The Hippo signaling pathway has emerged as a critical regulator for organ size control. The serine/threonine protein kinases Mst1 and Mst2, mammalian homologs of the Hippo kinase from Drosophila, play the central roles in the Hippo pathway controlling the cell proliferation, differentiation, and apoptosis during development. Mst1/2 can be activated by cellular stressors and the activation of Mst1/2 might enforce a feedback stimulation system to regulate oxidant levels through several mechanisms, in which regulation of cellular redox state might represent a tumor suppressor function of Mst1/2. As in Drosophila, murine Mst1/Mst2, in a redundant manner, negatively regulate the Yorkie ortholog YAP in multiple organs, although considerable diversification in the pathway composition and regulation is observed in some of them. Generally, loss of both Mst1 and Mst2 results in hyperproliferation and tumorigenesis that can be largely negated by the reduction or elimination of YAP. The Hippo pathway integrates with other signaling pathways e.g. Wnt and Notch pathways and coordinates with them to impact on the tumor pathogenesis and development. Furthermore, Mst1/2 kinases also act as an important regulator in immune cell activation, adhesion, migration, growth, and apoptosis. This review will focus on the recent updates on those aspects for the roles of Mst1/2 kinases. Drosophila through genetic screens for regulators of organ size. The loss of function (LOF) mutant of the protein kinase “Hippo” exhibits tissues overgrowth and tumorigenesis, in which the increased cell number is associated with the acceleration of cell cycle progression and a failure of developmental apoptosis [The Hippo pathway plays a very important role in controlling cell proliferation and differentiation, and monitoring organ size and oncogenesis. This pathway was first discovered in poptosis -5. The Hpoptosis ,7 and thpoptosis  as well poptosis ,10. Sav poptosis ,12, a trpoptosis ,14 , a cysteine-containing, highly conserved enzyme that reduces H2O2 to H2O and O2, interacts with Mst1 under conditions of oxidative stress and Prdx1 is required for Mst1 activation by H2O2, as knockdown of Prdx1 is associated with loss of Mst1 activity . ChernofIncreased production of ROS during pro-oxidant conditions would lead to Mst1/2 activation resulting in phosphorylation of GABP, inhibition of its transcription activity, and downregulation of YAP expression, consequently decreased the expression of a variety of genes that encode mitochondrial proteins and proteins with antioxidant properties, resulting in increased cellular ROS and a diminished GSH/GSSG ratio . On the Drosophila Hippo pathway.The Hippo signaling pathway is a tumor suppresser pathway. Mst1 or Mst2 single knockout mice are viable and do not exhibit obvious organ overgrowth or tumor development, whereas Mst1 and Mst2 double-knockout (DKO) mice exhibit early embryonic lethality ,47. To dWe and other groups have demonstrated that Mst1 and Mst2 are the most potent tumor suppressors in liver and a single copy of either Mst1 or Mst2 can significantly inhibit tumor formation in the liver ,49,50. EDrosophila, Lats1/2 does not serve as the Mst1/Mst2 activated YAP kinase in hepatocytes, indicating the existence of an novel, as yet unidentified intermediary kinase downstream of Mst1/Mst2 that is critical for YAP(Ser127) phosphorylation in the liver [Inactivation of Mst1/Mst2 in liver leads to the loss of YAP(Ser127) phosphorylation and increased YAP nuclear localization. Knocking-down YAP in Mst1/Mst2-deficient HCC cell lines results in massive cell death and cell cycle arrest, similarly, the restoration of Mst1 expression in these cells restores YAP(Ser127) phosphorylation and leads to cell cycle arrest and apoptosis. In contrast to he liver . Howeverhe liver . Neverthhe liver .Mst1−/−Mst2fl/fl-villin-Cre) with ablation of both Mst1 and Mst2 in intestinal compartment are born normal at birth, however they develop colonic adenomas within 3 months old and can only survive for about 13 weeks (median age) accompanied by severe wasting. Both the small and large intestine of Mst1−/−Mst2fl/fl-villin-Cre mice exhibit an expansion of stem-like undifferentiated cells expressing high levels of CD133, Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) and Achaete-scute complex homolog 2 (Ascl2), which are stem cell markers in the intestine, an increased number of cells expressing CD44 and CD24, markers associated with colon cancer stem cells, and an almost complete absence of all secretory lineages. The loss of Mst1/2 in intestine decreases phosphorylation of YAP(Ser127 and Ser384) and causes an increase in both YAP abundance and nuclear localization. The hyperproliferation and loss of differentiation caused by the Mst1/2 deficiency can be entirely reversed by deleting a single YAP allele in Mst1−/−Mst2fl/fl-villin-Cre mouse [Mst1−/−Mst2fl/fl-villin-Cre mouse exhibits similar phenotype to the transgenic mice overexpressing YAP(Ser127Ala) in the small intestinal compartment, wherein intestinal dysplasia and loss of goblet and Paneth cells are also observed [The intestines of Mst1 or Mst2 single knockout mice are indistinguishable from their wild-type counterparts. Mst1/2 intestinal DKO mice  and Wnt targets Lgr5 and Ascl2 [Mst1−/−Mst2fl/fl-villin-Cre mouse. Mst1/Mst2 deficient intestines develop colonic adenomas, and unlike the polyps described in the Sav1-deficient colon [The inactivation of Mst1/2 in the intestine compartment to promote the hyperproliferation of intestinal stem cells and to inhibit intestinal epithelial differentiation is attributed largely to an enhancement of β-catenin action and an activation of Notch signaling. The enhanced β-catenin transcriptional activity in the intestine compartment of nd Ascl2 . The expnd Ascl2 ,55, the nt colon , the polnt colon ,57.The Hippo pathway is necessary for proper development and to preserve homeostasis in the liver and intestines, both of which, as well as the pancreas, are developed from a primitive gut tube derived from the embryonic endoderm . Thus thIn mouse embryo, normal pancreatic differentiation is divided to two stages, the primary transition and the secondary transition. The primary transition occurring between embryonic days 9.5 and 12.5 (E9.5 and E12.5 respectively) marks the appearance of very low levels of acinar digestive enzymes and the first wave glucagon-gene and subsequently insulin-gene expressing cells. The secondary transition (between E13.5 and E16.5) characterized by intense proliferation and differentiation throughout the pancreas epithelium spans the geometric increase of acinar digestive enzymes and insulin . Mst1  cells and developmentally earlier thymocytes. Recently, patients bearing LOF mutations of Mst1 are reported with a primary immunodeficiency syndrome characterized by naïve CD4+and CD8+ T-cell lymphopenia in particular, as well as neutropenia, closely assembling with the major defect of Mst1 deficient mice in lymphocyte homeostasis. Those patients have recurrent bacterial infections, viral infections, and autoimmune manifestations with autoantibodies [Mst1−/−Mst2fl/fl-VavCre mouse) causes a marked exacerbation of the deficits seen in Mst1 deficient T cells, suggesting that Mst2 might play a redundant role in lymphoid tissues during the absence of Mst1 [The murine Mst1 and Mst2 kinases are most abundant in tissues of the lymphoid system. Mst1 kinase acts as an important regulator in T cell selection, adhesion, migration, growth, and apoptosis -73. The tibodies ,74,75. I of Mst1 . The kin+ ICAM-1hi mTECs in a negatively selecting environment. Thus, Mst1 might have a key role in regulating thymocyte self-antigen scanning in the medulla [Mst1-deficient naive T cells proliferate vigorously in response to TCR stimulation and have enhanced ongoing apoptosis in vivo. Mst1, but not Mst2, is greatly reduced in effector/memory T cells compared to that in naïve T cells, thus Mst1 might serves as a likely determinant of the threshold for activation of naïve T cells. Upon the T cell receptor (TCR) stimulation, the increase in tyrosine phosphorylation of CD3ζ, ZAP70, Lck, and PLCγ is similar in splenic T cells from wild-type and Mst1 deficient mice, whereas the phosphorylation of Mob1A/B observed in the wild-type T cells is lost entirely in the Mst1 deficient T cells. Elimination of Mst1 has little effect on the Lats1 carboxyl-terminal phosphorylation, Lats1/2 autophosphorylation and YAP phosphorylation in T cells. Thus the activation of Mob1A/B might serve as the effector of Mst1’s antiproliferative effect in naïve T cells ,71. The  medulla .The mammalian Hippo pathway has generated great interests and gained significant progress in the past few years. In addition to the conserved role of growth control and tumor prevention, the Hippo pathway has also been shown to integrate with other critical signaling pathways, such as Wnt and Notch pathways and extend its function in many other critical biological events. There are still many open questions in the Hippo pathway field remained to be fully elucidated, especially the mechanism by which upstream regulators of the Hippo pathway to initiate or terminate signaling, and how the cellular redox plays a role in this process. Advances in understanding the Hippo signaling pathway regulation may not only solve the scientific questions, such as organ size control and developmental regulations, but also provide new therapeutic targets for human diseases.DKO: Double-knockout; FoxO: Forkhead box protein; GABP: GA-binding protein; GSH: Glutathione; Lats1/2: Large tumor suppressor; LOF: Loss of function; MEFs: Mouse embryo fibroblasts; Mob1A/B: Mps one binder kinase activator-like 1; Mst1/2: Mammalian Ste20-like kinases; Prdx1: Peroxiredoxin-1; ROS: Reactive oxygen species; TCR: T cell receptor; Trx1: Thioredoxin-1; WW45: WW-domain scaffolding protein; YAP: Yes-associated protein.The authors declare that they have no competing interests.All authors drafted and approved the manuscript.


