyzed for HIV-1 transcription or phosphorylation of CDK9.CDK2-directed siRNA (siGENOME SMARTpool reagent M-003236-03-0005) and a control siRNA (D-001206-13-05) were purchased from Dharmacon . Control siRNA targets firefly luciferase gene. The siRNAs were transfected at final concentration of 100 nM using Lipofectamin reagent (Invitrogen) according to the manufacturer’s recommendations. The siRNAs were incubated with cells for 2 days before cells were lysed for Western blotting analysis or retransfected.399gcttaaggagctttaaccat418 (OS211957), 919ccaggagttacttctatgc937 (OS211958), 1010atggacggagcttgttatc1028 (OS211959) and 49aggcggcaacattgtttca67 (OS211960) sequences of CDK2 . Stable clones were selected with 10 μg/ml puromycin. Several clones transfected with OS211959 vector showed significant decrease in CDK2 expression and one of these clones, designated as 293T-CDK KD cells, was used for further studies.293T cells were transfected with HSH000225-1-LvH1 vectors expressing shRNA that targeted 293T and 293T-CDK KD cells were co-transfected with pNL4-3 Luc plasmids and CMV-LacZ expression vector. After 48 hours the cells were collected, washed in PBS, and lysed with Steady Lite Luciferase. The samples luminescence was determined in a Luminoskan Perkin-Elmer). The lysates were then used to measure β-galactosidase activity with ONPG-based assay . Lucifer293T cells were co-transfected with CDK9 WT and mutants and KB SP WT. Cells were also co-transfected with Tat-expressing vector. After 48 hrs the cells were collected, washed in PBS, lysed with Steady Lite Luciferase lysis buffer, and luminescence was determined in a Luminoskan. Luciferase activity was normalized to GFP expression.VSVG-HIV-1 was added to 293T and 293T-CDK2 KD cells that were seeded in 24-well plate at ~30 confluence. After 48 hours, the cells were washed in PBS, and lysed with Steady Lite Luciferase buffer. Light emission was analyzed in a Luminoskan. To adjust for the cell number, the MTT assay was performed. Control samples were supplemented with 0.5 mg/ml 3--2,5-diphenyltetrazolium bromide (MTT) and incubated for 1 hr at 37°C. Media was removed, formazan crystals were solubilized in dimethyl sulfoxide and absorbance was measured at 654 nm. Luciferase activity was normalized for MTT reading at 654 nm.t-test was used to test statistical significance.Total RNA was extracted from cultured 293T and 293T-CDK KD cells using TRIzol reagent according to the manufacturer’s protocol (Invitrogen Corp.). Total RNA (100 ng) was reverse transcribed to cDNA using Superscript™ RT-PCR kit , hexamers and oligo-dT were used as primers. Real-time PCR analysis was conducted on Roche LightCycler 480 detection system (Roche Diagnostics) with SYBR Green. The cDNA was amplified in 45 cycles of denaturation at 95°C for 10 seconds, annealing at 60°C for 10 seconds, and extension at 72°C for 10 seconds and primers for β-Actin, CDK2, Cyclin A and Cyclin E. Primer sequences for β-Actin, forward-AGGCTCAGAGCAAGAGAG, reverse-TACATGGCTGGTGTGTTGA, amplicon size 229; CDK2 forward- TTTGCTGAGATGGTGACTCG, reverse- CTTCATCCAGGGGAGGTACA, amplicon size 196 bp; Cyclin A, forward-GAAACTGCAGCTCGTAGGAA, reverse-ACTTTCAGAAGCAAGTGTTCCA, amplicon size 150bp; Cyclin E, forward –AGCACTTTCTTGAGCAACACC, reverse-CGCCATATACCGGTCAAAGA, amplicon size 161bp. Mean Cp values for β-Actin, CDK2, Cyclin A and cyclin E were determined and expression levels determined using ΔΔCp analysis using β-Actin as reference. Unpaired t-test was used to test statistical significance.Approximately 1 million cells were fixed in 70% ethanol at -20°C for 2 hours and stained with Propidium Iodide (10mg/ml) containing RNAse (1mg/ml) for 30 minutes. The data were acquired in BD FACSCalibur  and analysis was done using FlowJo software. Unpaired 2, 1 mM EDTA, 250 μM sucrose, 1 mM DTT, 0.5% NP-40 and protease inhibitors) added at 500 μl/107 cells. The mixture was incubated on ice for 10 min and centrifuged at 1000 x g for 5 min to pellet the nucleus. The supernatant was removed and saved as the large complex extract (LC). The remaining pellet was resuspended in Buffer B (20 mM HEPES-KOH (pH 7.9), 450 mM NaCl, 1.5 mM MgCl2, 0.5mM EDTA, 1 mM DTT and protease inhibitors) added at 500 μl/107 cells. The mixture was incubated on ice for 10 min and centrifuged at 10,000 x g for 1 hr. The supernatant was saved as the small complex extract (SC) (11). The LC and SC were resolved by SDS-PAGE and transferred to a PVDF membrane  for Western blotting. The membrane was probed with anti-CDK9 antibodies.293T cells, 293T –CDK2 KD cells that stably express CDK2 shRNA (OS211959) or 