A) and type B (ETB) receptors. The extracellular signal-regulated kinase 1 and 2 (ERK1/2) mitogen-activated protein kinases (MAPK) are involved in ET-1-induced VSMC contraction and proliferation. This study was designed to investigate the ETA and ETB receptor intracellular signaling in human VSMCs and used phosphorylation (activation) of ERK1/2 as a functional signal molecule for endothelin receptor activity.Endothelin-1 (ET-1) is a potent vasoactive peptide, which induces vasoconstriction and proliferation in vascular smooth muscle cells (VSMCs) through activation of endothelin type A  caused a time-dependent ERK1/2 activation with a maximal effect by less than 20% of the ET-1-induced activation of ERK1/2. Increase in bosentan concentration up to 10 μM further inhibited ET-1-induced activation of ERK1/2 and had a stronger inhibitory effect than BQ123 or the combined use of BQ123 and BQ788. To further explore ET-1 intracellular signaling, PKC inhibitors (staurosporin and GF109203X), PKC-delta inhibitor (rottlerin), PKA inhibitor (H-89), and phosphatidylinositol 3-kinase (PI3K) inhibitor (wortmannin) were applied. The inhibitors showed significant inhibitory effects on ET-1-induced activation of ERK1/2. However, blockage of L-type Ca2+ channels or calcium/calmodulin-dependent protein kinase II, chelating extracellular Ca2+ or emptying internal Ca2+ stores, did not affect ET-1-induced activation of ERK1/2.Subconfluent human VSMCs were stimulated by ET-1 at different concentrations (1 nM-1 μM). The activation of ERK1/2 was examined by immunofluorescence, Western blot and phosphoELISA using specific antibody against phosphorylated ERK1/2 protein. ET-1 induced a concentration- and time- dependent activation of ERK1/2 with a maximal effect at 10 min. It declined to baseline level at 30 min. The ET-1-induced activation of ERK1/2 was completely abolished by MEK1/2 inhibitors U0126 and SL327, and partially inhibited by the MEK1 inhibitor PD98059. A dual endothelin receptor antagonist bosentan or the ETA receptors predominate in the ET-1-induced activation of ERK1/2 in human VSMCs, which associates with increments in intracellular PKC, PKA and PI3K activities, but not Ca2+ signalling.The ET A) and endothelin type B (ETB), and plays an important role in hypertension, vascular remodelling, cardiac hypertrophy and coronary artery disease [A receptors locate on VSMCs and mediate vasoconstriction, while the ETB receptors primarily locate in vascular endothelial cells and mediate transient vasodilation in vivo [B receptors exist in the VSMCs and mediate vasoconstriction [A receptor activates G proteins of Gq/11 and G12/13, which results in the contractile and proliferation effects in VSMCs through activation of diverse signaling molecules such as phospholipase C (PLC), intracellular Ca2+, protein kinase C (PKC), and extracellular signal-regulated kinase 1 and 2 (ERK1/2). Whereas, the ETB receptor stimulates the Gi and the Gq/11 families in VSMCs and endothelial cells [A and ETB receptors, which may result in receptor signal cross-talk in vascular physiology and pathology. However, there is limited knowledge about this.In the human cardiovascular system, endothelin-1 (ET-1) is the most important isoform, which induces long-lasting vasoconstriction and stimulates proliferation of vascular smooth muscle cells (VSMCs) [ disease . The ETA in vivo . Howevertriction ,4. The Eal cells ,6. ET-1 A or the ETB receptor results in phosphorylation of ERK1/2, which is an important regulator for cellular proliferation, migration, differentiation and vascular smooth muscle constriction [2+ signals are required for MAPK/ERK1/2 activation induced by angiotensin II in VSMCs [2+-independent contraction is suggested to be associated with PKC, phosphoinositide 3-kinase (PI3K), Rho kinase and MAPK [A receptors predominate over ETB receptors in mediating ET-1-induced activation of ERK1/2 in human VSMCs. This activation is associated with PKC, PKA and PI3K activities, but not intracellular Ca2+ signalling.ERK1/2, also termed p44/42 MAPK (mitogen-activated protein kinase), is one of the members of MAPK superfamily, which includes a family of serine/threonine kinase associated with VSMCs contraction, proliferation, migration, differentiation, adhesion, collagen deposition and survival . Activattriction -12. A MAtriction . In the triction . Intracein VSMCs -17. Howein VSMCs . Thus, Cand MAPK ,11,19. TET-1-induced activation of ERK1/2 was examined in human aortic smooth muscle cells (HASMCs) at different time points and ET-1 concentrations. There was a 2.6 fold (p < 0.001) increase of phosphorylated ERK1/2 in cells exposed to 1 μM of ET-1 for 5 min; the enhancement reached a peak  at 10 min after exposure to ET-1 Figure . ThereafA and ETB receptors in mediating ET-1-induced activation of ERK1/2 were studied by using bosentan , BQ123 (a selective peptide antagonist for the ETA receptor), and BQ788 (a selective peptide antagonist for the ETB receptor). To clarify if the ETB receptors in HASMCs were involved in ET-1- induced activation of ERK1/2, sarafotoxin 6c (S6c), a selective ETB receptor agonist was employed and the phosphorylation of ERK1/2 was examined by immunofluorescence and western blot , PKC-delta inhibitor (Rottlerin), PKA specific inhibitor (H-89), and PI3K inhibitor (wortmannin) on ET-1-induced pERK1/2 activities Figure . The act2+, a second messenger, has a central role in activation of various key cellular responses, including muscle contraction, cell proliferation, migration and adhesion [2+signaling in mediating ET-1-induced activation of ERK1/2, nifedipine was used to block external Ca2+ influx through L-type Ca2+ channels, 5 mM of EGTA was employed to chelate extracellular Ca2+, and 1 μM of thapsigargin was used to cause intracellular Ca2+ stores to become depleted. KN-62, a calcium-calmodulin dependent protein kinase II (CAMKII) inhibitor was also examined  [2+and decrease Ca2+ influx in our experiments.Caadhesion . To evalr Figure , chelati+ Figure , abolishe Figure , or inhie Figure . Replaciy Figure . To idens (SOCC) . We founs (SOCC) . The acts (SOCC) . This suA receptors to induce phosphorylation of ERK1/2 in HASMCs. The ET-1-induced response requires intracellular signal molecule PKC, PKA and PI3K activities, while it is independent of intracellular calcium signaling.The present study has revealed that ET-1 acts primarily via the ET2+-dependent and Ca2+-independent contraction of VSMCs [ERK1/2 are important regulators of cell proliferation and migration in VSMCs ,9. Theseof VSMCs -12,19. Iof VSMCs -24. ET-1of VSMCs  in pulmoof VSMCs . In humaof VSMCs . This imof VSMCs , ET-1 inof VSMCs .A and ETB. In human vasculature, ETA receptors predominate on the smooth muscle cells and mediate constriction, whereas ETB receptors are expressed less than 15% on these cells [In-vivo studies suggest that both subtypes of endothelin receptors can mediate vasoconstriction in human resistance and capacitance vessels [A predominately mediated ET-1-induced activation of ERK1/2. Although some activation of ERK1/2 was obtained with the ETB-selective agonist, S6c, the maximum response produced to S6c was transient and less than 20% of the ET-1 effect. In addition, BQ123, a selective antagonist of the ETA receptor [B receptor antagonist BQ788, significantly inhibited the activation of ERK1/2 induced by ET-1, suggesting that ET-1-induced activation of ERK1/2 is predominately mediated by ETAreceptors. Compared to BQ123, a further inhibition of ET-1-induced activation of ERK1/2 was obtained in combination of BQ123 and BQ788. Bosentan, a dual ETA and ETB receptor antagonist had a significant stronger inhibitory effect on ET-1-induced activation of ERK1/2 than either BQ123 or the combination of BQ123 and BQ788. These results suggest that ET receptor dimerization [The physiological and pathological effects of ET-1 are mediated through two G protein-coupled receptors, ETse cells ,30. In-v vessels . In the receptor , but notrization  might alrization -36 and tERK1/2 activation requires a sequential activation of Ras, Raf and MEK signal cascades ,37. MEK 2  through G-protein coupled receptors, leading to the generation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), which are involved in intracellular Ca2+ mobilization and PKC activation [2+ signaling is critical for activation of ERK1/2 induced by angiotensin II in VSMCs [2+ signaling in ET-1-induced activation of ERK1/2 in human VSMCs remains unclear. It has been reported that the activation of L-type Ca2+ channels contributes to ET-1-induced sustained phase of the Ca2+ response and the ability to generate force [2+ influx through L-type Ca2+ channels did not participate in ET-1-induced activation of ERK1/2 in human VSMCs. To further investigate the involvement of intracellular Ca2+ through other Ca2+ channels, which are suggested to be involved in ET-1-mediated contractions of VSMC [2+ chelation by EGTA did not affect activation of ERK1/2 induced by ET-1. ET-1-induced Ca2+ release from intracellular stores is triggered by the binding of IP3 to receptors on the sarcoplasmic reticulum (SR). Depletion of intracellular Ca2+ stores can lead to a local Ca2+ flux through store-operated Ca2+ channels (SOCC), which has been reported to initiate the activation of ERK1/2 in RBL-1 cells [2+-ATPase pump, which results in Ca2+ release and depletion from internal stores, was applied together with 5 mM of EGTA. The results showed that ERK1/2 activation by ET-1 did not require the participation of intracellular Ca2+ release. Studies have indicated that the CAMKII pathway mediates G-protein coupled receptor ligand-depedent activation of ERK1/2 in cultured VSM cells [2+ channel blockers LOE 908 and SK&F 96365, and L-type Ca2+ channels blocker nifedipine, Kawanabe et al noted that ET-1-induced ERK1/2 activiation involved a Ca2+ influx-dependent cascade through Ca2+permeable nonselective cation channels (NSCCs) and SOCC, and a Ca2+influx-independent cascade in rabbit carotid artery VSMCs [2+.ET-1 stimulates phospholipase C-dependent hydrolysis of PIPin VSMCs -17. Howete force . Unlike  of VSMC  and mito of VSMC , 5 mM of-1 cells . TherefoSM cells ,45,46. Hry VSMCs . The stuA receptors through upstream signal molecule PKC, PKA and PI3K, while it is independent of CAMKII and intracellular Ca2+ signaling. The endothelin system plays key roles in hypertension, stoke and myocardial infarction. Understanding the intracellular signaling mechanisms of endothelin receptors may provide new strategies for developing new drugs for cardiovascular diseases.In conclusion, we have demontrated that ET-1-induced activation of ERK1/2 in human VSMCs is predominantly mediated by ETB receptor agonist [A antagonist BQ123 and ETB antagonist BQ788 [2+ channels inhibitor; EGTA (ethylene glycol tetraacetic acid), a Ca2+ chelator; thapsigargin, a sarco-endoplasmic reticulum Ca2+-ATPase pump inhibitor; KN-62, a CAMKII inhibitor, were applied to determine the involvement of Ca2+ signaling and CAMKII in activation of ERK1/2. The concentration of inhibitors was determined by recommendation from product data sheet and literatures. All drugs were purchased from Sigma-Aldrich Co. . ET-1 and S6c were dissolved in sterile water with 0.1% BSA; the other reagents were dissolved in DMSO as a stock solution and diluted in cell culture medium before use.ET-1 and S6c, a selective ET agonist , were usst BQ788 ,48 were A monoclonal antibody for