Mycoplasma pneumoniae (Mpn) is a human pathogen that causes acute and chronic respiratory diseases and has been linked to many extrapulmonary diseases. Due to the lack of cell wall, Mpn is resistant to antibiotics targeting cell wall synthesis such as penicillin. During the last 10 years macrolide-resistant Mpn strains have been frequently reported in Asian countries and have been spreading to Europe and the United States. Therefore, new antibiotics are needed. In this study, 30 FDA-approved anticancer or antiviral drugs were screened for inhibitory effects on Mpn growth and selected analogs were further characterized by inhibition of target enzymes and metabolism of radiolabeled substrates.-1, whereas trifluorothymidine, gemcitabine and dipyridamole had MIC values of approximately 2 μg ml-1. In wild type Mpn culture the presence of 6-thioguanine and dipyridamole strongly inhibited the uptake and metabolism of hypoxanthine and guanine while gemcitabine inhibited the uptake and metabolism of all nucleobases and thymidine. Trifluorothymidine and 5-fluorodeoxyuridine, however, stimulated the uptake and incorporation of radiolabeled thymidine and this stimulation was due to induction of thymidine kinase activity. Furthermore, Mpn hypoxanthine guanine phosphoribosyl transferase (HPRT) was cloned, expressed, and characterized. The 6-thioguanine, but not other purine analogs, strongly inhibited HPRT, which may in part explain the observed growth inhibition. Trifluorothymidine and 5-fluorodeoxyuridine were shown to be good substrates and inhibitors for thymidine kinase from human and Mycoplasma sources.Sixteen drugs showed varying inhibitory effects and seven showed strong inhibition of Mpn growth. The anticancer drug 6-thioguanine had a MIC (minimum inhibitory concentration required to cause 90% of growth inhibition) value of 0.20 μg mlMycoplasma nucleotide biosynthesis are potential targets for future design of antibiotics against Mycoplasma infection.We have shown that several anticancer and antiviral nucleoside and nucleobase analogs are potent inhibitors of Mpn growth and that the mechanism of inhibition are most likely due to inhibition of enzymes in the nucleotide biosynthesis pathway and nucleoside transporter. Our results suggest that enzymes in  Mycoplasmas are wall-less, gram-positive bacteria and are pathogenic to humans, animals, and plants -Gua (guanine), [3H]-Ura (uracil), and [3H]-Hx (hypoxanthine) was low (< 1%) as compared with that of [3H]-dT (thymidine) (> 7%). Dipyridamole strongly inhibited the uptake and incorporation of [3H]-Hx and [3H]-Gua into DNA and RNA but had no effect on uptake and metabolism of all other nucleobases and [3H]-dT, suggesting that dipyridamole is a specific inhibitor of purine transport. Similar to dipyridamole, 6-TG also strongly inhibited the uptake and incorporation of [3H]-Hx and [3H]-Gua into DNA and RNA but had no effect on any other nucleobases and dT. Pyrimidine nucleoside analogs, TFT, 5FdU (5-fluorodeoxyuridine) and dFdC, inhibited the uptake and incorporation of all nucleobases. However, [3H]-dT uptake was stimulated (2-fold) by TFT and 5FdU but inhibited by dFdC, and the percentage of radioactivity found in DNA was similar to that of control in all cases  cleavage site at the N-terminus, and was purified to >98% purity by metal affinity chromatography, as assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis (data not shown).The purine analog 6-TG strongly inhibited Mpn growth, which promoted further investigation of potential targets of this compound. HPRT is the first enzyme in the salvage pathway of purine bases for nucleotide biosynthesis, and is the enzyme responsible for metabolizing 6-TG in human patients treated with this drug . Mpn HPRm value of 100.1 ± 6.5 μM and Vmax value of 15.8 ± 0.8 μmol min-1 mg-1  and guanine (Gua) as substrates but not adenine or uracil. With Hx as substrate the reaction was linear with time for up to 25 min and the substrate saturation curve was hyperbolic, which indicated that the enzyme followed Michaelis–Menten kinetics with a K50) values were 3.5 ± 0.5 μM (Hx) and 3.2 ± 0.2 μM (Gua). The 6-TG also inhibited human HPRT, but with a much higher IC50 value  and 281.9 ± 21 μM (Gua). With human HPRT, 6-MP had similar IC50 values to those of 6-TG. Theophylline and caffeine were poor inhibitors of both Mpn and human HPRT , and two other purine analogs that did not inhibit Mpn growth on Mpn HPRT, and compared the results with human HPRT. The 6-TG strongly inhibited Mpn HPRT with either Hx or Gua as a substrate, and the half maximal inhibitory concentration , TK2 , and Ureaplasma TK. Because Ureaplasma TK and Mpn TK share 46% sequence identity and they also share 36% respective 32% sequence identity to human TK1 -hypoxanthine , [3H]-thymidine , [γ-32P]-ATP (2000 Ci/mmol), [3H]-Uracil , and [3H]-adenine  were purchased from PerkinElmer. [3H]-guanine  and [5-3H]-deoxyuridine 5’-monophosphate  were from Moravek Biochemicals, Inc. The nucleoside and nucleobase analogs library -dT (1 μCi ml-1) and different concentrations of TFT. After 70 hours at 37°C the cultures were harvested and divided to two aliquots, one was used to determine total uptake/metabolism of radiolabeled dT and total proteins were extracted from the other aliquot and used to measure TK and TS activity using [3H]-dT and [5-3H]-dUMP as substrates -Hx) or guanine ([3H]-Gua) as substrates, essentially as previously described -Hx or [3H]-Gua in a total of 50 μl. The reaction was initiated by addition of the enzyme, and at 0, 5, 10, and 15 min intervals, 10 μl reaction mixture was withdrawn and spotted onto the DE81 filter paper and dried. The unreacted substrate was washed and the products were eluted and counted in a liquid scintillation counter. With [3H]-Gua as substrate the reaction  was initiated by addition of the enzyme (10 μl), incubated at 37°C for 2 min, stopped by addition of 1 M HCl (10 μl), and placed immediately on ice. After neutralization, 15 μl of the mixture was spotted onto the DE81-filter paper. The filters were then washed, and the products were eluted and counted by liquid scintillation.The HPRT assay was performed by using the DE-81 filter paper assay with tritium labeled hypoxanthine  or [3H]-Gua (10 μM) and variable concentrations of the inhibitors.IC3H]-dT) as substrate and various concentrations of the inhibitors essentially as previously described [50 values of TFT and 5FdU.Thymidine kinase assay was performed using tritium labelled thymidine  and dried. The TLC plates were developed in isobutyric acid/ammonia/H2O (66:1:33). The reaction products were visualized and quantified by phosphoimaging analysis .Kinetic parameters for TFT were determined by using the phosphoryl transfer assays as previously described . Brieflyt-test (two tailed) using GraphPad Prism 5 software. P < 0.05 is considered as significant.The data were analysed by unpaired student’s Both authors declare that they have no competing interests.RS performed the kinetic and inhibitions studies with thymidine kinases, analyzed the data and created the figures; LW designed the study, performed growth inhibition studies, uptake and metabolism of labelled nucleosides, characterized Mpn HPRT; analyzed the data and wrote the manuscript. All authors have read and approved the manuscript.


While Clb1 phosphorylation is dependent on activity of both CDK and polo-like kinase Cdc5, its nuclear localization requires CDK but not Cdc5 activity. Furthermore we show that increased nuclear localization of Clb1 during meiosis enhances activation of FEAR (Cdc Fourteen Early Anaphase Release) pathway. We discuss the significance of our results in relation to regulation of exit from meiosis I.Cyclin-dependent kinases (CDK) are master regulators of the cell cycle in eukaryotes. CDK activity is regulated by the presence, post-translational modification and spatial localization of its regulatory subunit cyclin. In budding yeast, the B-cyclin Clb1 is phosphorylated and localizes to the nucleus during meiosis I. However the functional significance of Clb1's phosphorylation and nuclear localization and their mutual dependency is unknown. In this paper, we demonstrate that meiosis-specific phosphorylation of Clb1 requires its import to the nucleus but not  Meiosis is a specialised form of cell division required for sexual reproduction, in which a diploid cell divides to form four haploid gametes. Two nuclear divisions must be achieved without an intervening round of DNA replication, in order to halve chromosome number Alternation of DNA replication and nuclear division during the mitotic cycle is achieved by oscillations of Cyclin-Dependent Kinase (CDK) activity. As CDK activity rises following exit from G1, DNA replication is initiated. While CDK promotes initiation of DNA replication it also prevents re-initiation of DNA replication by phosphorylating components of the replication machinery Cdc55Cdc55 through a poorly understood mechanism. This results in increased Net1 phosphorylation by CDK Dephosphorylation of CDK substrates and the precipitous drop in CDK activity during anaphase in budding yeast are caused by the release of CDK-antagonising phosphatase Cdc14 The FEAR network-driven Cdc14 release is necessarily transient, as the FEAR network relies on CDK activity to maintain Cdc14 release. As the released phosphatase counters CDK activity, this causes its own resequestration into the nucleolus. MEN mutants, in which Cdc14 release is driven only by the FEAR network, arrest in anaphase with intact spindles Unlike mitosis, the nuclear divisions of meiosis I and II must be achieved without triggering an intervening round of DNA replication. During meiosis I the spindle must elongate, dividing the nuclei, and be disassembled, a task under the control of Cdc14. However, DNA replication complexes must not be relicensed, a process also dependent on Cdc14 release vice versa. We also provide evidence that increased nuclear localisation of Clb1 amplifies FEAR activation during meiosis. We suggest that the meiosis-specific nuclear localisation of Clb1 might facilitate FEAR-mediated disassembly of anaphase I spindles.Localisation changes in situ immunofluorescence were performed as previously described The genotypes of all strains used in the experiments are listed in 600 of 3 were used. For mitotic samples 75 ml samples of cell cultures at OD600 of 1 were used. Samples were bead beaten in lysis buffer  and cleared by spinning twice at 13 K rpm for 15 minutes at 4°C. The lysate was mixed with 1 µl of primary antibody (3F10: Rat-anti-HA Roche or 9E10: Mouse anti-myc Roche) and DTT  and incubated on a rotary wheel at 4°C for an hour. Pre-equilibrated Protein-G dynabeads were added to the lysate and incubated for further 2 hours. Beads were washed thrice in 1 ml lysis buffer. The washed beads were then used in phosphatase treatment or kinase assays.For meiotic samples, 25 ml samples of cell cultures at OD2), and split into 3 samples. 1 µl of Lambda Protein Phosphatase (NEB) was added to the first sample. To the second sample, 1 µl of Lambda Protein Phosphatase (NEB) was added along with a mixture of phosphatase inhibitors . The third sample served as a negative control. The three samples were then incubated at 30°C for 30 minutes with shaking. The reaction was stopped by adding 2x Laemmli sample buffer  and heating at 94°C for 5 minutes.50 µL of beads bearing bound Clb1 were washed and resuspended in 1x NEB buffer for Lambda phosphatase. The assay was performed at 30°C with 50 µM ATP (including 0.25 µCi/µl γ-32P-ATP) and 35 µM or 3.5 µM of Histone H1 for 40 minutes, with 25 µM 1-NM-PP1(+) or an equivalent volume of DMSO (-). Samples were taken at 0, 10, 20 and 40 minutes, and stopped by addition of Laemmli sample buffer and heating. Samples were run on 15% SDS-PAGE gels and analysed using an imaging plate and phosphorimager.35 µl of beads bearing bound protein were washed and resuspended in kinase assay buffer  either under the endogenous promoter or the mitosis-specific promoter CLB2PCLB2CDC20P cells remained mononucleate during the entire experiment  normally, we monitored Clb1 nuclear localization and modification in CLB2CDC55P cells CLB2CDC55 and PCLB2CDC55 PCLB2CDC20P strains , we examined whether Ime2 is required for nuclear localization and phosphorylation of Clb1-TAP. Addition of the inhibitor, 1-NA-PP1, selectively inhibits Ime2-as but not Ime2 CLB2CDC20P strain background to sustain Clb1 modification and nuclear localization. Inhibition of Ime2 by addition of the inhibitor to the cultures after 6, 7, or 8 hours into SPM did not reduce the nuclear concentration or phosphorylation of Clb1-TAP  or Nuclear Export Sequences (NES), and 6 copies of HA epitope. In each case, two sequences were added in tandem to overcome any endogenous localisation signals . The tanpe cells . Expresspe cells , suggestCLB2CDC20P strains, in which Clb1 phosphorylation is maintained Click here for additional data file.Figure S2Kinase activity of Ime2 is not required for nuclear localization and phosphorylation of Clb1. Cultures of CLB2-CDC20 CLB1-TAPIME2 P cells and CLB2CDC20 CLB1-TAPime2-as P cells were induced to enter meiosis by transferring them to SPM. The ATP analogue 1-NA-PP1 (20 µM) or an equivalent volume of DMSO was added to sporulating cultures at 5, 6, 7 and 8 hours into SPM. A) Modification of Clb1 was assayed by subjecting whole cell extracts from the above cultures to SDS-PAGE followed by Western analysis using an anti-TAP antibody. Blots were also probed with anti-myc (to detect Ime2) and anti-tubulin antibody. B) Cells were fixed and examined for Clb1-Myc localisation by in situ immunofluorescence. Proportion of cells with nuclear Clb1 (green), cytoplasmic Clb1 (red) and no Clb1 signals (blue) are indicated.(EPS)Click here for additional data file.Figure S3CDK activity is required for Clb1 phosphorylation and nuclear localization. Cultures of CLB2-CDC20 CLB1-myc9CDC28 P cells and CLB2CDC20 CLB1-myc9cdc28-as P strains were induced to enter meiosis by transferring them to SPM. Samples were taken hourly from 5 hours for whole cell extracts (A) and hourly throughout the time course for imaging (B). 10 µM 1-NM-PP1 was added to sporulating cultures at 6 and 8 hours into SPM.(EPS)Click here for additional data file.Figure S4Fusion of NES and NLS sequences to the C-terminus of Clb1 does not affect CDK-Clb1 activity. Tagged Clb1 was immunoprecipitated from mitotic cultures of CLB2CDC20 cdc28-asP, CLB2CDC20 cdc28-asCLB1-ha P , CLB2CDC20 cdc28-asCLB1-NES-ha P and CLB2CDC20 cdc28-asCLB1-NLS-ha P strains using an anti-HA antibody. A) Immunoprecipitated Clb1 from the three cultures was detected by western blotting. B) Purified Clb1 was incubated with 3.5 µM histone and 50 µM ATP (0.25 µCi/µL of γ-32-ATP) in the kinase buffer for 40 minutes and phosphorylation of Histone H1 after 0, 10, 20 and 40 minutes (in the absence of the inhibitor 1-NM-PP1) was determined. C) Sporulation efficiencies of diploid strains bearing Clb1 Nuclear Localization sequence or Nuclear Export sequences with or without clb1Δ or clb3Δclb4Δ are graphically presented.(EPS)Click here for additional data file.Table S1List of yeast strains. All yeast strains used are derivatives of SK1 and have the following markers, unless otherwise stated stated. ho::LYS2/ho::LYS2, ura3/ura3, leu2::hisG/leu2::hisG, trp1::hisG/trp1::hisG, his3::hisG/his3::hisG, lys2/lys2. The strains used for experiments described in the Main and Supplementary figures are listed in Table 1A and 1B respectively.(DOCX)Click here for additional data file.


