Inhibitory circuits play an important role in synaptic plasticity during development and adulthood. Changes in interneuronal activity induce structural and synaptic rearrangements of inhibitory interneurons, network oscillations, and homeostatic plasticity. In addition to epileptic seizures, deficits in the inhibitory system lead to aberrant information processing and cognitive impairment in various neurological disorders. Studies exploring the structural and functional plasticity of interneurons are essential, not only to understand the mechanisms underlying normal development and behavior but also, to identify the etiology of different psychiatric and neurological disorders to pursue new therapies. Here, it will be discussed the role of inhibitory circuit in the synaptic plasticity, and how cellular replacement strategies can remodel changes in circuit function and homeostasis in the context of brain repair.Most of the gamma-aminobutyric acid-containing (GABAergic) interneurons in the cerebral cortex and hippocampus originate from three progenitor regions in the embryonic subpallium: caudal ganglionic eminence (CGE), medial ganglionic eminence (MGE), and preoptic area (POA) –4. Each In the cortex and hippocampus, interneurons subtypes differ in their functional connectivity and generate differentially timed inhibition at distinct sites of postsynaptic cells –8. InterBesides the direct control of inhibition, synchrony and plasticity on principal cells, different interneuron subtypes exhibit a wide range of responses to different neuromodulatory inputs, leading to changes in net inhibition, synchronization, and synaptic plasticity . The innInterneuron subtypes differ in their functional connectivity to the principal cells in cortex and hippocampus , 8, provAs mentioned above, GABAergic interneurons targeting dendritic domains control the efficacy and plasticity of excitatory inputs onto principal neurons. This dendritic remodeling of inhibitory neurons, either in normal or pathological conditions, affects activity-dependent modulation of neuronal connectivity within local circuits . ReductiA receptor kinetics are likely to be needed to achieve precise timing gamma oscillations in cortex and hippocampus  (Ccnd2−/−)  has been performed in animal models. The studies had shown that MGE-derived cells are able to survive, differentiate in mature interneurons, migrate, and functionally integrate through the host brain parenchyma with low risk of promoting brain tumors. The integrated mature interneurons modify the neuronal network, rescuing the normal functional inhibition and the synaptic plasticity –44. SomeDlx1−/−)  and cyclcnd2−/−) . Dlx1−/−hibition , with coenotypes .A receptors containing the α1 subunit, abundant at synapses mediated by PV-expressing interneurons (The maturation of GABAergic interneurons, specifically PV- and SOM-expressing subtypes, has been strongly implicated in critical period of plasticity, such as the development of visual cortex , 46. It rneurons , 51.Changes in GABAergic function mediated by PV- and SOM-expressing interneurons impair synaptic plasticity and disrupt network organization and normal brain oscillations. However, by temporal and selective coordination of interneuronal activity, it is possible to modulate interneuronal network and increase GABA release on different subcellular domains of target cells to maintain or rescue normal network oscillations and synaptic plasticity. The investigation of PV- and SOM-expressing interneurons, using strategies that modify interneuronal network and regulate inhibitory input/output of such interneurons at different time points, as selective target them or using precursor cells grafts, are helping us to better understand the role of these specific interneuron subtypes in controlling and restoring synaptic plasticity and brain oscillations during development and adulthood.The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Birch pollen-allergic subjects produce polyclonal cross-reactive IgE antibodies that mediate pollen-associated food allergies. The major allergen Bet v 1 and its homologs in plant foods bind IgE in their native protein conformation. Information on location, number and clinical relevance of IgE epitopes is limited. We addressed the use of an allergen-related protein model to identify amino acids critical for IgE binding of PR-10 allergens.Norcoclaurine synthase (NCS) from meadow rue is structurally homologous to Bet v 1 but does not bind Bet v 1-reactive IgE. NCS was used as the template for epitope grafting. NCS variants were tested with sera from 70 birch pollen allergic subjects and with monoclonal antibody BV16 reported to compete with IgE binding to Bet v 1.N57/I58E/D60N/V63P/D68K) harboring an IgE epitope of Bet v 1. Bet v 1-type protein folding of the NCS variant was evaluated by 1H-15N-HSQC NMR spectroscopy. BV16 bound the NCS variant and 71% (50/70 sera) of our study population showed significant IgE binding. We observed IgE and BV16 cross-reactivity to the epitope presented by the NCS variant in a subgroup of Bet v 1-related allergens. Moreover BV16 blocked IgE binding to the NCS variant. Antibody cross-reactivity depended on a defined orientation of amino acids within the Bet v 1-type conformation.We generated an NCS variant (Δ29NCSOur system allows the evaluation of patient-specific epitope profiles and will facilitate both the identification of clinically relevant epitopes as biomarkers and the monitoring of therapeutic outcomes to improve diagnosis, prognosis, and therapy of allergies caused by PR-10 proteins. Betula verrucosa) pollen, Bet v 1 Millions of patients with allergies to tree pollen are sensitized (produce IgE antibodies) to the major allergen of birch  from the meadow rue Here we aimed at establishing a recombinant model protein system to specifically analyze epitopes of PR-10 allergens. For this purpose we used the truncated variant Δ29NCS. To study the impact of individual amino acids in IgE binding within NCS we generated variants of Δ29NCS, analyzed their IgE antibody binding with sera of birch pollen allergic subjects and determined the cross-reactivity of suspected IgE epitopes grafted onto NCS.A/L to birch pollen measured by ImmunoCAP  were included as serum donors. Patients were recruited at the Allergy Unit, Department of Dermatology, University Hospital Zürich, Switzerland, at the Hospital Borkum Riff, Borkum, Germany, and at the Paul-Ehrlich-Institut, Langen, Germany. Study participants provided written informed consent. Ethics approval by the local ethics committee ‘Kantonale Ethikkomission Zürich, Switzerland’ of the University Hospital in Zürich, Switzerland and the local ethics committee ‘Ethik-Kommission, Fachbereich Medizin der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany’ of the University Hospital in Frankfurt included consent form and consent procedure. Sera 52–69 were published elsewhere A/L against Δ29NCS_5x) comprised sera 11, 14, 20–23, 32, 42, 47, and 70. The negative serum pool (IgE<0.35 kUA/L against Δ29NCS_5x) comprised sera 4, 12, 18, 29–31, 33, 40, 43, and 46.Sixty-nine patients with a convincing history of pollinosis to early flowering tree pollen and specific IgE levels>0.35 kUSpecific IgE levels to recombinant proteins were determined by ImmunoCAP in Phadia 100 and 250 instruments  according to the manufacturer's instructions. In the case of Bet v 1, Api g 1.01 and Cor a 1.04, commercial ImmunoCAP tests were used, whereas for Pru av 1, Dau c 1.01, NCS and its variants, experimental ImmunoCAPs were manufactured by coupling the recombinant allergens individually to the solid phase, as described elsewhere N57/I58E/D60N/V63P, Δ29NCSN57/I58E/D60N/V63P/D68K, and Bet v 1N43A/E45S/N47A/K55A were sequentially introduced into pET29b-Δ29NCS 6-tag as described Mutations leading to the amino acid exchanges/insertion in Δ29NCSFar UV circular dichroism (CD) spectra of the Δ29NCS variants were acquired at 293 K using a Jasco J-810 spectropolarimeter  at a band width of 1 nm and a sensitivity of 100 mdeg in a 0.2 cm cell. All proteins were analyzed at a concentration of 2.5 µM in 5 mM sodium phosphate, pH 7.0. Each measurement comprised the average of 10 repeated scans between 260 and 190 nm.15N-labeled protein in 20 mM sodium phosphate, pH 7.0, 1 mM DTT, 0.04% sodium azide and 10% D2O. 1H-15N-HSQC spectra were recorded on a Bruker Avance 700 MHz spectrometer at 298 K. NMR data were processed using in-house software and were visualized with NMR view NMR samples were prepared by dissolving lyophilized N57/I58E/D60N/V63P, Δ29NCSN57/I58E/D60N/V63P/D68K, and Bet v 1N43I/E45S/N47D/K55A were performed by using the Phyre server Structure predictions of Δ29NCSNunc Maxisorp plates  were coated overnight at room temperature with 250 ng/100 µl proteins , with 50 ng/100 µl Api g 1, and with 25 ng/100 µl Bet v 1 and Bet v 1_4x in phosphate-buffered saline (PBS). After blocking with PBS containing 2% BSA a dilution series of the murine monoclonal anti-Bet v 1 antibody BV16 −5–3 µg/well) of recombinant inhibitors  for 3 hrs at room temperature with PBS containing 0.05% Tween 20 and 0.1% BSA. Allergen-specific IgE was detected with horseradish peroxidase-conjugated mouse anti-human IgE antibody  diluted 1∶1000 with PBS containing 0.05% Tween 20 and 0.1% BSA. The substrate for horseradish peroxidase was 3,3′,5,5′-tetramethylbenzidine  and the reaction was stopped by addition of 25% H2SO4. The absorbance was measured at 450 nm.For IgE-ELISA inhibition experiments, Nunc Maxisorp plates  were coated overnight at room temperature with 300 ng/100 µl Δ29NCS_5x with 10 mM potassium phosphate-buffered saline (PBS). After blocking with PBS containing 2% BSA, plates were incubated with the human serum pool (dilution 1∶10) and increasing concentrations  were coated overnight at room temperature with 25 ng/100 µl recombinant Bet v 1 and Bet v 1_4x, respectively with phosphate-buffered saline (PBS). Blocking and IgE detection was done as described for IgE ELISA above.2 for 1 h SDS-PAGE was performed with 15% separating gels and 5% stacking gels using a discontinuous buffer system The mediator release assay followed an established protocol 4HCO3; 10% aceto nitrile (ACN) and the digestion was stopped by adding 5% formic acid. The peptides were analyzed by using a nano-ultra performance LC system coupled to a nano-ESI- MS  with a 5 µm symmetry 180 µm×20 mm c18 pre-column and a 1.7 µm BEH 130 100 µm×100 mm c18 separation column. After 3 minutes of trapping (99% water at 5 µl/minute), a 30 minutes gradient (3–40% ACN at 500 nl/minute) was applied to separate peptides. MS was operated in V mode, acquiring MSE data and applying standard parameters. Data analysis was performed with protein lynx global server version 2.4 (Waters), searching an in house database consisting of the Uniprot database  and the amino acid sequences of the recombinant variants Δ29NCS, Δ29NCS_4x and Δ29NCS_5x. The identification of a protein was accepted at a false positive rate of less than 4%; peptide mass accuracy was 9 parts per million (ppm) or better.The identity of recombinant proteins was confirmed by liquid chromatography mass spectrometry (LC-MS). Recombinant proteins were excised from Coomassie stained SDS-PAGE gels and analyzed as described elsewhere Amino acid sequence alignments were carried out with ClustalO norcoclaurine synthase  revealed a conformation identical to that of Bet v 1, even though the sequence identity of these proteins is only 25% 1H15N-HSQC NMR spectra of Δ29NCSN57/I58E/D60N/V63P (Δ29NCS_4x) and Δ29NCSN57/I58E/D60N/V63P/D68K (Δ29NCS_5x) indicated that the amino acid substitutions in the NCS variants did not change the proteins' secondary or tertiary structures and confirmed that the native protein conformation is maintained in the variants  and a majority to the PR-10 food allergens Pru av 1.01 , Cor a 1.04 , Dau c 1.01 , and Api g 1.01 , respectively. In contrast, most of the sera  tested negative (<0.35 kUA/L) to Δ29NCS, whereas 25/70 (36%) showed significant IgE binding  to Δ29NCS_4x and 50/70 (71%) to Δ29NCS_5x . Thus, gradual substitution of certain amino acids of the low-IgE binding protein Δ29NCS with those of Bet v 1 