The Kaposi sarcoma associated herpesvirus (KSHV) latency associated nuclear antigen (LANA) is expressed in all KSHV associated malignancies and is essential for maintenance of KSHV genomes in infected cells. To identify kinases that are potentially capable of modifying LANA, in vitro phosphorylation assays were performed using an Epstein Barr virus plus LANA protein microarray and 268 human kinases purified in active form from yeast. Interestingly, of the Epstein-Barr virus proteins on the array, the EBNA1 protein had the most similar kinase profile to LANA. We focused on nuclear kinases and on the N-terminus of LANA (amino acids 1–329) that contains the LANA chromatin binding domain. Sixty-three nuclear kinases phosphorylated the LANA N-terminus. Twenty-four nuclear kinases phosphorylated a peptide covering the LANA chromatin binding domain (amino acids 3–21). Alanine mutations of serine 10 and threonine 14 abolish or severely diminish chromatin and histone binding by LANA. However, conversion of these residues to the phosphomimetic glutamic acid restored histone binding suggesting that phosphorylation of serine 10 and threonine 14 may modulate LANA function. Serine 10 and threonine 14 were validated as substrates of casein kinase 1, PIM1, GSK-3 and RSK3 kinases. Short-term treatment of transfected cells with inhibitors of these kinases found that only RSK inhibition reduced LANA interaction with endogenous histone H2B. Extended treatment of PEL cell cultures with RSK inhibitor caused a decrease in LANA protein levels associated with p21 induction and a loss of PEL cell viability. The data indicate that RSK phosphorylation affects both LANA accumulation and function. The Kaposi sarcoma associated herpesvirus (KSHV) is associated with cancers that have an increased incidence in individuals with compromised immune systems. KSHV expresses a protein, LANA, that is needed to maintain KSHV genomes in infected cells and also promotes the growth of KSHV associated tumors. Kinases regulate protein function through phosphorylation. To identify kinases that may affect LANA function, we performed a screen in which 268 human kinases were isolated and tested for the ability to phosphorylate LANA in vitro. We focused on the region of LANA that contains the chromatin binding domain, a motif essential for tethering KSHV genomes to the cell chromatin and maintaining latent infection. We identified serine 10 and threonine 14 as amino acids within the chromatin binding domain whose phosphorylation was important for histone binding. Serine 10 and threonine 14 were targets of the CK1, PIM1, GSK-3 and RSK3 kinases. Treatment with an inhibitor of RSK kinase reduced LANA binding to histones, decreased LANA protein levels and caused a loss of KSHV infected PEL cell viability. Our experiments show that phosphorylation affects LANA function and suggest that KSHV infected cells may be particularly vulnerable to kinase inhibitors. The Kaposi sarcoma associated herpesvirus (KSHV) LANA protein is essential for establishment of KSHV latency through its role in replicating the KSHV genome, tethering the episomal genomes to cell chromosomes, interfering with induction of the viral lytic program and creating an environment that is permissive for cell survival and proliferation. Deletion of LANA in KSHV or rhesus rhadinovirus results in a more actively replicating virus LANA serves as the origin binding protein for KSHV latency DNA replication and binds to sequences within the terminal repeats The LANA primary amino acid sequence includes 120 serine, threonine and tyrosine residues that could be subject to post-translational modification. The kinases glycogen synthase kinase 3, PIM1/3, ERK1/2 and DNA-PK, 32P]-ATP and individual kinases. As a negative control, two chips per experiment were incubated with kinase buffer containing [γ32P]-ATP but minus the protein kinase. Phosphorylation signals were detected by exposing the arrays to X-ray film. An example of a kinase assay performed on the protein array is shown in We have previously described a protein microarray displaying Epstein–Barr virus (EBV) proteins purified as GST-fusions from yeast and printed in duplicate Amino acids 5–22 are important for KSHV association with the cell chromatin To assess the potential contribution of phosphorylation of the chromatin binding domain, LANA variants were generated that carried phosphomimetic mutations of S10, S13 and T14. The ability of these LANA variants to immunoprecipitate histone H2B was examined. Conversion of S10 to glutamic acid (S10E) restored the ability to bind to H2B both in the context of an individual S10E mutation and in the context of an S10E, S13E, T14E triple mutation . LANA caThe LANA N-terminus contains the chromatin binding domain and we therefore chose to focus on this region of LANA and to limit subsequent experiments to the 63 kinases that phosphorylated both the 6xHis-GST and 6xHis-Biotin AviTag fusions of the N-terminus of LANA (aa 1–329) and are known or predicted to function in the cell nucleus. LANA has a nuclear localization and this subset of kinases is therefore more likely to be biologically relevant . IncludeThe possibility that phosphorylation of sites outside of the chromatin binding domain could complicate interpretation of the kinase assays using GST-LANA (1–50) as substrate led us to turn to peptide substrates that covered only the LANA chromatin binding domain. A synthetic peptide representing LANA amino acids 3–21 and covering the S10 and T14 amino acids of interest was used to retest 31 of the nuclear kinases that were positive for phosphorylation of GST-LANA (1–50). In this assay, 24 of these kinases also phosphorylated the amino acid 3–21 LANA peptide . ExampleWe next performed in vitro phosphorylation assays using synthetic peptides that carried individual mutations in S10A, S13A or T14A or combined mutations of S10A,S13A,T14A or S10A,S13A,T14A,T19A . In this32P]-ATP and a PIM1 inhibitor, SMI-4a, resulted in phosphorylation of the wild-type peptide but not of the S10 mutant peptide  treatment with CKI-7 at 30 and 100 µM had no effect on LANA interaction with histone H2B . InhibitThe effect of a longer exposure to RSK inhibitor was examined firstly in transfected 293 cells. Interestingly, when the cells were treated with 3.4 µM BRD 7389 for 24 hr versus 6 hr, there was not only a further decrease in LANA binding to histone H2B but also a decrease in LANA protein levels . A compaLoss of LANA would be expected to impact on LANA regulated events. LANA is known to bind to p53 and to block p53 mediated responses and release of this inhibition leads to induction of p53 target genes To determine the effect of the loss of LANA protein on PEL cell growth, duplicate cultures of BC3 PEL cells and KSHV negative BJAB cells were treated with RSK inhibitor and each sample was measured in duplicate for cell viability using the CellTiter-Glo assay. RSK inhibition has been reported to reversibly inhibit proliferation of tumor derived cell lines and indeed all three inhibitor concentrations stopped the growth of both BC3 PEL cells and BJAB B cells . While, To further evaluate the difference in loss of viability between inhibitor treated PEL cells and BJAB B cells, BC3, BCBL1 and BJAB cells were treated with 0.85 µM BRD 7389 for 1 day and a western blot was performed to detect PARP cleavage as a measure of caspase induction and apoptosis. The antibody used is specific for cleaved PARP. BRD 7389 treatment induced PARP cleavage that was substantially greater in the PEL cells than in BJAB cells . The assProtein microarrays and high throughput techniques for kinase expression and purification have previously been used to characterize the yeast kinome and analyze yeast phosphorylation networks In vitro kinase screens have limitations. Not all of the kinases that phosphorylate the substrate in vitro will have the appropriate intracellular localization, expression pattern or activating signals to perform this function in the cellular environment while kinases that require specialized reaction conditions or partners such as priming kinases for maximum activity will tend to be under represented. It should be noted that substrates purified from yeast exhibit a limited degree of phosphorylation that can partially support the priming activity