Hepatitis C virus (HCV) NS5A protein is phosphorylated on multiple residues; however, despite extensive study, the precise identity of these sites has not been determined unambiguously. In this study, we have used a combination of immunoprecipitation and mass spectrometry to identify these phosphorylation sites. This analysis revealed the presence of a major phosphorylated residue within NS5A from the genotype 1b Con1 isolate – serine 249 (serine 2221 in polyprotein numbering). However, mutation of this residue (or the corresponding threonine in the JFH-1 isolate) to either a phosphomimetic (aspartate) or a phosphoablative  residue resulted in no phenotype. We conclude that phosphorylation of this residue, in the context of a highly culture-adapted HCV genome, does not play a role in either viral RNA replication or virus assembly. It is possible that it might be important in an aspect of virus biology that is not recapitulated faithfully in the Huh-7 cell-culture system. HCV, a member of the family Flaviviridae, is an enveloped virus with a positive-sense RNA genome 9.6 kb in length and containing a single ORF. An internal ribosome entry site mediates cap-independent translation of the 3000 aa polyprotein, which is cleaved co- and post-translationally by host-cell and viral proteases to release the structural proteins  and non-structural proteins .Hepatitis C virus (HCV) is estimated to infect some 123 million individuals  replicon  approach. We showed previously that a recombinant baculovirus containing a tetracycline-responsive mammalian promoter  and 72 h (to assess RNA replication)  Fig. . RelativSerine 249 is almost completely conserved in all genotype 1 isolates of HCV; however, this residue is not conserved in other genotypes . The datcis–trans isomerization of the peptidyl–prolyl bond. Structural studies (e.g. NMR) on purified wild-type and mutant NS5A, phosphorylated in vitro, might shed light on this issue, but represent significant technical challenges.What, then, might be the role of phosphorylation of serine 249? It is possible that, in the context of the culture-adapted FK5.1 subgenomic replicon or the highly efficiently replicating JFH-1 genome, there is no requirement for phosphorylation of this residue. However, in the context of the non-culture-adapted infectious clones , the phosphorylation of serine 249 might be important in some aspect of NS5A function. Indeed, the acquisition of culture-adaptive mutations in NS5A has been shown to correlate with a loss of hyperphosphorylation, even when the mutations were not at serine residues (


Ca2.3 component of endothelium-dependent hyperpolarization (EDH) is lost following stimulation of thromboxane (TP) receptors, an effect that may contribute to the endothelial dysfunction associated with cardiovascular disease. In cerebral arteries, KCa2.3 loss is associated with NO synthase inhibition, but is restored if TP receptors are blocked. The Rho/Rho kinase pathway is central for TP signalling and statins indirectly inhibit this pathway. The possibility that Rho kinase inhibition and statins sustain KCa2.3 hyperpolarization was investigated in rat middle cerebral arteries (MCA).In rat middle cerebral and mesenteric arteries the KMCAs were mounted in a wire myograph. The PAR2 agonist, SLIGRL was used to stimulate EDH responses, assessed by simultaneous measurement of smooth muscle membrane potential and tension. TP expression was assessed with rt-PCR and immunofluorescence.Ca2.3 mediated hyperpolarization, an effect that was reversed by Rho kinase inhibitors or simvastatin. KCa2.3 activity was lost in L-NAME-treated arteries, but was restored by Rho kinase inhibition or statin treatment. The restorative effect of simvastatin was blocked after incubation with geranylgeranyl-pyrophosphate to circumvent loss of isoprenylation.Immunofluorescence detected TP in the endothelial cell layer of MCA. Vasoconstriction to the TP agonist, U46619 was reduced by Rho kinase inhibition. TP receptor stimulation lead to loss of KCa2.3 function. The ability of statins to prevent isoprenylation and perhaps inhibit of Rho restores/protects the input of KCa2.3 to EDH in the MCA, and represents a beneficial pleiotropic effect of statin treatment.Rho/Rho kinase signalling following TP stimulation and L-NAME regulates endothelial cell K Ca3.1  channels, irrespective of the agonist used to stimulate EDH Ca3.1 and KCa2.3  is necessary for block of EDH Ca2.3 Ca2.3 to EDH responses is restored in the middle cerebral artery by exposure to antagonists of thromboxane receptors (TP) Ca2.3 input to EDH in rat middle cerebral and mesenteric arteries Ca2.3 function in the vasculature. The mechanism that ‘protects’ KCa2.3 function during NO signalling or TP inhibition remains unclear.In rat middle cerebral arteries (MCA) endothelium-dependent hyperpolarization (EDH) responses  are observed in the presence of NO synthase (NOS) inhibitors, and can be abolished by inhibition of endothelial cell KCa2.3 channel function by direct interaction/stimulation of the channel Ca channels by binding to the heme groups of enzymes. For example, the cytochrome P450 metabolite 20-HETE inhibits EDH responses in coronary arteries Ca2.3 Ca2.3 function Ca2.3 function is restored by antagonizing TP Ca2.3 function by PKG dependent inhibition of these receptors NO could potentially protect KCa2.3 channels expressed selectively in these cells. The role of Rho kinase signalling on KCa2.3 channel function can be directly assessed using inhibitors of this kinase but the statin class of drugs are also reported to have effects on Rho mediated signalling. They improve endothelium-dependent relaxation via a mechanism that involves inhibition of Rho signalling A major signalling pathway associated with TP is activation of Rho kinase Ca2.3 functionality following TP stimulation; 2) to establish if inhibition of Rho kinase signalling might restore the KCa2.3 component of the EDH response suppressed by the presence of NOS inhibitors, and 3) to assess if statins had a similar effects to inhibitors of Rho kinase.The aims of the current study were 1) to investigate if disrupting the Rho kinase pathway could protect KMale Wistar rats (200–300 g) were humanely killed by cervical dislocation following institutional guidelines for animal welfare and schedule 1 of the Animals (scientific procedures) Act 1986. The brain was removed and immediately placed in ice-cold Krebs solution. Segments of the MCA (∼2 mm long) were dissected and stored in ice-cold Krebs for use within 30 min, with similar size vessels used in all experimental groups.3, 24; KCl, 3.6; KH2PO4, 1.2; MgSO4⋅7H2O, 1.2; glucose, 11.0; CaCl2, 2.5; gassed with 95% O2, 5% CO2 and maintained at 37°C. After equilibration for 20 min, vessels were tensioned to 1–1.5 mN . Smooth muscle tension was recorded with an isometric force transducer and Powerlab software . Vessel viability was assessed by adding exogenous K+ ; only vessels developing tension of ≥3 mN were used. Endothelial cell viability was assessed by the ability of the agonist of protease-activated receptor 2 (PAR2) SLIGRL  to relax spontaneous tone and to hyperpolarize the smooth muscle cell membrane by >15 mV. A concentration of 20 µM SLIGRL was used in all further experiments; our previous study characterized the potency of this agent to evoke endothelium dependent relaxation and this concentration evokes circa 75–80% of the maximal relaxation response Segments of MCA  were mounted in a Mulvany-Halpern myograph  in Krebs solution containing (mM): NaCl, 118.0; NaCO+) with varying concentrations of the TP receptor agonist U46619 (circa 50–100 nM). Under these conditions EDH was assessed in the presence of the KCa channel blockers used at concentrations that should cause full channel inhibition with no off target effects at other KCa channels with respect to their published Ki values, apamin  Ca3.1, 1 µM) Ca1.1, 100 nM) Ca blockers on EDH responses was also assessed after addition of inhibitors of Rho kinase  Ca3.1 contribute to EDH Ca channel blockers were added in the sequence detailed above only if the previous KCa blocker(s) failed to prevent hyperpolarization. KCa channel blockers were assessed against EDH responses to SLIGRL (20 µM) in the presence of: 1. the Rho kinase inhibitors Y27632 (10 µM) 50 for Rho kinase to ensure full inhibition with minimal effects on other, related kinases i values of low to high nanomolar, reported in various rat cell types m) and tension were measured simultaneously (except where stated) at a sample rate of 100 Hz as previously described, using glass microelectrodes  to measure EmIn some experiments endothelium-dependent responses to SLIGRL were obtained in vessels that were able to synthesize NO but were pre-constricted to approximately 80% of maximal tone  and KCa2.3 . Control reactions omitted reverse transcriptase. PCR products were separated by electrophoresis, stained with ethidium bromide, and sequenced to confirm identity.RNA from intact rat MCAs was isolated using Trizol  and was reverse-transcribed using standard protocols with random hexamers and TaqMan reverse transcription reagents (Life Technologies Ltd.). Subsequent PCR reactions used primers specific for rat TP receptor ; rabbit anti-mouse thromboxane A2 receptor  and donkey anti-mouse Cy5 and anti-rabbit Rhodamine Red-X .Arteries were cut longitudinally and pinned flat on sylgard plates and fixed with 100 mM PBS, pH 7.4 containing 4% paraformaldehyde . Artery whole mounts were washed in PBS containing 10% normal horse serum and 0.3% Triton-X-100  and proteins localized using primary antibodies: Thromboxane A2 receptor (1∶100) and PECAM-1  . Whole mounts were washed , incubated with secondary antibodies coupled to Rhodamine Red-X, or Cy5  and mounted in ProLong® Gold  which contains DAPI.Whole mounts were observed with a Zeiss laser-scanning confocal microscope (LSM Meta 510) using an EC Plan-Neofluar 40×/1.3 Oil DIC objective. Images were collected at a zoom of 1.5 and an iris of <3 µm, and at least five optical sections were taken at intervals of 0.5 µm. Single sections are shown. Images were processed using ImageJ and Adobe Photoshop software. The specificity of the immunostaining was evaluated by omission of the primary antibody and processed as above. Under these conditions, no staining was observed in any experimental situation.n animals. Tension values are given in mN  and Em as mV. Vasodilatation is expressed as percentage reduction of the total vascular tone (spontaneous tone plus vasoconstrictor response), quantified by relaxation with papaverine (150 µM). Graphs were drawn and comparisons made using one-way ANOVA with Tukeys’ post-test . P≤0.05 was considered significant. pEC50 values were calculated using non-linear regression fitted to the Hill equation with variable slope .Results are expressed as the mean±s.e.mean of + was added as an isotonic physiological salt solution in which all the NaCl was replaced with an equivalent amount of KCl. Concentrations of K+ used are expressed as final bath concentration. GGPP, L-NAME (NG-nitro-L-arginine methyl ester), papaverine HCl, TRAM-34 (1-[(2-Chlorophenyl)diphenylmethyl]-1H-pyrazole), U46619  and Y27632 ((R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexane carboxamide dihydrochloride) were all obtained from Sigma . Apamin and iberiotoxin, from Latoxan . SLIGRL-NH2 from Auspep . Simvastatin and Lovastatin were from Cayman-Europe . SR5037 ((R)-6-Methoxy-N-(2-(1-methylpiperidin-4-yloxy)-4-(1H-pyrazol-4-yl)phenyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide) was a generous gift from Professor Phil LoGrasso . All stock solutions (100 mM) were prepared in dimethylsulfoxide except L-NAME, apamin, iberiotoxin, papaverine and SLIGRL which were dissolved in 0.9% NaCl. Controls included appropriate vehicle. All blocking drugs were used at appropriate concentrations to ensure full inhibition of their target  and minimal off target effects and are the recommended selective inhibitors as classified by IUPHAR Exogenous KCa2.3 in the MCA by RT-PCR  and the subsequent application of the KCa2.3 blocker apamin (100 nM) had no further effect . However, the residual EDH was suppressed by the KCa1.1 (BKCa) blocker, iberiotoxin , despite a trend toward block, TRAM-34 did not significantly inhibit EDH . However, the subsequent addition of apamin significantly inhibited EDH . The residual hyperpolarization was again inhibited further by iberiotoxin, as in the absence of Y27632  the MCA constricts and EDH is blocked by TRAM-34 alone indicating a critical role for Krization . Thus, iCa2.3 function against TP receptor stimulation produced by U46619. In a separate group of experiments in vessels able to synthesise NO and stimulated with U46619 (50–100 nM) we assessed if statins shared this effect of Rho kinase inhibitors. SLIGRL (20 µM) evoked EDH of 30.9±5.9 mV (n = 7). This response was not modified by 100 nM simvastatin . Subsequently, EDH was not reduced with TRAM-34 , but was progressively and significantly reduced by adding apamin and then iberiotoxin EDH  was not altered by simvastatin  or by the combination of simvastatin and isoprenoid GGPP . However, with GGPP present EDH responses were virtually abolished by the addition of TRAM-34 alone  the statin, simvastatin (100 nM and 1 µM) restored the input of KCa2.3 to EDH  form to inhibit HMG-CoA. It is important to note we used the closed ring (lactone) form of simvastatin (and lovastatin) in this study. These require hydrolysis to the open ring NaCa2.3 channel input to EDH, it remains to be determined how Rho/Rho kinase inhibits KCa2.3. It is possible regulation involves direct phosphorylation of the channel by Rho kinase, or an associated kinase, similar to the regulation of KCa3.1 channels by kinases in immune cells Ca2.3 by remodelling the cytoskeleton; delayed rectifier channels in cerebral artery smooth muscle cells are inhibited by actin polymerization that is Rho kinase dependent Although our data strongly suggest a regulatory role for rho kinase on endothelial cell KCa function appears to involve isoprenoids and Rho-mediated signalling; such protection may help to explain the beneficial effect of statin treatment after stroke Ca2.3 channels located on the endothelium, our data support the rationale for treating endothelial dysfunction and related disorders by targeting Rho signalling.Whatever the precise mechanism, vasoconstrictors such as TP, endothelin-1 and angiotensin II signal via the Rho kinase pathway and receptors for these vasoconstrictors are present on endothelial cells Figure S1Histograms of the mean data for SLIGRL-induced EDH  in the presence of the NOS inhibitor L-NAME (100 and 300 µM). The EDH responses were completely abolished by the KCa3.1 blocker TRAM-34 (1 µM). *P<0.05 indicates a significant difference from the 100 µM L-NAME control group; n = 3.(TIF)Click here for additional data file.Figure S2Histograms of the mean data for SLIGRL-induced EDH mediated  in the presence of the NOS inhibitor L-NAME (100 µM) and the subsequent effect of the statin lovastatin (100 nM). Normally in the presence of L-NAME inhibition of KCa3.1 alone is sufficient to block the EDH response. However, lovastatin revealed a KCa2.3 component to the EDH response. *P<0.05 indicates a difference from control, φP<0.05 indicates a significant difference from lovastatin as determined by one-way ANOVA with Tukey’s post-test, n = 5.(TIF)Click here for additional data file.


Bothrops lanceolatus, a member of the Crotalidae family, is considered to be the only involved snake. B. lanceolatus, commonly named “Fer-de-Lance”, is endemic and only found on this Caribbean island. Envenomation local features include the presence of fang marks, swelling, pain, bleeding from punctures, and ecchymosis. Severe envenomation is associated with multiple systemic thromboses appearing within 48 h of the bite and resulting in cerebral, myocardial or pulmonary infarctions. Diagnosis requires first of all identification of the snake. Coagulation tests are helpful to identify thrombocytopenia or disseminated intravascular coagulation. A clinical score based on 4 grades is helpful to assess envonimation severity. A specific monovalent equine anti-venom  to neutralize B. lanceolatus venom is available. Its early administration within 6h from the biting in case of progressive local injures, general signs or coagulation disturbances is effective to prevent severe thrombosis and coagulopathy. Its tolerance is considered to be good. Despite an increasing incidence of bites, no deaths have been recently attributed to B. lanceolatus in Martinique, probably due to the currently recommended strategy of early antivenom administration when required.Approximately 20-30 declared snakebite cases occurin Martinique each year.  Bothrops lanceolatus , bradykinin, histamine, and serotonin [B. lanceolatus antivenom were shown to immunodeplete B. lanceolatus venoms in vitro and to be fully effective in mice in neutralizing the lethal, hemorrhagic, oedema-forming, myotoxic, and indirect hemolytic activities of the venom [molecule ,8,9. Themolabile . Composiactivity . The aciactivity . In miceerotonin . Both pohe venom ,14,15. B. lanceolatus is the only snake in the world whose venom produces significant systemic thrombotic complications [B. lanceolatus has no coagulant effect, even at concentrations as high as 100 µg/mL [B. lanceolatus venom vis-à-vis other Bothrops venoms. Interestingly, the monospecific B. lanceolatus antivenin is devoid of neutralizing capacity against the procoagulant and defibrinating activities of heterologous Bothrops snake venoms in mice [ications ,16. Deve00 µg/mL . This is in mice .B. lanceolatus leads to swelling and pain, and occasionally to systemic signs and/or coagulopathy. Severity of envenoming by B. lanceolatus depends on the injected amount of venom as well as on the victim’s conditions such as age, body weight, immunity, and past medical history, as described for other crotalidae bites [Envenomation by ae bites . The venClinically, local effects such as pain, bleeding from the fang punctures, swelling, erythema, ecchymoses, and bullae may occur. Local envenomation may increase over time, resulting in serious complications such as blistering, local necrosis, abscesses, and extensive swelling involving the whole bitten limb and the trunk. Severe envenomation is regularly associated with systemic multifocal thrombotic complications, usually occurring within two days after the bite. Rapidly progressive swelling is usually indicative of severe envenomation. Hypotension/hypertension, tachycardia, muscle fasciculation, weakness, lethargy, difficulty of breathing, chest pain, and (near)-syncope are predictive of further life-threatening complications. However, complications may develop in patients who initially have signs of only moderate envenomation associated with normal results of blood coagulation tests apart from decreased platelet counts ,18. B. lanceolatus bites lead to multiple vascular obstruction, in which the exact mechanism remains unknown [B. lanceolatus, thus enhancing its particularities. Interestingly, a fatal case with diffuse thrombotic microangiopathy causing multiple cerebral, myocardial, and mesenteric infarctions at autopsy was reported [B. lanceolatus venom of von Willebrand factor activators (like in inherited thrombotic thrombocytopenic purpura and hemolytic uremic syndrome) or vascular endothelial growth factor-type factors of the kind demonstrated in several Viperidae venoms.Approximately 30 to 40% of envenomed cases by  unknown . Vascula unknown ,3. Throm unknown ,21,22,23 unknown . Consistreported . Multi-fB. lanceolatus envenomation is based on the circumstances of the bite and identification of the snake, when possible, as well as the presence of bleeding, swelling, and pain from the fang punctures  was developed to measure venom antigen levels in the serum of patients bitten by ceolatus . A good Otherwise, plain radiographs may depict teeth or fangs retained in the wound. Chest X-rays, electrocardiography, and serum troponin I-c measurement may be indicated on the basis of envenoming severity. When suspecting thrombotic or embolic complications, additional radiological examinations may be useful. A head CT-scan and if available, a MRI should be considered in patients with headaches or altered level of consciousness if presenting severe thrombopenia or rarely any other coagulopathy, to rule out cerebral infarctions or haemorrhages.B. lanceolatus, the victim should be moved and placed at rest and transported to the emergency department of the nearest hospital [After a bite from hospital . The ABCB. lanceolatus envenomation. Analgesics and tetanus prophylaxis are systematic. In addition, grade 3 and 4 victims should receive empiric antibiotics, as more than one-third of these patients present with a primary bacterial infection [B. lanceolatus victims should be admitted, either to the emergency department for continuous and close observation for a minimum of 24h before discharge or to the intensive care unit if treated with antivenom.Management in the emergency department includes initial