293T cells transiently transfected with a combination of Flag-tagged CDK9 (WT or S90A mutant) and cyclin T1 were cultured in DMEM containing 10% fetal bovine serum. Cells were resuspended in Buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 10 mM MgCl293T cells were lysed in whole cell lysis buffer  supplemented with protease cocktail. CDK9 was precipitated as indicated with either anti-CDK9 antibodies or anti-Flag antibodies in the case of overexpression of Flag-CDK9 as we previously described . Briefly′-GGATGTGAGGCGATCTGGCTG-5′; reverse primer 3′-TAAAGAAAGGCAGACTGCCAC-5′. Theses primers were used in a PCR reaction that followed the RT reaction. Real-time PCR analysis was conducted on Roche LightCycler 480 detection system (Roche Diagnostics) with SYBR Green. The cDNA was amplified in 45 cycles of denaturation at 95°C for 10 seconds, annealing at 60°C for 10 seconds, and extension at 72°C for 10 seconds. To quantify the amount of 7SK RNA, serial dilutions of 7SK RNA expressing vector [RNA was isolated from complexes that co-immunoprecipitated with anti-CDK9 antibodies or nonspecific IgG as controls using TRIzol reagent according the Invitrogen’s protocol. RNA was also extracted from total cell extract or small and large complex extracts prepared as described above. Total RNA was reverse transcribed using SuperScript II kit (Invitrogen) with the 7SK reverse primer. The following primer sequences were used: forward primer 3g vector  was used2, 6 mM EGTA, 2.5 mM DTT) containing 100 ng of GST-CTD as substrate, 200 μM cold ATP and 5 μCi of (γ-32P) ATP. Kinase reactions were stopped with SDS-loading buffer and resolved on 10% PAGE. The dried gel was exposed to Phosphor Imager screen.Kinase assay was performed at 30°C for 30 min in a kinase assay buffer  ATP. Kinase reactions were stopped with SDS-loading buffer and resolved on 10% PAGE. The dried gel was exposed to Phosphor Imager screen.293T and 293T-59 cells were lysed in whole cell lysis buffer  supplemented with protease cocktail. CDK2 was precipitated with anti-CDK2 antibodies as described above. Kinase assay was performed at 30°C for 20 min in the kinase assay buffer containing 2 μg Histone H1 as substrate, 200 μM cold ATP and 5 μCi of (γ-21KLAKIGQGTFGEVFK35), Ser90 (88KASPYNRCKGSIYL101); and Ser175 and Thr186 (172RAFSLAKNSQPNRYTNRVV190). The peptides were phosphorylated by recombinant CDK2/cyclin E in a 10 μl reaction with 100 μM ATP (1μCi of γ-(32P) ATP) in the kinase assay buffer using 4 μg of a peptide per reaction for 20 min at 30°C. The reaction was stopped by the addition of 4X SDS-PAGE loading buffer and resolved on a 15% Tris-Tricine gel. The gel was stained with Coomassie blue for 10 min, destained for 1 hr, dried, and exposed to Phosphor Imager screen.We designed synthetic peptides containing CDK9 residues Thr29 . The obtained clones were sequenced. CDK9 mutants were expressed in 293T cells, immunoprecipitated with anti-FLAG or anti-Cyclin T1 antibodies and tested for phosphorylation of GST-CTD as described above.QuikChange XL Site-Directed Mutagenesis Kit (Stratagene) was used to generate mutants of CDK9 with the substitutions in the sites of CDK2 phosphorylation determined previously. Primers for S90A substitution were GATTTGTCGAACCAAAGCTGCCCCCTATAACCGCTGC (forward) and GCAGCGGTTATAGGGGGCAGCTTTGGTTCGACAAATC (reverse); and for S90D substitution - GATTTGTCGAACCAAAGCTGACCCCTATAACCGCTGC (forward) and GCAGCGGTTATAGGGGTCAGCTTTGGTTCGACAAATC (reverse). CDK9 S90D was created to mimic phosphorylation. PCR reactions were run for 18 cycles with the extension time of 8 min to allow the synthesis of the whole plasmid sequence. PCR products were digested with 86 RTKASPYNR94 peptide without or with Ser 90 phosphorylated was synthesized with an additional Cys residue at the N-terminus for coupling to Keyhole limpet hemocyanin (KLH) or BSA. The KLH-coupled phospho-peptide was injected in the rabbit. The polyclonal serum was double affinity- purified on the BSA-coupled phospho- and non-phosphopeptide linked to CNBr-activated Sepharose-4B .CDK9-derived The 8 amino acid residues of CDK9, Ala89-Lys96, that were missing in the CDK9 crystal structure , were moThe authors declare that they have no competing interests.DB, AK, TA, NK, AI, MB (Monique Beullens) and SN conducted experiments, discussed and analyzed data. AVI and MGP conducted modeling and discussed data. DB, PRR, MB (Mathieu Bollen), FK and SN designed the study, discussed the results and wrote the manuscript. SN performed overall design, general control and coordination of the study. All authors read and approved the manuscript.