required by kinases such as GSK-3. To ensure that we focused on the most biologically relevant LANA partners, kinases that phosphorylated LANA in the initial microarray screen were subjected to database searches for their intracellular localization and only those with a known or predicted nuclear activity were included in subsequent assays. An interesting observation from the initial microarray assay was that the number of kinases phosphorylating LANA and its functional homolog in latency viral DNA replication in EBV, the EBNA1 protein, was an order of magnitude greater than the number detected for most other EBV proteins on the array. This suggests that LANA and EBNA1 functions may be particularly sensitive to regulation by phosphorylation. The extensive overlap in phosphorylating kinases for these two proteins also suggests that they may be subject to regulation by common signaling pathways.Within the LANA chromatin binding domain, three of the amino acids that had been implicated as critical for histone and chromatin binding in mutagenesis studies were S10, S13 and T14. We tested S13 as a single mutation, S13A, and did not find any impact on LANA binding to histones. This placed the emphasis on S10 and T14 as residues potentially subject to post-translational modification. The ability of phosphomimetic S10E and T14E mutations to restore LANA binding to histone H2B is suggestive of a role for phosphorylation in the regulation of LANA chromatin binding function. Two of the kinases that we identified as phosphorylating these residues, PIM and RSK have been implicated in KSHV lytic replication. Overexpression of PIM1 and PIM3 led to reactivation of KSHV from latency and the role of these kinases was linked to phosphorylation of LANA at serines 205 and 206, an event that abolished LANA mediated repression of RTA We saw no effect on LANA interaction with histone H2B after a short treatment with CK1, PIM1 or GSK-3 inhibitors. However, a short treatment with the RSK inhibitor BRD 7389 decreased LANA binding to histone H2B. All of the RSK family members, RSK1, 2, 3 and 4 are inhibited by BRD 7389. Interestingly, a longer BRD 7389 treatment resulted in a decrease in LANA protein levels in LANA transfected cells and in PEL cells. Treatment of cancer cell lines with RSK inhibitors results in growth inhibition Overall, our data provide evidence that phosphorylation affects LANA interaction with chromatin and that inhibition of the RSK kinases that phosphorylate the chromatin binding domain has an additional effect on LANA protein levels. The dual outcomes of reduced histone binding and loss of LANA protein stability imply that chromatin binding protects LANA from degradation and contributes to the long half-life demonstrated by LANA in latently infected cells. ERK signaling positively regulates transcription of LANA upon KSHV infection of endothelial cells EBV proteins were purified as 6x-His-GST fusion proteins from yeast using a high-throughput protein purification protocol described previously 32P]-ATP and a mixture of histone H3, myelin basic protein (MBP), and casein.Human kinases from 268 recombinant yeast strains were purified as 6xHis-GST-fusion proteins using a previously described high-throughput protein purification protocol 32P]-ATP, 1 mM DTT, 10 mM MgCl2, 1 mM MnCl2, 1 mM EGTA, 1 mM NaVO4, 1 mM NaF, and 0.1% NP-40) for 30 minutes at 30°C. Control slides were incubated with kinase reaction mixture without kinase and processed in parallel. The arrays were subjected to three ten minute washes in, firstly, TBS, 0.1% Tween-20 (TBST) and secondly in 0.5% SDS. Arrays were then rinsed briefly with double-distilled H2O and dried by centrifugation. Each slide was then exposed to BioMax high resolution X-ray film (Kodak) for 16 hours. Finally, developed films were scanned using an office scanner at a resolution of 4,800 dpi. Captured images from the autoradiographic films were processed using Photoshop. The substrate profile for each image was acquired using GenePix software (Axon). Each protein was printed in duplicate and a protein was scored as a positive hit only if both spots of the same protein showed signal intensity higher than the cutoff value of three standard deviations above the mean. Negative control experiments identified proteins that underwent autophosphorylation on the protein microarrays.To identify potential LANA phosphorylating kinases, a protocol similar to that described by Zhu et al. was used The Flag-LANA plasmid pMF24 that is deleted for the central repeat region was cleaved with XmaI and AscI and repaired with oligonucleotide insertions to introduce G5A, M6A, R7A, L8A, R9A, S10A, G11A, R12A, S13A or T14A mutations (pMS30 to pMS39 respectively). Glutamic acid substitutions were introduced by site-directed mutagenesis on plasmid DY52 (FLAG-LANA) to generate S10E (pGL621), S13E, T14E (pGL628), and S10E, S13E, T14E (pGL629). These constructions were then used as PCR templates to generate GST-LANA (aa1–329) wt, S10A, S13A and T14A plasmids (pGL485 to pGL488 respectively) and GST-LANA (aa1–50) wt, S10A, S13A and T14A plasmids (pGL502 to pGL505 respectively). The GST-LANA (aa1–50) triple mutant S10A/S13A/T14A (pGL515) was generated using XmaI and AscI cleavage and repair with an oligonucleotide insertion carrying mutated sequences. Full length 3xFlag-LANA carrying the triple mutations S10A/S13A/T14A (pGL628) and S10E/S13E/T14E (pGL629) were generated by PCR mutagenesis in the vector p3xFlag-CMV (Sigma). Full length Flag-LANA (pDY52) has been described previously 32P]-ATP for 30 min at 30°C. Reactions were terminated by heating the mixture at 90°C for 5 min, and the proteins were separated on NuPAGE gels . The gels were dried and exposed to MP Hyperfilm . For characterization of GSK-3β phosphorylation, peptide substrates were incubated for 30 min at 30°C in (i) kinase buffer with 10 µCi of [γ32P]-ATP, (ii) kinase buffer with 10 µCi of [γ32P]-ATP and Pim1 , (iii) kinase buffer with 10 µCi of [γ32P]-ATP and GSK-3β (Millipore) and for primed phosphorylations (iv) kinase buffer with 20 µM cold ATP and Pim1  followed by incubation with Pim1 inhibitor SMI-4a  for 30 min at 30°C (Millipore). Samples were then reincubated in kinase buffer with 10 µCi of [γ32P]-ATP or reincubated for 30 min at 30°C in (v) kinase buffer with 10 µCi of [γ32P]-ATP and GSK-3β. Samples were separated by SDS-PAGE  and analyzed by autoradiography.LANA amino acid 3–21 polypeptides and variants carrying S/T to A mutations were purchased from Peptide 2.0. The purified GST-LANA fusion proteins (0.1 µg) or synthetic polypeptides (250 µM) were incubated with kinase in 25 µl kinase buffer containing 33.3 nM [γHEK 293T cells grown in 10-cm dishes were transfected with 10 ug of total DNA using calcium phosphate precipitation. 48 h after transfection, cells were lysed in 1 ml of lysis buffer (50 mM Tris (pH 7.9), 100 mM NaCl, 0.5 mM EDTA, 2% glycerol, 0.2% NP-40), plus protease inhibitors  and phosphatase inhibitors , sonicated for 10 s, and cleared by centrifugation. Extracts were precleared using protein A/G PLUS-agarose  and immunoprecipitated with anti-FLAG M2-agarose (Sigma). Beads were washed six times with precipitation buffer and bound H2B was detected by western blotting using rabbit anti-H2B antibody (Abcam). Flag-LANA was detected using rabbit anti-Flag antibody (Sigma) or rat anti-LANA antibody (ABI Advanced Biotechnologies). The effect of kinase inhibition on H2B interaction was examined using the inhibitors CKI-7 (Sigma Aldrich), LiCl (J.T. Baker), SMI-4a (Enzo Life Sciences) and BRD 7389 (Tocris Bioscience). Cells were treated with the proteasome inhibitor lactacystin (Peptide Institute Inc) for 6 hr at a final concentration of 1 µM. PARP cleavage was detected by western blotting using cleaved PARP (ASP214) specific and total PARP antibodies . LANA and Actin were detected by rat anti-LANA (Advanced Biotechnologies Inc) and mouse anti-Actin (Sigma).2 at 37°C. Cell growth in cultures treated with BRD 7389 was measured using the Cell Titer-Glo luminescence assay kit (Promega) and luciferase activity was quantified using a Glomax Multi Detection System (Promega).BC3 and BCBL1 PEL cells and BJAB B cells were cultured in RPMI 1640 plus 15% fetal bovine serum in 5% COTable S1Supporting (DOC)Click here for additional data file.