non-specific supportive measures. Usually, victims require adequate intravenous hydration to correct hypotension and electrolyte disorders. Tracheal intubation and mechanical ventilation are mandatory in case of coma, severe collapse, cardiac arrhythmia, or respiratory distress. Shock is treated with fluids, followed by vasopressors to improve haemodynamic function or inotropic drugs to counteract conduction disturbances. However, such life-threatening presentations remain exceptional with nfection ,18,22. Anfection . Prophylnfection . All B. B. lanceolatus toxin neutralization is available since 1993 in Martinique [B. lanceolatus venoms. It contains 97% antivenom F(ab)2  and 3% antivenom Fab (antigen-binding fragment). Alternatives  should be considered as well if available.The most important variable affecting the severity and outcome of envenoming from most viperid and crotalid snakes is the time elapsed between the bite and treatment initiation with a specific antivenom . Usuallyrtinique ,14.BothrB. lanceolatus [Initial strategy was to reserve antivenom for the most severe cases and use anticoagulants to prevent thromboses in all other patients. However, this approach was modified after the observation of serious thromboses in patients with moderate poisoning . A prospceolatus . BecauseAntivenom administration is indicated in case of progressive signs of envenomation or imminent risk of acute complication. Ideally, it should be intravenously infused less than 30 min after hospital admission: its effectiveness is a function of how rapidly it is administrated ,27. It rB. lanceolatus were recently reported to have developed cerebral infarctions [In a recent study, among a total of thirty-three patients who did not receive antivenom serum or who received it eight hours after being bitten, fourteen (42%) developed severe thrombotic complications such as cerebral infarctions, myocardial infarctus, myocardial and cerebral infarctions and pulmonary embolisms ,18. Fourarctions . These 3B. lanceolatus venom antigens may have not been represented in the initial venom pool which was derived from only 7 snakes used to immunise horses for antivenom production. The World Health Organisation currently recommends a pool from at least 50 snakes of each species to obtain a reliable antivenomen serum [B. lanceolatus and whose venom produces a similar thrombotic syndrome [Researchers are now working to identify the main causes for treatment failure. Sanofi-Pasteur announced a reduction of the antivenom power of their marketed serum up to 20-fold the lethal dose 50% per millilitre. To our opinion, all en serum . Thus, asyndrome . HoweverEarly intravenous infusion of antivenom serum is well-tolerated . UndesirBothrops lanceolatus, notoriously named “Fer-de-Lance”, is the only endemic snake in Martinique. It is responsible of about 20-30 declared bites per year. Envenomation generally leads to swelling and pain, while occasionally, systemic signs and/or coagulopathy may appear. Severe multifocal vascular thromboses may result in life-threatening symptoms leading to permanent disabilities or death. An equine monovalent antivenom serum (Bothrofav®) was shown to be safe and effective for the treatment of B. lanceolatus bites when used within 6 hours. However, in recent cases, thrombotic stroke occurred despite antivenom early administration, questioning the necessity to obtain an improved antivenom.


S. cerevisiae cell cycle from S-phase until cytokinesis. However, much of the regulatory mechanisms that govern Cdc5 remain to be determined. Cdc5 is phosphorylated on up to 10 sites during mitosis. In this study, we investigated the function of phosphorylation site T23, the only full consensus Cdk1 (Cdc28) phosphorylation site present.Cdc5 (polo kinase/Plk1) is a highly conserved key regulator of the T23A Cdc5introduces a degron that reduces its cellular amount to undetectable levels, which are nevertheless sufficient for normal cell proliferation. The degron acts in cis and is reversed by N-terminal GFP-tagging. Cdk1 kinase activity is required to maintain Cdc5 levels during G2. This, Cdk1 inhibited, Cdc5 degradation is APC/CCdh1 independent and requires new protein synthesis. Cdc5T23E is hyperactive, and reduces the levels of Cdc5 (in trans) and drastically reduces Clb2 levels.CLB2 cluster protein accumulation, preventing potential protein toxicity, which may arise from their overexpression or from APC/CCdh1 inactivation.Phosphorylation of Cdc5 by Cdk1 is required to maintain Cdc5 levels during G2. However, phosphorylation of T23 (probably by Cdk1) caps Cdc5 and other  CLB2 cluster transcription (including itself) [Cdh1 inhibition [Cdc5/polo kinase is a crucial player in cell-cycle regulation from yeast to man, and the processes and substrates it regulates have been extensively investigated. Cdc5 regulates numerous cell cycle events, including promoting the destruction of the Cdk1-Clb2 inhibitor Swe1 [ itself) , spindle itself) , microtu itself) , APC/C a itself) , mitotic itself) , cytokin itself) ,10, Cdc1 itself) ,12 and Ahibition . Howeverhibition ,15. Phoshibition , though hibition , of whichibition . Four ofCdh1 ubiquitin ligase is mediated by two D-boxes (RxxL) located within the N-terminal 70 amino acid region. Cdc5 levels oscillate during the cell cycle, from low in G1 and S-phase to elevated in G2/M [cdh1Δ cells are invariable throughout the cell cycle and comparable to levels of Cdc5 in wt metaphase cells. , nocodazole (metaphase arrest), or rapamycin (early G1 arrest). Curiously, T23A Cdc5protein was undetectable under all these conditions . Examination of whole blots with anti-Cdc5 ruled out proteolytic processing . Moreover ΔN70cdc5, which lacks this phosphorylation site [T23A cdc5cells still express it, albeit at an undetectable level.The abundance of wild type Cdc5 and s Figure . Examinag Figure . Moreoveion site , migrateWT [cdc5T23A from the Cdc5 promoter either as the sole Cdc5 or in addition to an endogenous copy of Cdc5. Surprisingly GFP-cdc5T23A was detectable, both by anti-GFP and by anti-Cdc5  and accumulated normally. This indicates that the T23A mutation indeed introduced a degron and that the degron was masked by the N-terminal fusion of the GFP protein.The lack of detection of this essential protein could be due to two reasons - the first is that the antibody used does not bind to this mutant. We considered this unlikely, as the antibody used is a polyclonal antibody against the entire C-terminus of Cdc5. The other possibility is that the T23A mutation is, or has, introduced a degron. To test these ideas we expressed GFP-Cdc55 Figure  and accuT23A cdc5has an effect in trans - on other copies of Cdc5, cdc5T23A was expressed in cells containing endogenous Cdc513myc, which has reduced electrophoretic mobility. Accumulation of endogenous Cdc513myc, and of Clb2, was unaffected by the presence of cdc5T23A , suggesting that this mutation acts only in cis.To determine whether the instability of A Figure , suggestcdc5T23A protein levels were undetectable, we used Swe1 levels as a measure for Cdc5 activity. Cells were synchronized with hydroxyurea and released into nocodazole. Surprisingly, Swe1 levels were reduced in cdc5T23A cells, even in hydroxyurea, and Swe1 was rapidly eliminated upon release from S-phase arrest  in cells. Accumulation of Clb2 was normal and, as we have already shown above, T23A cdc5was not detected.The degradation of Swe1, a kinase that phosphorylates Cdk1 on Y19 and inhibits its activity, requires Cdc5 . As cdc5t Figure . Phosphocdk1as1 [Cdh1 mediated destruction of Cdc5 [ and is phosphorylated during mitosis . Althougcdk1as1  to preve of Cdc5 ,14. Upon [Figure . Swe1 gr is completely stable in G1 [The decline in Cdc5 levels was prevented by addition of cycloheximide Figure , indicat1Δ cells , whilst le in G1 .cdc5T23E mutant [cdc5T23E cells (as their sole Cdc5) grew with normal morphology, separation, tubulin and septin dynamics (data not shown). S-phase arrested cdc5T23E cells had reduced Swe1 levels and activity, and upon release Swe1 levels declined more rapidly than in wild-type cells, suggesting increased Cdc5 activity . In contrast to T23Acdc5, the presence of T23E cdc5affects Cdc5 levels in trans, reducing Cdc5 accumulation by approximately 50% . Mutating a threonine (T) to glutamic acids (E) is generally considered a phospho-mimicking mutation. It is however hard to assess whether the effect of glutamic acid is like genuine threonine phosphorylation. In many cases such a mutation has no such effect, and behaves like a non-phosphorylatable alanine. The effect we observed here is clearly not like that of non-phosphorylatable alanine, as cdc5T23E is considerably more stable than the undetectable cdc5T23A. However as cdc5T23E is not as stable as wt cdc5 and leads to some other non-wt effects like clb2 disappearance, we might observe here an intermediate effect. It is also possible that phospho-mimicking of this site has one effect on stability and another on activation. The T23 site does not fall within the kinase domain of Cdc5 and its enhanced apparent activity is likely due to modified interactions with other proteins, rather than intrinsic changes in its kinase activity.We next examined the phospho-mimicking E mutant . cdc5T23 [Figure . In cont% Figure . N-termie Figure . MutatinCdh1 substrate proteins accumulate to maximal levels in metaphase and are either absent or present at much lower levels during the rest of the cell cycle. In cdh1Δ cells the level of these proteins is, in most cases, uniformly high throughout the cell cycle. Strikingly this level is comparable to their metaphase level in wild type cells. This observation suggests that some additional mechanisms are likely to cap the level of these proteins, in addition to APC/CCdh1 specific degradation. In this report, we have uncovered a potential mechanism that provides a negative feedback regulation of Cdc5 levels, a toxic protein when overexpressed [Cdh1 independent mechanism that requires protein synthesis and which may account for the residual instability of D-box mutated Cdc5 [CLB2 cluster proteins. A potential mechanism could be via further phosphorylation of Ndd1/Fkh2 [cdc5T23E to switch off CLB2 cluster transcription when a desirable level of CLB2 cluster proteins is reached.APC/Cxpressed , enablinted Cdc5 . Secondlted Cdc5 , limits The authors declare that they have no competing interests.KJSL planned and performed the experiments and wrote the manuscript, MB wrote the manuscript and planned experiments. All authors read and approved the final manuscript.