Differentiation and fusion of skeletal muscle myoblasts into multi-nucleated myotubes is required for neonatal development and regeneration in adult skeletal muscle. Herein, we report novel findings that protein kinase C theta (PKCθ) regulates myoblast differentiation via phosphorylation of insulin receptor substrate-1 and ERK1/2.shRNA) myotubes had reduced inhibitory insulin receptor substrate-1 ser1095 phosphorylation, enhanced myoblast differentiation and cell fusion, and increased rates of protein synthesis as determined by [3H] phenylalanine incorporation. Phosphorylation of insulin receptor substrate-1 ser632/635 and extracellular signal-regulated kinase1/2 (ERK1/2) was increased in PKCθshRNA cells, with no change in ERK5 phosphorylation, highlighting a PKCθ-regulated myogenic pathway. Inhibition of PI3-kinase prevented cell differentiation and fusion in control cells, which was attenuated in PKCθshRNA cells. Thus, with reduced PKCθ, differentiation and fusion occur in the absence of PI3-kinase activity. Inhibition of the ERK kinase, MEK1/2, impaired differentiation and cell fusion in control cells. Differentiation was preserved in PKCθshRNA cells treated with a MEK1/2 inhibitor, although cell fusion was blunted, indicating PKCθ regulates differentiation via IRS1 and ERK1/2, and this occurs independently of MEK1/2 activation.In this study, PKCθ knockdown (PKCθCellular signaling regulating the myogenic program and protein synthesis are complex and intertwined. These studies suggest that PKCθ regulates myogenic and protein synthetic signaling via the modulation of IRS1and ERK1/2 phosphorylation. Myotubes lacking PKCθ had increased rates of protein synthesis and enhanced myotube development despite reduced activation of the canonical anabolic-signaling pathway. Further investigation of PKCθ regulated signaling may reveal important interactions regulating skeletal muscle health in an insulin resistant state. Skeletal muscle development and the regeneration of adult muscle tissue requires the completion of myogenesis: activation, proliferation, differentiation, and fusion of muscle specific stem cells, known as satellite cells . Myogenein vivo phenylalanine incorporation  . In response to wortmannin, PKCθshRNA protein synthesis rates remained 35% higher in PKCθshRNA compared to scramble myotubes . Thus, PKCθshRNA cells are able to complete the myogenic program independent of PI3-kinase signaling. These results support our protein expression data . shRNA-mediate reduction of PKCθ protected muscle cell differentiation in the presence of both PI3-kinase and MEK1/2 inhibition, but cell fusion was protected only in the presence of PI3-kinase inhibition. Take together, these data show that MEK1/2 signaling is required for cell fusion independently of differentiation and the expression of PKCθ. Furthermore, our data suggests a PKCθ-specific myogenic regulatory pathway involving IRS1 and ERK1/2 phosphorylation events in the regulation of muscle cell differentiation.PKCθ Figures C,D. Cell Figures C,E. ThershRNA myotubes treated with a MEK1/2 inhibitor suggests that MEK signaling is required for fusion independent of PKCθ. Additionally, abrogation of PKCθ promoted full completion of the myogenic program and increased rates of protein synthesis, despite reduced IR phosphorylation and maintained higher protein synthesis rates when treated with a PI3-kinase inhibitor. These findings demonstrate that PKCθ may be a viable therapeutic target to promote increases in protein synthesis and promote the maintenance of skeletal muscle health in conditions with impaired insulin signaling.The objective of this study was to investigate the contribution of skeletal muscle cell PKCθ to signaling events that regulate protein synthesis and myogenesis. Taken together, our data supports a model in which PKCθ regulates IRS1 and ERK1/2 signaling that controls myoblast differentiation and protein synthesis. Our findings that cell fusion is equally inhibited in scramble and PKCθ2C12 mouse muscle cells were provided by Francis X. Pizza . To identify an siRNA to knockdown mouse PKCθ (mPKCθ) a free Web-based tool (http://www.genelink.com/sirna/shRNAi.asp) was used to design a putative siRNA against the mPKCθ gene and to design oligonucleotides that encode a corresponding small hairpin RNA (shRNA) as previously described  phenylalanine . After incubation, cells were washed with ice-cold PBS (pH 7.5) and proteins were precipitated with 10% trichloroacetic acid (TCA). An aliquot of the culture medium was saved to determine the specific activity of the medium (c.p.m). TCA homogenates were incubated on ice for 30 minutes; plates were thoroughly scraped followed by centrifugation at 4500 g for 5 min. The supernatant was discarded and the TCA insoluble fraction was resuspended in 10% TCA, followed by centrifugation at 4500 g for 5 min. This was repeated four consecutive times, and the TCA insoluble fraction was homogenized in 0.15 M NaOH at 55°C for 1 hour with frequent vortex mixing. Aliquots of each sample were analyzed to determine the incorporated radioactivity (c.p.m) via liquid scintillation counting using a Beckman Coulter LS 6500. Protein content was analyzed using the DC protein assay . The rate of protein synthesis was calculated as femtomoles [3H] phenylalanine per milligram of protein per hour.Following 4 days in differentiation media in the presence or absence of wortmannin or U0126 replaced daily, cells were incubated for 1 hour in serum-free DMEM containing 2.5 mM phenylalanine and 2.5 Statistical analyses were performed using Graph Pad Prism 5 software. Student’s t-Test was used to determine differences between two means. One-Way ANOVA was used to determine differences when more than 2 variables were compared, followed by a Bonferroni multiple comparisons post-hoc test. Analysis of gene expression between cell types across time was performed using a two-way ANOVA. Significance required an alpha level