Further analysis of three hits, Y-27632, Fasudil and TPCA-1, revealed low micromolar IC50 values for MRCKα and MRCKβ. We also describe the crystal structure of MRCKβ in complex with inhibitors Fasudil and TPCA-1 bound to the active site of the kinase. These high-resolution structures reveal a highly conserved AGC kinase fold in a typical dimeric arrangement. The kinase domain is in an active conformation with a fully-ordered and correctly positioned αC helix and catalytic residues in a conformation competent for catalysis. Together, these results provide further validation for MRCK involvement in regulation of cancer cell invasion and present a valuable starting point for future structure-based drug discovery efforts.MRCKα and MRCKβ (myotonic dystrophy kinase-related Cdc42-binding kinases) belong to a subfamily of Rho GTPase activated serine/threonine kinases within the AGC-family that regulate the actomyosin cytoskeleton. Reflecting their roles in myosin light chain (MLC) phosphorylation, MRCKα and MRCKβ influence cell shape and motility. We report further evidence for MRCKα and MRCKβ contributions to the invasion of cancer cells in 3-dimensional matrix invasion assays. In particular, our results indicate that the combined inhibition of MRCKα and MRCKβ together with inhibition of ROCK kinases results in significantly greater effects on reducing cancer cell invasion than blocking either MRCK or ROCK kinases alone. To probe the kinase ligand pocket, we screened 159 kinase inhibitors in an  Furthermore, we determined the structure of MRCKβ in complex with two ATP-competitive inhibitors, namely Fasudil and TPCA-1. These results and crystal structures provide valuable starting points for the development of compounds that could potentially be used as anti-metastatic therapeutics.In this study we have confirmed that the greatest inhibition of 3-D ECM invasion by MDA MB 231 breast cancer cells occurs with the combined inhibition of MRCK and ROCK. To examine the structural basis of MRCK activity and to explore the potential for developing specific inhibitors, we screened a collection of kinase inhibitors and identified several that inhibited MRCK with low micromolar ICThe contribution of MRCK to tumor cell invasion was examined by knocking down both MRCKα and MRCKβ in MB 231 breast cancer cells and determining the effects in a 3-dimensional inverse matrigel invasion assay in vitro kinase assays at the enzyme Km for ATP. We found that 15% (24/159) of the inhibitors resulted in >80% inhibition at 30 µM , consistent with the high homology between the MRCK and ROCK kinase domains. Interestingly, the reportedly IKK2-selective inhibitor TPCA-1 10 drug concentrations were carried out for Y-27632, TPCA-1 and Fasudil for MRCKα and MRCKβ. These assays revealed comparable rank orders of potencies with Y-27632>TPCA-1>Fasudil for both MRCKα and MRCKβ .In order to identify tool compounds and to begin building structure-activity relationship information on MRCK inhibitors, we screened 159 kinase inhibitors at 30 µM and 3 µM in MRCKβ at 30 µM  and 7% . The crystallographic asymmetric unit contains two molecules of the MRCKβ kinase domain, locked in a conserved intermolecular interaction that represents the biological dimer interface. As in the structures of the related AGC kinases, the regions of MRCKβ involved in the dimer interface are the four N-terminal helices as well as the C-terminus of the kinase domain  and 4B.2, while those of ROCK1 and DMPK are 2100 Å2 and 1650 Å2 respectively . The difference between MRCKβ and DMPK can be explained by the extended α1-helix seen in the former, spanning 14 residues in comparison to only 6 residues in DMPK. The differences between ROCK1 and MRCKβ are more subtle, and the length of the α1-helix is very similar in both proteins. The C-terminus of MRCKβ can be traced further in electron density than that of ROCK1, thus contributing to the increased buried surface at the interface /T137 (MRCKβ) and M144 (ROCK2)/L128 (MRCKβ). These differences could be used to design selective inhibitors for MRCKβ . ROCK1 aTo conclude, the results shown in this study indicate that development of highly potent and specific inhibitors of these AGC kinases could be challenging, but the methods now available for structural studies of both MRCK and ROCK kinases should allow iterative drug development approaches.2 in a humidified incubator.MDA-MB-231-luciferase  were grown in DMEM with 10% fetal bovine serum (FBS), 2 mM L-glutamine plus 10 Units/ml penicillin and 10 µg/ml streptomycin at 37°C in 5% COAll siRNA reagents were obtained from Dharmacon RNA Technologies. All siRNA sequences available on request. Oligofectamine was used for transfection of siRNA on sub-confluent cells, according to manufacturer's instructions.RIPA lysates were prepared as described previously 5 cells per ml from each condition were prepared in their normal growth medium. When the Matrigel had set, the Transwell inserts were inverted and 100 µl of cell suspension pipetted onto the filter. Transwell inserts were then carefully covered with the base of the 24 well tissue culture plate, making contact with each cell suspension droplet, and the plate then incubated in the inverted state for 4 hours to allow cell attachment. Following this time, plates were turned right-side-up and each Transwell insert washed with 3×1 ml serum-free DMEM and finally placed in a well in containing any additional inhibitors as indicated. DMEM plus 10% FBS was gently pipetted on top of the set Matrigel/PBS mixture and incubated for 5 days at 37°C. Propoidium iodide (PI) was used to visualize nuclei following fixation with paraformaldehyde, cells were imaged by confocal microscopy with a 20× objective with optical Z-sections scanned at 15 µm intervals moving up from the underside of the filter into the matrigel. ImageJ software was used for quantification. Statistical analysis was performed by ANOVA followed by Bonferroni's post-hoc test of significance, with p>0.05 selected as the cut-off.Cell invasion assays were as previously described 6 tag and TEV protease cleavage site was codon optimized for expression in E. coli, synthesized and cloned into the Nde I and EcoR I restriction sites of pET21a (Novagen) by Blue Heron. MRCKβ 2–417 was expressed in E. coli BL21 (DE3). Transformed cells were grown in Terrific Broth at 30°C until A600 nm reached 0.6–0.9, and induced with 1 mM IPTG, followed by incubation at 18°C for 12–15 hrs. Pelleted cells were resuspended in lysis buffer  and lysed by sonication. Following centrifugal clarification , supernatant was incubated with Ni-NTA Sepharose 6 FF beads  for 2.5 hr at 4°C. Weaker binding components were sequentially removed with 20 mM imidazole, 40 mM imidazole or 80 mM imidazole in ‘wash buffer’ . Beads were packed into a XK16/60 column  before bound protein was step eluted with 500 mM imidazole in ‘Wash Buffer’. Fractions were analyzed by UV, pooled and purified further by size exclusion chromatography (HiLoad 26/60 Superdex) using crystallization buffer . Fractions containing dimeric MRCKβ were pooled, concentrated , flash frozen and stored at −80°C.The cDNA encoding the human MRCKβ kinase domain (residues 2–417) with an N-terminal (His)Crystals of MRCKβ 2–417 were obtained by incubating the protein (∼15 mg/mL concentration) with 1 mM of inhibitors (prepared from 50 mM DMSO stocks) overnight and setting up commercial crystallization screens the following morning. Plate-like crystals were observed after the protein was incubated at 4°C in hanging drops over well solution consisting of 15–25% PEG3350, 0.1 M BisTris pH 5.6, 0.2 M Ammonium Sulfate and 0.1 M Potassium/Sodium Tartrate. Crystals were frozen in well solution supplemented with 20% ethylene glycol. Data was collected at Diamond and Soleil.Data were processed using Mosflm 2+ in 20 mM Tris buffer (pH 7.4) containing 0.01% Tween-20 and 1 mM DTT. Dose response inhibition analyses were performed over a drug concentration range from 0.002–40 µM, and single point inhibition was assessed at 3 or 30 µM drug. Kinase reactions were stopped with 2 assay volumes of 0.25% (v/v) IMAP binding reagent in 1× IMAP binding buffer A (Molecular Devices Inc.). After two hours incubation to allow binding reagent to bind phosphorylated peptide, fluorescence polarization was measured on a PHERAstar plate reader (BMG Labtech GmbH) at excitation and emission wavelengths of 485 nm and 520 nm respectively. Percent inhibition was calculated using no inhibitor and no enzyme controls for 0 and 100% inhibition, respectively.Activity assays for MRCKα and β were performed using an IMAP fluorescence polarization assay kit (Molecular Devices Inc.). 8–12 nM MRCKα or MRCKβ (Millipore) was incubated for 60 minutes at room temperature with 100 nM FAM-S6-ribosomal protein derived peptide (Molecular Devices Inc.) in the presence of 0.7 µM ATP and 0.5 mM Mg