at the corresponding positions induced significant binding of serum IgE from subjects with birch pollinosis.To assess potential cross-reactive IgE binding to rΔ29NCS variants, 70 sera of birch pollen allergic subjects were analyzed for IgE binding to a range of PR-10 proteins using ImmunoCAP tests . All ser_5x was inhibited by Δ29NCS_5x itself and Bet v 1 in a dose-dependent manner, suggesting the presence of an IgE epitope shared by the two proteins.Since the majority of the patients' sera IgE bound Δ29NCS_5x, we asked whether this interaction was due to a Bet v 1-specific IgE epitope presented by the Δ29NCS variant. Therefore we used serum with sIgE to both Bet v 1 and Δ29NCS_5x and tested its IgE interaction with the Δ29NCS_5x variant in the presence of Bet v 1 . As expeNext, we asked whether the IgE epitope presented by Δ29NCS_5x is cross-reactive with PR-10 food allergens. Thus, we performed competitive IgE binding experiments (ELISA) with the PR-10 food allergens Cor a 1.04, Gly m 4, Dau c 1.01, Api g 1.01, and Pru av 1, respectively . Bet v 1Δ29NCS harbors 47% and Δ29NCS_5x 76% of the structurally resolved IgG epitope  of Bet v 1, as defined earlier N43I/E45S/N47D/K55A). As for Δ29NCS_5x and Δ29NCS_4x, the amino acid substitutions did not alter the protein conformation according to CD and 1H15N-HSQC NMR spectroscopy  does not interact with patients' IgE, ii) has a stable allergen-like protein fold, and iii) allows the analysis of individual antibody binding by amino acid substitutions.Here we analyzed a non IgE-binding variant of the PR-10 enzyme Δ29NCS of the common meadow rue with Bet v 1-type protein structure 1H15N-HSQC NMR spectroscopy. The CD spectra of the Δ29NCS variants were virtually identical and typical for PR-10 proteins. Furthermore, the 1H15N-HSQC spectra of the Δ29NCS variants were superimposable for the majority of resonances, and the spectra are in agreement with the notion of only minor local differences caused by the amino acid substitutions  neither sequential IgE binding peptides of Bet v 1 nor IgE binding truncated variants of Bet v 1 have been described so far ions cf. , but othN43I/E45S/N47D/K55A (Bet v 1_4x) which showed only little competition for serum IgE binding to Δ29NCS_5x and did not bind the Bet v 1-reactive murine monoclonal antibody BV16. Interestingly, residue E45 of Bet v 1 has already been identified as being critical for both IgE and BV16 interaction When we tested the impact of specific amino acids on serum IgE binding, we found that amino acid substitutions in Δ29NCS with structurally corresponding residues of Bet v 1 induced a significant increase in IgE interaction of the model protein to up to four CAP classes cf. . As the Since Bet v 1-specific IgE cross-reacts with PR-10 food allergens 2 and comprises 10–25 amino acids, only a small subset of these residues dominates the energetics of the antibody-antigen interaction and thus constitutes the functional (active) epitope Whereas the structural epitope of an antigen usually spans a molecular surface area of about 400–1000 ÅSome information on functional IgE epitopes of Bet v 1 and other PR-10 allergens has emerged in recent years. These studies analyzed the interaction of IgE with either native Bet v 1 isoforms or recombinant allergen variants No mediator release in humanized rat basophil leukemia cells sensitized with sera of birch pollen allergic subjects has been observed with the Δ29NCS variants, indicating that no additional IgE epitope that would mediate IgE receptor cross-linking is displayed in the variants cf. . In contIn summary, we have established a protein model system which enables the specific analysis of functional IgE and IgG epitopes and their homologies among PR-10 allergens. Upon single amino acid substitutions of the non-allergenic protein variant Δ29NCS, individual epitopes are identified in a residue-specific manner by detection of newly generated immunoglobulin interactions of the model protein. Our system allows the evaluation of patient-specific epitope profiles and will facilitate both the identification of clinically relevant epitopes as biomarkers and the monitoring of therapeutic outcomes to improve diagnosis, prognosis, and therapy of allergies caused by PR-10 proteins.Figure S11H-15N-HSQC spectra of different NCS variants. Spectra of (A) 400 µM Δ29NCS (black) (B) 250 µM Δ29NCSN57/I58E/D60N/V63P (Δ29NCS_4x) (blue), and (C) 400 µM Δ29NCSN57/I58E/D60N/V63P/D68K (Δ29NCS_5x) (red). The proteins were measured in 20 mM sodium phosphate, pH 7.0, 1 mM DTT, 0.04% sodium azide and 10% D2O at a 700 MHz spectrometer at 298 K. Due to its solubility limit Δ29NCSN57/I58E/D60N/V63P (Δ29NCS_4x) was measured at a lower protein concentration.(TIF)Click here for additional data file.Figure S2Folding and structural integrities of Bet v 1 and Bet v 1_4x variant. (A) Circular dichroism of Bet v 1 and Bet v 1_4x. (B) 1H-15N-HSQC spectra of Bet v 1 variants. Overlay of the spectra of 100 µM Bet v 1 (petrol) and 100 µM Bet v 1_4x (purple). The proteins were measured in 20 mM sodium phosphate, pH 7.0, 1 mM DTT, 0.04% sodium azide and 10% D2O at a 700 MHz spectrometer at 298 K.(TIF)Click here for additional data file.


Angiogenic sprouting, EC migration, cytoskeleton re-organization, and signaling events regulating matrix adhesion were studied. Sustained inactivation of mTORC1 activity up-regulated mTORC2-dependent Akt1 activation. In turn, ECs exposed to mTORC1-inhibition were resistant to apoptosis and hyper-responsive to renal cell carcinoma (RCC)-stimulated angiogenesis after relief of the inhibition. Conversely, mTORC1/2 dual inhibition or selective mTORC2 inactivation inhibited angiogenesis in response to RCC cells and VEGF. mTORC2-inactivation decreased EC migration more than Akt1- or mTORC1-inactivation. Mechanistically, mTORC2 inactivation robustly suppressed VEGF-stimulated EC actin polymerization, and inhibited focal adhesion formation and activation of focal adhesion kinase, independent of Akt1. Endothelial mTORC2 regulates angiogenesis, in part by regulation of EC focal adhesion kinase activity, matrix adhesion, and cytoskeletal remodeling, independent of Akt/mTORC1.Tumor neovascularization is targeted by inhibition of vascular endothelial growth factor (VEGF) or the receptor to prevent tumor growth, but drug resistance to angiogenesis inhibition limits clinical efficacy. Inhibition of the phosphoinositide 3 kinase pathway intermediate, mammalian target of rapamycin (mTOR), also inhibits tumor growth and may prevent escape from VEGF receptor inhibitors. mTOR is assembled into two separate multi-molecular complexes, mTORC1 and mTORC2. The direct effect of mTORC2 inhibition on the endothelium and tumor angiogenesis is poorly defined. We used pharmacological inhibitors and RNA interference to determine the function of mTORC2  Drug therapy to inhibit tumor neovascularization is used clinically as an adjuvant in chemotherapy–resistant cancers, including renal cell carcinoma, recurrent glioblastoma, and bowel cancer. The rapalog mammalian target of rapamycin (mTOR) inhibitors are used after failure of pro-angiogenic growth factor–receptor tyrosine kinase inhibitors, and in some cases as first line therapy . RapalogIn mammalian cells, mTOR is assembled in two distinct signaling complexes: mTOR complex-1 (mTORC1), sensitive to inhibition by rapalog drugs, and mTOR complex-2 (mTORC2) . In addiPro-angiogenic cues are recognized by activation of several growth factor receptors displayed on the vascular endothelium, and the diverse signals are integrated to recruit key signal transduction pathways in the endothelial cell (EC). For example, the principal endothelial VEGF receptor, VEGF-receptor 2, is coupled to phosphatidylinositide 3 (PI3)-kinase, signaling to the downstream mTOR kinase . In pre-in vitro. Mechanistically, we identify that mTORC2 activity in ECs is needed for cell migration, development of mature matrix adhesion structures, and specifically regulates VEGF-stimulated Src and focal adhesion kinase activity.The effect of disrupted signaling of the mTORC2 branch point on the PI3 kinase pathway in the endothelium is poorly understood, but may contribute anti-angiogenic effects . In thisS473, anti-phospho-S6KT389, anti-FAK, anti-phospho-FAKY397, anti-raptor, anti-rictor, and rictor siRNA were from Cell Signaling Technology . ON-TARGETplus human raptor siRNA-SMARTpool was from Thermo Scientific .Medium 199 (M199), Hank's Balanced Salt Solution (HBSS), fetal bovine serum (FBS), and endothelial cell growth supplement were purchased from Invitrogen . VEGF-A was from Peprotech . Rapamycin, PP242, a highly specific mTOR active-site inhibitor , and antHuman umbilical vein ECs (HUVECs) were isolated as described previously . The proHUVECs were seeded at 70–80% confluency and transfected serially twice over two days with either 50 nM non-silencing (siNS) or specific siRNA using Hiperfect transfection reagent according to the manufacturer’s protocol. Protein expression was evaluated by Western blot.HUVEC monolayers were washed once with ice cold phosphate buffered solution (PBS) and then lysed immediately on ice by RIPA buffer  followed by boiling at 95°C for 5 minutes. The lysates were resolved by SDS-PAGE, blotted on nitrocellulose membranes (Biorad), then immunostained overnight at 4°C in blocking buffer (5% BSA/TBS-Tween20). Proteins were detected using Luminata forte  and a Fluorchem FC2 CCD camera (Alpha Innotech). Protein bands were equally contrast enhanced by Adobe Photoshop CS3 then quantified by ImageJ.in vitro was done as previously described [versus mTORC1/2, rapamycin (5 nM) or PP242 (1–10 μM) were added, respectively, to both the fibrin gel and the growth media. To study tumor angiogenesis in vitro, HUVECs were pre-treated with PP242 (1 uM), ku-0063794 (50 nM) or rapamycin (5 nM) for 24 hours, then labeled with CellTracker Green (Life Technologies). Caki-1 human renal cell carcinoma cells  were labeled with CellTracker Red. Cytodex beads were coated with HUVECs or Caki-1 (~400 cells/bead) then embedded in fibrin gel. Growth media  containing Caki-1  in suspension was then added on top. At least 90 beads per treatment from each experiment were imaged after 18–20 hours incubation in 5% CO2 at 37°C, using a 20X objective lens and a CCD camera equipped inverted fluorescence microscope . Scoring was done using OpenLab .A 3D angiogenesis assay escribed . Brieflyin vivo was performed as described previously [Assay of angiogenesis eviously . BrieflyCell-matrix adhesion and cell migration were tested using the ECIS device . HUVECs were treated with PP242, siAkt1, siRictor, or siRaptor as indicated. Equal numbers of cells were seeded into gelatin-coated ECIS plates with VEGF (20 ng/ml). Adhesion and migration were then assessed by continuous resistance measurements in optimum growth conditions over 12 hours.Apoptosis was measured in HUVECs in optimum growth conditions and after inducing apoptosis. To induce apoptosis, EC monolayers were washed, then incubated with cycloheximide  and tumor necrosis factor-α  for 4 hours. HUVECs were incubated with the FITC-conjugated active caspase-3 reporter, DEVD-FMK, for 30 minutes at 37°C as directed by the manufacturer . Cells were trypsinized and combined with the floating cells in the medium. After brief washes, cells were analyzed by flow cytometry (LSR-Fortessa).HUVECs were cultured on gelatin-coated glass bottom microwell dishes . Cells were fixed for 10 minutes in 3% fresh paraformaldehyde in PBS, washed with PBS, then permeabilized and blocked in 3% BSA + 0.1% Triton X-100 in PBS for 30 minutes. Cells were washed in PBS then incubated with phalloidin-FITC or anti-vinculin-FITC, and DAPI in 1% BSA for 1 hour in room temperature. The cells were washed 3 times with PBS. Filamentous actin and vinculin were visualized at 40X using an inverted fluorescence microscope, and focal adhesions were enumerated as described .The separation of filamentous actin (F-actin) and globular actin (G-actin) was done according to the manufacturer’s instruction . The ratios of F-actin to G-actin in ECs were estimated by Western blot.t-test using Prism 5 . P values <0.05 were considered significant. Each