of p<0.05.PKCθ: Protein kinase C theta; PKCΔ: Protein kinase C delta; IR: Insulin receptor; IRS1: Insulin receptor substrate-1; AKT: Also known as protein kinase B (PKB); ERK: Extracellular signal-related kinase; MEK1/2: Mitogen activated ERK kinase; PI3-kinase: Phosphoinositide 3-kinase; mTOR: Mammalian target of rapamycin; shRNA: Short hairpin RNA.The authors declare no competing interests.in vitro cell staining. TJM, ERS and JWH contributed to experimental design, interpretation of data and manuscript revising. JWH gave final approval for publication. All authors read and approved the final manuscript.JSM contributed to experimental design, growth and treatment of cells, protein and gene expression analysis, and wrote the manuscript. TDH developed the scramble and PKCθ knockdown cell lines and contributed to experimental design. RAH performed protein synthesis experiments. EO and JLO contributed by quantifying cell differentiation and cell fusion. AD performed


Ras is frequently mutated in a variety of human cancers, including lung cancer, leading to constitutive activation of MAPK signaling. Despite decades of research focused on the Ras oncogene, Ras-targeted phosphorylation events and signaling pathways have not been described on a proteome-wide scale.By functional phosphoproteomics, we studied the molecular mechanics of oncogenic Ras signaling using a pathway-based approach. We identified Ras-regulated phosphorylation events (n = 77) using label-free comparative proteomics analysis of immortalized human bronchial epithelial cells with and without the expression of oncogenic Ras. Many were newly identified as potential targets of the Ras signaling pathway. A majority (∼60%) of the Ras-targeted events consisted of a [pSer/Thr]-Pro motif, indicating the involvement of proline-directed kinases. By integrating the phosphorylated signatures into the Pathway Interaction Database, we further inferred Ras-regulated pathways, including MAPK signaling and other novel cascades, in governing diverse functions such as gene expression, apoptosis, cell growth, and RNA processing. Comparisons of Ras-regulated phosphorylation events, pathways, and related kinases in lung cancer-derived cells supported a role of oncogenic Ras signaling in lung adenocarcinoma A549 and H322 cells, but not in large cell carcinoma H1299 cells.This study reveals phosphorylation events, signaling networks, and molecular functions that are regulated by oncogenic Ras. The results observed in this study may aid to extend our knowledge on Ras signaling in lung cancer. HRAS, NRAS, and KRAS) and corresponding biological processes 2,4], the downstream phosphorylation targets and pathways regulated by oncogenic Ras-mediated signaling remain to be elucidated in lung cancer.Constitutive activation of Ras-mediated signaling and its downstream components, such as MAP family kinases, play an important role in the pathogenesis of human cancers 1,2]. Lung cancer represents a good choice for investigating the molecular mechanisms of Ras-mediated MAPK signaling, because the activation of Ras oncogenes by mutation or amplification has been reported most frequently in lung cancer 1,3]. Among the post-translational modifications, reversible protein phosphorylation is a dominant regulatory mechanism involved in the oncogenic signaling process. Simultaneous identification and quantification of phosphorylation events induced by oncogenic signaling not only would provide insight into signaling mechanisms but also are essential for understanding the molecular determinants of disease progression. Despite decades of intensive analysis of the Ras family of proto-oncogenes  using IDEAL-Q -based quantitation proteomics, followed by computational methods to infer Ras-mediated signaling pathways and molecular functions. Furthermore, the interpretation of Ras-mediated phosphorylation targets and related pathways allowed us to demonstrate the involvement of Ras-mediated signaling in lung adenocarcinoma (AD), but not in large cell carcinoma (LCC). Our findings on the phosphorylation events, kinomes, and pathways regulated by oncogenic Ras and differential activation of Ras downstream signaling in lung AD and LCC could serve as a basis for future investigations elucidating the molecular mechanisms involved in the pathogenesis of human cancers.Apart from traditional molecular biological approaches, alternative strategies are needed to identify downstream phosphorylation targets and regulated pathways of oncogenic signals. Presently, quantitative phosphoproteomics is being widely used and is emerging as a key technology in signal transduction research 5]. The V12-transformed HBEC3-KT cells (3KTR) were maintained in K-CFM medium containing 50 µg/mL bovine pituitary extract (BPE) and 5 ng/mL EGF under 5% CO2 and 37°C, as previously described 9,10]. These cells were the kind gift of Dr. John D. Minna . The human non-small-cell lung cancer (NSCLC) H1299, H322, and A549 cell lines were purchased from the American Type Culture Collection. H1299 and H322 cells were grown in RPMI medium supplemented with 10% fetal bovine serum and 1% antibiotics under tissue culture conditions. A549 cells were grown under similar conditions in DMEM supplemented with 10% fetal bovine serum and 1% antibiotics.Cdk4 (cyclin-dependent kinase 4)/hTERT (human telomerase reverse transcriptase)-immortalized human bronchial epithelial cells (HBEC3-KT or 3KT) and K-RASluciferase and with sequences targeting human KRAS  purchased from the National RNAi Core Facility, Taiwan, were introduced into HEK293T cells with lentiviral packaging vectors pMD.G and pCMV 8.91. Viruses were collected from the medium 60 h after transfection. For knockdown experiments, cells were infected with the collected viruses over 24 h in the presence of Polybrene (at 3 MOI for H322 and 5 MOI for A549 and H1299). Cells were cultured in designated medium for 24 hr prior to lysis. To prepare cell lysates, cells