The aim of this study was to analyze whether tyrosine phosphorylation in tumoral arteries may modulate their vascular response. To do this, mesenteric arteries supplying blood flow to colorectal tumors or to normal intestine were obtained during surgery and prepared for isometric tension recording in an organ bath. Increasing tyrosine phosphorylation with the phosphatase inhibitor, sodium orthovanadate produced arterial contraction which was lower in tumoral than in control arteries, whereas it reduced the contraction to noradrenaline in tumoral but not in control arteries and reduced the relaxation to bradykinin in control but not in tumoral arteries. Protein expression of VEGF-A and of the VEGF receptor FLT1 was similar in control and tumoral arteries, but expression of the VEGF receptor KDR was increased in tumoral compared with control arteries. This suggests that tyrosine phosphorylation may produce inhibition of the contraction in tumoral mesenteric arteries, which may increase blood flow to the tumor when tyrosine phosphorylation is increased by stimulation of VEGF receptors. Tyrosine kinases are a family of protein kinases present in metazoans, which are mainly associated with receptors of growth factors such as vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF) . TyrosinTyrosine phosphorylation may also regulate contraction of vascular smooth muscle. Vascular smooth muscle cells show levels of tyrosine kinase activity which are relatively high . InhibitIn tumoral tissues, tyrosine phosphorylation is often related to activation of growth factor receptors which have tyrosine kinase activity, including VEGF receptors which have a central role in cancer development . VEGF acRegulation of tumoral vasculature is an important factor in the development of solid tumors, as blood flow must increase in parallel with the growth of the tumor. The hypothesis of the present study is that tyrosine phosphorylation may play a role in the regulation of vasoconstriction and/or vasodilatation of the arteries supplying blood flow to the tumors. Therefore, we have studied the effect of increasing tyrosine phosphorylation on vasoconstriction and vasodilatation of arteries supplying colorectal tumors, comparing them with arteries supplying normal tissue. The inhibitors of tyrosine phosphatases such as vanadate increase tyrosine phosphorylation and reproduce the vascular effects of receptors with tyrosine kinase activity .In this study, arteries  from 13 patients diagnosed with colorectal tumour were used . The study was approved by the local ethics committee and the informed consent from all the patients was obtained before they were allowed to participate. Arteries supplying blood flow to the tumour as well as arteries supplying the normal colon were dissected out at surgery from each patient and frozen on dry ice for RT-PCR and western blot techniques, or stored in cold isotonic saline solution for in vitro vascular response. 2PO4, 1.2; MgSO4, 1.2; CaCl2, 2.5; NaHCO3, 25; glucose, 11. The solution was equilibrated with 95% oxygen and 5% carbon dioxide to a pH of 7.3-7.4. Briefly, two fine steel wires (100 μm in diameter) were passed through the lumen of the vascular segment, one wire was fixed to the organ bath wall while the other was connected to a strain gauge for isometric tension recording . This arrangement permits passive tension to be applied in a plane perpendicular to the long axis of the vascular cylinder. The changes in isometric force were recorded on a Macintosh computer using Chart v 3.6/s software and a MacLab/8e data acquisition system (ADInstruments). An optimal passive tension of 1 g was applied to the vascular segments, and then, they were allowed to equilibrate for 60–90 min. Before beginning the experiment, the vascular segments were stimulated with potassium chloride (50 mM) to determine the contractility of smooth muscle, and the segments which failed to contract at least 0.5 g were discarded.After collection and once transported to the laboratory, the arteries were cut into 2 mm long segments and each segment was prepared for isometric tension recording in a 4 mL organ bath containing modified Krebs-Henseleit solution at 37°C (mM): NaCl, 115; KCl, 4.6; KH−4–10−2 M) in the vascular segments from arteries supplying the tumour  and from those supplying normal colon (control) in the same patients. This effect was also recorded in the presence of the inhibitor of tyrosine kinase genistein (10−4 M) to analyze whether the response was due to tyrosine phosphorylation. To study the effect of tyrosine phosphorylation in response to vasoconstrictor of vasodilator stimuli, the contraction to noradrenaline (10−9–10−4 M) and to the endothelium-dependent vasodilator bradykinin (10−9–10−5 M) was studied after treatment with orthovanadate (10−3 M) in control and tumoral arteries. Orthovanadate was added to the organ bath 30 min before the concentration-response curve to noradrenaline or bradykinin, and before recording the relaxation to bradykinin, the arteries were precontracted with U46619 (10−9–10−8 M) to reach a contractile tone of about 3–3.5 g.To analyze the direct effect of tyrosine phosphorylation on the vascular tone of mesenteric arteries, cumulative dose-response curves were recorded for sodium orthovanadate , whereas the relaxation in response to bradykinin was expressed as the percentage of the active tone achieved with U46619. In each concentration-response curve, the pDTotal RNA was isolated from frozen vascular segment by using TriPure Isolation Reagent  protocol, as suggested by the manufacturer. RNA concentration and integrity were determined using RNA 6000 Nano LabChip in an Agilent 2100 Bioanalyzer. The reverse transcription was performed with the High Capacity RNA-to-cDNA Master Mix (Applied Biosystems). The primers used for amplification of cDNA were obtained from TaqMan Gene Expression Assays  for VEGFA (Hs03929005_m1), VEGFR1 (FLT1) (Hs01052961_m1), VRGFR2 (KDR) (Hs00911700_m1), and GAPDH (endogenous control 4352934E). Amplification of target cDNA was done using a 7900HT Fast Real-Time PCR System with the following standardized thermal cycling conditions: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. All RT-PCR reactions were run in duplicate. Threshold cycle (Ct) was determined for both target gene and GAPDH for each sample, and relative quantifying of both genes was determined with the 2-DDCt method .