phospho-ERK1/2 (phospho T183 + Y185) and a polyclonal antibody for total-ERK1/2 were obtained from Abcam plc. . Polyclonal β-actin was purchased from Cell Signaling Technology, Inc. .HASMCs at the end of the tertiary culture stage were obtained as a commercially available product from Cascade Biologics Inc. . Cells were plated in 75 cm2 tissue culture flasks at a density of 2.5 × 103 viable cells/cm2 in Medium 231 supplemented with 5% smooth muscle growth supplement (SMGS). Medium 231 and SMGS were purchased from Cascade Biologics Inc. The cells were incubated in a 5% CO2 incubator at 37°C and the medium was replaced every other day until the culture was approximately 80–90% confluent. Then the cells were removed from the flasks with accutaseTM Enzyme Cell Detachment Medium  and seeded onto 100-mm tissue culture dish .All experiments were performed with the cells of passages 6 to 9. HASMCs were allowed to grow to 70%–80% confluence within 2 to 3 days, and maintained in medium 231 with 0.05% SMGS for 24 h, then we added vehicle or ET-1, S6c at different concentration from 1 nM to 1 uM, or with a time course at 5 min, 10 min, 15 min, 30 min, 1 h, 6 h and 24 h. Inhibitors or DMSO were treated for 30 min prior to addition of ET-1.3/well in 4 well NUNC Lab-Tek II Chamber Slides for 3 days and were starved in medium 231 with 0.05% SMGS for 24 h. The cells were stimulated with ET-1 or S6c at above indicated time points after treatment with vehicle or inhibitors for 30 minutes, and then washed, fixed in 4% paraformaldehyde, permeabilized in PBS containing 4% Triton X-100. The monoclonal primary antibody against phospho-ERK1/2 (phospho T183 + Y185) was added to the cells at 1: 1000 dilution and incubated at room temperature for 1 h or overnight at 4°C, followed by adding fluorescein isothiocynate (FITC)-conjugated goat anti-mouse secondary antibody at 1:5000 dilution in dark according to the recommendation of the manufacturer. In the control experiments, either the primary antibody or the secondary antibody was omitted. After washing with PBS, ProLong Gold antifade mounting reagent  was added and the cells were sealed with cover slip on the slide. The immunofluorescence stained cells were observed under a laser scanning confocal microscope  and analysed by ImageJ software . The fluorescence intensity of cells was measured at 4 preset areas of per sample and at least three independent experiments were performed. The fluorescence intensity of each treated group was determined as the percent increase over control, with the control normalized to 100%. There was no change of fluorescence intensity after cells were treated with inhibitors compared with vehicle treatment [see Additional file HASMCs were seeded at a density of 5 × 10About 70%–80% confluent HASMCs in 100-mm tissue culture dishes were made quiescent by placing them in medium 231 supplemented with 0.05% SMGS for 24 h and harvested in cell extract denaturing buffer  with addition of a phosphatase inhibitor cocktail and protease inhibitor cocktail  after treatment. Incubating cells at 4°C for 30 min, whole cell lysates were sonicated for 2 min on ice, centrifuged at 15,000 × g at 4°C for 30 min, and the supernatants were collected as protein samples. The protein concentrations were determined using the protein assay reagents  and stored at -80°C until immunoblotting assay. The protein homogenates were diluted 1:1(v/v) with 2 × SDS sample buffer . 25–50 ug of total proteins were boiled for 10 min in SDS sample buffer and separated by 4–15% SDS Ready Gel Precast Gels  for 120 min at 100 v, and transferred electrophoretically to nitrocellulose membranes  at 100 v for 60 min. The membrane was then blocked for 1 h at room temperature with phosphate buffered saline (PBS) containing 0.1% Tween-20  and 5% non-fat dried milk, and incubated with primary antibodies diluted 1:1000 overnight at 4°C, followed by incubation with ECL anti-mouse or anti-rabbit IgG, horseradish peroxidase-conjugated secondary antibodies  diluted 1:10000 for 1 h at room temperature. The probed proteins were developed by LumiSensor Chemiluminescent HRP Substrate ECL Western Blot Detection Reagent . To detect multiple signals using a single membrane, the membrane was incubated for 5–15 min at room temperature with restore plus western blot stripping buffer . The membranes were visualized using a Fujifilm LAS-1000 Luminiscent Image Analyzer , and then quantification of band intensity was analyzed with Image Gauge Ver. 4.0 . Three independent experiments were performed in duplicate.3/well in 96-well plate for 3 days and starved in medium 231 with 0.05% SMGS for 24 h. The cells were treated with vehicle or different inhibitors for 30 min prior to the addition of ET-1. After 10 min of ET-1 stimulation, the cells were fixed and stored at 4°C until the performance of experiments. Phosphorylated ERK1/2 was measured using a cell-based ELISA Assay Kit  following the manufacturer's instructions. Phosphorylated ERK1/2 activity was presented as a relative extent to the level of total ERK1/2. Independent experiments were done in duplicate or triplicate and were repeated at least three times.HASMCs were seeded at a density of 3 × 10Comparison between two groups was performed using two-tailed unpaired Student's t-test with Welch's correction. For more than two groups one-way ANOVA followed by Dunnett's post test was used. A p-value, less than 0.05 was considered to be significant. Results were presented as mean ± SEM. At least 3 different samples or independent experiments were analyzed in each group.QC carried out the main part of the experiments, participated in the design, statistical analysis, drafting and writing of the manuscript. LE participated in the writing of the manuscript. CX conceived the study and the design, coordinated the work and the writing of the manuscript. All authors have read and approved the final manuscript.Inhibitory effects of BQ123 and U0126 on pERK1/2 activity in HASMCs. The data provided represent the Western Blot analysis of inhibitory effects of ETA receptor inhibitor BQ123 and MEK inhibitor U0126 on ET-1-induced phosphorylation of ERK1/2. 24 h Serum-starved cells were stimulated with 10 nM of ET-1 for 10 min after cells were treated with inhibitors for 30 min. Phosphorylated ERK1/2 activity was determined by western blot with an anti-phospho-ERK1/2 antibody, and presented as a relative extent to the level of β-actin. A, bar graph shows inhibitory effects of 5 μM of ETA/ETB receptor inhibitors on phosphorylated ERK1/2 activity induced by ET-1. B, bar graph shows inhibitory effect of 1 μM of U0126 on phosphorylated ERK1/2 activity induced by ET-1. The upper panels of A and B indicate representative autoradiographs of western blot showing phosphorylated ERK1/2 and β-actin. Data represent mean ± S.E.M. * p < 0.05, ** p < 0.01 compared with the ET-1-stimulated states after DMSO treatment. p = phosphorylation, ns = non-significant.Click here for file2+Effect of ET-1 on activation of ERK1/2 in HASMCs in the absence of external Ca. The data provided represent the immunofluorescence analysis of ET-1-induced phosphorylation of ERK1/2 in the absence of external Ca2+by replacing culture medium with PBS. Serum-starved cells were placed in the presence or absence of external Ca2+ for 3 min by replacing culture medium with PBS plus 1 mM EGTA prior to addition of ET-1. Phosphorylated ERK1/2 was determined at 10 min after the addition of 10 nM of ET-1 by immunofluorescence with an anti-phospho-ERK1/2 antibody. The bar graph shows effect of ET-1 on phosphorylated ERK1/2 in the absence of extracellular Ca2+. The fluorescence intensities of phosphorylated ERK1/2 are expressed relative to the quiescent state in the presence of external Ca2+. The upper panel indicates representative images of immunofluorescence showing the phosphorylated ERK1/2 from samples given the different treatments. Data represent the mean ± S.E.M. *** p < 0.001. ns = non-significant.Click here for file2+ chelator EGTA abolished thapsigargin-induced activation of ERK1/2 in ET-1 untreated starved cellsThe Ca. The data provided represent the immunofluorescence analysis of inhibitory effect of the Ca2+ chelator EGTA on extracellular Ca2+influx through thapsigargin-induced store-operated Ca2+ channels. Serum-starved cells were treated with 1 μM of thapsigargin with or without 5 μM of EGTA for 15 min. Phosphorylated ERK1/2 was determined by immunofluorescence with an anti-phospho-ERK1/2 antibody. The bar graph shows effect of thapsigargin on phosphorylated ERK1/2 in the presence or in the absence of EGTA. The upper panel indicates representative images of immunofluorescence showing the phosphorylated ERK1/2 from samples given the different treatments. Data represent mean ± S.E.M. *** p < 0.001 compared with the vehicle value.Click here for fileEffects of the inhibitors used in the present study on the activities of ERK1/2 in ET-1 untreated cells. The data provided represent the immunofluorescence analysis of the stability of fluorescence intensity after cells were treated with inhibitors compared with vehicle treatment. Serum-starved cells were treated with variety of inhibitors indicated or DMSO for 30 min. Phosphorylated ERK1/2 was determined by immunofluorescence with an anti-phospho-ERK1/2 antibody. The bar graph shows no significant effects of the inhibitors on phosphorylated ERK1/2 in ET-1 untreated control cells. The upper panel indicates representative images of immunofluorescence showing the phosphorylated ERK1/2 from samples treated with different inhibitors. Data represent mean ± S.E.M.Click here for file


The glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for several neuronal populations in different brain regions, including the hippocampus. However, no information is available on the: (1) hippocampal subregions involved in the GDNF-neuroprotective actions upon excitotoxicity, (2) identity of GDNF-responsive hippocampal cells, (3) transduction pathways involved in the GDNF-mediated neuroprotection in the hippocampus. We addressed these questions in organotypic hippocampal slices exposed to GDNF in presence of N-methyl-D-aspartate (NMDA) by immunoblotting, immunohistochemistry, and confocal analysis. In hippocampal slices GDNF acts through the activation of the tyrosine kinase receptor, Ret, without involving the NCAM-mediated pathway. Both Ret and ERK phosphorylation mainly occurred in the CA3 region where the two activated proteins co-localized. GDNF protected in a greater extent CA3 rather than CA1 following NMDA exposure. This neuroprotective effect targeted preferentially neurons, as assessed by NeuN staining. GDNF neuroprotection was associated with a significant increase of Ret phosphorylation in both CA3 and CA1. Interestingly, confocal images revealed that upon NMDA exposure, Ret activation occurred in microglial cells in the CA3 and CA1 following GDNF exposure. Collectively, this study shows that CA3 and CA1 hippocampal regions are highly responsive to GDNF-induced Ret activation and neuroprotection, and suggest that, upon excitotoxicity, such neuroprotection involves a GDNF modulation of microglial cell activity. Considerable interest has been devoted to neurotrophins as candidate neuroprotective agents for several neurodegenerative disorders since they promote neuronal survival, neuritic growth, and differentiation of several, but selective, neuronal populations. One such candidate is glial