were grown to 85% confluence and subjected to lysis using modified RIPA buffer containing 0.5% NP40 , 300 mM NaCl, 1 mM EDTA, 0.5 mM DTT, 1 mM NaVO3, 10 mM NaF, and a cocktail of protease inhibitors . After centrifugation at 15,000× g for 5 min, the cell lysates were collected. Western blotting was performed as previously described The lentivirus-based knockdown approach has been previously described TM; Waters Corp., Milford, MA, USA). The MS was operated in ESI positive V mode with a resolving power of 10,000. Data were acquired via Data Directed Analysis (DDA). The method included a full sequential MS scan  and 3 MS/MS  of the three most intense ions present in the full-scan mass spectrum. The details of the MS analysis, peptide identification, and label-free quantitation using the IDEAL-Q algorithm Purified phosphopeptide samples were desalted and analyzed by LC-MS/MS (Waters Q-Tof Premierpa) was derived using t-test statistics to discriminate between two classes and formulated.Based on the report of Lee et al. Pathways were clustered based on their functional similarity. The functional similarity was computed for each pathway pair based on the Gene Ontology (GO) annotations of proteins in each pathway. The details of pathway functional clustering are available in V12-transformed 3KT (3KTR) cells were subjected to quantitative phosphoproteomics analysis. Three non-small cell lung cancer (NSCLC)-derived cell lines, A549 , H322 (AD), and H1299 , were also studied. The flowchart in 49FQpSEEQQQTEDELQDK63) from β-casein, an internal standard, and it was consistent in the triplicate runs of each cell lysate and comparable with those from other cell lysates, demonstrating the efficiency of tryptic digestion and the IMAC enrichment process  and KRAS process . PhosphoOf the phosphopeptides identified (n = 1645), 1508 peptides from 696 proteins were quantified based on their concurrent detectability in the individual pairs of cell lines to be compared . For anyin vivo kinase-substrate relationships based on not only motif sequences but also various contextual factors Given that the regulated phosphopeptides consisted of motif sequences with distinctive features, we further explored the potential upstream kinases accountable for the regulated phosphorylation events by NetworKIN analysis. The NetworKIN algorithm predicts Supporting the above observations, both the LC-MS/MS and IDEAL-Q quantitative results and Western blot analysis showed increased phosphorylation and activation of proline-directed MAPK1 in Ras-transformed HBECs and AD cells, but not in LCC cells . Furtherluciferase mRNA. In addition, knockdown of KRAS resulted in downregulation of lamin pS392 in adenocarcinoma A549 and H322 cells but not in large cell carcinoma H1299 cells. These results are in agreement with the mass spectrometry data, where we observed the upregulated lamin pS392 in A549 and H322 and but not in H1299 . For each signaling pathway, an activity level was summarized from the phosphorylation levels of the subset of proteins in the pathway, following the method reported by Lee et al. with modifications In the subgroup of upstream kinases targeting proline-directed sites, the MAPK family represented the major upstream species, followed by CDK and GSK . The subWe next established protein interaction networks to identify the physical and functional interactions among the proteins that are phosphorylated by Ras-mediated signaling. The proteins (n = 584) harboring the phosphopeptides (n = 1288) that were subjected to comparative proteomics analysis between normal HBECs and Ras-transformed HBECs were further analyzed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database of physical and functional interactions. This yielded a phosphoprotein interaction network consisting of 1279 known plus predicted protein interactions among 401 proteins . In thisIn a large-scale proteomics study, identifying kinase substrates is difficult owing to the unavailability of antibodies. The NetworKIN algorithm provides a highly accurate alternative approach for the prediction of site-specific upstream kinases Finally, the identified Ras-regulated pathways were clustered based on their biological processes and function similarities. Using mass spectrometry-based comparative phosphoproteomics, we established the phosphorylation profiles of five lung epithelial cell lines. Comparison of HBECs upon Ras-transformation revealed that proline-directed kinases, which include the MAPK family, are the major mediators of Ras downstream signaling. The comparisons of the upstream kinases in three NSCLC cell lines demonstrated that proline-directed and basophilic kinases play important roles in the regulation of phosphorylation events in AD and LCC cells. Further, we inferred Ras-regulated pathways and their functions by introducing an algorithm, which supports the stimulation of Ras/MAPK signaling in lung adenocarcinoma but not in LCC. Our data demonstrate Ras-regulated phosphorylation events and pathways, as well as the upstream kinomes, and thereby provide new insights into Ras-mediated signaling networks in lung adenocarcinoma.G12DKRAS mutation) and H322 (KRAS amplification) cells, but not in LCC H1299 (Q61KNRAS) cells  cells , in agreK) cells  and immuK) cells . In the K) cells . The incK) cells . Our datK) cells .Seventy-seven phosphorylation events in 52 proteins were significantly altered by oncogenic Ras signaling in HBECs. Among these proteins, 14 were identified as novel targets of MAPK family kinases by NetworKIN analysis . These pUnderstanding altered signaling pathways that regulate cellular processes is a basis for drug discovery 32]. The identification of deregulated pathways, rather than genes, as biomarkers would give precise information of clinical value. Several studies have demonstrated pathway signatures based on the transcriptome, transcription factors, or proteome 33,34]. Given that phosphorylation plays a