experiment was done at least three times.Data are shown as mean ± SEM. Statistical analysis was performed by 1-way ANOVA as appropriate followed by the Bonferroni post-hoc test. Pairwise comparisons were done by paired Student in vivo exposure of ECs to rapamycin by treatment with 5 nM rapamycin for 24 hours. Paradoxically, sustained exposure to rapamycin increased Akt S473 phosphorylation, the mTORC2-dependent site, in a dose-dependent manner  phosphorylation and full activation of Akt. Inhibition of mTORC1 relieves this inhibition, but has a variable and cell-type specific effect on upstream growth factor receptor signaling ,19. We st manner . Rapamyct manner . Similart manner .To determine if mTORC2- has the same effect as mTORC1- inactivation, we specifically disrupted EC mTORC2 or mTORC1 using siRNA targeted against rictor or raptor, respectively ,21. We oNext, we sought to study the effect of dual mTORC1 and mTORC2 inhibition on VEGF-stimulated EC Akt activation. Selective mTOR kinase inhibitors are active against mTOR in both complexes . Pretreaversus mTORC1 inactivation on EC apoptosis. We challenged the EC with pro-apoptotic factors, then quantified active cleaved caspase 3 expression among rapamycin-, PP242-, or Ku-0063794-pretreated ECs. We observed that extended mTORC1 inhibition increased EC resistance to apoptosis versus mock-treated, or mTORC1/2 inhibited EC  to disrupt mTORC2, and confirmed the effect of the knock-down in EC by Western blot of rictor expression, and mTORC2 activity by Akt S473 phosphorylation  is a principal regulator linking growth factor signals to cell-matrix adhesion and cytoskeleton remodeling . Therefoated ECs . Similarated ECs  or Ku-00ated ECs  treated ated ECs .versus control ECs  has been previously identified both outside and as a component of the mTORC2, and shown to participate in regulation of Akt . Our datin vitro independent of downstream Akt-regulated events. These data indicate that mTORC2 in the endothelium is an attractive target to inhibit pathologic neoangiogenesis.In summary, our results show that sustained mTORC1 inhibition activates maladaptive PI3 kinase signaling to Akt and responsiveness to proangiogenic growth factors in primary human ECs. In contrast, mTORC2 inactivation prevents Akt and downstream FOXO1/3, or S6 kinase hyper-stimulation. Moreover, mTORC2 regulates EC matrix adhesion, motility, and angiogenesis S1 FigA) HMEC-1were treated with the mTORC1/2 inhibitor Ku 0063794 (50 nM), or Rapamycin, and stimulated with VEGF overnight. A representative Western blot of EC phospho-FAK, total FAK, phospho-Akt, total Akt and actin (n = 3 independent experiments).(TIF)Click here for additional data file.S2 FigHUVECs were treated with PP242 overnight. A representative Western blot of EC phospho-Akt, phospho-FOXO1/2, phospho-eNOS and actin (n = 3 independent experiments).(TIF)Click here for additional data file.S3 FigP<0.05 by ANOVA, scale bar = 95 um).HMEC-1 mounted on Cytodex beads were embedded in a fibrin gel as in (TIF)Click here for additional data file.S4 FigCollagen gels containing VEGF 100 ng/mL, or VEGF + PP242 at the indicated concentration, were cultured on chick CAMs as described in Methods. Newly formed vessels growing into the implant (arrows) are identified.(TIF)Click here for additional data file.S5 FigHUVECs were transfected with non-silencing (siNS) or siRNA against Akt1 (siAkt1). Representative Western blot of EC Akt1 and tubulin.(TIF)Click here for additional data file.S6 FigHUVEC were transfected with siRNA against rictor, Akt, or raptor, then seeded on gelatin-coated electrodes at high density and grown to confluence as described in Methods. The monolayer was focally disrupted by an electrical pulse. Continuous electrical impedance values are shown. Representative of 3 independent experiments.(TIF)Click here for additional data file.S7 FigHUVEC were transfected with siRNA against rictor, Akt, or raptor, or treated with PP242 as indicated. The EC were then seeded on gelatin- coated electrodes as described in Methods. Continuous electrical impedance values are shown. Representative of 3 independent experiments.(TIF)Click here for additional data file.


A simple and accurate, label-free quantitative method was used to localize and quantitate thousands of phosphorylation events. We also correlated changes in the phosphoproteome with the proteome to more accurately determine changes in the activity of regulatory kinases determined by kinase landscape profiling. The abundance of phosphopeptides with sites that function in cytoskeletal regulation, GTP/GDP exchange, protein kinase C, IGF signaling, and melanosome maturation were highly divergent after transition to a drug resistant phenotype.The treatment of melanoma by targeted inhibition of the mutated kinase BRAF with small molecules only temporarily suppresses metastatic disease. In the face of chemical inhibition tumor plasticity, both innate and adaptive, promotes survival through the biochemical and genetic reconfiguration of cellular pathways that can engage proliferative and migratory systems. To investigate this process, high-resolution mass spectrometry was used to characterize the phosphoproteome of this transition  In melanoma, coding mutations in the mitogen-activated kinase pathway (MAPK)  are common and contribute to disease severity . In cutain vivo and in vitro and this has been extensively reviewed  and motif analysis (NetworKIN) of the flanking linear amino acid sequences predicted several regulatory kinases that are most likely to be responsible for differential phosphorylation detected during long-term exposure to BRAF inhibition in LM-MEL-28. Key regulatory sites that control actin and microtubule-based cytoskeleton and cellular GTP/GDP ratio exhibited large changes in phosphorylation. Phosphorylation of the melanosome G-protein coupled receptor (GPCR) OA1 (GP143) indicated a direct role for the melanocyte maturation pathway. While sites of phosphorylation of the insulin receptor substrate IRS-2 and IGFR2 indicated novel points of regulation in the IGF-1R pathway previously identified to mediate drug resistance in melanoma.The discovery of these mechanisms and others [reviewed in Ref. ] has estsistance , 32. Bot2. Cells were treated with PLX4720  for a 1-month period in 5 μM PLX4720 to generate a drug resistant line referred to LM-MEL-28R1. Cells were tested for authenticity by short tandem repeat profiling according to the ANSI/ATCC ASN-0002-2011 standards. For phosphoproteomic analysis, six biological replicates were generated by sub-culture and cells were grown to 80–90% confluence with the LM-MEL-28-R1 continuously cultured in the presence of 5 μM PLX4720 and LM-MEL-28 in the presence of vehicle. Cells were washed three times in PBS and harvested by gentle enzyme-based release (TrypLE), pooled and centrifuged at 400 × g, cell pellets frozen on dry ice and stored at −70°C. Cells were lysed by boiling for 5 min in 1% (w/v) sodium deoxycholate (Sigma), 50 mM triethylammonium bicarbonate (TEAB) (Sigma), and 1 mM MgCl2 (Sigma). Lysates were cooled to 4°C, sonicated to complete lysis, and DNA was digested by incubation with benzonase (Sigma) . Lysate were centrifuged at 20,000 × g for 10 min and protein amounts determined by the micro-BCA assay (Pierce). Samples were stored at −80°C. Mutational testing was performed by MelCarta assay and all cell lines were tested for mycoplasma and appropriate consent from all patients had been obtained.The melanoma cell line LM-MEL-28 was selected from the Ludwig Institute for Cancer Research Melbourne Melanoma Cell Line Panel . LM-MEL-2 beads  were washed once in 0.1% (v/v) TFA, 70% (v/v) acetonitrile (ACN), and 80 mg/ml glycolic acid, added directly to the sample and incubated with shaking for 1 h. A C8 stage-tip was prepared and washed with methanol (Sigma), then 0.1% (v/v) TFA, 70% (v/v) ACN, and 80 mg/ml glycolic acid (40 μl). TiO2 beads were added to the C8 stage-tip and tips were centrifuged 1000 × g until all liquid was dispensed. Beads were washed on tip with 300 μl of 0.1% TFA, 70% ACN, 80 mg/ml glycolic acid (300 μl) twice then thrice with 0.1% (v/v) TFA and 70% (v/v) ACN. Phosphopeptides were eluted from TiO2 tip with consecutive 100 μl additions of 1% (v/v) ammonia (Sigma) with 0, 30, and 50% (v/v) ACN. Samples were immediately dried and resuspended in 1% (v/v) TFA and 5% (v/v) ACN for LC-MS/MS.Five hundred milligrams of total protein lysate were reduced with 5 mM DTT (Sigma) for 30 min at 60°C and alkylated with 10 mM of iodoacetamide (Sigma) in the dark at room temperature for 30 min. Trypsin (Promega) was added at ratio of 1:50 ratio for 18 h at 37°C. Samples were adjusted to 1% (v/v) trifluoroacetic acid (TFA) (Sigma), 80 mg/ml glycolic acid (Sigma), and the precipitated deoxycholate was removed by centrifugation. Five milligrams of TiOProteolytic digestion of 100 μg total protein was carried out as described above and samples were labeled by reductive dimethylation using formaldehyde isotopologues  with sli® 2.7 μm 160 Å ES-C18, Advanced Materials Technology) and desalted for 10 min with buffer A [0.1% (v/v) formic acid], peptide separation was carried out using a self-packed 75 μm × 10 cm  column. A buffer B  gradient (5–40% in 120 min) was used to elute peptides. Phosphopeptides were ionized by electrospray ionization and data-dependent MS/MS acquisition carried out using a Q-Exactive consisting of 1 full MS1 (R = 70 K) scan acquisition from 350 to 1500 m/z, and 10 HCD type MS2 scans (R = 15 K). Dimethylated peptides were analyzed on an Orbitrap Elite (Thermo Fisher Scientific) consisting of 1 full MS1 (R = 120 K) scan acquired from 350 to 1500 m/z, and 10 CID type MS2 scans. On both instruments, monoisotopic precursor selection, charge state screening, and dynamic exclusion were enabled, charge states of +1, >4, and unassigned charge states were not subjected to MS2 fragmentation. Raw mass spectra were identified using Maxquant 1.3 using a 1% peptide and protein FDR. Searches were conducted against the uniprot complete proteome reference database downloaded on June 06, 2014. The database was supplemented with common contaminants often found in cell culture and proteomics experiments these were later removed. Searches specified for tryptic peptides with four missed cleavages, 7 ppm precursor ion mass tolerance, 0.05 Da fragment ion mass tolerance, fixed modifications of carbamidomethylation (C), and variable modification of oxidation (M), acetylation , and phosphorylation (STY). For phosphopeptides, quantitation was performed using peptide intensity for modified (STY) P-sites and for proteins using the protein intensity ratio from the protein groups detected in the dimethylated data-set generated by Maxquant . The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium  alone or in combination with 1.25 or 2.5 μM of the CK2 inhibitor (CX-4945). After 72 h, cell viability for each cell line was assessed by Presto Blue Assay (Life Technologies).BRAF(V600E) mutant cell line (LM-MEL-28) was cultured in media containing 5 μM of the selective BRAF inhibitor PLX-4720 for 1 month to generate the stable cell line, LM-MEL-28R. The resistant line LM-MEL-28R was threefold less sensitive to the growth inhibitory effects of PLX4720 than the parental line LM-MEL-28 as shown in a viability assay  sequences, mapping to 1164 distinct protein groups and 16,713 none-phosphorylated peptide spectral matches mapping to 3556 protein groups at a FDR of 1% using Maxquant; 836 phosphoproteins (72%) had dual P-site and protein quant estimates providing added confidence in this dataset for detecting changes in phosphorylation occupancy . Using a probability function, 76% (2395 of 3162) p[S], p[T], p[Y] sites could be localized with high confidence (>75%) by MS/MS spectra  database reflects this and is reported in Table To more accurately determine change in phosphorylation after BRAF drug resistance the proteome of LM-MEL-28 and LM-MEL-28R1 cell populations was compared using isotope coded quantitative proteomics and LC-MS . Analysis of these data alone indicated widespread regulation of protein biosynthesis occurs during the development of resistance to BRAFi. Using a twofold cut-off, the majority of proteins were found down-regulated (317) and fewer (151) up-regulated. 