μg of total protein was resolved in 12% SDS-PAGE and electrophoretically transferred onto a PVDF-membrane using Mini Trans-Blot cell . Membranes were blocked in 5% skim milk for 1 h at room temperature. After an overnight incubation at 4°C using diluted monoclonal antibodies from Sigma (1 : 1000), the membranes were incubated with 1 : 2500 diluted horseradish peroxidase-conjugated secondary antibody (Sigma-Aldrich). The blots were then visualized using ImmunoStar HRP Substrate Kit (Bio-Rad) according to manufacturer's instructions. Relative densities of the bands were analyzed using Image Gauge v 4.0, Fujifilm. The proteins were normalized with tubulin. The expression of VEGF-A, KDR, and FLT1 in vascular samples was measured by densitometry quantitation of immunoblots. Briefly, vascular tissues were homogenized in a lysis buffer (in mM): 50 Tris-HCl, 125 NaCl, 1 EDTA, 1 EGTA, 1% Nonidet (NP-40) containing 5% complete mini-tab cocktail protease inhibitor  and centrifuged at 10000 rpm for 15 min at 4°C. Protein concentration was determined using a modified Lowry method . Then, 5Antibodies are monoclonal anti-VEGF-A, monoclonal anti-VEGF receptor 1 (FLT1), monoclonal anti-VEGF receptor 2 (KDR), diluted at (1 : 1000), polyclonal anti-tubuline (1 : 1000) (Sigma Aldrich).t-test.The data are expressed as mean ± standard error of the mean. The responses to orthovanadate, noradrenaline, and bradykinin in tumour and control vascular segments, treated and untreated with genistein or orthovanadate, were compared by two-way ANOVA followed by Bonferroni test to determine which comparisons were statistically significant. Genic and protein expression of VEGF, FLT1, and KDR in control and tumoral arteries were compared by paired Student's 3VO4); genistein -4H-benzopyran-4-one, 4,5,7-trihydroxy-isoflavone); L-noradrenaline ((1R)-4-(2-amino-1-hydroxyethyl)-1,2-benzenediol); bradykinin acetate (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg), U46619 .The compounds used were  sodium orthovanadate (Nan = 10) compared to control (n = 9) arteries (n = 4 in both groups).Stimulation with potassium chloride (50 mM) produced a similar contraction in tumoral (3.27 ± 0.22 g) and control (3.18 ± 0.28 g) arteries. Sodium orthovanadate produced concentration-dependent contraction, which was smaller in tumoral  , whereas in tumoral arteries, the contraction to noradrenaline in the segments treated with orthovanadate was smaller  than that in untreated segments.Noradrenaline also produced concentration-dependent contraction, which was similar in control and tumoral arteries  . In cont2 = 6.89 ± 0.30, n = 7 versus 7.11 ± 0.24, n = 7, resp.) ; however, in tumoral arteries, this treatment did not modify the relaxation to bradykinin . The contractile tone produced by U46619 was similar in control and tumoral arteries, with and without pretreatment with sodium orthovanadate.In the segments precontracted with U46619, bradykinin produced concentration-dependent relaxation, which was similar in the control and tumoral arteries  . TreatmeP < 0.05). We have also determined the expression of KDR and FLT1, the main receptors of VEGF-A in the arteries [P < 0.05). As a control, the expression of GAPDH was measured in parallel using the same mRNA samples (Quantitative PCR-RT analysis revealed a significant decrease in VEGF-A expression in tumor samples compared to the control (1.18 ± 0.20 versus 0.36 ± 0.06,  samples .Next we investigated the VEGF-A, FLT1, and KDR protein expression by western blot analysis. We found that protein expression of VEGF-A and FLT1 was similar in control and tumor vascular tissues. However, in samples from tumors, there is a significant increase in the protein expression of KDR .2+ influx [2+ of the intracellular contractile mechanisms [This study finds that tyrosine phosphorylation may underlie a differential regulation of vascular function in colorectal tumors. Tyrosine phosphorylation is known to produce vasoconstriction of vascular  and nonv+ influx , 25 or ichanisms , 27.Although orthovanadate contracted both control and tumoral arteries, the contraction was smaller in tumoral arteries, suggesting a reduced contractile effect of tyrosine phosphorylation in these arteries. Also, we have found that orthovanadate reduced the contraction to noradrenaline in tumoral arteries but not in control arteries. This contrasts with observations in rabbit aorta and mesenteric arteries, in which tyrosine phosphorylation increases adrenergic vasoconstriction ; therefoOrthovanadate also reduced endothelium-dependent vasodilatation in control mesenteric arteries. The effects of tyrosine phosphorylation on the endothelium may be mixed. Tyrosine kinases may enhance the release of endothelial nitric oxide , 29–33. This possible inhibitory effect of tyrosine phosphorylation in tumoral arteries may be surprising, as the activation of tyrosine phosphorylation produces vasoconstriction in normal arteries , 36–39. These results may have relevance, as the medium in the vicinity of the tumour may be rich in factors that activate tyrosine kinases and inhibitors of tyrosine kinase are used as antitumoral agents. Our results suggest that in normal arteries, tyrosine phosphorylation may produce vasoconstriction, whereas in the arteries supplying the tumor, it may produce inhibition of contraction. This may redistribute blood flow to the tumor, thus facilitating its growth.


Glycogen synthase kinase-3 (GSK-3) is a key regulator of many cellular signaling pathways. Unlike most kinases, GSK-3 is controlled by inhibition rather than by specific activation. In the insulin and several other signaling pathways, phosphorylation of a serine present in a conserved sequence near the amino terminus of GSK-3 generates an auto-inhibitory peptide. In contrast, Wnt/β-catenin signal transduction requires phosphorylation of Ser/Pro rich sequences present in the Wnt co-receptors LRP5/6, and these motifs inhibit GSK-3 activity. We present crystal structures of GSK-3 bound to its phosphorylated N-terminus and to two of the phosphorylated LRP6 motifs. A conserved loop unique to GSK-3 undergoes a dramatic conformational change that clamps the bound pseudo-substrate peptides, and reveals the mechanism of primed substrate recognition. The structures rationalize target sequence preferences and suggest avenues for the design of inhibitors selective for a subset of pathways regulated by GSK-3.DOI:http://dx.doi.org/10.7554/eLife.01998.001 Cells need to be able to respond to changes in the body, such as changes in hormone levels or the arrival of a pathogen such as a virus. Proteins acting in signaling pathways—where one protein switches ‘on’ or ‘off’ the next protein in the pathway—allow the detection of different changes or signals to be translated in the appropriate response.The properties of a protein often depend on its shape, and many proteins change shape when they are switched ‘on’ and ‘off’. Moreover, the ability to change shape allows a protein to interact with many other proteins and to be involved in many different signaling pathways.The enzyme GSK-3 is a protein that is involved in several pathways, and it controls other proteins by adding chemical tags, called phosphate groups, to them. Unlike many other enzymes, GSK-3’s default state is to be permanently switched on, and it can be switched off in a number of different ways. When a cell detects the hormone insulin, for example, another enzymes adds a phosphate group to a site near one end of GSK-3 to switch it off. Alternatively, when a cell recognises a different signaling molecule, called Wnt, a phosphate group is added to yet another protein, which then binds to and switches off GSK-3.To explore the workings of GSK-3 in greater detail, Stamos et al. solved the three-dimensional structure of the enzyme that had been switched off in these two ways. In the insulin pathway, the region near to one end of GSK-3 that contains the added phosphate group was shown to bind to and block the site on the enzyme that usually binds to its target . In the Wnt pathway, remarkably, the same site on GSK-3 was blocked in a very similar way by the piece of the other protein with the phosphate group added.GSK-3 is a potential drug target for the treatment of several diseases, such as diabetes and neurological disorders. However, as this enzyme is involved in multiple pathways, it has been hard to find drugs to treat any one condition without side effects. Uncovering subtle differences in how GSK-3 can be controlled in different pathways could, in the future, help with efforts to develop more specific drugs to target GSK-3 to treat these diseases.DOI:http://dx.doi.org/10.7554/eLife.01998.002 Glycogen synthase kinase-3 (GSK-3) is a highly conserved kinase  that in The overall structure of the GSK-3 catalytic domain is similar to other protein kinases, containing an N-terminal lobe composed primarily of β-strands in a β barrel conformation, and a C-terminal lobe composed