major role in regulating signaling pathways that dominate the pathogenic development of malignancies, we demonstrated a new strategy to identify pathways altered at the phosphorylation level. We inferred pathway activities, which revealed the activation of MAPK signaling in Ras-transformed HBECs. We further showed that MAPK signaling was upregulated in A549 and H322 cells, but not in H1299 cells . These rFigure S1Identification of phosphopeptides, coefficient of variation (CV) of phosphopeptide abundances, and normalization process. (A) Whole cell lysates were prepared from each cell line, mixed with β-casein, digested by trypsin, and phosphopeptides were enriched by IMAC. The enriched phosphopeptides from each cell line was analyzed by LC-MS/MS for three times. % of identified phosphopeptides in triplicate runs is shown. (B) % of CV of quantifiable phosphopeptide abundances in three runs are shown for five cell lines. (C) The observed abundance of phosphopeptide (49FQpSEEQQQTEDELQDK63) derived from β-casein in three LC-MS/MS runs is shown for five cell lines. (D) Normalization process is shown for a case comparison, 3KTR vs 3KT. Phosphopeptide ratios  were obtained in the comparison of 3KTR and 3KT cells after normalized against β-casein, internal standard intensity. The phosphopeptide ratios are shown on X-axis and the number of phosphopeptides is shown on Y-axis. (E) The ratios normalized with internal standard were then log-centered.(TIF)Click here for additional data file.Figure S2MAPK phosphorylation observed by LC-MS/MS. Quantitative assessment of the relative phosphorylation status of ERK at T185 and Y187 residues in 3KTR, A549, H322, and H1299 cells in comparison to 3KT cells by LC-MS/MS and IDEAL-Q.(TIF)Click here for additional data file.Figure S3http://www.pantherdb.org/.(A) Classification of Ras-regulated phosphoproteins according to their molecular function. (B) Classification of Ras-regulated phosphoproteins according to their biological processes. The classification was made using the database, Panther Classification System, available on (TIF)Click here for additional data file.Figure S4Functional interaction network of the phosphoproteins in Ras-transformed HBECs. Phosphoproteins identified in Ras-transformed HBECs were subjected to STRING database with medium confidence (≥0.4) applied. The upregulated and downregulated phosphoproteins involved in oncogenic Ras signaling are marked in pink and green boxes, respectively. The known and novel substrates of MAP kinases identified by NetworKIN analysis are marked in boxes circumscribed by blue and red, respectively.(TIF)Click here for additional data file.Figure S5Clustering of Ras-regulated signaling pathways. Hierarchical agglomerative average-linkage clustering analysis of oncogenic Ras-regulated pathways identified in HBECs was performed based on their gene ontology functional similarity. Pearson's correlation coefficient was derived as the distance matrix and visualized by Generalized Association Plots (GAP). The color bar indicates the increasing association among the pathways with increasing color intensity from blue to red. The rows and columns show the pathways in similar order, and the association between two identical pathways is shown as a red line throughout the map. Four pathway clusters were identified, depicted in thick red lines on the right side, using a correlation threshold of 0.5.(TIF)Click here for additional data file.Table S1The list of quantifiable phosphopeptides across five cell lines are shown here. Upon comparisons, phosphopeptides showing up- and down-regulated levels are highlighted in red and green colors, respectively. Data corresponding to each peptide identified by mass spectrometry and Mascot search, including peptide sequence, m/z value, charge, modification site, international protein index (IPI) accession number, average abundance and standard deviation, are shown. Few peptides were not quantified by IDEAL-Q and such peptides are indicated as NaN, -9999, and 9999. In the case of 3KTR vs 3KT, if the peptide was not identified in both cell lines or identified in 3KTR only or identified in 3KT only then they are indicated as NaN, 9999 and -9999, respectively.(XLS)Click here for additional data file.Table S2The list of site specific upstream kinases of regulated phosphorylation events predicted by NetworKIN. The regulated phosphorylated peptides identified in the cases of 3KTR or A549 or H322 or H1299 vs 3KT were subjected to NetworKIN analysis. List of site specific upstream kinases predicted by NetworKIN analysis are shown in this table. For each regulated peptide, Swiss-Prot accession number of the protein, phosphorylation site, upstream kinase family, kinase ID, kinase symbol, NetPhorest posterior probability, String score, NetworKIN score of the kinase, are shown.(XLS)Click here for additional data file.Table S3Frequency of upstream kinases of regulated phosphorylation events identified in 3KTR, A549, H322 and H1299 cells. The upstream kinases of regulated phosphorylation events identified in individual pairs of cell lines were predicted by NetworKIN analysis. According to their target sequences, the upstream kinases were grouped into subsets of proline-directed, basophilic, acidophilic, and other kinases. The kinases targeting other than proline-directed, basophilic and acidophilic S/T sites are shown. The frequencies of subsets of kinases are listed.