cell line-derived neurotrophic factor (GDNF) given its spectrum of demonstrated activities which includes, but is not limited to, potent trophic actions on a wide variety of neuronal populations of the central and peripheral nervous systems In vitro studies have shown that following GDNF binding to GFRα1 the resulting complex recruits Ret, leading to its activation by dimerization and autophosphorylation at specific cytoplasmic tyrosine residues, thus initiating a number of downstream intracellular pathways NCAM isoform of the neural cell adhesion molecule (NCAM) and subsequent activation of Fyn and FAK kinases, has been as well demonstrated to take place in primary glial cells and neurons in vivo studies indicated that GDNF and its receptors are widely expressed in the rat hippocampus In vitro, GDNF has been found to protect hippocampal slice cultures and primary cortical neurons against toxic activation of the N-methyl-D-aspartate (NMDA) receptor In the last years a large number of studies demonstrated that GDNF provides potent neuroprotection in animal models of Parkinson's disease In the present study, by combining Western blotting, immunofluorescence and confocal analysis on rat organotypic hippocampal slice cultures (OHSCs), we first investigated the intracellular signalling mechanisms triggered by acute or chronic GDNF exposure in the different subfields of the hippocampus. Then, we used the NMDA-induced neurotoxicity in OHSCs as experimental model to insight the neuroprotective actions of GDNF in the different hippocampal subregions. Finally, we explored the identity of hippocampal cells that respond to GDNF stimulation following NMDA-induced neurodegeneration.in vitro (DIV) 2, 7, and 14 following dissection. As shown in NCAM-dependent mechanism, we stimulated hippocampal slices at 7 and 14 DIV with 200 ng/ml GDNF for 30 minutes and looked for Ret and FAK tyrosine phosphorylation, the latter being an upstream effector of NCAM-dependent GDNF signalling NCAM-dependent, Ret-independent, pathway. Since both the basal and GDNF-induced levels of Ret phosphorylation  a clear increase of both Ret and ERK phosphorylation occurred essentially in the CA3 region  uptake, that was detectable after at least 4–8 h of NMDA exposure and was further increased after 24 and 48 h A marked neuroprotective effect was observed when GDNF was added to the incubation medium 48 h before NMDA exposure . ShorterTo verify whether GDNF prevention of NMDA-induced PI uptake involved neuronal cell protection, immunohystochemical analysis for the neuron-specific nuclear protein NeuN was performed. Organotypic hippocampal slices exposed for 24 and 48 h to NMDA showed an evident loss of NeuN staining intensity in CA1 and CA3 neurons , that waGiven the widespread observed neuroprotective actions of GDNF against NMDA-induced neurodegeneration even in the subregions that poorly respond to GDNF stimulation, cfr , we examIn the present study, by using rat hippocampal organotypic slices subjected to NMDA-induced neurotoxicity as model system, we investigated the neuroprotective role of GDNF in the hippocampus.NCAM isoform in the presence of GFRα, but independently of Ret, thus leading to activation of the cytoplasmic protein tyrosine kinases Fyn and FAK, two signaling mediator downstream of p140NCAMNCAM-dependent pathway.First, we demonstrated that GDNF stimulation induces activation of Ret tyrosine kinase receptor and its downstream effector ERK in a dose and time dependent manner. By combining immunoblotting and immunofluorescence experiments performed on separated CA1, CA3 or DG subregions, we established that a GDNF treatment either if rapid (30 min) or prolonged (48 h) induces a strong activation of the Ret/ERK signalling preferentially in the CA3 subfield. Recently, NCAM has been identified as an alternative signalling receptor for GDNF In line with our results, in situ hybridization and immunohystochemical experiments demonstrated that both Ret mRNA and protein are much more abundant in CA2 and CA3 pyramidal sector of rat postnatal Furthermore, with the attempt to elucidate the identity of hippocampal cells that respond to GDNF stimulation, we found activated both Ret and ERK proteins in a subpopulation of cells of the pyramidal layer that by immunofluorescence co-localization experiments with specific markers, NeuN, GFAP, OX-42, Olig-2 and NF-200 do not resemble neither mature neurons, astrocytes, microglia, oligondendrocyte precursors nor neuronal cells. Intriguingly, these cells double-labelled for both phosphorylated ERK and Ret proteins, resulted positive for the staining with IB4, a lectin that although largely used as a marker for microglia We found that GDNF displayed a broad neurotrophic action following NMDA treatment that resulted in protecting, even if at different degree, CA1 and the less vulnerable CA3 subfields. Interestingly, we found that, while in the absence of the excitotoxicity insult, GDNF stimulates Ret-dependent signalling mainly in the subpopulation of IB4-positive cells of the CA3 region (see above), the pattern of GDNF-dependent activation of Ret changes following NMDA treatment. Indeed, we observed that a pre-treatment of hippocampal slices with GDNF before inducing the insult, caused a pronounced increase of Ret phosphorylation in both CA1 and CA3 region. More specifically, we observed an enhanced activation of Ret in IB4-positive microglial cells in the CA1, and with a lesser degree in the CA3 region. In accordance with our findings, it has been demonstrated that the GDNF receptors, Ret and GFRα1, are expressed in rat primary cultured microglia Collectively, this study shows that CA3 and CA1 hippocampal regions are highly responsive to GDNF-induced Ret receptor activation and neuroprotection, and suggest that, upon excitotoxicity, such neuroprotective action involves a GDNF modulation of microglial cell activity. Our results open new insights on the mechanisms involved in the neuroprotective actions of GDNF.Propidium iodide was from Molecular Probes Europe BV . Recombinant GDNF was from Alomone Labs . All media and sera for OHSCs were purchased from Gibco . NMDA was dissolved directly in serum-free medium (stock solutions of 10 mM) and then diluted to the desired concentration; this stock solutions was stored at 4°C for 1 month. Stock solutions were diluted to achieve the desired drug concentration immediately before use.2 atmosphere. Experiments and animal use procedures were in accordance with the National Institutes of Health guide for the care and use of Laboratory animals ; the experimental protocol was approved by the Animal Care Committee of the “Federico II” University of Naples.OHSCs were prepared as previously described in detail Cultures were exposed to GDNF in two ways. To examine the acute effects of GDNF treatments in OHSCs we used the “drop application”, in which a 5 µl drop of 100–200 ng/ml GDNF solution was applied to the top of each slice, as described by Marty et al. NMDA exposure was performed in OHSCs as previously described Cell injury was assessed using the fluorescent dye propidium iodide. PI is a very stable and highly polar compound which only enters cells with damaged or leaky plasmamembranes, binds to DNA, and emits a brightly red fluorescence when exposed to blue-green light. PI is non-toxic to neurons and has been used as a marker of neuronal membrane integrity and cell damage. Briefly, at the beginning of the experiment, 5 µg/ml PI was added to the culture medium for 30 minutes to check for slice viability; OHSCs in which significant PI fluorescence was detected were excluded from further studies. PI uptake was recorded by a digital camera  mounted on a Nikon Eclipse 400 fluorescence microscope . Preliminary tests were carried out on maximally fluorescent OHSCs to determine a combination of illumination intensity and integration time which would fully exploit the 12-bit dynamic range of the imaging system without saturating it. For densitometric measurements, the digital photos were analyzed with the Image Pro-Plus software , after freehand outlining of the CA1, CA3 and DG neuronal layers, as previously described 3VO4. The solution was centrifuged at 16000 g for 30 min at 4°C and the residue was discarded. Protein concentration was determined by the Bradford assay using bovine serum albumin (BSA) as the standard and the crude extracts were subjected to 10% SDS-PAGE. Gels were electroblotted into polyvinylidene difluoride membranes , and filters were probed with the indicated primary antibodies: anti-(Tyr1062-phosphorylated) Ret (indicated as pRet), anti-ERK1 (C-16) ; anti-(Ser473-phosphorylated), anti-phosphorylated-p44/42 mitogen-activated protein kinase E10 ; anti-α-tubulin ; anti- (Tyr397-phosphorylated) FAK . Proteins were visualized with peroxidase-conjugated secondary antibodies using the enhanced chemiluminescence system . When indicated, membranes were stripped in 62.5 mM Tris-HCl pH 6.7, 0.1 M 2-mercaptoethanol, 2% SDS for 30 min at 55°C.In some experiments the slices were transferred to a glass slide at ice temperature, and the CA3 areas were dissected out from CA1/DG subregions under a stereo microscope and stored at ±80°C until assay. The slices were lysed in 50 mM Tris-HCl pH 8.0 buffer containing 150 mM NaCl, 1% Nonidet P-40, 2 µg/ml aprotinin, 1 µg/ml pepstatin, 2 µg/ml leupeptin, 1 mM NaBandeiraea Simplicifolia B4 (IB4) directly conjugated to FITC  at 4°C.Immunohistochemistry in OHSCs was performed as described by Boscia et al. 2O2. After the final wash, sections were dehydrated, coverslipped, and processed for microscope analysis. Images were acquired by a digital camera  mounted on a Nikon Eclipse 400 microscope.After 48 h, the slices processed for light microscopy were first incubated in biotinylated horse anti-mouse or goat anti-rabbit IgG  for 2 h and then in avidin–biotinylated horseradish peroxidase complex  for 1.5 h, always at room temperature. The peroxidase reaction was developed using 3,3-diaminobenzidine 4-HCl (DAB) as a chromogen and 0.05% HFollowing 48 h of primary antibody incubation, the slices processed for single or double-labelling fluorescence experiments were incubated in single or in a mixtures of fluorescent-labelled secondary antibodies Alexa 488-conjugated anti-mouse IgG and Alexa 568-conjugated anti-rabbit IgG  for 3 h at RT. Finally, the sections were washed in TBS, transferred onto microscope slides and covered with Vectashield mounting medium . Immunofluorescence images were observed using a Zeiss LSM 510 META Laser-Scanning Confocal Microscope  by which single images were taken with an optical thickness of 0.7 µm and a resolution of 1,024×1,024 pixels. Controls of the methods in the present experiments included replacement of the primary antisera with normal serum (1∶200). To control for a possible cross-reactivity between IgGs in double immunolabeling experiments, some sections were processed through the same immunocytochemical sequence except that primary antisera were replaced with normal serum, or only one primary antibody was applied, but the full complement of secondary antibodies was maintained. In addition, the secondary antibodies utilized were highly pre-adsorbed to the IgGs of numerous species. Tissue labelling without primary antibodies was also tested to exclude autofluorescence. No specific staining was observed under these control conditions, thus confirming the specificity of the immunosignals.