The purpose of this study was to determine whether anti-IgE therapy, by blocking facilitated Ag presentation in vivo, attenuates allergen specific Th2 cell responses.Anti-IgE therapy inhibits mast cell and basophil activation, blocks IgE binding to both FcεRI and CD23 and down regulates FcεRI expression by antigen (Ag) presenting cells (APCs). In addition to its classical role in immediate hypersensitivity, IgE has been shown To test this hypothesis, food allergen specific T cell responses were examined during a 16-week clinical trial of omalizumab in nine subjects with eosinophilic gastroenteritis and food sensitization. Allergen specific T cell responses were measured using carboxyfluorescein succinimidyl ester dye dilution coupled with intracellular cytokine staining and polychromatic flow cytometry. Four independent indices of allergen specific T cell response  were determined.Eight of the 9 subjects had measurable food allergen specific responses, with a median proliferation index of 112-fold. Allergen specific T cell proliferation was limited to CD4 T cells, whereas CD8 T cell did not proliferate. Food allergen specific responses were Th2 skewed relative to tetanus specific responses in the same subjects. In contradistinction to the original hypothesis, anti-IgE treatment did not diminish any of the four measured indices of allergen specific T cell response.in vivo.In sum, using multiple indices of T cell function, this study failed to demonstrate that anti-IgE therapy broadly or potently inhibits allergen specific T cell responses. As such, these data do not support a major role for IgE facilitated Ag presentation augmenting allergen specific T cell responses NCT00084097ClinicalTrials.gov identifier  FcεRI i1000-fold,3.Activation of DC by cross-linking FcεRI has a number of additional consequences. Activation of human plasmacytoid DCs (pDCs) via FcεRI induces TNF and IL-10 expression, as well as downregulates TLR9 expression and CpG oligonucleotide induced IFN-α expression. Conversin vivo immunomodulatory activity on T cell responses[Omalizumab is a humanized anti-IgE monoclonal antibody indicated for use in allergic asthma. Anti-IgE therapy reduces the concentration of circulating free IgE, blocks IgE binding to both FcεRI and CD23, and down regulates surface FcεRI on mast cells, and basophils. Individresponses.To test the hypothesis that anti-IgE therapy affects allergen specific T cell responses, we assessed food allergen specific T cell responses in patients with allergic eosinophil associated gastrointestinal disorders (EGID) during a clinical trial of omalizumab. Using carboxyfluorescein succinimidyl ester dye dilution coupled with intracellular cytokine staining and polychromatic flow cytometry, four diNine subjects with allergic EGID were enrolled in a 16-week open label clinical trial of omalizumab, the results of which were previously published. The diaFor each subject, 2 allergens were selected for study, with a preference for food allergens yielding the highest CFSE proliferation index. Six of the 8 subjects were studied with peanut and shrimp, one with peanut and dust mite, and one with egg yolk and egg white extracts. Food antigens were saline extracts prepared by the investigators as previously described; mixed d5 cells/ml in RPMI with 10% autologous serum and cultured at 2 ml per well in a 24 well plate with the indicated concentration of allergen. After 4-5 days, 1 ml of culture supernatant was replaced with fresh media. After 7 days, ionomycin (1 μM), phorbol myristate acetate (20 ng/mL) and brefeldin A (10 μg/mL) were added and the cells incubated an additional 6 hours, at which point DNAse  final concentration 3,500 Dornase U/ml was added for an additional 5 minutes. Cells were removed from each well, stained with LIVE/DEAD® Fixable Violet Dead Cell Stain Kit (Invitrogen) according to the manufacturers instructions, washed once in PBS and fixed with 4% paraformaldehyde[Allergen specific T cell responses were measured using a polychromatic adaptation of published flow cytometry methods utilizing carboxyfluorescein succinimidyl ester (CFSE) dye dilution,15.The lmaldehyde.+, violet LIVE/DEAD negative and CD4+, CD8- gates  was determined using Prism software . Precursor frequency calculations  was noted in the media control  and the 16-week omalizumab time points , IL-5:IFN-γ   Figure  or of ei) Figure .Inhibition of IgE facilitated Ag presentation by APCs has been hypothesized to be a mechanism by which anti-IgE therapy may decrease allergen specific T cell responses and thus have immunomodulatory activity beyond immediate hypersensitivity. Additioin vivo vs. those occurring in the in vitro culture system.We used an established Ag specific CFSE based proliferative assay to examiin vivo IgE blockade and were of similar duration. Notably, the two studies used very different methods to examine allergen specific T cell responses; the previous study used purified CD4 T cell and DC populations, thymidine incorporation and cytokine ELISA, whereas we used unfractionated mononuclear cells and studied proliferation and cytokine expression using flow cytometry. The studies also examined different allergic diseases  and allergen . Differences in the APC populations, assay systems, T cell to APC ratio, allergen, or disease state are likely factors that account for the divergent results of these two studies.In contrast to our findings, Schroeder and colleagues recently demonstrated that Omalizumab treatment significantly decreased cat allergen specific T cell proliferation by 20-33% and Th2 cytokine expression by 50%. The reaIn contrast to the above, Noga and colleagues examined allergic asthmatic subjects treated with omalizumab for 12 weeks and using ionophore and phorbal ester activated mononuclear cells demonstrated decreased T cell cytokine expression. GM-CSF In vitro IgE facilitated Ag presentation shifts the T cell proliferation dose response 100-1000-fold to the left[in vivo on T cell function showed a relatively modest effect[in vitro data, suggesting that IgE facilitated Ag presentation plays a relatively modest role in vivo. the left,3. Simil the left. In contst effect. An altein vivo activation and clonal expansion of allergen specific T cells, as well as decreased in vitro allergen specific T cell proliferation. Second, anti-IgE may inhibit mast cell and basophil activation in vivo,[We hypothesized three potential mechanisms whereby anti-IgE therapy could inhibit allergen specific Th2 responses. First, anti-IgE may block IgE facilitated Ag presentation, resulting in decreased allergen specific T cell responses. Through this mechanism, anti-IgE inhibition of Ag presentation could have multiple consequences, including decreased  in vivo,, which m in vivo,. A limitRecently, in a number of murine model systems, basophils have been shown to be the dominant APC population initiating Th2 responses. HoweverGreater than 90% of EGID patients respond to an elemental  diet, demonstrating that it is clearly a food allergen driven disease,22. EGIDin vivo, such change would be read out by the various endpoints examined, irrespective of the APC population. Notably, this report largely consists of negative results that do not show a statistically significant effect. The substantial results within this work and the academic and ethical issues inherent in non-publication of results supports the value of these findings[This study is notable for several limitations. This study used PBMC, which contains a mixed APC population that may not include specific APC populations that are more IgE dependent. However, if anti-IgE therapy substantially modified T cell responses  findings,26. The  findings. This suWe have recently reported that Th2 cells are composed of two major subpopulations obtained using a short term 6-hour assay to identify antigen specific T cells. In thisin vivo.In conclusion, examining multiple indices of T cell function, this study failed to demonstrate that anti-IgE therapy has an immunomodulatory or inhibitory effect on allergen specific T cells. As such, these data do not support a major role for IgE facilitated Ag presentation augmenting allergen specific T cell responses 50: Concentration of Ag yielding half maximal proliferation; PBMC: Peripheral blood mononuclear cells; PE: Phycoerythrin;Ag: Antigen; APC: Antigen presenting cell; Cy: Cyanine; CFSE: Carboxyfluorescein succinimidyl ester; ECThe authors declare that they have no competing interests.BF performed flow cytometric and data analyses, SF performed the clinical trial and participated in the design of the study, YY performed additional data analyses and contributed to writing the manuscript, CP conceived of and designed the clinical trial and study, and wrote the manuscript. All authors read and approved the final manuscript.


Growth factor receptor-bound protein 14 (Grb14) is an adapter protein implicated in receptor tyrosine kinase signaling. Grb14 knockout studies highlight both the positive and negative roles of Grb14 in receptor tyrosine kinase signaling, in a tissue specific manner. Retinal cells are post-mitotic tissue, and insulin receptor (IR) activation is essential for retinal neuron survival. Retinal cells express protein tyrosine phosphatase-1B (PTP1B), which dephosphorylates IR and Grb14, a pseudosubstrate inhibitor of IR. This project asks the following major question: in retinal neurons, how does the IR overcome inactivation by PTP1B and Grb14?Akita mouse retinas. Decreased retinal IR activation has previously been reported in this mouse line.Our previous studies suggest that ablation of Grb14 results in decreased IR activation, due to increased PTP1B activity. Our research propounds that phosphorylation in the BPS region of Grb14 inhibits PTP1B activity, thereby promoting IR activation. We propose a model in which phosphorylation of the BPS region of Grb14 is the key element in promoting IR activation, and failure to undergo phosphorylation on Grb14 leads to both PTP1B and Grb14 exerting their negative roles in IR. Consistent with this hypothesis, we found decreased phosphorylation of Grb14 in diabetic type 1 Ins2Our results suggest that phosphorylation status of the BPS region of Grb14 determines the positive or negative role it will play in IR signaling. Grb14-/- animals have also revealed positive effects of Grb14 on receptor tyrosine kinase signaling, in a tissue specific manner  ATP was purchased from New England Nuclear . Generation of a polyclonal Grb14 antibody was described earlier  ATP to reach a final concentration of 200 μM, and the reaction was terminated by adding 10 μL of 50% (v/v) acetic acid. Twenty-five microliters of assay mixture was spotted onto P81 phosphocellulose filter paper disks (1.5 cm × 1.5 cm), which were immersed in a solution containing 0.75% phosphoric acid (v/v). The filter paper disks containing the bound phosphorylated peptide were washed three times with phosphoric acid and rinsed in acetone. Radioactivity was quantified in 5 mL of liquid scintillation cocktail and counted .The phosphorylation reaction was performed essentially as described  in a totin vitro PTP1B activity assay was conducted based on a previously published protocol using the peptide RRLIEDAEPYAARG (Upstate Biotechnology) , 50 ng of vector, BPS-SH2 (R466A), and 125 ng of sense and antisense primers with mutations in a total volume of 50 μl, followed by the addition of 2.5 units of pfu DNA polymerase. The primers used in the site-directed mutagenesis are as follows: E373Q . The sequence of each mutation was verified by DNA sequencing. The phosphorylation of each fusion protein in E. coli (BL21) was achieved by coexpressing a tyrosine kinase VSRC under the control of different replicons  and blocked with either 5% bovine serum albumin or non-fat dry milk powder (Bio-Rad) in TTBS for 1 h at room temperature. Blots were then incubated with anti-Grb14 (1:1000), anti-pTyr-416-Src (1:1000), anti-Src (1:1000), anti-His (1:1000), anti-PY99 (1:1000), anti-arrestin (1:1000), anti-Tα (1:1000), at 4°C overnight or anti-GST (1:5000), anti-opsin  for 1 h at room temperature. Following primary antibody incubations, immunoblots were incubated with HRP-linked secondary antibodies (mouse or rabbit) and developed by enhanced chemiluminescence according to the manufacturer’s instructions.t test for unpaired data. The significance level was set at p < 0.05 or p < 0.001.Data were analyzed and graphed using GraphPad Prism software . The data were expressed as the mean ± S.E. and compared by Student's Grb14: Growth factor receptor-bound protein 14; PTP1B: Protein tyrosine phosphatase 1B; Shp2: Src-homology phosphotyrosyl phosphatase 2; IR: Insulin receptor.The authors declare that they have no competing interests.Project planning, data analysis, and manuscript composition were performed by RR. Experimental work was performed by RR, DKB and AR. All authors read and approved the final manuscript.