(XLS)Click here for additional data file.Table S4The oncogenic Ras-regulated phosphorylation events were identified in 3KTR and their status is shown in NSCLC cells. The regulated phosphorylation events in HBECs upon oncogenic Ras-transformation (3KTR vs 3KT) are shown in Log2 scale. The relative levels of these phosphorylation events in NSCLC cells (A549 or H322 or H1299 vs 3KT) in relation to 3KT are shown. The regulated phosphosites were listed as proline-directed, basophilic, acidophilic and others. The relative phosphorylation levels with 2-fold increase or decrease are indicated by bold font. Few peptides were not quantified by IDEAL-Q and such peptides were indicated as NaN, -9999, and 9999. In the case of 3KTR vs 3KT, if the peptide was not identified in both cell lines or identified in 3KTR only or identified in 3KT only then they were indicated as NaN, 9999 and -9999, respectively. The protein symbol, full name, and Swiss-Port database protein accession number corresponding to each phosphopeptide, along with phosphorylated residue location, and NetworKIN assessment of upstream kinases are shown. In the table, (A) indicates peptides listed here only when they regulated in 3KTR in relation to 3KT with exception for MAPK1 T185, MAPK3 Y204 and MAPK14 Y182 peptides. (B) indicates MEK1/2 kinases were not covered by NetworKIN. (C) indicates MAP kinases are listed for the regulated sites according to the NetworKIN specificity. (I) indicates isoform.(XLS)Click here for additional data file.Table S5The list of pathway activities inferred in Ras-transformed HBECs and NSCLCs. The expression values of up- and down-regulated phosphorylation events were integrated in Pathway Interaction Database to examine the up- and down-regulated pathway activity signatures respectively. Pathway activities observed in four comparisons (3KTR or A549 or H322 or H1299 vs 3KT) were listed. For each identified regulated pathway, pathway ID, number of phosphoproteins identified, and number of regulated phosphoproteins identified, pathway activity score, are shown. The pathways consisting more than five identified phosphoproteins were considered for pathway activity analysis.(XLS)Click here for additional data file.Table S6The expression levels of bonafide MAPK targets and MAP kinases in lung adenocarcinomas with and without KRAS mutations. The relative expression levels of bonafied MAPK targets and MAP kinases are shown by comparing lung adeoncarcinomas with (n = 12) and without (n = 84) KRAS mutations to normal tissues (n = 5). This data is derived from Agilent Homo sapiens 21.6K custom array, the data values were corrected for background and normalized, and the data was deposited on NCBI's GEO oligo microarray database (Data series GSE11969). Upon comparisons, proteins showing up- and down-regulated levels are highlighted in red and green colors, respectively.(XLS)Click here for additional data file.Table S7Identification of pathways associated with Ras-signaling by pathway clustering. Upregulated pathways (n = 23) identified in Ras-transformed HBECs in relation to normal HBECs (3KTR vs 3KT) were clustered based on their gene ontology functional similarities. Correlation values observed among the upregulated pathways in Ras-transformed HBECs are shown. The maximum correlation score was one, which is seen between the same pathways. The rows and columns show the pathways in similar order therefore association between two identical pathways shown a red line throughout the map (see  map see .(XLS)Click here for additional data file.Methods S1Supplementary Methods.(DOC)Click here for additional data file.


Plasmodium merozoites is vital for replication and survival of the parasite and, as such, is an attractive target for therapeutic intervention. Merozoite invasion is mediated by specific interactions between parasite ligands and host erythrocyte receptors. The P. vivax Duffy-binding protein (PvDBP) is heavily dependent on the interaction with the human Duffy blood group antigen/receptor for chemokines (DARC) for invasion. Region II of PvDBP contains many allelic polymorphisms likely to have arisen by host immune selection. Successful vaccine development necessitates a deeper understanding of the role of these polymorphisms in both parasite function and evasion of host immunity. A 3D structure of the homologous P. knowlesi DBP predicts that most variant residues are surface-exposed, including N417K, which is a dimorphic residue change that has previously been shown to be part of a linked haplotype that alters DBP sensitivity to inhibitory antibody. In natural isolates only two residues are found at this site, asparagine (N) and lysine (K). Site-directed mutagenesis of residue 417 was used to create a panel of 20 amino acid variants that were then examined for their binding phenotype and response to immune sera. Our results suggest that the observed dimorphism likely arose due to both structural requirements and immune selection pressure. To our knowledge, this is the first exhaustive examination of this kind of the role of a single amino acid residue in antigenic character and binding ability. Our results demonstrate that a single amino acid substitution can dramatically alter both the ability of the PvDBP to bind to human erythrocytes and its antigenic character.Invasion of human red blood cells by  Plasmodium vivax is responsible for 70–80 million cases of clinical malaria annually and has a wide distribution that causes more than 50% of malaria cases outside of Africa P. vivax emphasize the need for better prevention and treatment strategies P. vivax merozoites Influenza hemagglutinin (HA) and Plasmodium apical membrane antigen 1 (AMA-1) reveals a pattern of polymorphisms located adjacent to and surrounding their putative receptor binding sites. A consensus viewpoint interprets these substitutions as making it more difficult for host inhibitory antibodies, elicited by previous exposure to the pathogen, to recognize new variant epitopes and block the interaction between the pathogen ligand and the host receptor P. vivax DBP homology model based on the P. knowlesi DBPα crystal structure reveals that this residue is immediately adjacent to a motif identified by mutational analysis to be important for DARC receptor recognition In previous studies we analyzed the variant alleles of field isolates from Papua New Guinea and determined that several polymorphisms  formed a linked haplotype pvdbp in clinical isolates has shown