Leishmania genus can rapidly alter several macrophage (MØ) signalling pathways in order to tame down the innate immune response and inflammation, therefore favouring their survival and propagation within their mammalian host. Having recently reported that Leishmania and bacterial LPS generate a significantly stronger inflammatory response in animals and phagocytes functionally deficient for the Src homology 2 domain-containing protein tyrosine phosphatase (SHP-1), we hypothesized that Leishmania could exploit SHP-1 to inactivate key kinases involved in Toll-like receptor (TLR) signalling and innate immunity such as IL-1 receptor-associated kinase 1 (IRAK-1). Here we show that upon infection, SHP-1 rapidly binds to IRAK-1, completely inactivating its intrinsic kinase activity and any further LPS-mediated activation as well as MØ functions. We also demonstrate that the SHP-1/IRAK-1 interaction occurs via an evolutionarily conserved ITIM-like motif found in the kinase domain of IRAK-1, which we named KTIM (Kinase Tyrosyl-based Inhibitory Motif). This regulatory motif appeared in early vertebrates and is not found in any other IRAK family member. Our study additionally reveals that several other kinases  involved in downstream TLR signalling also bear KTIMs in their kinase domains and interact with SHP-1. We thus provide the first demonstration that a pathogen can exploit a host protein tyrosine phosphatase, namely SHP-1, to directly inactivate IRAK-1 through a generally conserved KTIM motif.Parasites of the  Leishmania developed several methods to seize control of macrophage signalling pathways in an effort to inactivate their killing abilities. One effective method utilized by the parasite is the activation of host protein tyrosine phosphatases, specifically SHP-1. This increased phosphatase activity contributes to the inactivation of signalling molecules involved in critical macrophage functions such as NO and cytokine production. Interestingly, the absence of SHP-1 results in stronger macrophage inflammatory responses to a bacterial cell wall component known as LPS, a molecule detected by macrophages through Toll-like receptors (TLRs). This observation suggested a role for SHP-1 in the regulation of TLR signalling. Our study reveals that upon Leishmania infection, SHP-1 is able to rapidly bind to and inactivate a critical kinase (IRAK-1) in this pathway. This regulatory binding was shown to be mediated by an evolutionarily conserved motif identified in the kinase. This motif was also present in other kinases involved in Toll signalling and therefore could represent a regulatory mechanism of relevance to many kinases. This work not only reports a unique mechanism by which Leishmania can avoid harmful TLR signalling, but also provides a platform on which extensive investigation on host evasion mechanisms and regulation of cellular kinases can be gained. Leishmania evolved strategies to avoid phagocyte activation by seizing control of key signalling pathways, therefore favouring their invasion and survival within the host cell Leishmania infection Innate inflammatory responses play a critical role in controlling pathogens Leishmania to block the macrophage (MØ) inflammatory response could result from rapid IRAK-1 kinase inactivation through SHP-1 action. This hypothesis is further reinforced by the fact that several LPS-mediated MØ functions , critical for the containment of pathogens and adaptive immune response development, are inhibited upon Leishmania infection As SHP-1 can interact with various members of the JAK and MAP kinase families in physiological, immune response, and infection contexts Leishmania- and LPS-induced innate inflammatory response in the absence of SHP-1 (PTPN6) PTPN6 gene coding for SHP-1 in humans has been recently linked to Sezary syndrome Whereas invertebrates depend mainly on the evolutionarily conserved innate immune system to fight off pathogens, vertebrates have developed a sophisticated adaptive immune system, hence the need to regulate the innate immune response. The TLR family has been shown to play a key role in triggering innate immunity as well as the subsequent induction of adaptive immune responses in vertebrates Leishmania. For instance, until recently there was no indication that SHP-1 could interact with IRAK-1. However, a recent study by Cao's laboratory From these observations, and given the fact that IRAK-1 serves as a crucial kinase in all MyD88-dependent pathways leading to the activation of innate inflammatory responses, we hypothesised that SHP-1 is a critical player in the negative regulation of this kinase that can be exploited by Leishmania infection through binding to an evolutionarily conserved ITIM-like motif located within IRAK-1's kinase domain. In addition, it is important to stress that this is the first mention of this motif to be found within a kinase, as to date it has only been found within the intracytoplasmic portion of immunoglobin (Ig)-like receptors. Of interest, we also discovered that this ITIM-like motif was present in several other kinases. Finally, our study also provides evidence from in silico sequence analyses that both IRAK-1 and SHP-1 evolutionarily emerged in vertebrates concomitantly with the development of a better-controlled innate immune response. Therefore the appearance of this key interaction in early vertebrates may have also contributed to the development of the more complex adaptive immune response.Here, we provide evidence that SHP-1 negatively regulates IRAK-1 intrinsic kinase activity in its resting state and upon −/−) and LM-1 (WT) bone marrow-derived MØs (BMDMs) were generated from motheaten mice  and their respective wild-type littermates (C3HeBFeJ me/+) as described r mice E.coli were purchased from Alexis Biochemicals, San Diego, CA. Flagellin and CpG DNA were purchased from Invivogen, San Diego, CA. IRAK1/4 inhibitor (N-(2-Morpholinylethyl)-2-(3-nitrobenzoylamido)-benzimidazole) was purchased from Calbiochem, La Jolla, CA.The immortalized me-3 , L. major Friedlin strain (MHOM/JL/80/Friedlin), and L. tarentolae strain TAR II promastigotes were kept in SDM medium (10% FBS), and stationary phase parasites were used to infect cells in a parasite to MØ ratio of 20∶1. Non-internalized parasites were removed by washing the plates with phosphate-buffered saline (PBS), after which MØs were collected for subsequent experiments.Western blotting was performed as previously described 6 MØs were lysed in cold lysis buffer (20mM Tris (pH 7.5), 1mM EDTA, 150mM NaCl, 1% Igepal, 10mM β-glycerophosphate, 1mM sodium orthovanadate, 25 µg/ml aprotinin and 25 µg/ml leupeptin). Lysates were precleared with protein A/G agarose beads . Samples were then centrifuged  and supernatants kept. IRAK-1 or IRAK-4 antibody and protein A/G agarose beads were added to the supernatant and samples were incubated O/N at 4°C. Beads were spun down and washed with the lysis buffer described above, followed by washes with the kinase assay buffer . Kinase assay buffer (20 µl) containing 10 µCi of γ-32P (Amersham) was then added to the beads and samples incubated . The reaction was stopped by the addition of 4× sample loading buffer (12.5% Tris-HCl (pH 6.8), 10% glycerol, 10% SDS, 5% β-mercaptoethanol, 0.05% bromophenol blue). Samples were boiled and ran on SDS-PAGE. Bands were detected using X-ray Kodak films (Amersham) or by image analyzer .6×106 MØs were lysed as described previously for the IRAK-1 kinase assay without the addition of sodium orthovanadate to the lysis buffer. Cell lysate controls (25 µg) were obtained using a PTP lysis buffer (50mM Tris (pH 7.0), 0.1mM EDTA, 0.1mM EGTA, 0.1% β-mercaptoethanol, 1% Igepal, 25 µg/ml aprotinin and 25 µg/ml leupeptin). Samples were loaded on a gel containing a γ-32P-labelled poly(Glu4Tyr) peptide (Sigma-Aldrich) and the SHP-1 band was observed by in gel PTP assay as previously described For immunoprecipitation samples, 6×10Samples were lysed in the western blot lysis buffer (no sodium orthovanadate was added when immunoprecipitating SHP-1) and immunoprecipitated using protein A/G agarose beads (Santa Cruz) and 4 µg of the IRAK-1, SHP-1 antibody, or anti-rat antibody (Sigma-Aldrich) for non-specific binding. Beads were spun down and washed three times with lysis buffer. Beads were resuspended in the 4× western sample loading buffer previously described and boiled supernatants were loaded on SDS-PAGE and western blot analysis was performed as described above.in vitro translated using the Promega TNT Quick Coupled Transcription/Translation kit  using 20 µCi 35S (Amersham). The active or the trapping mutant of GST SHP-1 was produced in BL21 bacteria. Bacterial lysates were extracted using the BugBuster Protein Extraction Reagent , and the GST protein (5 µg) was pulled down from bacterial lysates using glutathione sepharose beads (30 µl) (Amersham). The active/trapping mutant of GST-SHP-1 bound to glutathione beads was left to interact with immunoprecipitates (IPs) or in vitro translated IRAK-1 protein in a PTP reaction buffer (50mM Hepes (pH 7.5), 0.1% β-mercaptoethanol) for 1 h at RT. When in vitro translation of IRAK-1 was performed, GST-SHP-1 was allowed to interact with IRAK-1 in a 5∶1 ratio. Beads were then spun down, washed 3× with the PTP lysis buffer, then resuspended in 4× sample loading buffer (20 µl), boiled, and loaded on SDS-PAGE. IRAK-1 bands were revealed by exposing to X-ray film (Amersham).Wildtype mouse IRAK-1 gene and the IRAK-1 genes of the different KTIM mutants  were 32P-labelled IRAK-1 was performed as previously described Kinase assays were run on SDS-PAGE as described above, pre-treatment image is taken by exposing the gel to a phospho-imager screen. Next, gels were fixed overnight at RT in a 10% methanol/7% acetic acid solution. Gels were then soaked in a 10% glutaraldehyde solution  with gentle shaking and rinsed in water prior to incubation with KOH. The alkali treatment of The mouse IRAK-1 gene cloned into a PCDNA3 plasmid was mutated at different sites within the KTIM using the QuikChange site-directed mutagenesis kit  as instructed by the manufacturer. The primers  designed to create the mutants were:For the tyrosine to phenylalanine mutation;5′GGCTTATACTGCCTTGTTTTTGGCTTCTTGCCCAATGG3′;sense: 5′CCATTGGGCAAGAAGCCAAAAACAAGGCAGTATAAGCC3′.anti-sense: For the leucine to methionine mutation;5′GGCTTATACTGCCTTGTTTATGGCTTCATGCCCAATGG3′;sense: 5′CCATTGGGCATGAAGCCATAAACAAGGCAGTATAAGCC3′.anti-sense: For the glycine to alanine, phenylalanine to tyrosine, leucine to methionine triple mutation, sequential mutagenesis was performed where the above-mentioned leucine mutation was used as the template to generate an additional glycine to alanine mutation using the primers:5′GGCTTATACTGCCTTGTTTATGCCTTCATGCCCAATGG3′;sense: 5′CCATTGGGCATGAAGGCATAAACAAGGCAGTATAAGCC3′.anti-sense: Finally, a phenylalanine to tyrosine mutation was generated using the previously described double mutation as a template using the primers:5′GGCTTATACTGCCTTGTTTATGCCTACATGCCCAATGG3′;sense: 5′CCATTGGGCATGTAGGCATAAACAAGGCAGTATAAGCC3′.anti-sense: 5′TTCCTCCACCAAGTCAAG3′ and 5′CCTGAGGAGTACATCAAGAC3′.All mutations were verified by sequencing the entire plasmid using the T7 and SP6 primers  and the internal primers 5′-CGG ATT TGG CCG TAT TGG GCG CCT-3′ and 3′- ACA TAC TCA GCA CCG GCC TCA CCC-5′; IL-12: 5′- GGA AGC ACG GCA GCA GAA TA-3′ and 3′-AAC TTG AGG GAG AAG TAG GAA TGG-5′.RNA was extracted from MØs using TRIzol reagent . Reverse transcription was performed using oligodT. Quantitative Real-Time PCR (qRT-PCR) was performed with a Corbett Research Rotorgene , using Invitrogen Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen) and 