1-D gene set enrichment analysis using the log2 Phospho − log2 Protein) specific post-translational activity (kinase or phosphatase) was inferred. Figure x/y scatter plot of the significant P-sites  where x is log2 P-site ratio (R1/S1) and y is log2 protein ratio (R1/S1). Data were well correlated indicating a large effect of protein abundance on P-site abundance   P-sites were differentially regulated by a minimum of twofold after protein abundance was subtracted, and these were selected for kinome analysis using Phosphosite.org and NetworKIN databases  were available and using the following equation (Phospho rate = log) Figure A. Forty-atabases , 42  mutated cells  mutant melanoma cell line that occur after the development of drug (PLX4720) resistance in vitro are described here. Using a single step phosphopeptide enrichment followed by LC-MS analysis and label-free quantitation using the freeware Maxquant, we accurately detected and measured ~2700 phosphorylation events and 3556 proteins. Initially, we quantitated the viability of both unexposed and drug resistant populations in the presence of BRAFi. We observed that although viability was reduced after drug adaption, stable growth was maintained and cells were able to propagate in the presence of 5 μM PLX4720. Our in vitro system provided a suitable model to measure phosphorylation in drug resistance in vitro; and through kinases landscape analysis the activity of several kinases regulating these events was predicted.Changes in the phosphoproteome of a Drug resistance in melanoma often occurs through reactivation of MAPK signaling despite continued exposure to the inhibitor . ThroughCytoskeletal changes are central to the phenotypic transitions that occur in tumor progression, altering invasiveness, metastasis, and resistance to therapy and this was reflected in our data. P-sites that are key regulatory residues in proteins controlling both actin and microtubule-based filaments were altered beyond protein metabolic control. Destrin  is responsible for actin stability  and Ser-The regulation of cytoskeletal dynamics by Rho/ROCK and GEF signaling is a key driver of the phenotypic transitions or switching that can change the migratory phenotype of cells . In melal-DOPA, and dopamine it also localizes to intracellular melanosomes and forms a key component of melanosome biogenesis and transport  mutant cell lines  increased in expression and phosphorylation in drug resistant cells. T497 in PKCα is located in the activation loop and phosphorylation is essential for full catalytic activity of PKCα . Phospholl lines . The recEvidence for potential upstream mechanisms for ERK reactivation is provided by a change in phosphorylation of insulin receptor substrate (IRS2), a downstream effector of insulin-like growth factor receptor 1 (IGF-1R). IGF-1R signaling in cancer cells results from up-regulation of the receptor or its ligands (IGF-I and IGF-II) and contributes to the emergence of chemotherapeutic resistance. Insulin receptor substrate (IRS1/2) proteins transmit oncogenic signals through PI3K and ERK signaling modules Figure . IRS1/2 This study demonstrated a simple and effective approach to detect kinase activity important in the transition of cells from a BRAF sensitive to BRAF resistant phenotype. Once detected these kinase present themselves as potential targets for futureBRAF(V600E) mutant melanoma  associated with established pathways of drug resistance in melanoma and other cancers (in vivo. The diversity of drug resistance mechanisms discovered in melanoma so far indicates a need to develop an individualized approach to multi-targeted cancer treatment. The MS-driven phosphoproteomic method described here can be readily applied to the analysis of tumors biopsied before, during, and after treatment to provide a direct readout for kinases that are drug-able targets in relapsed patients.A central paradigm of acquired drug resistance in BRAF mutant melanomas is the reactivation of MAPK signaling . In this cancers , 80. PhoThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.http://journal.frontiersin.org/article/10.3389/fonc.2015.00095The Supplementary Material for this article can be found online at Click here for additional data file.Click here for additional data file.


Background and aims. Because of compromised angulations of implants, the abutments are sometimes prepared. The purpose of this study was to investigate the effect of removing one wall of the implant abutment on the retention of cement-retained crowns.Materials and methods. Four prefabricated abutments were attached to analogues and embedded in acrylic resin blocks. The first abutment was left intact. Axial walls were partially removed from the remaining abutments to produce abutments with three walls. The screw access channel for the first and second abutments were completely filled with composite resin. For the third and fourth abutments, only partial filling was done. Wax-up models were made by CAD/CAM. Ten cast copings were fabricated for each abutment. The copings of fourth abutment had an extension into the screw access channel. Copings were cemented with Temp Bond. The castings were removed from the abutment using an Instron machine, and the peak removal force was recorded. A one-way ANOVA was used to test for a significant difference followed by the pairwise comparisons.Results. The abutments with opened screw access channel had a significantly higher retention than the two other abutments. The abutment with removed wall and no engagement into the hole by the castings exhibited the highest retention.Conclusion. Preserving the opening of screw access channel significantly increases the retention where one of the axial walls of implant abutments for cement-retained restorations is removed during preparation. Therefore, the use of cement-retained implant-supported restorations has increased.7Implication of dental implants in the rehabilitation of partially edentulous patients has become a well–recognized, accepted clinical method with predictable long-term success.9 enhancement of the esthetics in areas that would otherwise be the locations of screw access holes,9 providing a passive fit,9—which may actually improve loading characteristics,9 and a reduction of complications such as elimination of occlusal screw loosening,8 as well as fracture of porcelain, cost, and time.5 Many of the factors influencing the retention of cement-retained, implant-supported restorations can be inferred from previous studies on natural abutments. The majority of abutment preparation designs and cementation techniques now imitate conventional fixed prosthodontic procedures for natural teeth.10The advantages of cement-retained, implant supported restorations are their ability to optimize occlusal interdigitation,11 It has been recommended that the ideal tapering and the longer walls of implant abutments support the use of provisional cements. There is not enough evidence regarding the most suitable type of cement and the behavior of provisional cements over time.12Ideally, the cement on an implant-retained restoration would provide sufficient retention to prevent it from loosening and allow the restoration to be removed without damaging the abutment, the restoration or the peri-implant tissues.2 Tan et al2demonstrated that the number and the position of the axial walls of implant abutments can be designed or amended to improve the retentive strength of cemented crowns which is unlike natural abutments. Emms et al10 also state that the method employed to fill the screw access channel of implant abutments can have an influence on the retention of coronal restorations cemented with Temp Bond. Naik et al7 confirmed that engagement of a casting cemented with Temp Bond into the screw access channel of an implant abutment considerably increases the retention. Therefore, a study deemed necessary to evaluate if there were any relationships between axial wall modification or coping design and the retention of the cement-retained implant-supported crowns.The presence of a screw access channel and having varying number of axial walls are the main geometrical difference between an implant abutment and a natural tooth preparation. Despite the stated differences, there are only a limited number of publications evaluating the effect of modified screw access channel on the retention of cemented restorations.The purpose of the present study was to investigate the effect of three different methods on the retention of cement-retained crowns when one wall of the implant abutment is removed. The null hypothesis was: there would be no significant difference in the retention of cemented crowns whether the implant abutments are intact or have lost one wall with engaging the screw access channel or without it.Four prefabricated straight abutments  were attached onto four corresponding implant analogues. Each abutment was tightened with digital pressure with a hex driver. The interface diameter for all implant abutments was 4.1 mm. The height of the selected abutments was 5.5 mm. The abutment-analogue complexes were vertically mounted in individual acrylic resin blocks  with a dental surveyor . The acrylic resin was left 1 mm lower than the implant-abutment joint.The first abutment was left intact without any modification as control group. The three other abutments were prepared by using a tapered carbide bur to remove 4 mm of the height of the flat walls of them.For the first abutment, a cotton pellet was placed on top of the abutment screw and the screw access channel was completely filled with composite resin  and CAD/CAM machine  to make the wax copings  were used for the cementation of the castings on the implant abutments and mixed for 30 seconds in proportions according to the manufacturer’s instructions. The mixed cement was placed in the castings and the castings were seated onto the abutments with finger pressure, and placed under a 5-kg pressure for 5 minutes. Excess cement was removed with a plastic scaler. The assemblies were then stored in 100% humidity at 37ºC for 24 hours.The castings were attached to a universal testing machine  by clamping them directly onto the loop attachment. The machine was used to apply vertical tensile forces at a crosshead speed of 5 mm per minute, to dislodge the castings from the abutments. The peak load to dislodgement was documented (N) and used to indicate the retentive values. Before testing each new casting, abutments were cleaned of remaining cements. To compare the groups, one-way analysis of variance (one-way ANOVA) and pairwise comparisons test (LSD) were used.The results of one-way ANOVA revealed a significant difference between the groups (P = 0.019). To achieve the homogeneity of variances in one-way ANOVA, the data of peak loads (retention) was changed to natural logarithm Ln; .Pairwise comparisons test (LSD) revealed that the abutments of the third and fourth groups in which one wall was removed and the screw access channel openings were preserved, had a significantly higher mean peak load to dislodgement than two other groups that their screw access channels were filled with composite .There was no significant difference between the first and second groups and between the third and fourth groups. The abutment of the third group in which one wall of the abutment was prepared and there was no extension of the castings in to the screw access channel exhibited the highest retention. The lowest mean retention value was seen in the second group that after removing one wall of the abutment, the screw access channel was restored to the original contour with composite .13 Another cause would be the surface roughness of the internal walls, as according to Tan et al,2 unlike the external axial walls, the internal walls are not coated with the smooth titanium nitride. This may have contributed to the observed results because the roughness of the surface preparation can have a pronounced effect on the retention.15The results of this study support rejection of the null hypothesis that there would be no significant