that variation at residue 417 is often linked to residues 437 and 503 Our previous study of genetic analysis of in vitro COS7 cell assay for DBP-erythrocyte binding function    [27]. Fresidues) . In the esidues) .A bias in codon usage for residue 417 may also affect amino acid substitutions. The codons for variant R that has a binding phenotype significantly different from naturally occurring residue N (p<0.05) but not from naturally occurring residue K (p>0.05), were analyzed . MutatioP. vivax DBP offer the potential of vulnerable epitopes associated with functionally sensitive motifs required for receptor recognition. However, the large number and diversity of polymorphisms found in the regions of the DBP necessary for ligand recognition and binding suggest that the parasite is very capable of adapting to evade any inhibitory host immune response Vaccine development against malaria has achieved limited success so far. A number of challenges need to be overcome in designing a highly effective vaccine, especially the challenge of strain-specific immunity. Some antigens like the P. knowlesi α DBL we found that residue 417 is located adjacent to a motif identified in two separate studies as important for recognition of the erythrocyte receptor Consistent with this possibility is the previous work that demonstrated that strain-specific antibody inhibition can be mediated by polymorphic residues found in a linked haplotype PvDBPII to bind to the human erythrocyte. The poor erythrocyte binding properties of these amino acid substitutions would seem to be sufficient to preclude their occurrence on this critical parasite ligand.Binding analysis of variant constructs representative of all 20 amino acid possibilities indicates a functional constraint for nine of the non-naturally occurring residues , indicatP. vivax strain containing N417 We performed inhibition analysis on the remaining eleven constructs using antisera raised against the Sal I P. vivax CDS  was significantly different in its inhibition phenotype from N (p<0.05), but not from K (p>0.05). We examined this residue further to try to determine its absence from natural isolates. The much lower frequency at which this amino acid appears in isolates . In addiisolates . AlthougP. vivax and is genetically more difficult to achieve spontaneously. Examination of the codons for these amino acids indicates that K is the simplest change to achieve with a single nucleotide substitution necessary to alter the codon. Other factors that may affect the absence of multiple polymorphisms at this site include the possibility that the partially linked residues at site 437 and 503 limit the appearance of certain residues at site 417. To our knowledge, this is the first exhaustive work of this kind to examine the impact of substitutions at a single residue on binding and antigenic character. These data provide experimental support for the hypothesis that positive immune selection pressure plays a role in the appearance of polymorphisms in functionally important residues of P. vivax DBP.In conclusion, these data demonstrate the impact a single amino acid substitution can have on both the ability of the PvDBPII to bind to human erythrocytes and the antigenic character of this vital invasion protein. In addition, these data provide a partial explanation for the absence of multiple polymorphisms at residue 417. A number of residues are eliminated from natural isolates because of functional constraints in their ability to bind to the red blood cell receptor. Other residues bind to erythrocytes in a similar fashion to the naturally occurring variants, but are not antigenically distinct from naturally occurring residue N, so there may be a lack of positive immune selection driving their appearance in natural isolates. Residue R, like naturally occurring residue K, has a more extreme difference in antigenic character as compared to residue N, but is a less abundant residue in P. vivax (containing N at the 417 location) was used as the parent template. Single residue changes were performed at the 417 site to create a panel of variants representative of all 20 amino acids all on the same genetic Sal I background. Recombinant plasmid DNA was purified using an endotoxin-free plasmid DNA purification system .Salvador I  DBPII was cloned into the pEGFP-N1 plasmid with flanking signal sequences from the herpes simplex virus glycoprotein D1 allowing expression of a GFP fusion protein on the surface of transiently transfected COS7 cells . Mutagenesis to create a panel of N417 variants was performed using the Stratagene Quickchange mutagenesis kit  as previously described COS7  cells were maintained in Dulbecco's modified Eagle's medium  containing 10% fetal bovine sera (FBS). Only cells between the passage numbers of 5 and 20 were used for binding and inhibition assays.7 cells/well, previously washed three times with incomplete DMEM). Wells were washed three times with PBS to remove nonadherent erythrocytes and binding was scored by counting the number of rosettes per 30 fields of view at 200× magnification. GFP surface expression was observed to be consistent between all constructs suggesting comparable expression levels. Inhibition assays were carried out in the same manner except that transfected COS7 cells were preincubated for 1 hour at 37°C, 5% CO2 with antiserum diluted in incomplete DMEM prior to addition of the erythrocyte suspension. Antiserum against recombinant Sal I DBPII was produced as previously described COS7 cells were plated in 24-well plates at a density of 35,000 cells per well and were transiently transfected with endotoxin-free pEGFP-DBPII DNA using Lipofectamine or Lipofectamine 2000  according to the manufacturer's instructions. Forty-two hours post-transfection, the transfected COS7 cells were incubated with DARC-positive human erythrocytes for 2 hours  Modeling was done using the MacPymol Molecular Graphics System  and SWISS-MODEL (