mostly of α-helices . At the GSK-3 differs from most kinases in that it is constitutively active and controlled by inhibition, rather than by specific activation conferred by phosphorylation of the activation loop. Phosphorylation of a serine present in a conserved sequence near the amino terminus of the protein (Ser9 in GSK-3β) is the major regulatory checkpoint in most pathways, including insulin signaling . For exaGSK-3 also plays a critical role in the Wnt/β-catenin pathway. Binding of a Wnt growth factor to its cell surface receptors Frizzled and LRP5/6 results in the stabilization of the transcriptional co-activator β-catenin and activation of target genes. In the absence of Wnt, β-catenin is found in a ‘destruction complex’ that includes GSK-3, casein kinase 1 (CK1), the Adenomatous Polyposis Coli protein, and Axin. Axin has binding sites for β-catenin, GSK-3 and CK1, and thereby scaffolds efficient phosphorylation of the N-terminus of β-catenin, which marks β-catenin for ubiquitylation and proteasomal degradation . The assUpon Wnt activation, Axin-bound GSK-3 translocates to the plasma membrane, where phosphorylated peptide repeat motifs in the cytoplasmic tail of the activated LRP5/6 receptor interact with the β-catenin destruction complex to inhibit its activity . The fivA major challenge has been to understand how GSK-3 is controlled such that its effects in one pathway are insulated from others. Some specificity is achieved through association with pathway-specific scaffolding proteins that bind GSK-3 and its substrate, such as the β-catenin destruction complex, which sequester GSK-3 from other pathways and lead to highly efficient phosphorylation. However, the inhibition by the LRP5/6 cytoplasmic domain demonstrates that other modes of inhibition can operate. We set out to assess the mechanistic similarities and differences between inhibition by N-terminal phosphorylation and inhibition mediated by the phosphorylated LRP5/6 motifs.There are only a handful of crystal structures of kinases bound to peptide substrates , and onlInhibition constants of approximately 700 µM and 13 µM have been reported for the pS9 auto-inhibitory peptide and LRP6 a-motif peptide (NPPPpSPApTERSH), respectively, but these were measured with different substrates and with different assays . To compi = 1.4 ± 0.2 μM, 40x better than the pS9 peptide  forms part of the binding site for substrate peptides. In addition to the basic side chains noted above, the backbone amide of Val214 also forms a hydrogen bond with the phosphate in the priming site. To accommodate the backbone of the inhibitory peptides, the side chain of Val214 adopts a rotamer that avoids steric clash with the phosphate. Residues in the P+1, P+2, and P+3 positions  form an arch over the conserved Ile217, and the P+1 residue packs against Tyr216 (discussed in more detail below). In addition to these interactions, the LRP6 peptide complexes also reveal a water-mediated connection between the backbone carbonyl oxygens of the P+2 proline and Tyr216.Comparison of the inhibited and peptide substrate-free GSK-3 structures shows that the loop immediately preceding the C-helix, designated here as the C-loop, undergoes a large movement toward the C-terminal lobe of the kinase and clamps down on top of the bound pS9 or LRP6 peptides . The α-cIn all three inhibitory peptide complexes, Phe93 is sandwiched in between the P+2 and P+5 residues of the peptide . HydrogeThe movement of the C-loop toward the peptide is associated with a small ‘closing’ movement  of the N-terminal lobe toward the C-terminal lobe relative to the non-substrate bound structures of GSK-3 . Directl3 and a peptide , with significant increases in KM that suggest a loss of affinity for the peptide substrate. These data indicate that the interaction with Phe93 contributes strongly to substrate binding, and the reductions in kcat are consistent with the notion that peptide binding is required for formation of a catalytically competent enzyme.Approximately 50% of the total buried surface area of each inhibitory peptide is involved in interactions with the C-loop, suggesting that the C-loop is a key element of target recognition specificity. In addition to the main-chain interactions observed between GSK-3 and the inhibitory peptides, GSK-3β Phe93 packs against residues at the P+2 and P+5 positions of the inhibitor peptides. Given that the inhibitor peptides appear to bind as pseudo-substrates and stabilize an active conformation of GSK-3, we tested the importance of Phe93 for GSK-3 activity by determining steady-state kinetic parameters for wild-type GSK-3β and the F93A and F93G mutants using peIF2b as the substrate . The mutUnlike most loops in the N-terminal lobe of GSK-3, the C-loop has been highly conserved throughout evolution . The posThe interactions of the P+2 site with Phe67 and Phe93, and the packing of the P+5 residue against Phe93, are consistent with the sequence preferences of these positions seen in experimentally confirmed GSK-3 substrates . The P+2E. coli, and we do not see evidence of phosphorylation of Tyr216 by mass spectrometry of the purified protein (data not shown) or in the electron density maps. Although partial phosphorylation of this residue has been observed previously in material produced in E. coli  is present at a similar position on the pS9 auto-inhibitory peptide, which we also do not observe. It is possible that the negative charge present at this position contributes to a relatively non-specific electrostatic interaction in a flexible region. However, previous experiments indicated that there was no increase in inhibitory activity toward GSK-3 of the doubly phosphorylated LRP6 peptide vs a peptide bearing only the first phosphorylated S/T . The secGSK-3 represents an important drug target for a variety of diseases, including diabetes, Alzheimer’s disease, and some cancers . InhibitGST-Axin-GBD (GSK-3 Binding Domain) fusion protein was constructed by inserting the region encoding residues 383–402 of human Axin into a modified pGEX-KG vector containing a TEV cleavage site between the GST protein and the insert. The use of EcoRI at the 5′ end for the cloning inserts the sequence GGIL between the TEV cleavage site and residue 383 of Axin.Human LRP6 c- and e-motif–mouse GSK-3β  fusion proteins were constructed by replacing the first 12 residues of GSK-3β with 8 residues of the motif (PPPPTPRS for c-motif or PPPPSPCT for e-motif) by PCR and extending through residue 383 of GSK-3β. The construct also contains a non-cleavable 6xHis tag fused directly to the C-terminus of GSK-3β via PCR. This gene was then cloned into pET29b(+) using NdeI-NotI restriction sites.E. coli via the pET system in the pET21 vector . Purification was carried out with Ni-NTA agarose beads  according to the manufacturer’s protocol. Purified protein was stored at −20°C in 40% glycerol prior to use.A shortened form of CK1ε (residues 1–321) was constructed with a 6x-His tag fused directly to the C-terminus via PCR, and expressed in 595 of 1.0–1.2 at 37°C. Cultures were then cooled to 16°C and induced with 0.1 mM IPTG. Fresh 100 μg/ml ampicillin was added and the cultures grown for 24 hr. Cell pellets were harvested via centrifugation and stored at −80°C until purification.Expression vectors for LRP6–GSK-3 fusion proteins, GSK-3 1-383, or GSK-3 26-383  were co-transformed with GST-Axin-GBD into chemically competent BL21(DE3) Codon-plus RIL cells  and plated onto LB plates with 50 μg/ml kanamycin and 100 μg/ml ampicillin. A single colony was used to inoculate a 2L LB shaking culture containing the same antibiotics as listed above and grown to an approximate ODg for 1 hr at 4°C to remove insoluble material. The clarified lysate was then added to a 10 ml bed volume of Glutathione-Agarose beads (Sigma) pre-equilibrated in Lysis Buffer and run under gravity flow. The beads were then washed with 200 ml of Wash Buffer containing 20 mM Tris pH 7.5, 300 mM NaCl, 5% glycerol, and 0.1% β-mercaptoethanol. The GSK-3/GST-Axin complex was then eluted from the beads by adding 6 × 5 ml aliquots