difference in the retention of cemented crowns whether the implant abutments are intact or have lost one wall with engaging the screw access channel or without it. Removing one wall of the abutment and leaving the screw access channel opened, either with engaging or without engaging the screw access channel, had significant influences on the retention. The higher retention of these abutments may be contributed to increasing the surface area due to the penetration of the cement into the screw access channel. The presence of internal axial walls compensated for the decrease in retentive surface area as a result of removing one wall. These findings are in line with the general consensus that retention is positively correlated with the surface area of the abutment—whether a natural tooth or an implant.2 This venting effect may explain the higher retention of the abutments with opened screw access channel and no engaging of the channel in comparison to abutments with opened screw access channel and engaged channel. Contrary to this finding, however, Naik et al7 concluded that engaging the screw access channel of an implant abutment can significantly improve retention.Another possible explanation for the improved retention seen in the abutments with one removed wall compared to abutments with 4 walls was the presence of an open screw access channel that may act as an internal vent, allowing for more complete seating of the castings, as explained previously in the literature.10 that complete filling of the screw access channel before cementation could reduce the removal force of a coronal restoration cemented with Temp Bond. The results were very similar between the intact abutment group and the group in which the screw access channel was filled with composite resin. Clinically, this finding means that if one wall of the abutment is removed, filling the hole with composite to the original contour produces the same retention as the intact abutment.This study also confirmed the explanation of Emms et alThe limitations of this study should be noted, since it only investigated retention and not resistance. Clinically, the removal of prosthodontic castings might not apply forces along a single withdrawal path.in vitro study, when one of the axial walls of implant abutments for cement-retained restorations is removed during preparation, preserving the opening of screw access channel significantly increases retention. This finding is consistent regardless of engaging the opening with casting into the abutment screw access channel or not.Within the limitations of this


N-phenylpyrazine-2-carboxamide), has been reported to have a significant radio- and chemo-sensitizing effect delimited to cancer cells (largely p53-deficient) without affecting normal cells. In this study, we employed SILAC-based quantitative phosphoproteomics to describe the mechanism of the radiosensitizing effect of VE-821 in human promyelocytic leukemic cells HL-60 (p53-negative). Hydrophilic interaction liquid chromatography (HILIC)-prefractionation with TiO2-enrichment and nano-liquid chromatography—tandem mass spectrometry (LC-MS/MS) analysis revealed 9834 phosphorylation sites. Proteins with differentially up-/down-regulated phosphorylation were mostly localized in the nucleus and were involved in cellular processes such as DDR, all phases of the cell cycle, and cell division. Moreover, sequence motif analysis revealed significant changes in the activities of kinases involved in these processes. Taken together, our data indicates that ATR kinase has multiple roles in response to DNA damage throughout the cell cycle and that its inhibitor VE-821 is a potent radiosensitizing agent for p53-negative HL-60 cells.DNA damaging agents such as ionizing radiation or chemotherapy are frequently used in oncology. DNA damage response (DDR)—triggered by radiation-induced double strand breaks—is orchestrated mainly by three Phosphatidylinositol 3-kinase-related kinases (PIKKs): Ataxia teleangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK) and ATM and Rad3-related kinase (ATR). Their activation promotes cell-cycle arrest and facilitates DNA damage repair, resulting in radioresistance. Recently developed specific ATR inhibitor, VE-821 (3-amino-6-(4-(methylsulfonyl)phenyl)- Intensity threshold for triggering MS/MS was set at 1 × 105 for ions with z ≥ 2 with a 3 Da isolation window. Precursor ions were accumulated with AGC of 1 × 105 (maximum fill time was 100 ms) and normalized collisional energy for HCD fragmentation was 27 units. MS/MS spectra were acquired with 17,500 resolution (at 200 m/z).On-line LC-MS analyses were performed on Thermo Scientific Dionex Ultimate™ 3000 RSLCnano system  coupled through Nanospray Flex ion source with Q Exactive mass spectrometer . TiO2 peaks, respectively. Only proteins, peptides and phosphorylation sites with FDR lower than 0.01 were accepted. For protein quantification only unmodified peptides, peptides oxidized at methionine residues, acetylated at N-terminus, or deamidated were accepted, both razor and unique peptides were used for the calculation of protein H/L ratios.The raw files were processed with MaxQuant software version 1.3.0.5 . Peak liet al. [Contaminants and reversed hits were removed before further data processing and data were further manually inspected. The GRT was used to find differentially regulated phosphorylation sites. Phosphorylation sites quantified in all three replicates were included in GRT and the FDR was estimated non-parametrically as described by Zhou et al. . The sigGO and signaling pathways over-representation analyses were performed using ConsensusPathDB, over-representation analysis web tool ,15. Only- or down-regulated phosphorylation sites (determined using GRT) were tested against a background reference set composed of sequences surrounding all phosphorylation sites revealed in our study that reached the minimal localization probability 0.75 .To analyze and visualize sequence motifs surrounding phosphorylation sites identified and quantified in our study, we employed the iceLogo tool and motif-x algorithm. In both motif analyses, the amino acid sequences (±7 residues) surrounding either significantly uphosites” ). In the-specific kinases together with the evaluation of significance based on the Wilcoxon-Mann-Whitney test . The significance threshold for the Wilcoxon-Mann-Whitney test was set to p < 0.05.The changes in kinase activities were evaluated using three different tools for the predictions of phosphorylation site1.4.0.20 ). Input database ) we empldatabase  and iGPSdatabase . Kinase-Defects in the DSB-induced DDR such as ATM and/or p53 deletion/mutation are common in human tumors, occur in up to 70% of cancer cells ,48 and h


However, there is considerable evidence for their specific association with the IC, including its recently identified pericentrosomal domain (pcIC), where many of the effectors turn out to be present, thus being able to exert their functions at the pre-Golgi level. The IC localization of these proteins is of particular interest based on the imaging of Rab1 dynamics, indicating that the IC is a stable organelle that bidirectionally communicates with the ER and Golgi, and is functionally linked to the endosomal system via the pcIC.Two conserved Rab GTPases, Rab1 and Rab2, play important roles in biosynthetic-secretory trafficking between the endoplasmic reticulum (ER) and the Golgi apparatus in mammalian cells. Both are expressed as two isoforms that regulate anterograde transport via the intermediate compartment (IC) to the Golgi, but are also required for transport in the retrograde direction. Moreover, Rab1 has been implicated in the formation of autophagosomes. Rab1 and Rab2 have numerous effectors or partners that function in membrane tethering, but also have other roles. These include the coiled-coil proteins p115, GM130, giantin, golgin-84, and GMAP-210, as well as the multisubunit COG (conserved oligomeric Golgi) and TRAPP (transport protein particle) tethering complexes. TRAPP also acts as the GTP exchange factor (GEF) in the activation of Rab1. According to the traditional view of the IC elements as motile, transient structures, the functions of the Rabs could take place at the two ends of the ER-Golgi itinerary, i.e., at ER exit sites (ERES) and/or  Saccharomyces cerevisiae, in which many of the molecular players have been characterized, differs considerably from that of mammalian cells, as it consists of tubular networks, but lacks the characteristic Golgi stacks. However, whether yeast cells—or fungi and plants in general—are equipped with a distinct organelle, comparable to the intermediate compartment (IC) that operates in bidirectional trafficking between the ER exit sites (ERES) and the Golgi stacks in mammalian cells, remains an open question  and the Golgi apparatus in mammalian cells   Figure . Namely,In conclusion, due to its division into vacuolar (saccular) and tubular subdomains, spatial complexity and connection with the centrosome, the IC shares striking similarity with the endosomal system  and active, GTP-bound (membrane-associated) states they are thought to function as master regulators of membrane trafficking and to ensure the directionality of transport  that share a high degree of sequence similarity . They were first shown to function in anterograde ER-Golgi transport and suggested to operate in a sequential manner -dependent, bidirectional movements throughout the cytoplasm  are capable of tethering membranes prior to their eventual fusion Munro, . As the The first identified Rab1 effector was p115, a myosin-like coiled-coil protein that interacts with several other transport factors and COPI coats and appears to play multiple roles in bidirectional ER-Golgi trafficking and Golgi biogenesis , multiple tethering factors could operate in a sequential—or possibly redundant—fashion at the same transport step. It has been suggested that an additional role of the tethers is to couple antero- and retrograde trafficking , anterograde transport from ERES to cis-Golgi is expected to involve two transport steps. Accordingly, the IC elements arriving from the peripheral ERES first fuse with the pcIC, which most likely represents a dynamic membrane system normally located close to the Golgi ribbon. Consequently, a second transport step would be required for trafficking between the pcIC and the Golgi stacks. This new model is supported by the observed localization the Rab1 isoforms and COPI coats to the pcIC . The localization of GM130 and GMAP210 to the pcIC . This new scenario raises the possibility that the COPI vesicles in the vicinity of the Golgi apparatus—defined by the tethers golgin-84 and p115 .The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The role of JNK inhibitors in cardioprotection against IR still remains controversial, in part, due to spill-over effects of non-specific inhibitors. In the present study, we sought to examine whether inhibition of JNK by SU3327, a specific JNK inhibitor that inhibits upstream JNK signaling rather than the kinase activity of JNK, improves cardiac function and reduces heart damage during IR. Hearts of male Sprague-Dawley rats perfused by Langendorff were subjected to 25 min of global ischemia followed by 30 min reperfusion in the presence or absence of SU3327. Cardiac function was monitored throughout the perfusion period. Myocardial damage was extrapolated from LDH activity in the coronary effluent. At the end of reperfusion, mitochondria were isolated and used to measure respiration rates and mitochondrial permeability transition pore opening. Protein analysis of mitochondria predictably revealed that SU3327 inhibited JNK phosphorylation. Although SU3327 significantly reduced cell damage during the first minutes of reperfusion, it did not improve cardiac function and, furthermore, reduced the mitochondrial respiratory control index. Interestingly, SU3327 activated the other stress-related MAPK, p38, and greatly increased its translocation to mitochondria. Mitochondrial P-JNK and P-p38 were co-immunoprecipitated with complex III of the electron transfer chain. Thus, JNK plays an essential role in cardiac signaling under both physiological and pathological conditions. Its inhibition by SU3327 during IR aggravates cardiac function. The detrimental effects of JNK inhibition are associated with reciprocal p38 activation and mitochondrial dysfunction. Heart diseases due to myocardial ischemia, including myocardial infarction and heart failure, are the major causes of death in developed countries, and their prevalence continues to grow in vivo and ex-vivo models of IR JNK, a member of the mitogen-activated protein kinase (MAPK) family, has been implicated in reactive oxygen species (ROS)- and other stress-induced apoptosis 2O2- Translocation of JNK to mitochondria was observed in response to DNA damage In this study, we investigated whether inhibition of JNK offers cardioprotection against IR using a Langendorff-mode perfusion of the isolated rat heart. We employed SU3327, which, in contrast to other JNK inhibitors, such as SP600125, inhibits upstream JNK activation rather than the kinase activity of JNK. We found that SU3327 aggravated the recovery of isolated hearts from IR. Moreover, the inhibitor elicited different effects depending on the presence or absence of stress and the timing of administration. Our findings imply the existence of crosstalk between the JNK and p38 pathways in response to oxidative stress, in which downregulation of JNK stimulates p38, which, in turn, aggravates cardiac function. Furthermore, inhibition of JNK during IR enhances interaction of p38 with complex III of the electron transport chain (ETC), which itself can cause cardiac dysfunction.Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health .Male Sprague-Dawley rats weighing 225–275 g were purchased from Charles River . All experiments were performed according to protocols approved by the University Animal Care and Use Committee of the UPR Medical Sciences Campus  and conformed to the AVMA Guidelines for the Euthanasia of Animals: 2013 Edition. The rationale for the use of decapitation of conscious rats was to avoid side effects of anesthesia on cardiovascular system, particularly cardiac function, which was an important end-target of the present study. The hearts were rapidly removed, immersed in Krebs solution, and retrogradely perfused via a non-recirculating Langendorff perfusion system at constant flow On the day of the experiment, the rats were euthanized with a guillotine in accordance to the To isolate mitochondria, the ventricles were cut, weighed and homogenized with a Polytron homogenizer at 1500 rpm for 5 sec in the ice-cold sucrose buffer containing 300 mM sucrose, 10 mM Tris-HCl, and 2 mM EGTA, at pH 7.4. Mitochondria were isolated from the homogenate by centrifugation at 2000×g for 2 min in a benchtop centrifuge to remove cell debris, followed by centrifugation of the supernatant at 10,000×g for 5 min to sediment the mitochondrial suspension. The pellet was then washed two times at 10,000×g for 5 min in 40 mL of BSA-free sucrose buffer. The final pellet was resuspended in 300 µL of sucrose buffer. The yield of mitochondria was 12.1±0.31 mg/mL. A 200-µL sample of the suspension was then used for measurement of mitochondrial respiration rates and MPTP opening. A 100-µL sample was mixed with protease inhibitor cocktail  and phosphatase inhibitor cocktail , then immediately frozen in liquid nitrogen and stored at −80°C for western blot analysis.2PO4, at pH 7.2, supplemented with either of the following substrates to measure complex I- and complex II-mediated respiration rates, respectively: (i) 2.5 mM 2-oxoglutarate and 1 mM L-malate or (ii) 2.5 mM succinate and 1 µM rotenone. Respiration rates were measured in the absence (state 2) and presence (state 3) of 1 mM ADP. At the end of each run, 0.5 µM antimycin A followed by 10 mM ascorbate and 0.3 mM N,N,N′,N′-tetramethyl-p-phenylendiamine (TMPD) were added, and the new rate of respiration was measured. The respiratory control index (RCI) was calculated as ratios of respiration in the presence of ADP (state 3) to the absence of ADP (state 2).Measurement of mitochondrial respiration was performed at 37°C using a YSI Oxygraph  model 5300 equipped with a Clark-type oxygen electrode 2+ was determined by monitoring the decrease in light scattering at 545 nm as described previously 2 was added to 100 µM and read for another 2 min. Rates of swelling were calculated as the ratio of decrement of absorbance with CaCl2 to decrement of absorbance without CaCl2.Swelling of de-energized mitochondria as an indicator of MPTP opening in the presence or absence of CaImmunoblotting was performed as described previously Protein carbonyls were analyzed according to the method described previously Co-immunoprecipitations of P-JNK and P-p38 with the specific antibody to each protein were conducted by following the recommended protocol of Dynabeads . A separate aliquot of mitochondrial proteins (0.3 mg each) was incubated with 2 µg of antibody-conjugated beads overnight. The immunoprecipitated proteins were dissolved in Laemmli buffer for immunoblot analysis using the specific antibody against adenine nucleotide translocase (ANT), voltage-dependent anion channel (VDAC), cyclophilin D (CyP-D), and mitochondrial ETC complexes.2 at 37°C. Prior to all experiments, cells were serum-starved for 24 hrs. Cells with 85∼90% confluence from passages 3∼20 were used for experiments.H9c2 embryonic rat cardiomyocytes were purchased from the American Type Culture Collection  and cultured according to the manufacturer's recommendations. In short, the cells were cultured in modified DMEM/Ham's F-12  media supplemented with 10% fetal bovine serum, and maintained in 95% air and 5% CO5 cells on 100-mm dishes and exposed to 75 µM H2O2 for 30 min, with or without inhibitors. After treatment, cells were rinsed with PBS, detached with Hyclone Trypsin  and counted using the TC20 Automated Cell Counter . The per cent of viable and dead cells was calculated from a total number of counted cells.Cell viability was determined using the trypan blue exclusion method. The cells were cultured at a density of 5×10Cells were trypsinized with a 0.25% trypsin–EDTA solution  and centrifuged at 500×g for 5 min. at room temperature. The resulting pellets were resuspended in culture medium, and cells were incubated with 20 µM 2′7 dichlorofluorescein diacetate  for 30 min at 37°C. The cells were again centrifuged at 500×g for 5 min. to remove medium with excess dye, and the pellets were resuspended in PBS and added to a 96-well plate. Fluorescence intensity was measured using a Spectramax M3 plate reader  at an excitation of 485 nm and emission of 530 nm.5 cells/well) were incubated for 30 min. with the membrane potential-sensitive dye 5,5′,6,6′-tetraethyl-benzimidazolylcarbocyanine iodide . Afterwards, the intensity of fluorescence was immediately measured using a Spectramax M3 microplate reader (Molecular Devices) at 527 and 590 nm for emission, and 488 nm for excitation.Cells plated in a 24-well culture plate  at reperfusion when compared to pre-ischemia . In the IRS group, LVEDP was elevated by 30 mmHg   .P<0.05, IR vs IRSR; vs IRSP).Hearts subjected to IR showed increased LDH activity in the coronary perfusate, indicating increased cell death. Interestingly, administration of SU3327 during reperfusion only decreased LDH activity by 40%  carbonylation of mitochondrial proteins compared to the IR group. There was no difference between IR, IRSP and IRSR groups.Elevated levels of carbonylated proteins are a marker of increased oxidative stress. The levels of oxidized proteins in the mitochondria of the IR group were increased by 1.2 fold compared to control P<0.05, . The IRSP<0.05, C vs IR) and p38 by 2.5 fold . SU3327 added during reperfusion only induced a 37%  reduction of JNK phosphorylation. Administration of the inhibitor before ischemia and during reperfusion did not affect activation of JNK (IR vs IRS). Furthermore, SU3327 was less able to inhibit JNK when it was added during pre-ischemia only (IR vs IRSP).To determine the effect of SU3327 on MAPKs, we examined the phosphorylation of JNK, as well as p38 and ERK1/2 in IR-hearts treated with SU3327. IR induced activation of JNK and p38, and slightly inhibited ERK. As shown in , IR actiP<0.05, C vs IR). All three JNK inhibitor-treated groups demonstrated more activated p38 than the IR group  (vs IRS), while ERK1/2 activation was only partially restored in other SU3327 treatment groups (IR vs IRSP and IRSR).IR increased p38 activation by 2.5 fold  . On the P<0.05) and reduced ERK1/2 activity by 0.7 fold (P<0.05), but did not affect p38 activity. Although IR reduced phosphorylation of mitochondrial P-ERK1/2, SU3327 increased P-ERK1/2 levels. Treatment with the inhibitor before ischemia and during reperfusion did not reduce total mitochondrial JNK compared to IR (IR vs IRS). We also found that IR increased mitochondrial total JNK and p38 . Treatment with SU3327 further increased the interaction, except in the IRSR group, which showed no significant difference. The IRS and IRSP groups showed a 2.0- and a 2.7-fold increase (P<0.05 for both) in JNK-UQCRC2 interactions, respectively, compared to the IR group. UQCRC2 was also co-immunoprecipitated with p38, although the differences between groups were not statistically significant  aggravated the recovery of heart function and respiration of mitochondria during IR, ii) inhibited JNK and activated cytosolic and mitochondrial p38, and iii) stimulated interaction of JNK and p38 with complex III of ETC.JNK, a stress-related MAPK, is involved in apoptotic cell death, in addition to inflammatory responses and cell proliferation, and thus has therapeutic potential in the treatment of inflammatory diseases and cancer. Since the most widely used pharmacological JNK inhibitor SP600125 became available in vivo IR in vitro studies of cultured cardiomyocytes showed that p38 inhibition could alleviate the aggravation caused by JNK inhibition, implying that p38 activation may be the main cause of the aggravation. It is notable that recovery of the mitochondrial membrane potential was not observed along with alleviation, although total ROS was significantly reduced. Activation of p38 was also observed in mitochondria isolated from IR hearts treated with SU3327. Surprisingly, hearts perfused with SU3327 during pre-ischemia only (IRSP group) exhibited activation rather than inhibition of JNK. Because SU3327 only targets JIP1-JNK interaction, pathways other than JIP1 may activate JNK. We also observed the highest activation of p38 in the IRSP group. It is tempting to speculate that inhibition of JNK by SU3327 prior to ischemia stimulates a complementary response of both stress-activated protein kinases which remain activated throughout the entire IR period.Previous studies showed that inhibition of JNK can confer both adverse In addition, our studies confirm an essential role of JNK in physiological cell signaling; JNK inhibition aggravated the function of isolated hearts treated with IR. JNK isoforms could respond differently to oxidative stress through stimulation survival or death pathways due to the distinct role they play in cell signaling. JNK has 10 isomers expressed from three different genes, and at least three JIPs have been reported. However, pharmacological inhibition of individual isoforms by inhibitors is remarkably difficult 0F1 ATPase, complex V of ETC, may serve as an essential structural component of the pore The role of mitochondrial JNK in the regulation of mitochondrial function and metabolism is still unclear. JNK is known to interact with Sab, a mitochondrial scaffold of JNK, and the interaction can lead to mitochondrial dysfunction In conclusion, the present study demonstrates that specific inhibition of JNK by SU3327 does not protect the heart from IR injury. Moreover, SU3327 actually aggravates cardiac and mitochondrial dysfunction in hearts exposed to IR, further highlighting the important roles of JNK in cell signaling. SU3327 both stimulates activation of p38 in the cytoplasm and mitochondria and promotes its interaction with the complex III of ETC, suggesting that a reciprocal relationship exists between stress-related MAPKs in response to oxidative stress.