0.4 µM primer in 25 µl. qPCR program is: 50°C 2 min; 95°C 3 min;  for 40 cycles followed by a melting curve. All primers annealing temperature was 60°C. Oligo sequences are: GAPDH: 4 cells/100 µl/well in RPMI-1640 (5% FBS) medium and incubated for 24 h until obtaining a monolayer. Supernatants from designated experiments were added to L929 cells and serially diluted in the presence of actinomycin D (2 µg/ml). After incubation , the L929 monolayers were stained with crystal violet, washed with distilled water, and left to dry. Then, methanol was added to dissolve the stain and cytotoxicity was determined by measuring absorbance at 595 nm. One unit of TNF was referred to as the reciprocal of the dilution that induced 50% of L929 cell lysis.TNF bioassay was performed as previously described NO production was evaluated by measuring the accumulation of nitrite in the culture medium by the Griess reaction, as previously described 32P] dATP radiolabeled double-stranded DNA oligonucleotide for 20 min at room temperature. The DNA binding consensus sequence used for NF-κB was (5′-AGTTGAGGGGACTTTCCCAGGC-3′). Sp1 consensus oligonucleotide was used as non-specific control (5′-ATTCGATCGGGGCGGGGCGAGC-3′) (Santa Cruz). DNA-protein complexes were resolved by electrophoresis in native 4% (w/v) polyacrylamide gels. The gels were then dried and autoradiographed.Nuclear extracts were prepared by a standard protocol, and EMSAs were performed as previously described MØs were collected, lysed in the PTP lysis buffer described previously and kept on ice for 45 min. Lysates were cleared by centrifugation, and protein content was determined by Bradford reagent followed by IP. Equal amounts of IPs were incubated in a phosphatase reaction mix (50mM Hepes (pH 7.5), 0.1% β-mercaptoethanol, 10mM pNPP) overnight at 37°C. OD was then read at 405 nm.http://www.ebi.ac.uk/Tools/emboss/index.html). The accession numbers of the protein sequences included in the study are: human (Homo sapiens) IRAK-1 (P51617), chimpanzee (Pan troglodytes) IRAK-1 (XP_521332), dog (Canis familiaris) IRAK-1 (XP_549367), bull (Bos Taurus) IRAK-1 (Q2LGB3), mouse (Mus musculus) IRAK-1 (Q62406), rat (Rattus norvegicus) IRAK-1 (XP_001057078), tropical frog  IRAK-1 (NP_001006713), zebrafish (Danio rerio) IRAK-1 (XP_697688), human IRAK-4 (Q9NWZ3), chimpanzee IRAK-4 (XP_001166114), rhesus monkey (Macaca mulatta) IRAK-4 (XP_001091707), dog IRAK-4 (XP_543727), bull IRAK-4 (Q1RMT8), mouse IRAK-4 (Q8R4K2), rat IRAK-4 (XP_217026), chicken  IRAK-4 (NP_001025909), zebrafish IRAK-4 (AAT37635), squid (Euprymna scolopes) IRAK-4 (AAY27972), sea urchin (Strongylocentrotus purpuratus) IRAK-4 (XP_784716), Caenorhabditis elegans IRAK-4 (NP_502587), honeybee (Apis mellifera) pelle-like protein (XP_624002), Drosophila melanogaster pelle (NP_476971), chicken IRAK-2 (NP_001025776), mouse JAK2 (Q62120), mouse JAK3 (Q62137), mouse TAK1 (Q62073), mouse Erk1 (Q63844), mouse Erk2 (P63085), mouse JNK (CAC88132), mouse p38 (P47811), mouse IKK-α (Q60680), mouse IKK-β (O88351), mouse LYN (AAH31547).Sequences were obtained from the NCBI protein database. Sequence alignments used to calculate identity and similarity percentages were generated by EMBOSS local pair-wise alignment algorithms program , and immortalized as cell lines. However, experiments done on the animals used in that study (reference 7) adhered to McGill University's guidelines for animal husbandry and was approved by the institutional research ethics committee.−/− MØs and their wildtype (WT) counterparts and subjected the IP to an IRAK-1 kinase assay. Results indicated that IRAK-1 kinase activity in SHP-1−/− cells was significantly higher compared to WT  located in its kinase domain (in vitro binding assays (Kinase Tyrosyl-based Inhibitory Motif). Importantly, these experiments also suggest that the SHP-1-mediated regulation of IRAK-1 is a mechanism not shared with IRAK-4.At the view of our observations, we screened the mouse IRAK-1 sequence for possible SHP-1 binding sites. We discovered that IRAK-1 contains an ITIM-like motif -2-(3-nitrobenzoylamido)-benzimidazole, a potent IRAK-1 inhibitor ll lines . In addiLeishmania as an infectious model, we studied its ability to inhibit key MØ LPS-mediated functions namely: IL-12 expression, TNF production, and NO generation. Our results confirmed that infection with Leishmania caused a significant inhibition of LPS-mediated expression of IL-12 .As cted MØs . To inveectively . InteresLeishmania-mediated IRAK-1 kinase inactivation could alter LPS-mediated functions in infected MØs. Our results indicated that unlike LPS stimulation per se that activates IRAK-1, infection with Leishmania rendered IRAK-1 activation refractory to this TLR4 agonist , flagellin (TLR5), and CpG (TLR9)  . These ron assay .Leishmania can rapidly induce host PTP SHP-1 to inactivate JAK and MAP kinase pathways Leishmania-induced IRAK-1 inactivation observed was associated with an increased SHP-1/IRAK-1 interaction. We indeed noticed by Western blot that a significantly greater amount of SHP-1 was co-immunoprecipitated with IRAK-1 upon Leishmania infection . Interestingly, this Leishmania-induced inactivation was not detected in the absence of SHP-1 (2±6% SD decrease in IRAK-1 activity)  . This re in response to LPS in naïve and mulation .In silico sequence comparisons of available IRAK-1 sequences revealed that KTIM (LVYGFL) was fully conserved from rodents to human , it was absent in zebrafish due to the presence of a methionine at the last position (VIYVYM). Next, we addressed the origin of IRAK-1 and SHP-1 as they are only present in vertebrates. Sequence similarity analyses, including available IRAK-4 sequences from vertebrates and invertebrates  did not contain KTIMs in their amino acid sequence (data not shown), we were intrigued to discover that several kinases from the JAK, MAP, Src, and IKK kinase families  contained one or more potential KTIMs, the majority located within their kinase domains . This fiLeishmania has been reported to inhibit critical LPS-mediated MØ functions such as NO and pro-inflammatory cytokines (e.g. IL-12 and TNF) production Leishmania in response to IFN-γ have been well explored Leishmania-infected cells is limited. Herein, we provide the first demonstration that the Leishmania parasite can rapidly inactivate IRAK-1 kinase activity with the participation of SHP-1, therefore inhibiting MØ LPS-mediated functions. We further reveal that the mechanism by which this inactivation occurs is through the binding of SHP-1 to an evolutionarily-conserved ITIM-like motif located in the kinase domain of IRAK-1. This is the first demonstration that a pathogen can use a host PTP to inactivate IRAK-1 and therefore block signalling pathways ultimately leading to free radicals and pro-inflammatory cytokines production known to be detrimental to its survival.Leishmania, it is not surprising that the parasite has evolved means to block the production of these molecules Leishmania phosphoglycans (PG) has been proposed in the inhibition of NO Leishmania-induced alterations in the Erk MAPK Leishmania. In this study, we confirmed that all three LPS-mediated MØ functions were inhibited by Leishmania. Importantly, looking at NO production as a key function involved in the killing of Leishmania parasites, we were able to show that IRAK-1 signalling is key for its production. In fact, our finding that Leishmania inactivates IRAK-1 kinase activity and that this inactivation is persistent upon subsequent LPS-stimulation supports the fact that the parasite is able to successfully block LPS-mediated NO production in MØs. Interestingly, consistent with the fact that IRAK-1 signals downstream of many TLRs, we showed that IRAK-1 was also unresponsive in Leishmania–infected cells subjected to stimulation with TLR2, TLR5, and TLR9 ligands. This result suggests that the parasite causes wide range unresponsiveness to TLR signalling upon infection possibly allowing Leishmania to avoid any harmful MØ activation involving TLR engagement. Interestingly, L. donovani has been shown to activate IRAK-1 in IFN-γ-primed MØs Leishmania inactivates IRAK-1, we were able to identify SHP-1 as a key player in this process as there was almost a complete rescue of IRAK-1 activity in SHP-1−/− MØs infected with Leishmania. This rescue was corroborated by the parasite's inability to block LPS-mediated NO production in SHP-1−/− MØs. These results suggest a new evasion mechanism whereby Leishmania can avoid detrimental MØ functions driven by MyD88-dependent pathways by blocking IRAK-1, a key kinase in this pathway.Given that TNF is a potent MØ activator, NO is leishmanicidal, and IL-12 is a critical cytokine that drives Th1 responses essential for the development of immunity against Leishmania–mediated IRAK-1 inactivation was associated with enhanced SHP-1 binding to IRAK-1 fits with our finding that SHP-1 binds to and regulates IRAK-1 at resting state. We clearly showed that IRAK-1's intrinsic kinase activity was higher in SHP-1−/− compared to WT MØs identifying SHP-1 as a novel regulator of IRAK-1 activity, a finding supported by recent work of Cao and colleagues Our observation that the Kinase Tyrosyl-based Inhibitory Motif).Having identified an ITIM-like motif in the kinase domain of IRAK-1 as the binding site of SHP-1, its functionality was demonstrated by generating mutations within the motif providing valuable information about the role of its amino acid components in the binding affinity of SHP-1. Firstly, the tyrosine to phenylalanine (Y288F) mutation suggested that the phosphorylation of the motif's central tyrosine is not necessary for the binding of SHP-1 to occur. Indeed it has been previously reported that tyrosyl phosphorylation within ITIMs is not always required for the binding of SH2-domain containing proteins Leishmania-induced SHP-1 had to interfere somehow with the integrity of the previous signalling events. Of utmost interest, we have been able to show that although IRAK-1 was still able to bind MyD88 in Leishmania-infected MØs in response to LPS stimulation, the kinase was unable to detach from the MyD88 complex and bind to TRAF6 as the stimulation persisted. This is the first demonstration that a pathogen can interfere with Toll signalling by altering IRAK-1's capacity to dissociate from the MyD88 complex. This inability of IRAK-1 to detach from MyD88 is supported by our observation that the binding of Leishmania-induced SHP-1 to the kinase domain of IRAK-1 causes a strong inactivation of this kinase seen by its inability to autophosphorylate, a process required for IRAK-1 to detach from the receptor complex and activate downstream signalling cascades.In MyD88-dependent signalling pathways, binding of TLR ligand to its corresponding receptor causes a rearrangement of the receptor complex and triggers the recruitment of the adaptor protein MyD88, which in turn recruits the kinases IRAK-4 and IRAK-1 to the receptor complex Finally, it was remarkable to find out that the KTIM in IRAK-1 was evolutionarily conserved from human to rodents. The absence of KTIM in fish and its appearance in amphibians suggests that this motif emerged rapidly after the appearance of the ancestral IRAK-1 gene in early branching vertebrates (amphibians) and was highly conserved thereafter . Our finLeishmania-activated SHP-1 binds to an evolutionarily conserved KTIM located in IRAK-1's kinase domain leading to its inactivation. This abrogation was associated with the inability of IRAK-1 to detach from the MyD88 complex to bind TRAF6, consequently resulting in the unresponsiveness of Leishmania-infected macrophages to several TLR ligand stimulation including LPS. By doing so, the parasite is not only able to block LPS-mediated MØ production of NO and pro-inflammatory cytokines known to be involved in Leishmania killing, but also terminate the extremely important roles played by these molecules in the development of an effective adaptive immune response. At the evolutionary level, we propose that the appearance of SHP-1 as a key regulator of IRAK-1 kinase activity represented a pivotal evolutionary step that could have favoured the development of the adaptive immune response in vertebrates.In conclusion, we have identified a new evasion mechanism whereby Figure S1IRAK-1 contains a KTIM motif in its kinase domain. The full amino acid sequence of mouse IRAK-1 has been obtained from the NCBI protein database (Ref. no. Q62406). The newly identified KTIM is in violet. Bottom drawing is a schematic representation of the IRAK-1 protein showing the locations of the different domains and critical residues. KTIM motif is shown as a violet rectangle. ProST, Proline/Serine/Threonine -rich.