Glomerular filtration rate (GFR) is widely estimated by serum creatinine based equations such as Cockcroft-Gault (CG) standardized for body surface, and an abbreviated formula derived from MDRD  study. However, some studies suggested that creatinine based estimation of GFR formula can be replaced by cystatin C based formula.The aim of this study was to determine whether cystatin C based equation could be used as an indicator for renal function in hemodialysis patients compared to MDRD equation; and whether cystatin C, a dialyzable molecule, was related to Kt/V, the marker for dialysis adequacy.In this cross-sectional study, 98 patients on chronic hemodialysis were included. Plasma levels of urea and creatinine were measured before and after dialysis, and cystatin C was measured before dialysis. GFR was calculated and compared.The mean age of patients was 55.50 ± 16.10 (24-86) years and 66 cases were male (67.3%). The GFR was estimated at 6.05 ± 2.36 and 5.83 ± 2.19 cc/min by MDRD and cystatin C based formulas, respectively, with a significant correlation . Serum cystatin C level was 9.74 ± 2.47 mg/L which showed significant reverse correlation with both MDRD  and cystatin C based formulas . Neither creatinine nor serum cystatin C showed correlation with Kt/V, as the marker of dialysis adequacy.Serum cystatin C may be considered as an indicator of renal function in patients under maintenance hemodialysis. Glomerular filtration rate (GFR), one of the most important parameters for assessment of renal function, is widely estimated by serum creatinine based equations of Cockcroft-Gault (CG) standardized for body surface, and modification of diet in renal disease (MDRD) formula. Both equations take parameters of serum creatinine, age, and gender into account. As creatinine production is affected by age, muscle mass, gender, medications, and catabolic state, the serum cystatin C based equations were proposed for GFR estimation , 2, espeCystatin C, a medium size molecule , is a cysteine proteinase inhibitor. It is synthesized by all nucleated cells with constant production rate, freely filterable through the glomerulus due to its small size, and metabolized by proximal tubular cells , 4. It sRanders et al. studied serum cystatin C, serum and urine creatinine, and GFR by 99mTc-DTPA clearance technique in 76 patients with various kidney diseases and serum creatinine less than 500 mmol/L, and 61 dialysis patients  in 1998. Similar to all plasma clearance techniques, 99mTc-DTPA clearance technique is reliable in GFR values higher than 20-30 mL/min. They found its significant linear relationship with 1/cystatin C and 1/creatinine in those with GFR higher than 30 mL/min  -0.203 × [1.212 if Black ] × [0.742 if Female]) and eGFR cysC was calculated by chronic kidney disease epidemiology collaboration (CKD-EPI) equation: eGFR = 127.7 (cystatin C in mg/L) - 1.179 (age) - 0.139 .In this cross-sectional study, 98 patients on maintenance hemodialysis in dialysis ward of Imam Khomeini hospital, Tehran, Iran, during April 2010 to June 2010 were enrolled. All patients were stable at the time of study and on regular thrice-weekly 4-hour sessions hemodialysis with synthetic polysulfone dialyzers for at least 4 months. Venous blood samples were collected from each patient, before and after dialysis. The collected samples before the dialysis were used to measure urea, creatinine, cystatin C, and albumin. The plasma urea samples taken after the dialysis were used to measure single pool (sp) Kt/V. Blood samples were centrifuged at 1500 × g for 10 min at 4 ºC. Creatinine was measured by enzymatic-colorimetric kinetic using Elitech diagnostic kit from ELITech Group France. Urea was measured by Enzymatic-UV kinetic using Elitech diagnostic kit from ELITech Group France. Cystatin C was measured by immunoassay method using Gentian kit Gentian Services LTD. The mean values of creatinine for each patient were used in MDRD formula (eGFR = 186 × Serum Creatinine (mg/Dl) All statistical analyses were conducted with SPSS, version 19. Baseline characteristics were presented as the mean ± SD. The Spearman correlation coefficient was used to study correlations between variables. P < 0.05 was regarded as statistically significant.The mean age of patients was 55.50 ± 16.10 (24-86) years. Sixty-six patients were male (67.3%) and thirty-six were diabetic (36.7%). The GFR was estimated at 6.05 ± 2.36 and 5.83 ± 2.19 cc/min by MDRD and cystatin C based formulas, respectively. There was a significant correlation between the two formulas . Serum cystatin C level was 9.74 ± 2.47 mg/L which showed significant reverse correlation with both MDRD  and cystatin C based formula . Mean serum Creatinine before dialysis and eq Kt/V (eq) were 10.02 ± 3.16 mg/dL and 1.19 ± 0.40, respectively. Neither creatinine nor cystatin C showed correlation with Kt/V, as the marker of dialysis adequacy. The negative correlation between serum cystatin C level and age was shown  .In this study, accuracy of two equations derived from prior studies was examined. According to our basic knowledge, the range of GFR in functionally anephric patients is less than 10 mL/min. This study showed that cystatin C based equation can be used similar to MDRD formula for estimating GFR in dialysis patients.Hoek et al. investigated in cohorts of both hemodialysis and peritoneal dialysis patients whether or not cystatin C based equation was able to estimate GFR more accurately than MDRD formula. This study showed that cystatin C measured before the dialysis was significantly correlated with mean creatinine  . ConsistOur study had some limitations: the first was absence of measured GFR by a gold standard such as 99mTc-DTPA scintigraphy to assess relationship of cystatin C or creatinine based formulas. Nevertheless, it was shown that chronic kidney disease epidemiology collaboration (CKD-EPI) creatinine based formula and Hoek cystatin C based formula had the best correlation with 99mTc-DTPA in GFR of less than 15 cc/min . SecondlSerum cystatin C is related to creatinine, significantly; therefore, it can be considered as an indicator of renal function in patients with reduced GFR. According to significant correlation of GFR estimation by cystatin C and MDRD formulas, this study verifies its capability to be used interchangeably. However, serum cystatin C does not seem to be able to show dialysis adequacy.


Abl kinase inhibitors targeting the ATP binding pocket are currently employed as potent anti-leukemogenic agents but drug resistance has become a significant clinical limitation. Recently, a compound that binds to the myristate pocket of Abl (GNF-5) was shown to act cooperatively with nilotinib, an ATP-competitive inhibitor to target the recalcitrant “T315I” gatekeeper mutant of Bcr-Abl. To uncover an explanation for how drug binding at a distance from the kinase active site could lead to inhibition and how inhibitors could combine their effects, hydrogen exchange mass spectrometry (HX MS) was employed to monitor conformational effects in the presence of both dasatinib, a clinically approved ATP-site inhibitor, and GNF-5. While dasatinib binding to wild type Abl clearly influenced Abl conformation, no binding was detected between dasatinib and T315I. GNF-5, however, elicited the same conformational changes in both wild type and T315I, including changes to dynamics within the ATP site located approximately 25 Å from the site of GNF-5 interaction. Simultaneous binding of dasatinib and GNF-5 to T315I caused conformational and/or dynamics changes in Abl such that effects of dasatinib on T315I were the same as when it bound to wild type Abl. These results provide strong biophysical evidence that allosteric interactions play a role in Abl kinase downregulation and that targeting sites outside the ATP binding site can provide an important pharmacological tool to overcome mutations that cause resistance to ATP-competitive inhibitors. Protein kinases are now avidly pursued as therapeutic targets for a host of human ailments, especially cancers Abl kinase is an important inhibitor target due to the role of the Bcr-Abl fusion protein in the development of Chronic Myleogenous Leukemia (CML). Imatinib  We previously reported on the discovery of GNF-2, a small molecule inhibitor of Bcr-Abl dependent cell proliferation in vitro and in vivo. The ability of the T315I mutation to activate Abl In the current work, we set out to understand mechanistically how GNF compounds inhibit kinase activity. In addition to potentially changing the conformation of the αI helix, GNF-2 binding could allosterically influence the catalytic site resulting in kinase inhibition through an as of yet unknown mechanism. This hypothesis has validity because there appears to be more to the effect of the GNF inhibitor than enhancement of SH2 docking to the kinase domain. GNF-2 and GNF-5 are not potent cellular inhibitors of the T315I mutant but combinations of nilotinib E. coliHydrogen exchange (HX) mass spectrometry (MS) was used to study the conformational effects that result from binding of dasatinib and GNF-5, both individually and in combination, to wild-type Abl core  and a T315I mutant version of Abl core. All experiments were performed with the Abl core expressed in The results show that dasatinib binding to Abl induced dynamic changes near the ATP binding site only. There were no significant changes in hydrogen exchange for Abl T315I in the presence of dasatinib, illustrating a lack of interaction between dasatinib and this mutant. As shown previously, GNF-5 binding to the myristic acid site caused a reduction of hydrogen exchange in both the myristate and the ATP site 96% formic acid, deuterium oxide (99.9%), sodium chloride and Tris-hydrochloride were purchased from Sigma-Aldrich . Acetonitrile and water (W5-4 HPLC grade) were purchased from Fisher Scientific. Potassium phosphate was purchased from EMD Biosciences. All chemicals were used without further purification unless otherwise specified. GNF-5 600 of 1.2 at 37°C, cooled for 1 h with shaking at 18°C prior to induction for 16 h at 18°C with 100 µM IPTG. Cells were harvested by 10 min centrifugation at 7000 g at 4°C and resuspended in 50 mM Tris (pH 8.0), 500 mM NaCl, 5% glycerol, 25 mM imidazole (buffer A). Insoluble protein and cell debris of cell lysate were sedimented by centrifugation at 40,000 g for 40 minutes at 4°C. The supernatant was loaded onto a Ni affinity column , equilibrated with buffer A. The loaded column was washed with five column volumes of buffer A, and protein was eluted with a linear gradient of 0–50% of buffer B (Buffer A plus 0.5 M imidazole). The peak fractions were analyzed by SDS-PAGE. Fractions containing the kinase were pooled and dialyzed against 20 volumes of buffer QA  using a 13-kDa molecular weight cutoff membrane. Tobacco etch virus protease TEV (expressed in bacteria using a plasmid kindly provided by M. J. Eck lab at DFCI) was added to the pooled protein to cleave the His tag . Dialysis and protease digestion were carried out overnight at 4°C. Subsequent anion exchange chromatography  at room temperature was used to remove protease and phosphatase contaminants. Proteins were eluted with a linear gradient of 0–35% buffer QB (buffer QA plus 1 M NaCl), and peak fractions were analyzed by SDS-PAGE.The bacterial expression of human c-Abl kinase - 2-propenoic acid, Sigma-Aldrich, cat. P-7002) and 20 µM Abl peptide substrate , fixed or varied (to determine inhibitor kinetic parameters) concentration of inhibitor applied, 1/50 of the final reaction mixture volume of PK/LDH enzyme , 160 µM NADH, 0.16 µM Abl, and ATP added last to start the reaction. Absorbance data were collected every 20 s at 340 nm using a SpectraMax