In response to environmental signals, kinases phosphorylate numerous proteins, including RNA-binding proteins such as the AU-rich element (ARE) binding proteins, and the GU-rich element (GRE) binding proteins. Posttranslational modifications of these proteins lead to a significant changes in the abundance of target mRNAs, and affect gene expression during cellular activation, proliferation, and stress responses. In this review, we summarize the effect of phosphorylation on the function of ARE-binding proteins ZFP36 and ELAVL1 and the GRE-binding protein CELF1. The networks of target mRNAs that these proteins bind and regulate include transcripts encoding kinases and kinase signaling pathways (KSP) components. Thus, kinase signaling pathways are involved in feedback regulation, whereby kinases regulate RNA-binding proteins that subsequently regulate mRNA stability of ARE- or GRE-containing transcripts that encode components of KSP. Protein kinases are central components of intracellular networks that regulate a vast number of cellular processes . Kinase-In addition to the role of transcriptional regulatory mechanisms in the regulation of kinase gene expression, kinases and other kinase signaling pathway (KSP) components are regulated at the posttranscriptional level through mRNA degradation or stabilization. The stability of KSP mRNAs can be regulated in the cytoplasm where kinase expression is often induced by different stimuli through transcript stabilization and increased translation. KSP mRNAs can also be rapidly degraded by RNA-binding proteins (RNA-BPs) that bind mRNAs through specific motifs, usually located in the 3’ untranslated regions (3’UTRs) ,7. AU-riThe level of an mRNA within an individual cell depends on both its rate of synthesis and the rate of decay . The recIn this article, we review our understanding of the mechanisms by which kinases control the function of RNA-BPs that bind to AREs or GREs, highlighting studies regarding ZFP36, ELAVL1 and CELF1. These proteins influence many aspects of mRNA metabolism, including regulation of the degradation of networks of target transcripts that encode multiple KSP components. Not only are these RNA-BPs regulated thorough phosphorylation by kinases, these RNA-BPs function as feedback regulators of kinase expression by controlling stability of transcripts encoding KSP components. We describe how phosphorylation affects binding by ZFP36, ELAVL1 and CELF1 to target transcripts and thereby changes the expression of networks of target transcripts that encode components of KSPs that control important cellular processes including immune activation, proliferation, and apoptosis. It was noted over a quarter of a century ago that mRNAs exhibit substantial variations in turnover rates upon exposure to different cell stimuli . More reARE or GRE-containing transcripts in clusters I and II contain 4 or more overlapping AUUUA or GUUUG pentamers and are each represented by only a few hundred transcripts. Most of the transcripts in these clusters are cytokines, transcription factors and early response genes. Clusters III through V contain shorter sequences with less sequence repetition, and these clusters contain up to several thousand members. Several functional classes of mRNAs contain clusters III-V, including transcripts encoding proteins involved in cell signaling, cell to cell communication, cell cycle and apoptosis. With gradually increasing overall U-richness in these clusters, the destabilizing potency decreases . NeverthARE-binding proteins regulate the expression of a large network of genes that control a variety of cellular processes including immune activation, cytokine expression, and stress responses . Over thCELF1 is an RNA-BP that regulates a network of transcripts that control important cellular functions such as cell growth and apoptosis by binding to GREs in the 3’UTR of target transcripts . Along wCELF1 is a known phosphoprotein with multiple predicted phosphorylation sites A. CELF1 CELF1 phosphorylation seems to be important in regulating immune responses. T cell activation through the T cell receptor and CD28 co-receptor leads to phosphorylation of CELF1, followed by inhibition of mRNA binding and stabilization of numerous GRE-containing target transcripts. In this case, the site of phosphorylation is not known, but this type of T cell activation is driven by PKCθ and other kinases, including MAP kinases and PI3 kinases (reviewed in Reference ). PossibThrough microarray-based mRNA decay profiling and identification of CELF1 target transcripts, we identified a number of GRE-containing transcripts that were CELF1 targets in normal but not malignant T cells even though they were expressed in both types of cells. We found CELF1 phosphorylation at serine 28 in malignant T cell lines, but not in resting or activated normal T cells, suggesting that a kinase capable of phosphorylating CELF1 at this site is active in malignant but not in normal T cells. In addition to phosphorylation at S28 in malignant T cells, we identified hyperphosphorylation of CELF1 at multiple unmapped sites. Overall, phosphorylation of CELF1 at S28 or other sites may lead to the altered CELF1 function that we observed in malignant T cells . Hyperphosphorylation of CELF1 has been reported as a major contributor to pathogenesis in myotonic dystrophies, such as myotonic dystrophy type 1 (DM1) . In DM1,Overall, a sizable body of evidence shows that phosphorylation affects many functions of CELF1 in posttranscriptional gene regulation. The effects of different kinase signaling may even cause a switch in cytoplasmic CELF1 functions , or causes a switch in CELF1 localization from cytoplasmic to nuclear, since different functions of CELF1 are carried out in different compartments . PhosphoELAVL1 is another member of the CELF family of RNA-binding proteins whose function has been extensively studied in cellular models. ELAVL1 can regulate alternative splicing, processing, stabilization or mRNA translation (reviewed in Reference ). ELAVL1.In stressed cells, phosphorylation of ELAVL1 appears to represent a protective mechanism to help stressed cells survive, making this protein, and upstream kinases, attractive pharmacological targets ,84. ELAVELAVL1 phosphorylation also changes its ability to shuttle between the nucleus and cytoplasm. During the normal cell cycle, ELAVL1 is phosphorylated by CDK1 or CDK5 at S202 ,92,93. PAnother example where ELAVL1 phosphorylation regulates its cytoplasmic localization and increased ARE-binding activity is the phosphorylation by PKC isozymes ,95,96. PNumerous transcripts encoding kinase signaling pathways, including kinases and phosphatases, are targets of RNA-BPs, suggesting that feedback regulation of KSP components occurs through regulated mRNA stability. It is becoming clear that KSP components are regulated at posttranscriptional levels by RNA-BPs that bind to AREs or GREs. For example, we have observed this type of feedback inhibition following activation of primary human T cells . NumerouThe increased expression of these transcripts appears to be due to increased transcription as well transcript stabilization as a result of inactivation of destabilizing RNA-BPs such as ZFP36 and CELF1 through transient phosphorylation ,61. SubsIn this example, phosphorylation of key RNA-BPs, such as ZFP36 and CELF1, appears to coordinate the transient increased expression of multiple KSP components following T cell activation to allow T cell activation signals to be successfully transmitted. Thus, RNA-BP phosphorylation increases the expression of a network (or regulon) of transcripts encoding KSP components to further promote T cell activation. Later in the cellular activation program, ZFP36 and CELF1 become dephosphorylated, allowing the network transcripts encoding KSP components to undergo rapid mRNA decay. The downstream result would be to down-regulate components of KSP signaling to turn off cellular activation and bring the cell back to a state of quiescence.versus decay. We propose a model whereby phosphorylation of ZFP36 or CELF1 following activation of kinase signaling pathways, shifts the balance toward ELAVL1 binding to target transcripts, promoting transient stabilization of ARE- or GRE-containing mRNAs, including transcripts encoding KSP components. We compared GRE-containing transcripts that were CELF1 targets  with ELAThis review highlighted the role of kinase signaling pathways in the regulation of phosphorylation and function of RNA-binding proteins such as ZFP36, CELF1 and ELAVL1, that in turn function as posttranscriptional regulators of ARE- and GRE-containing mRNAs which encode components of KSPs. Such feedback inhibition mechanism is important for many cellular processes e.g. cell activation, limited proliferation and stress responses. A major priority for future research should be to design integrative studies to further elucidate the mechanisms by which AREs, GREs, RNA-BPs and also small regulatory RNAs coordinate signaling pathways involved in health and disease. For example, system level approaches should be applied to look at the interplay between differentially phosphorylated RNA-BPs and target transcripts to better understand functional outcomes of specific phosphorylation events. Immunoprecipitation of multiple RNA-BPs and identification of co-purified transcripts in single cell using high throughput sequencing technology would allow computational approaches to characterize a composite of regulatory regions within mRNAs and to provide information on how combinations of RNA-BPs function together. Proteomics studies will make possible identification of subcellular RNA-protein complexes, their interactions and trafficking. Animal models, to evaluate gain- or loss-of-function mutations on the functions of RNA-BPs, should be expanded to also assess the effect of phosphomimic or nonphosphorylatable mutations in RNA-BPs. These and similar genetic manipulations in mouse models should shed light on the functional relevance of feedback regulation of kinase signaling pathways by AREs and GREs. Finally, studies should be pursued to understand RNA-BP phosphorylation and downstream posttranscriptional networks in disease states, such as autoimmunity, immunodeficiency, and cancer.