of Wash Buffer plus 20 mM reduced L-glutathione (pH adjusted to 7.5 with NaOH). Elution fractions were analyzed by SDS-PAGE. TEV protease was added to the fractions containing the GSK-3/GST-Axin complex and allowed to incubate overnight at 4°C. Removal of the GST tag was confirmed by SDS-PAGE and samples were run over PD-10 columns pre-equilibrated with Wash Buffer according to manufacturer’s protocol  to remove glutathione at room temperature. Samples were then passed over 10 ml of glutathione-agarose beads again and the flow-through collected in order to remove cleaved GST and any uncleaved GST-Axin complex. The resulting GSK-3/Axin complex was >95% pure as analyzed by SDS-PAGE, and its concentration was approximately 0.3–0.5 mg/ml.Cell pellet from 4 l of culture was lysed in 40 ml of cold Lysis Buffer containing 20 mM Tris pH 7.5, 300 mM NaCl, 5% glycerol, 0.01% Triton X-100, 1 mg/ml lysozyme, 5 mg/l DNaseI , EDTA-free Protease Inhibitor Cocktail , and 0.2 mM PMSF with an Emulsiflex homogenizer . Lysate was centrifuged at 39000×2 overnight at 4°C, then for 2 hr at room temperature. Removal of the lambda phosphatase, TEV cleavage, and the rest of the purification were carried out as for the AKT-phosphorylated GSK-3β 1-383. Analysis of the purified protein by western blotting showed little phosphorylation on pS9 compared to the protein before phosphatase treatment (data not shown).GSK-3β 1-383 used for the kinetics assays was produced by incubating 10 ml of the uncleaved protein with 16,000 units of lambda phosphatase  and 2 mM MnCl2, and 400 μM ATP (inhibitor-free structure), 30 μg of active AKT (Millipore), 10 mM MgCl2, and 200 μM ATP (pS9 inhibitory peptide), or 50–100 μg of purified CK1, 10 mM MgCl2, and 200 μM ATP (LRP6 c- and e-motif structures) at room temperature and injected into a 3–12 ml size Slide-a-Lyzer cassette . The cassette was placed into a 250 ml reservoir solution of 10% PEG 35,000, 20 mM Tris pH 7.5, 300 mM NaCl, 5% glycerol, 10 mM MgCl2, 200 µM ATP, and 5 mM DTT at room temperature to facilitate phosphorylation, then incubated at 4°C. Crystals formed inside the cassette after approximately 72 hr. To form the pS9–AlF3 complex, the cassette was transferred to the same solution, with 200 μM ADP, 200 μM MgCl2, 200 μM Al(NO3)3, and 1.2 mM NaF replacing the ATP, and incubated for 1 week at 4°C. Crystals ranging from 20 to 750 µm in diameter were harvested by excising one side of the dialysis membrane on the cassette with a razor blade and adding a cryoprotectant solution composed of 10% PEG 35,000, 20 mM Tris pH 7.5, 300 mM NaCl, 20–22% glycerol, 10 mM MgCl2, 200 µM ATP 3, and 1.2 mM NaF were used instead of ATP) and 5 mM DTT directly to the inside of the cassette. Crystals were mounted on cryoloops  and frozen directly into liquid nitrogen.Crystallization of the various GSK-3/Axin complexes was accomplished by dialysis. Purified GSK-3/Axin complex (12 ml) was combined with 20 mM MgClpS9) used in kinase assays was produced by incubating GST-Axin/GSK-3β 1-383 with 15 µg AKT (Millipore) for approximately 48 hr in a dialysis cassette as described for crystallization, but prior to TEV cleavage. The protein solution was removed from the cassette when its volume reached ∼1 ml and diluted back to 12 ml with wash buffer supplemented with 10 mM MgCl2 and 200 µM ATP. The AKT was removed by applying the solution to glutathione-agarose beads. TEV cleavage and subsequent purification then proceeded as described above. Analysis of the purified protein by mass spectrometry showed that Ser9 was 24% phosphorylated.GSK-3β 1-383 phosphorylated at Ser9 (GSK-3β 1-3831/2 was approximately 0.5 and Mn(I/σ) was approximately 1.3 or better in the best direction . The structures were solved and refined in the Phenix package  beamlines 12-2 or 11-1, or beamline 23ID-B of the Advanced Photon Source. Integration of images was performed with XDS , and sca package . The LRP in Coot . Data co32P into a primed, synthetic eIF2b peptide . A typical reaction mixture contained 50 mM Tris pH 7.5, 10 mM MgCl2, 0.1% β-mercaptoethanol, and 112.5 μM γ32P-ATP at 0.02 μCi/μL  and 224 nM GSK-3β. All measurements were performed at 30°C. The substrate peptide concentration was 20 μM or 80 µM for the inhibition assays and was varied for the determination of KM and kcat values. Inhibitor peptide was added at concentrations ranging from 50–1000 μM for the pS9 peptide and 5–20 μM for the LRP6 a-motif. For each measurement, a 20 μl sample was removed from the reaction and mixed immediately with 20 μl of 150 mM phosphoric acid. 35 μl of the resulting solution was then blotted onto a 96-well plate containing P81 cellulose phosphate paper and washed 5 or 10 times with 0.5% phosphoric acid. Scintillation fluid was then added, and the plate was counted in a Wallac MicroBetta 1450 liquid scintillation counter. Control reactions performed in the absence of substrate peptide showed that signal from GSK-3 autophosphorylation was not significant. Rates were calculated from the slope of the linear regression fit of 32P incorporation vs time.GSK-3β activity was measured by incorporation of i values were calculated from initial rates by the method described in i was calculated from the equation0 is the rate in the absence of inhibitor, vi is the rate in the presence of the inhibitor, [I] is the concentration of the inhibitor, and Kiapp is the apparent Ki. The actual Ki was then calculated from Kiapp asKi values obtained from each independent experiment (n = 27 for pS9 and n = 11 for LRP6 a-motif) were averaged to obtain the reported value of Ki, with the error propagated from the errors in v0, vi, and KM.Individual K review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  X-ray crystal structures of the important signaling kinase GSK3. These structures are noteworthy as they contain phosphorylated inhibitory motifs from the N-terminus of GSK3 itself or derived from the Wnt signaling pathway protein LRP6. These structures reveal that the inhibitory motifs bind as predicted for primed pseudosubstrates to the enzyme in its active conformation. A special feature of the complex is the presence of an extensive interaction between the C-loop and the substrate/inhibitory motif which undergoes a significant conformational change compared to the inhibitor-free structure. These structures may also help us understand how GSK3 substrate recognition occurs and in fact how Tyr216 phosphorylation may modulate substrate specificity. This study gives insight into how Tyr216 phosphorylation can modestly activate the kinase. The conserved nature of GSK3 Phe93, from the C-loop, is now explained and tested here by mutagenesis, showing its importance. A role of a conserved Arg in GSK3 interacting with the phospho-site of the primed substrate is suggested. Some technically impressive aspects of the study include the use of chimeric LRP6-GSK3 to capture the inhibited conformation and the use of kinases to obtain cleanly phosphorylated GSK3 for crystallization, and the use of ALF3- to visualize the “transition state”. Overall, this is a well-done and significant manuscript that should be of broad interest to Principal concerns to be addressed:cat values, in the range of 0.04 s-1 (-1). It would be helpful if the authors could compare their rate constants with any other GSK3 rate constants recorded in the literature to see if this has been found in other labs. The background phosphate values (time zero) are quite high at low inhibitor peptide as shown in M determinations? It doesn't look like the authors used a standard model for substrate competitive inhibition to fit their data for inhibition by the pS9 and LRP6 peptides. Was non-linear regression used? What software? What inhibition model?1) There were several concerns raised about the kinetic measurements which should be addressed. The k0.04 s-1  are on t2) It does not appear that the catalytic activity of the phospho-GSK3 enzymes were measured (or at least they are not reported). These would presumably show reduced activity compared to the non-N-terminally phosphorylated enzyme but it would be nice to demonstrate this and report the quantitative data. If the activity of these forms is below the level of detection, that could be stated (and the detection threshold mentioned).Additional comments for consideration:- While it certainly seems reasonable that the present work provides insight regarding recognition of substrate peptides , it may be excessive to claim that the structures presented here reveal the basis of primed substrate recognition. These peptides are inhibitors, not substrates, and indeed it seems that none of the structures reveal an ordered residue in the position expected for a phosphoacceptor. Further, intermingled discussion of the peptides as inhibitors and substrates is potentially confusing to the reader. Our advice would be to discuss implications for substrate recognition in a separate section, with clear acknowledgement of the limitations of the interpretation.