(1.56 MB TIF)Click here for additional data file.Figure S2Among the IRAK family, KTIM is unique to IRAK-1. All other IRAK family members  whose sequences are available for various invertebrate and vertebrate organisms lack a KTIM. A sequence comparison in the KTIM region among the different IRAK family members is shown. Mouse was chosen as a representative organism.(0.38 MB TIF)Click here for additional data file.Figure S3TLR ligands activate NF-κB in stimulated MØs. Gel represents an electromobility shift assay (EMSA) showing NF-κB nuclear translocation in response to a 2 h stimulation with the different TLR ligands used in (0.19 MB TIF)Click here for additional data file.Figure S4Macaca mulatta; Chicken: Gallus gallus; Squid: Euprymna scolopes; Sea urchin: Strongylocentrotus purpuratus; Worm: Caenorhabditis elegans; Honeybee: Apis mellifera; Fly: Drosophila melanogaster.IRAK-4 shows homology to IRAK-1 but does not bear a KTIM due to a single amino acid substitution. IRAK-4 sequence comparison of various vertebrates and invertebrates reveal that IRAK-4 has no KTIM due to a single leucine to methionine/isoleucine substitution. All IRAK-4 homology percentages were calculated using the human IRAK-4 sequence as a reference. IRAK-1/IRAK-4 homology percentages were calculated within the same species. Rhesus monkey: (1.72 MB TIF)Click here for additional data file.Figure S5Regulation of IRAK-1 by SHP-1 through its binding to KTIM is unique to vertebrates and may have favoured the development of their adaptive immune response. Schematic representation of the emergence of IRAK-1 and SHP-1 from IRAK-4 and SHP-2, respectively. Unlike TOLLIP and SHP-2 which are found in invertebrates, SHP-1 arose in vertebrates just like IRAK-1 and KTIM, coinciding with the emergence of the adaptive immune response.(1.54 MB TIF)Click here for additional data file.Alternative Language Abstract S1Translation of the Abstract into Arabic by Issa Ayoub Abu-Dayyeh(0.02 MB DOC)Click here for additional data file.Alternative Language Abstract S2Translation of the Abstract into French by Marceline Côté(0.02 MB DOC)Click here for additional data file.Alternative Language Abstract S3Translation of the Abstract into Spanish by Maria-Adelaida Gomez and Irazu Contreras(0.02 MB DOC)Click here for additional data file.Alternative Language Abstract S4Translation of the Abstract into Farsi (Persian) by Kasra Hassani(0.02 MB DOC)Click here for additional data file.


We previously reported that combined treatment with melphalan and mild hyperthermia (1 h at 42 degrees C) caused a synergistic cytotoxic effect in JR8 melanoma cells, paralleled by a stabilisation of a melphalan-induced G2-phase cell block. In this study, we investigated the effect of melphalan and hyperthermia on proteins that regulate G2-M transition. Neither hyperthermia nor melphalan at a concentration of 2.5 micrograms ml-1, which had no antiproliferative effect at 37 degrees C, interfered with cyclin B1 expression or p34cdc2 kinase activity. At a concentration of 8.5 micrograms ml-1, which reduced cell growth by 50% at 37 degrees C, melphalan inhibited p34cdc2 kinase activity as a consequence of an increased tyrosine phosphorylation of the protein. A similar inhibitory effect on p34cdc2 kinase was obtained when the lowest melphalan concentration (2.5 micrograms ml-1) was used under hyperthermic conditions. Our results indicate that thermal enhancement of melphalan cytotoxicity could be mediated at least in part by an inhibition of p34cdc2 kinase activity, which prevents cell progression into mitosis.


In the visual cortex, as in many other regions of the developing brain, excitatory synaptic connections undergo substantial remodeling during development. While evidence suggests that local inhibitory synapses may behave similarly, the extent and mechanisms that mediate remodeling of inhibitory connections are not well understood.Using scanning laser photostimulation in slices of developing ferret visual cortex, we assessed the overall patterns of developing inhibitory and excitatory synaptic connections converging onto individual neurons. Inhibitory synaptic inputs onto pyramidal neurons in cortical layers 2 and 3 were already present as early as postnatal day 20, well before eye opening, and originated from regions close to the recorded neurons. During the ensuing 2 weeks, the numbers of synaptic inputs increased, with the numbers of inhibitory (and excitatory) synaptic inputs peaking near the time of eye opening. The pattern of inhibitory inputs refined rapidly prior to the refinement of excitatory inputs. By uncaging the neurotransmtter GABA in brain slices from animals of different ages, we find that this rapid refinement correlated with a loss of excitatory activity by GABA.Inhibitory synapses, like excitatory synapses, undergo significant postnatal remodeling. The time course of the remodeling of inhibitory connections correlates with the emergence of orientation tuning in the visual cortex, implicating these rearrangements in the genesis of adult cortical response properties. Inhibitory circuitry in the cortex is generated through a diverse array of specific types of inhibitory neurons. Local inhibitory interactions target specific regions of neurons and act in particular layers of cortex . GeneratPyramidal neurons form roughly 80% of neurons in layers 2/3 and 5 and send long axon collaterals horizontally to interconnect functionally similar domains. During development of layers 2 and 3 of primary visual cortex, an initially crude pattern of horizontal connections gives rise to a lattice of intrinsic excitatory connections that specifically links iso-orientation columns . The devInhibitory interneurons in layers 2 and 3 comprise a diverse class of cells with specific patterns of local axonal arborizations and connections ,15. AnatOne mechanism that may contribute to the formation of the mature pattern of inhibitory inputs is the ability of gamma-aminobutyric acid (GABA) to act as an excitatory neurotransmitter early in development . In a nuTo examine the mechanisms underlying the development and rearrangement of functional inhibitory synaptic connections, we used scanning laser photostimulation in tangential brain slices of ferret visual cortex to map the patterns of inhibitory and excitatory inputs within layer 2/3 onto single neurons at different times during development. By distinguishing excitatory and inhibitory inputs, we find that both types of local connections undergo developmental remodeling. Refinement of inhibitory connections to a mature pattern occurs before the refinement of excitatory connections and is correlated with the timing of the emergence of direction selectivity. Building on findings that remodeling of GABAergic connectivity may, in part, rely on GABA acting as an excitatory neurotransmitter, we used photostimulation to uncage GABA and explore when during development it acted as an excitatory transmitter. Consistent with previous reports in many developing systems, during early development GABA uncaging causes excitation. Interestingly, the change from excitatory to inhibitory transmitter by GABA correlates with a period of rapid refinement in inhibitory inputs. Taken together, these findings provide a detailed description of the maturation of excitatory and inhibitory inputs in the super granular layers of ferret visual cortex.A total of 76 cells, at different postnatal ages, were analyzed using glutamate uncaging and photostimulation Table . Here tht-test, P < 0.02). Similar increases were seen at all distances greater than 250 μm . In both age groups the density of inputs declined with increasing distance from the recorded neuron 20 to P22), inhibitory synaptic inputs onto each recorded neuron could be evoked by photostimulation at numerous sites . Within 250 μm of the cell body, the decline in sites evoking IPSCs was not significant . Thus, during the period immediately after eye opening a significant fraction of the inhibitory synapses onto cells disappears , the percentage of stimulated sites that evoked inhibitory inputs at distances greater than 250 μm from the recorded neurons declined by half , but the three-fold decline at longer distances (>250 μm) was highly significant . In mature animals, most sites generating IPSCs were located within 250 μm of the recorded neurons and almost all were within 500 μm. Local inhibitory inputs formed a largely radially symmetric distribution around the recorded neurons, and patchy patterns of long-range inhibitory inputs were robust at any age. We interpret these findings to indicate that inhibitory inputs transition through a period during which longer-range inhibitory connections are preferentially eliminated and that long-range inhibitory influences are sparse. Finally, although nearby inputs also decline during this period, they are more likely to be maintained as neurons attain the mature patterns of inhibitory inputs.While the pattern of inhibitory inputs seen by P36 to P40 was adult-like, the percentage of sites generating IPSCs continued to decrease after P41. The decline close to the cell bodies was not significant . After P41, the number of both local and longer-range inputs decreased; inputs from regions beyond 500 μm, although sparse, were frequently found in groups. This probably reflects the emergence of clustered horizontal connections , both excitatory and inhibitory inputs (green), or inhibitory inputs only (blue) Figure . The strBetween P20 and P22, excitatory inputs dominated the regions close to the recorded cells (<250 μm), and more distant locations were dominated by inhibitory inputs Figure . The rat2, P < 0.05). Second, the region dominated by inhibitory inputs in younger animals (beyond approximately 300 mm from the cell body) becomes sparse with inputs but is dominated by excitation in older animals .The relationships between inhibition and excitation undergo two major shifts between P20 and P22 and older ages. First, in the youngest animals a much larger area close to recorded neurons was dominated by excitatory inputs , we found numerous locations that gave rise to synaptic currents Figure . Betweenin vitro and changes in and the rapid refinement of inhibitory inputs observed.Using a functional assay for the organization of synaptic inputs, we found that both inhibitory and excitatory synaptic currents were present as early as P20 in layers 2 and 3 of ferret visual cortex. Both excitatory and inhibitory inputs were initially sparse, but their numbers increased steadily to reach a maximum number and spatial extent by P30 to P35. The number