A receptors are important for inhibition in the CNS where neurosteroids and protein kinases are potent endogenous modulators. Acting individually, these can either enhance or depress receptor function, dependent upon the type of neurosteroid or kinase and the receptor subunit combination. However, in vivo, these modulators probably act in concert to fine-tune GABAA receptor activity and thus inhibition, although how this is achieved remains unclear. Therefore, we investigated the relationship between these modulators at synaptic-type α1β3γ2L and extrasynaptic-type α4β3δ GABAA receptors using electrophysiology.GABAS408A,S409Aγ2L receptors, and unaltered by PKC inhibitors or activators, indicating that phosphorylation of β3 subunits is important for regulating neurosteroid activity. To determine whether extrasynaptic-type GABAA receptors were similarly modulated, α4β3δ and α4β3S408A,S409Aδ receptors were investigated. Neurosteroid potentiation was reduced at both receptors by the kinase inhibitor staurosporine. By contrast, neurosteroid-mediated potentiation at α4S443Aβ3S408A,S409Aδ receptors was unaffected by protein kinase inhibition, strongly suggesting that phosphorylation of α4 and β3 subunits is required for regulating neurosteroid activity at extrasynaptic receptors. Western blot analyses revealed that neurosteroids increased phosphorylation of β3S408,S409 implying that a reciprocal pathway exists for neurosteroids to modulate phosphorylation of GABAA receptors.For α1β3γ2L, potentiation of GABA responses by tetrahydro-deoxycorticosterone was reduced after inhibiting protein kinase C, and enhanced following its activation, suggesting this kinase regulates neurosteroid modulation. In comparison, neurosteroid potentiation was reduced at α1β3A receptors in vivo, and into the mechanisms by which GABAergic inhibitory transmission may be simultaneously tuned by two endogenous neuromodulators.Overall, these findings provide important insight into the regulation of GABAThis article is part of the Special Issue entitled ‘GABAergic Signaling in Health and Disease’. •A receptors is modulated by protein kinase activity.Neurosteroid potentiation at GABA•Synaptic-type α1β3γ2L receptor potentiation is up or down-regulated by PKC activity.•PKC-mediated modulation occurs via β3 subunit S408 & S409 phosphorylation.•S443.Potentiation at extrasynaptic-type α4β3δ receptors is additionally regulated by α4•A receptors via βS408,S409.Neurosteroids facilitate phosphorylation of GABA These r C (PKC)  and hypo C (PKC) . Howevernal cord  and the nal cord , enhanceto THDOC .A receptor. Although the neurosteroids appear to display only modest changes in potency across most GABAA receptor subtypes  to a total of 4 μg): 20 μl CaCl2 (340 mM) plus 24 μl of 2× HBS  per 22 mm coverslip. Cells were used for electrophysiology 24–48 h later. For biochemistry (60 mm culture dishes) cells were transfected with a total of 9 μg of the appropriate cDNA mix.HEK293 cells were cultured in Dulbecco's modified Eagle medium  supplemented with 10% v/v foetal calf serum, 2 mM glutamine, 100 U/ml penicillin G, 100 μg/ml streptomycin and incubated at 37 °C in 95% air/5% CO2.32, 2.52 mM Glucose, 11 mM HEPES and 5 mM CaCl2 (pH 7.4). Whole-cell recordings were performed using patch pipettes (4–5 MΩ) filled with an internal solution  in conjunction with an Axopatch 200B amplifier (Axon Instruments). Cells were voltage clamped at −10 mV and currents filtered at 3 kHz (8 pole Bessel filter), digitized  and viewed and analysed using Clampex and Clampfit ver 9.2, respectively (Molecular Devices). Cells were continually monitored for access resistance and discarded if this changed by >20%. Drugs were rapidly-applied using a modified U-tube system with 10–90 % response time of 100–150 msec. THDOC was prepared as a 10 mM stock solution in DMSO, and diluted to the appropriate final concentration in Krebs. The effect of the DMSO vehicle alone was negligible. The EC20 GABA concentration was pre-determined by constructing GABA concentration–response curves and new curves were generated regularly to adjust for any drift in EC20. The EC20 GABA currents were recorded at 2 min intervals. The neurosteroid-mediated potentiation was measured by co-application of EC20 GABA with 50 nM THDOC, followed by a recovery EC20 GABA response. Recording ceased if the responses did not return to baseline GABA-activated levels. Subsequently, cells were perfused with a protein kinase inhibitor or activator diluted in Krebs solution, and applied continuously via the bath. The exceptions to this were the protein kinase G (PKG) inhibitor (KT5823) and protein kinase A (PKA) activator (cAMP), which were applied via the patch electrode. Stocks of staurosporine (1 mM), BIM-I (1 mM), KT5823 (3 mM) and phorbol-12-myristate-13-acetate  were prepared in DMSO. cAMP (10 mM) was dissolved in distilled water.GABA-activated currents were recorded from transfected HEK293 cells continuously perfused with Krebs solution containing: 140 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl2.4A receptor β3 and phosphorylated β3 subunits (on S408 and S409 residues). Total protein was isolated from each transfected cell culture by homogenisation in ice-cold RIPA buffer, followed by cell disruption with repeated freeze–thaw cycles. Equal amounts of total protein were subjected to sodium-dodecyl-sulphate (SDS) polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Blotting was performed using 5% w/v milk, or 0.2% BSA, in 0.1% v/v TWEEN-supplemented TRIS-buffered saline (TBS), with blocking for 1 h at room temperature (RT), exposure to primary antibody overnight at 4 °C, and secondary horseradish peroxidase (HRP)-conjugated antibody for 1 h at RT. Following incubation with each antibody, membranes were washed 3×  with phosphate buffered saline (PBS) and imaged using the ECL detection system . Images were quantified using the Western blot plug-in on ImageJ software .Transfected HEK293 cells were treated with 50 nM THDOC or 100 nM PMA as appropriate and were lysed to isolate total protein. Lysis buffer was supplemented with a combination of protease inhibitors (phenylmethyl sulfonyl fluoride and benzamidine) and phosphatase inhibitors . Equal amounts of total protein were subjected to Western blotting to assess the expression of GABA2.5A receptor , Rabbit anti-phospho-β3-GABAA receptor . A donkey anti-rabbit  secondary antibody (IgG (H&L)) conjugated to HRP was subsequently used for detection.The following primary antibodies were used for Western blotting: Rabbit anti-β3-GABA2.620 GABA. The potentiation caused by a neurosteroid was measured relative to the preceding response to EC20 GABA and expressed as a percentage. All concentration response curves were constructed by plotting mean peak response amplitude against GABA concentration and the data subsequently fitted with the Hill equation:50 = concentration of GABA inducing 50% of the maximal current response and n is the Hill coefficient. Data were graphically represented and analysed using Origin version 6.0 . Statistical analyses were undertaken using GraphPad Instat (v.3) employing either a student's t-test  or an ANOVA  followed by an appropriate post-hoc test (as stated in the text) to compare selected data sets.Peak whole-cell GABA-activated currents were analysed using Clampfit ver 9.2. For protein kinase inhibitor or activator experiments, current responses were normalized to the first response elicited by EC3A receptors by neurosteroids, HEK293 cells were transfected to express α1β3γ2L subunit-containing GABAA receptors. Whole-cell recording was used to assess the magnitude of neurosteroid-mediated potentiation before and after cells were treated with modulators to inhibit or activate protein kinase activity. Peak currents were recorded in response to brief (3 s) applications of either EC20  GABA alone, (baseline control responses), or EC20 GABA co-applied with 50 nM of the neurosteroid, THDOC (the potentiated GABA responses). At this concentration neurosteroids act to potentiate GABAA receptor currents. Higher concentrations in the region of 1 μM and above induce proportionately more direct activation of the receptor in the absence of agonist.To examine the effects of phosphorylation by protein kinases on the potentiation of typical synaptic GABA420 GABA alone and the potentiated response after co-application of EC20 GABA and 50 nM THDOC was established under basal conditions , selected as it is broad-spectrum non-specific protein kinase inhibitor. Staurosporine was also chosen because it is cell permeable, which allows the potentiation induced by THDOC to be measured before and after treatment within the same cell, thus reducing any potential variation in results that could arise from using separate populations of treated and untreated cells. For each cell, the baseline response to EC5A receptor is a target for phosphorylation by a number of serine–threonine protein kinases, including PKA, PKC, PKG and CaMKII . Cells treated with 100 nM PMA (for 36 min) resulted in a significant increase in THDOC-mediated potentiation , 66.2 ± 10.0 (50 nM), 111.5 ± 17.2 (100 nM); P < 0.05, paired t-test; n = 5–11; P > 0.05; n = 8; P < 0.05, paired t-test; n = 5–11; in vivo. Interestingly, treatment with PMA revealed that potentiation could be elicited by just 1 nM THDOC under conditions of increased PKC activity, which previously was subthreshold for potentiation  with 200 nM staurosporine . By contrast, cells expressing α1β3S408A,S409Aγ2L GABAA receptors showed no change in the magnitude of the potentiation elicited , the magnitude of potentiation elicited by 50 nM THDOC at α1β38A receptors is specifically modulated by PKC  or BIM-I , indicating that these residues are important for the modulatory effects of PKC observed previously. These results support those obtained using staurosproine and BIM-I, demonstrating that PKC-mediated phosphorylation specifically at β3 S408 and S409 is sufficient to modulate the magnitude of potentiation elicited by 50 nM THDOC at α1β3γ2L GABAA receptors.We have shown that THDOC-mediated potentiation at α1β3γ2L GABAd by PKC . We therS408A,S409Aγ2L GABAA receptors is significantly reduced compared to that observed at wild-type receptors . This supports the idea that basal phosphorylation at the wild-type receptor, which is eliminated from the double mutant receptor, results in an apparent enhancement in the level of potentiation elicited by 50 nM THDOC. In support of this, it is notable that in the PKC activation experiment, there was a trend for the THDOC (50 nM) potentiation elicited at wild-type receptors (70.2 ± 17.3% enhancement of the EC20 GABA-activated current) to be greater than that at α1β3S408A,S409Aγ2L GABAA receptors , indicating that phosphorylation by protein kinases is not required for the potentiation of GABAA receptor function by neurosteroids.To further explore the requirement for protein kinase activation in the modulation of GABA10A receptors. However, this reflects just one aspect of this relationship and does not address the potential for this modulation to be bi-directional, i.e., can neurosteroids affect the extent of phosphorylation of the GABAA receptor? To investigate this, Western blot analysis was used to determine the extent of β3 subunit phosphorylation in HEK293 cells expressing α1β3γ2L GABAA receptors, before and after exposure to a neurosteroid. Cells either remained untreated or were exposed to 50 nM THDOC for 5, 10 or 20 min. Lysates were probed in duplicate with both β3 and phospho-β3 anti-sera and, for each treatment group, the level of phosphorylated β3 was normalized to the corresponding amount of total β3 present. As expected, no phosphorylated β3 was detected in cells expressing α1β3S408A,S409Aγ2L  was examined. THIP has been shown to exhibit super-agonist activity at δ subunit-containing receptors compared to its partial agonism at αβγ receptors  for 14 min. Staurosporine significantly reduced the potentiation induced by 50 nM THDOC  prevented any modulation of THDOC potentiation by the both PMA and BIM-I. Clearly, both these residues are targeted by PKC for phosphorylation under both basal and enhanced phosphorylation conditions, and in order to establish the enhanced potentiation observed with 50 nM THDOC. The specificity of this synergistic interaction for this synaptic receptor was confirmed by Western blotting following substitution of S408 and S409, and the potentiating neurosteroid binding site, α1Q241. Clearly, the phosphorylation status of the synaptic receptor (α1β3γ2) is very important in revealing the full effect of THDOC, as increased phosphorylation enhances the actions of THDOC, whereas inhibition of phosphorylation impairs it.By searching for a mechanism, using specific kinase inhibitors and activators, we establish that PKC is largely responsible for these actions on synaptic receptors. Importantly, the activity of PKC is able to modulate THDOC potentiation over a broad spectrum of physiological concentrations of this neurosteroid. Candidate residues for phosphorylation by PKC have previously been identified as neighbouring serines 408 & 409 on the β3 subunit . MutatinA receptors (α4β3δ). However, for this isoform, modulation of THDOC potentiation by protein kinase not only occurs via phosphorylation of S408 and S409 on the β3 subunit, but also occurs simultaneously via S443 on the α4 subunit, as only on the triple mutant receptor  is staurosporine ineffective. In fact, the α4S443 site is exclusively phosphorylated by PKC, rather than by other kinases  currents in hippocampal slices  in the α subunits (location of a neurosteroid binding site) with the large intracellular region between M3 and M4 (sites for phosphorylation). We may speculate that although the M3-M4 loop lacks a defining physical structure to date, the interaction between neurosteroids and protein kinases may suggest this domain has a much closer association with the membrane based M1-M4. It also argues that other interacting molecules with the M3-M4 loop (receptor-associated proteins) and post-translational modifications  may also have as yet undisclosed effects on neurosteroid potentiation.In conclusion, we find that phosphorylation of residues β3t.smart@ucl.ac.uk).The authors declare no competing financial interests. Correspondence and request for materials should be addressed to TGS (