M5 Microplate Reader. The two-substrate kinase reaction was simplified to two one-substrate reactions to determine ATP kinetic parameters and inhibitor parameters separately. When determining ATP parameters, the inhibitor concentration was kept the constant. When determining inhibition parameters, the ATP concentration was fixed at 20 µM. Steady-state initial velocity data were drawn from the slopes of the A340 curves and fit to the Michaelis-Menten equation to determine Vmax and Km values. Data were fitted globally using GraphPad Prism (GraphPad Software) and Excel XLfit 4.0 to fit velocity equations for competitive and mixed inhibition. At a concentration of 3 uM, GNF-5 does not achieve 100% inhibition of Abl. A concentration range of 0–3 µM was the range where positive additive effects of GNF-5 and dasatinib were apparent. Full inhibition of WT Abl could be achieved at concentrations of in excess of 3 uM.The ATP/NADH-coupled assay system in a 96-well format was used to determine the initial velocity of Abl tyrosine kinase catalyzed peptide phosphorylation. The reaction mixture contained 20 mM Tris-HCl, (pH 8.0), 50 mM NaCl, 10 mM MgCl3 wildtype or Bcr-Abl transformed Ba/F3 cells per well were plated in duplicate in 384-well plates, in RPMI 1640 media supplemented with 10% FBS, (addition of 10 ng/ml of IL-3 for wide type Ba/F3 cells). Test compounds in DMSO stocks were serially diluted in DMSO and added using a 384 pin tool. After 48 h of growth, Bright-Glo reagent  was added to each well to determine cell viability as a percentage of growth in the absence of the compound. Luminescence was read as counts/sec. XL-fit (IDBS) was used for IC50 analysis. For the resistant colony formation experiment, cultures containing 1×104 Ba/F3 cells in 96-well plates were incubated with various concentrations of GNF-5, dasatinib or both. The cells were continuously exposed to compound in RPMI 1640 with 10% FBS medium and medium was changed every three days with fresh inhibitor added each time. The number of resistant colonies was counted at day 12.4×10d of 0.1 µM, 98% of the proteins were bound to the inhibitor. All mixtures were incubated for 30 min at room temperature before deuterium labeling. As a control, proteins were incubated in 20 mM Tris, 100 mM NaCl (pH 8.3) buffer and treated exactly as the inhibitor- bound proteins.Hydrogen exchange experiments were performed essentially as described in Iacob et al 2O buffer at room temperature. At each deuterium exchange time point (from 10 s to 4 hours) an aliquot from the exchange reaction was removed and labeling was quenched by adjusting the pH to 2.6 with an equal volume of quench buffer . Quenched samples were immediately frozen on dry ice and stored at −80°C until analysis. The same procedure was used for the labeling of all proteins used in this study.Deuterium exchange was initiated by dilution of the free or bound protein with 15-fold 20 mM Tris, 100 mM NaCl (pD 8.3), DEach frozen sample was thawed rapidly to 0°C and injected into a custom Waters nanoACQUITY UPLC system and analyzed as described previously m/z range of 100 to 2000. Mass accuracy was ensured by calibration with 100 fmol/µL GFP, and was less than 10 ppm throughout all experiments. The mass spectra were processed with the software HX-Express EE was performed by a series of low-high collision energies ramping from 5–25 V, therefore ensuring proper fragmentation of all the peptic peptides eluting from the LC system.Mass spectra were obtained with a Waters QTOF Premier equipped with standard ESI source . The instrument configuration was the following: capillary was 3.5 kV, trap collision energy at 6 V, sampling cone at 37 V, source temperature of 100°C and desolvation temperature of 250°C. Mass spectra were acquired over an Resistance to ATP-competitive kinase inhibitors limits the duration of response that can be achieved with this class of therapeutics. The most common resistance mechanism involves the acquisition of mutations in the ATP-binding site that prevent drug binding or that increase the affinity for ATP. The most frequent site of mutation occurs to the so-called gatekeeper residue which is located towards the rear of the ATP-binding cleft. Many clinically relevant kinases such as Bcr-Abl, c-Kit, EGFR and PDGFR possess a threonine residue at the gatekeeper position that is involved in critical interactions with ATP competitive drugs. Mutation of this residue to a large hydrophobic amino acid such as isoleucine or methionine disrupts inhibitor binding without disrupting the kinase activity of the enzyme. The T315I gatekeeper mutation is resistant to the three Bcr-Abl inhibitors that are currently approved for the treatment of Chronic Myelogenous Leukemia: imatinib, nilotinib and dasatinib. There are two approaches for developing new drugs that can overcome ATP-site mutations such as the gatekeeper mutation. The first is to make new ATP-competitive inhibitors that exploit binding modes that can circumvent the existing mutations. This approach has been successfully exploited and several ATP-competitive T315I Bcr-Abl inhibitors are currently in preclinical development 50 = 0.22 µM, dasatinib IC50 = 0.12 µM using an ATP concentration of 20 µM, which is close to the apparent Km under our assay conditions. The myristate site mutant E505K was inhibited by dasatinib with an IC50 = 0.02 µM, but not by GNF-5 (IC50 >10 µM). The T315I mutant was partially inhibited by dasatinib or GNF-5 in this assay. We next examined whether combinations of GNF-5 and dasatinib resulted in additive inhibition of wild-type, T315I, or E505K recombinant Abl proteins. Strong positive cooperativity was observed for combinations of GNF-5 and dasatinib on T315I Bcr-Abl with a calculated combination index of 0.34. As expected no additivity was observed with the E505K myristate site mutant.Here we sought to determine whether cooperative inhibition of wild-type and T315I Bcr-abl could be achieved between the clinically approved inhibitor dasatinib and GNF-5. Dasatinib is an ATP-competitive inhibitor but it binds to Bcr-Abl in the active ‘type I’ conformation in contrast to nilotinib which binds to the inactive conformation. We first evaluated the ability of dasatinib and GNF-5 (or combinations) to inhibit recombinant Abl using a pyruvate kinase-lactate dehydrogenase detection system, that couples the production of ADP to oxidation of NADH which can be monitored spectrophotometrically 50 = 20 nM . Much stronger synergism was observed with the T315I form (CI = 0.32), Moderate synergism was observed for combinations of GNF-5 and dasatinib on the wild-type Abl Confident that biologically there was strong evidence for cooperativity between the two binding sites, we utilized hydrogen exchange mass spectrometry to try to understand how this synergy worked mechanistically. The conformational dynamics of each protein were measured in the presence of various drug combinations. We previously used this methodology to investigate conformational changes in Abl upon removal of the myristic acid group and upon mutation in a down-regulated form of Abl E. coli and purified to homogeneity. The purity and mass of the proteins were verified by electrospray mass spectrometry , including all regions of both SH3 and SH2. However a few areas showed differences in deuterium uptake in the presence of dasatinib or GNF-5. The deuterium incorporation data for regions where changes were observed are summarized in d of 0.1 µM. The control experiment in which no dasatinib was present was performed at the same time, under identical conditions so the exchange differences could be compared without the need for back-exchange correction (explained in detail in Wild type Abl was incubated with dasatinib in a 1∶7 molar ratio (protein:inhibitor), so that >98% of the Abl molecules were bound, based on a KThe location of the changes in wild type Abl when bound to dasatinib are shown in It was shown previously that the P-loop is very flexible even when dasatinib is bound indicating that for the most part the P loop does not form a critical interaction with dasatinib In contrast to what was observed for dasatinib binding to wild type Abl, when dasatinib was incubated at the same molar ratio with the T315I mutant, no major changes in HX were detected. For example, compared with wild-type Abl, residues 336–343 displayed no changes in deuterium incorporation when dasatinib was incubated with T315I . Subtle We previously showed that GNF-5, a compound representing a novel class of allosteric Bcr-Abl inhibitors, specifically binds to the myristic acid binding pocket 2O for various periods of time as described above. Changes in deuterium incorporation in the presence of GNF-5 were observed in several peptides  at 100 μL/min for 2 minutes, and eluted at 50 μL/min with a 4 minute linear 15%–75% (v/v) acetonitrile gradient directly into an LCT‐Premier mass spectrometer  equipped with a standard electrospray source. The instrument was calibrated with 500 fmol/µL myoglobin and the mass accuracy was less than 10 ppm. Phosphorylation (+80 or +160 Da) was observed in the intact protein spectra. The location of each phosphorylation was determined by trypsin digestion followed by LC‐MS/MS (data not shown). Each recombinant protein (50 pmol each) was incubated with trypsin  for 16 hours at 37°C. The resulting peptides were analyzed with a Waters nanoAcquity UPLC system (1.0×100.0 mm ACQUITY C18 BEH column) coupled to a Waters QTof Premier mass spectrometer. Peptide mass spectra were acquired over an m/z range of 100 to 2000. Mass accuracy was ensured by lock‐mass calibration with 100 fmol/µL Glu‐Fibrinogen peptide, and was less than 10 ppm throughout all experiments. MS(TIF)Click here for additional data file.Figure S2A. Dasatinib antiproliferative EC50 in the presence of 0.4 to 10 μM GNF‐5 on Ba/F3 cells expressing T315I and E505K Bcr‐Abl. B. Inhibition of Bcr‐Abl autophosphoryl‐ation was determined by Bcr‐Abl immunoprecipitation, followed by a immunoblot for phospho‐Tyr (Tyr412) GNF‐5 binds to the myristic pocket and inhibits activity in Bcr‐Abl T315I. (TIF)Click here for additional data file.Figure S3Comparison of deuterium exchange in wild‐type Abl and T315I. The deuterium uptake curves for six representative peptides are shown in the left . All other deuterium uptake curves for all other regions showed no significance difference between wild‐type and T315I and are therefore not shown. The location of each peptide, according to the labels A‐H, is shown on the crystal structure at the right (PDB 1OPL). Coloring is as in (TIF)Click here for additional data file.Figure S4A. Residues 287‐298 (peptide m/z =682.3+2). B. Residues 325‐335 (peptide m/z =683.4+2). C. Residues 525‐534 (peptide m/z =543.8+2). A dotted line is provided at the same m/z in both free or bound data to guide the eye.Example mass spectra for selected regions, intended to illustrate the quality of data for all experiments. (TIF)Click here for additional data file.Figure S5Location key for (TIF)Click here for additional data file.Figure S6A. Ribbon diagram, B. space filling model, in the same orientation as A. This model was created with two crystal structures: PDB 2FO0 and PDB 2F4J were overlaid and aligned. Then, only the αI helix is shown for the 2F4J structure as the rest of the structure was essentially identical to 2FO0. The αI helix for 2F4J is shown in green/blue. In 2FO0, the αI helix is broken into two smaller helices, αI and αI' where an almost 90 degree bend is introduced between αI and αI'. The peptide spanning residues 516‐524 is shown in red (2FO0) or blue (2F4J). Changes in HX are colored as in Expanded view of the myristic acid pocket. (TIF)Click here for additional data file.