and dispersion of sites generating inhibitory inputs quickly refined to the mature pattern, indicating that inhibitory circuits, like excitatory ones, undergo a period of remodeling during which some connections are functionally lost. The data also suggest that the refinement of inhibitory connections precedes the refinement of excitatory connections, but further work is needed to show this definitively. In the case of inhibitory connections, most of the loss is from sites distant from the neuronal cell body, implying that the region of inhibitory influences becomes increasingly focused as visual behavior begins. Finally, the timing of refinement of inhibitory inputs correlates with a switch in the activity of GABA from excitatory to inhibitory, suggesting a potential link between the ability of GABA to act as an excitatory neurotransmitter We focused on understanding how inhibitory and excitatory inputs develop in the simplified ferret brain slice system. By making tangential slices through the superficial layers of visual cortex, we examined the development of these inputs onto pyramidal cells. By leaving intralaminar and local connections intact, but removing interlaminar connections, we could focus on the emergence of the pattern of inputs just within layers 2/3. The main sources of inhibition in layers 2/3 are basket and chandelier type neurons and inhibitory neuron axons are typically restricted to a single layer -28. TherOur results are consistent with reports that indicate inhibitory connections are formed just before or as excitatory connections begin to develop ,19,24,25The substantial reduction in the number of physiologically observed synaptic inputs seems, at first glance, inconsistent with the well-documented increase in the number of anatomically observed synaptic contacts during development -37. FactOur experiments indicate that during development inhibitory connections in striate cortex undergo a sequence of events similar to that of excitatory connections: they are initially sparse, undergo a period of rapid elaboration, and finally refine to their adult pattern. In systems in which significant remodeling of excitatory projections occurs, neuronal activity has been implicated as a driving force in the process . Theet al. [Emergence of the adult pattern of inhibitory connections coincides closely with the appearance of the mature map of orientation selectivity ,49. Usinet al. , our res2PO4, 1.3 mM MgSO4, 2.1 mM CaCl2, 26 mM NaHCO3, 10 mM dextrose) [2PO4, 1.3 mM MgSO4, 2.1 mM CaCl2, 26 mM NaHCO3, 10 mM dextrose). Slices were transferred to the recording chamber following at least one additional hour of recovery.Slices were prepared using methods previously described ,21. Brieextrose) . Visual 2, 1 mM CaCl2, 10 mM HEPES, 3 mM ATP, 1.8 mM GTP; pH = 7.2) containing 0.1 to 0.4% biocytin . The response to each stimulus and the location of the stimulated site were recorded using custom software.The method for scanning laser photostimulation has been described in detail elsewhere ,51-53. BAfter recording, slices were fixed with 4% paraformaldehyde, cryoprotected, and resectioned at 60 to 80 μm on a freezing microtome. Biocytin labeled cells were visualized by standard immunocytochemical staining techniques  intensified with heavy metals . To faciPSCs were counted throughout each 360-ms recording trace made following photolysis of caged glutamate, using software that discriminates PSCs by monitoring zero crossings in the derivative, the amplitude of the event, and length of the rise and decay time of the event. Inhibitory and excitatory events were discriminated by the sign of the rising phase of the derivative, and by an amplitude threshold. The results of the computer driven analysis were verified by a scientist blind to the position within the map of traces being analyzed. The program recorded the times at which events occurred within the trace, the number of events within the trace, and the location of the stimulus. A second program was then used to score whether a given location produced inhibitory responses only, a mixture of inhibitory and excitatory responses, or excitatory responses only. Maps from both programs were then combined to form a final validated excitatory and inhibitory map. Both sets of analyzed data were then discriminated so that only traces with events occurring within 70 ms of the stimulus were counted. This time was chosen based on the dynamics of the response to photo-uncaging, as the majority of photostimulation evoked spikes occur within this time ,20,21. IACSF: artificial cerebrospinal fluid; EPSC: excitatory post-synaptic current; GABA: gamma-aminobutyric acid; IPSC: inhibitory post-synaptic current; P: postnatal day; PSC: post-synaptic current.The authors declare that he has no competing interests.MBD conducted the experiments and wrote the paper.


Proof-of-concept data is provided for cellular AKT, B-RAF and CamK2δ assays. Importantly, comparable activity profiles were found for well characterized B-Raf inhibitors in TR-FRET assays relying on either promiscuous peptide substrates or a MEK1(WT) protein substrate respectively. Moreover, IC50-values correlated strongly between cellular TR-FRET assays and a gold standard Ba/F3 proliferation assay for B-Raf activity. Finally, we expanded our initial assay panel by screening a kinase-focused cDNA library and identified starting points for >20 cellular Ser/Thr-kinase assays.High-throughput cellular profiling has successfully stimulated early drug discovery pipelines by facilitating targeted as well as opportunistic lead finding, hit annotation and SAR analysis. While automation-friendly universal assay formats exist to address most established drug target classes like GPCRs, NHRs, ion channels or Tyr-kinases, no such cellular assay technology is currently enabling an equally broad and rapid interrogation of the Ser/Thr-kinase space. Here we present the foundation of an emerging cellular Ser/Thr-kinase platform that involves  The channels , 4, repochannels , 6 or Bachannels , 8 have channels -12.Recent advances in automated uHTS technologies have enabled the quantitative and parallel interrogation of arrays of cellular assays , 14. In In vitro, a minimal reagent set of three peptide substrates in conjunction with one phospho-specific antibody [TM-platform, Cisbio International). We have translated this biochemical platform into a cellular format while maintaining the original homogeneous addition-only protocol as well as the HTS-friendly TR-FRET readout [Here, we present the foundation of a cellular Ser/Thr-kinase assay platform that relies on promiscuous peptide substrates for the quantification of intracellular kinase activities. antibody  is suffi readout -21. In tEuropium-conjugated pSTK (61PSTKAY), pAkt (61P15 KAYC) pMEK1/2 (61P17KAYC), cMyc (61MYCKLB) antibodies and XL665-conjugated FLAG (61FG2XLB) and His6 (61HISXLB) antibodies were purchased from Cisbio, US. Unconjugated B-Raf antibody was from Santa Cruz Biotechnology (sc-5284), pMEK1/2 antibody from Cell Signaling Technology (9121), anti-FLAG M2 from Sigma. TR-FRET assays were performed in white tissue culture 384-well or 1536-well plates (Greiner Bio One). pcDNA5TO plasmid, HEK293 T-Rex cells, hygromycin, zeocin, and blasticidin were purchased from Invitrogen. Plasmid pSEMS1-26m was obtained from Covalys. Wortmannin and LY294002 were from Calbiochem and staurosporine from Sigma. Other reference compounds were synthesized at GNF.i.e. the SNAP-tag [Peptide substrate sequences were epitope-tagged and fused to AGT, SNAP-tag -24. GeneSNAP-tag . For simFull-length B-Raf wild-type (WT) and B-Raf(V600E) (NM_004333) cDNAs were subcloned from pcDNA3.1 into pcDNA5TO (Invitrogen) using HindIII/ Not1 restriction enzyme sites. The kinase-inactive (KD) double mutant B-Raf  was generated by site-directed mutagenesis. Akt1 (NM_005163) cDNA was subcloned from pcDNA3.1 into pcDNA5TO (Invitrogen) using BamHI/XbaI restriction enzyme sites and a C-terminal His-tag was added to the amino-terminus by PCR. The kinase-inactive AKT-KD (K179M) point mutant was generated by site-directed mutagenesis. HEK293 T-Rex cells (Invitrogen) were cultured in DMEM supplemented with 10% fetal bovine serum (Hyclone) and 5μg/ml blasticidin. Stable pools of cells constitutively expressing either cSTKpeptide or MEK1 protein substrates were generated by transfection of cDNA plasmids and selection in G418 (1 mg/ml) or zeocin (200 μg/ml), respectively. Double stable cell lines expressing tetracycline-inducible kinases were subsequently generated by transfection of these “substrate-only” HEK293 T-Rex cells and selection in medium containing 175μg/ml hygromycin B.HEK293 cells were transfected using Fugene 6 (Roche) at a density of 8000 cells/well in 384-well plates and a final transfection volume of 30μl. The amounts of kinase and substrate plasmids per well varied for each experiment and are specified in the figure legends. Cell lysis and detection of phosphorylated or total substrate levels was performed 48h post transfection by addition of 30μl detection buffer  containing anti-FLAG XL665 (2ng/ul) and Eu-pSTK (0.18ng/μl) or Eu-pMEK1 (0.24ng/μl). For the relative determination of total substrate levels 30μl detection buffer was added per well containing anti-FLAG XL665 (2ng/μl) and Eu-cMyc (0.3ng/μl). Plates were incubated for 3-6h at room temperature and TR-FRET readings taken on Acquest, Analyst GT (Molecular Devices Corp.), Envision or Viewlux (Perkin Elmer) plate readers. Emission readings at 671nm and 618nm were expressed as a ratio and multiplied by a constant value of 100 to obtain “TR-FRET Ratios”.cDNA libraries and screening protocols were applied essentially as previously described , 26. BriFor LMW screening purposes in 384-well plates HEK293 T-Rex cells were either transiently transfected with tetracycline-inducible kinase and substrate constructs at a previously optimized ratio following the above mentioned reverse transfection protocol. Alternatively, double stable cells were plated directly at a density of 8,000cells/well in a volume of 30μl. 24h post plating, tetracycline (1μg/ml) was added to induce kinase expression for approximately 16h prior to compound addition. Kinase inhibitors were resuspended in DMSO and 12 point 1:3 serial dilutions were prepared. 100nl of compound stocks were pintool transferred into the assay plates resulting in a final DMSO concentration of 0.3%. After a 2h incubation period TR-FRET detection reagents detection reagents were added. For 1536-well plate screening cells were either transiently transfected in bulk or stable cells were seeded directly. For transient bulk transfections the transfection reagent mixture was prepared according to the following ratios: plasmid DNA: fugene 6: serum Free media = 1μg: 3μl: 21μl. For every 1ml of cells 1.25μg of kinase plasmid, 0.156μg of substrate plasmid (8:1), 4.22μl of Fugene 6 and 30μl of serum free media was prepared. The transfection mixture was then incubated at room temperature for 30 min and mixed into a HEK293 cell suspension (400 cells/μl). 2,000 cells/well were dispensed in 1536-well plates in 5μl media volumes and incubated at 37˚C. Kinase expression was induced in stable cells after 24h incubation through the addition of 1μl tetracycline (5μg/ml) followed by an additional 4-16 h incubation time before compound addition. Compound treatment in transient transfection experiments occurred 48h post seeding. In either case, 50nl DMSO stocks were pintool transferred into each well and incubated at 37˚C for a 2h period before addition of 4μl detection reagents. TR-FRET readings were taken after another incubation time of 4-24h. Z’ scores were determined as described .2EDTA, 1 mM EGTA, 1% Triton, 2.5mM sodium pyrophosphate, 1 mM Beta-glycerophosphate, 1 mM Na3VO4, 1ug/ml leupeptin, supplemented with 1X protease inhibitors (Sigma). Cellular extracts were resolved by SDS-PAGE , transferred to nitrocellulose membranes, and immunoblotted with the indicated antibodies. Immunolabeled proteins were visualized through Odyssey infrared imaging , with IRDyeTM800  and AlexaFluor® 680 (Molecular Probes)-conjugated fluorescent secondary antibodies.HEK293 cells were harvested 48h post transfection in cell lysis buffer -Ba/F3 cell line was generously provided by Markus Warmuth (GNF). Ba/F3 cells were counted and diluted to a final concentration of 75,000 cells/ml in RPMI media and 50μl/well were plated into 384-well white solid bottom plates. 50nl of a 10mM compound stock in DMSO were pintool transferred followed by a 48h incubation time at 37°C for 48h. Finally, 25μl Cell-Titer-Glo (Promega) detection reagent was added each and luminescence read were taken on a CLIPR (Molecular Devices Corp.) reader.1A, [In vitro, the incubation of purified kinases with biotinylated peptide substrates leads to substrate phosphorylation quantifiable by TR-FRET [Cisbio’s KinEASE platform utilizes a minimal reagent set of three promiscuous peptide substrates (STK-peptides) sharing a common Ser-phosphorylation site and one corresponding phospho-specific antibody (pSTK) to support a panel of >100 biochemical Ser/Thr-kinase assays . In vit TR-FRET . We soug1B). The added epitope tags enable the quantification of either total substrate expression or substrate phosphorylation. To have a reference for peptide based cellular Ser/Thr-kinase assays we developed MEK1 as a model full-length protein substrate of kinases such as B-Raf or B-Raf(V600E), an activated oncogenic kinase mutant and established cancer drug target [To this end, we flanked the STK peptide sequences with N-terminal Myc- and C-terminal Flag-epitopes. The resulting oligopeptides were fused to hAGT carrier proteins (“SNAP-tag”) -24 to sug target , 32.2A, C and E). Similar observations were made with peptide substrates cSTKpep-1 and cSTKpep-2 .When the cSTKpep-3 substrate was expressed alone in HEK293 cells the observed basal phosphorylation levels derived from endogenous Ser/Thr-kinase activities were low, barely detectable by TR-FRET with weak phospho-specific bands being detectable by western blotting . Moreover, B-Raf(V600E)-induced cSTKpep-3 phosphorylation was both dependent on the kinase activity as well as the presence of the target phosphorylation site since no cSTKpep-3 phosphorylation was detected when using a kinase-inactive mutant version of B-Raf(V600E), B-Raf(V600E)-KD or a Ser/Ala-mutant of cSTKpep-3 (cSTK pep-3-SA) .Cotransfection of cSTKpep-3 with B-Raf(V600E) resulted in a several fold increase in substrate phosphorylation by both western blotting and TR-FRET . Comparable results were obtained when using kinase-inactive, MEK1(KD), as a substrate ; cotransfection of MEK1 with B-Raf(V600E) resulted in increased MEK1 phosphorylation as detected by both western blotting and TR-FRET.As expected, cSTKpep-3 phosphorylation could be modulated by both the amounts of substrate and kinase plasmids transfected as well as by the activation state of the transfected kinase .For the pharmacological evaluation we generated double stable cells constitutively expressing either cSTKpep-3, wild-type MEK1(WT) or kinase-inactive MEK1(KD) substrates in combination with Tet-inducible B-Raf(V600E). Kinase inhibitors were then tested in 384-well or 1536-well formats. In all cases short compound treatment times of around 2h were employed to minimize cytotoxicity related compound interference . Good agreement was also observed when comparing TR-FRET assay results with historical Ba/F3-proliferation data for B-Raf(V600E) . None of these B-Raf inhibitors affected basal cSTKpep-3 and MEK1(WT) background phosphorylation in the absence of prior B-Raf(V600E) induction (data not shown). Finally, a selection of unrelated kinase inhibitors, including Wortmannin and LY29002 did not show any activity in either the cSTKpep-3 or MEK1(WT) TR-FRET assays (Table 1).Overall, cellular TR-FRET assays performed very robustly with B-Raf inhibitor A in both 384-well and 1536-well plates for MEK(WT), MEK(KD) or c-STKpep-3 substrates with Z’=0.6-0.9. Moreover, the observed potencies of a panel of selective in-house B-Raf inhibitors correlated very well in cSTKpep-3 and MEK1(WT) TR-FRET-assays (R1), pronounced discrepancies were reproducibly observed for the activities of the highly selective MEK1/2 inhibitor PD0325901 [in vitro. Both compounds had comparable activities when using cSTKpep-3 or MEK1(WT) as B-Raf(V600E) substrates, but were inactive when using MEK1(KD). Similar observations of substrate dependent inhibitor profiles in cellular MEK-phosphorylation assays were recently made using an ELISA readout [While B-Raf selective compounds compared very well in the above assays . In the absence of other efficacious AKT inhibitors staurosporine was used as a tool compound and found to effectively inhibit AKT-dependent cSTKpep-3 phosphorylation . Its cellular potency (IC50 ~100nM) agreed with the in vitro activity against AKT (IC50~100nM). Comparable IC50 values were found for incubation periods ranging from 30min (IC50~240nM) to 2h (IC50~100nM) (data not shown).For a second case study we turned to AKT and its upstream activation pathway. AKT-dependent intracellular cSTKpep-3 phosphorylation could be detected by western blotting and by TR-FRET . After short 2h treatment staurosporine exclusively affected cSTKpep-3 phosphorylation, but not cSTKpep-3 expression levels. After an extended 48h treatment stauros-porine affected phospho- as well as total-cSTKpep-3 levels with equal apparent potency, suggesting that the occurrence of cytotoxicity inappropriately interfered with the assay readout.To determine if staurosporine-induced cytotoxicity and the subsequent loss of kinase substrate expression affected the outcome of the cellular assays, we aimed to separate the effect of staurosporine on cSTKpep-3 phosphorylation from its effect on total cSTKpep-3 expression . To this end we developed another TR-FRET assay using His-tagged AKT to monitor Ser473 phosphorylation.Next, we aimed to interrogate the AKT pathway in greater detail by separating AKT activation as indicated by Ser473 phosphorylation, from signaling events depending on activated AKT such as cSTKpep-3 phosphorylation  and cSTKpep-3 phosphorylation  assays. Moreover, the observed potencies of Wortmannin and LY294002 in the low nanomolar and low micromolar concentration ranges respectively were in line with their reported in vitro and cellular activities on PI3K [50~6μM) versus p-cSTKpep-3 (IC50~60nM). These cellular activities mirror the in vitro potency of staurosporine on PI3K (IC50~9μM) and AKT (IC50~100nM).As predicted for inhibitors of AKT activation, the PI3K inhibitors LY294002 and Wortmannin displayed comparable potencies in both AKT phosporylation . Of these 22 Ser/Thr-kinases, 13 were already known members of the biochemical KinEASE-panel, whereas 9 kinases were newly identified. Nine out of the 13 previously known kinases preferred the same STK peptide substrate in vitro and in cells (Table 2). No Tyr-kinases were found to stimulate cSTKpep phosphorylation, suggesting that no indirect Tyr-kinase induced activation of endogenous Ser/Thr-kinase pathways triggered cSTKpep phosphorylation. Overall, these observations are consistent with the idea that the intracellular kinase-cSTKpep interactions were direct.Having shown proof-of-concept for two important drug targets AKT and B-Raf, we sought to identify additional Ser/Thr-kinases amenable to our cellular assay approach through cDNA screening. A kinase focused sublibrary of GNF’s genome scale cDNA collection , 26 compvia protein engineering, e.g. through the deletion of autoinhibitory domains or activating mutations, may be required beyond their plain overexpression to induce cSTKpep phosphorylation. The subcellular localization of kinases or their substrates, cell cycle dependency, cellular backgrounds and the dynamics of signaling pathways all may play a role. By rescreening our kinase cDNA library under modified assay conditions, e.g. in the presence of external stimuli, nuclear targeted substrates, different cell types, or alternatively through the design of an entirely new cDNA library of activated kinase constructs we aim to further expand the current panel of cellular Ser/Thr-kinase assays in the future. In the case of CamK2δ we found that transient activation of kinase activity and subsequent cSTKpep-1 phosphorylation was ionomycin inducible . We believe that assay conditions can be found to activate additional Ser/Thr-kinases in a similar manner.While a number of kinases already represented on the biochemical KinEASE panel were picked up through DNA screening, others were missed. Several reasons might help explain these findings. Preactivation of Ser/Thr-kinases either through physiologic stimuli or 50 values. This specific feature of the presented cellular TR-FRET kinase platform tremendously benefits HTS applications as it avoids artifactual cytotoxic hits or non-specific transcriptional inhibitors which tend to interfere with traditional proliferation or reporter gene assays of Ser/Thr-kinase activity. The builtin option to quantify relative substrate amounts as well as substrate phosphorylation by TR-FRET and thereby to normalize for substrate expression levels further helps to sort out non-specific compound interference. Finally, having validated cellular TR-FRET assays for two important drug targets, we expanded our assay concept to seed a panel of currently >20 cellular Ser/Thr-kinase assays through cDNA screening. While this initial panel will mainly target full-length wild-type kinases which are constitutively activated upon overexpression in HEK293 cells, a future generation of assays can be envisioned to include also engineered Ser/Thr-kinases or to involve kinase activation through physiologic stimuli. Wherever high-quality phospho-specific antibodies are available, protein kinase substrates will be incorporated into our panel as well. However, due to the very limited availability of such reagents it is expected that a rapid expansion and a significant coverage of the Ser/Thr-kinome will be initially achieved through TR-FRET assays relying on more broadly applicable peptide substrates like cSTKpep-1, 2 and -3 in conjunction with the corresponding universal p-STK antibody [Here we present a starting point for the construction of a new cellular Ser/Thr-kinase assay platform that is tailored towards automated high density compound profiling and primary uHTS. We demonstrated that generic peptide substrates can form a viable basis for the determination of intracellular Ser/Thr-kinase activities and that a homogeneous TR-FRET protocol can be formulated to detect peptide phosphorylation in a sensitive, quantitative and automation-friendly manner. For B-Raf we showed that peptide- and protein-based cellular TR-FRET assays returned well correlated activity profiles for a panel of tool compounds ranging from highly selective to relatively promiscuous kinase inhibitors. Despite the overexpression of target kinases high-potency inhibitors could be readily identified as such. In addition, overexpressed target kinases like AKT and CamK2δ remained sensitive to upstream activating signals. Importantly, short compound incubation times on the order of 1-2 hours were sufficient to obtain robust ICantibody , 21. In