- The authors refer to the phosphorylated residue as P+4. This is understandable, given the P+4 priming phenomenon. However, is this a structurally sound interpretation? In the superposition with PKA bound to PKI, does the “P+1” in the peptides studied here superimpose with the P+1 in PKI? Are there backbone hydrogen bonds between peptide and activation loop in common between the structures described here and other kinase/peptide substrate complexes that allow the authors to define the register and conclude that the phosphorylated Ser/Thr is indeed in a P+4 position.- Were the C-loop mutants studied in the context of inhibition by LRP6 or pSer9?- The authors state that this is the first kinase complexed to a primed substrate. While this may be true for Ser/Thr kinases, the authors should cite the following, “Structural recognition of an optimized substrate for the ephrin family of receptor tyrosine kinases” :4395-404), as a structure that shows a pTyr primed substrate complexed to an ephrin receptor kinase.- The authors state that, “Most kinase inhibitors in use today stabilize the inactive conformations...” While lapatinib, sorafenib, and imatinib are examples of this, erlotinib, gefitinib, dasatinib, stutent, and vemurafinib bind to active conformations of kinases. Thus, we would suggest modifying this point.- Is it clear from the structures that an N-tail from one enzyme is not extending into the active site of a neighbor in the crystal lattice (ie intermolecular interaction)? The authors could comment on this. If there is any doubt in solution, they could measure the rate of GSK3 kinase activity at different phospho-GSK3 concentrations.- The authors state in their Abstract and ultimate sentence that their results will aid design of GSK-3β inhibitors that are selective for specific pathways, but it is not obvious how. The authors may wish to better flesh out this claim or consider omitting it. Principal concerns to be addressed:1) There were several concerns raised about the kinetic measurements which should be addressed. The kcatvalues, in the range of 0.04 s-1 are on the low side compared to many other active kinases . It would be helpful if the authors could compare their rate constants with any other GSK3 rate constants recorded in the literature to see if this has been found in other labs.cat values  smaller by a factor of two.Note: Some small errors in data analysis were corrected in the revised manuscript. The final values of kinetic constants have changed slightly, but the conclusions are unaffected. In particular, the LRP6 a-motif peptide inhibits GSK-3 approximately 40x more strongly than the S9 peptide, rather than 16x reported in the previous version. The other notable error was in conversion of rates from pmol/sec to 1/sec, which resulted in kn-4 sites). Nonetheless, we can compare values from the handful of studies that report kinetic constants in the table below.The activity is indeed considerably lower than PKA and some other kinases. There have been very few measurements of rate constants reported for GSK-3, as the vast majority of studies use simple qualitative endpoint assays, for example western blotting to check for formation of phosphorylated substrates, to measure activity. Also, as we note in the text, direct comparison with other GSK-3 studies is not straightforward because different groups have used different substrate sequences, most of which are processive . Fiol et al (1987) also gives a kcat for what the authors later determined is rabbit GSK-3α  that is more than an order of magnitude lower than ours . Dajani et al. also reported a kcat of 0.7, but no units were provided. The assay referenced in that paper uses rate units of pmol min-1, so if we assume that the kcat is reported in units of min-1 then the value is 0.012 s-1, somewhat smaller than our value of 0.079 s-1.Fiol et al (1987 and 1990)  Wang et al (1994) [3] and Dajani et al (2003) [4] all give KReferences:262, 14042-14048.1. Fiol, C.J., Mahrenholz, A.M., Wang, Y., Roeske, R.W., and Roach, P.J. (1987). Formation of protein kinase recognition sites by covalent modification of the substrate. Molecular mechanism for the synergistic action of casein kinase II and glycogen synthase kinase 3. J Biol Chem 265, 6061-6065.2. Fiol, C.J.; Wang, A.; Roeske, R.W.; and Roach, P.J. (1990). Ordered Multisite Protein Phosphorylation. Analysis of glycogen synthase kinase 3 action using model peptide substrates. J Biol Chem 269, 14566-14574.3. Wang, Q.M., Fiol, C.J., DePaoli-Roach, A.A., and Roach, P.J. (1994). Glycogen synthase kinase-3 beta is a dual specificity kinase differentially regulated by tyrosine and serine/threonine phosphorylation. J Biol Chem 22, 494-501.4. Dajani, R., Fraser, E., Roe, S.M., Yeo, M., Good, V.M., Thompson, V., Dale, T.C., and Pearl, L.H. (2003). Structural basis for recruitment of glycogen synthase kinase 3β to the axin-APC scaffold complex. EMBO J The background phosphate values (time zero) are quite high at low inhibitor peptide as shown inand get lower with increasing inhibitor peptide additions. It would be helpful if the authors could comment on why this might be the case and also indicate how they dealt with this in calculating rates. For example, were the rates based on slopes (probably best) or were they product/time without background subtraction? Why such large error bars for the KMdeterminations? It doesn't look like the authors used a standard model for substrate competitive inhibition to fit their data for inhibition by the pS9 and LRP6 peptides. Was non-linear regression used? What software? What inhibition model?32P incorporated into the substrate peptide versus time. Unfortunately the solid black symbols for the inhibitor free data in the original Rates were calculated as the slopes of M measurements. First, filter binding is itself a somewhat noisy assay, and individual rates were not always perfectly consistent across different days. Second, the inherent noise in the assay was compounded for the mutants by the low signal.There are two principal reasons for the large error bars in the KAs described in the Materials and methods section, the inhibition constants were obtained using the method in the 2) It does not appear that the catalytic activity of the phospho-GSK3 enzymes were measured (or at least they are not reported). These would presumably show reduced activity compared to the non-N-terminally phosphorylated enzyme but it would be nice to demonstrate this and report the quantitative data. If the activity of these forms is below the level of detection, that could be stated (and the detection threshold mentioned).This proves to be an extremely difficult experiment, which is probably why it has not been reported previously despite 20 years of knowledge that phosphorylation of Ser9 inhibits GSK-3β activity. The reviewers reasonably assumed that this would be straightforward given the crystal structures, but in fact is very difficult to obtain 100% phosphorylated material. We attempted in vitro phosphorylation using AKT for native GSK-3, or CK1 for the LRP6 chimeras, but we obtained only a few percent phosphate incorporation (measured by mass spectrometry). As GSK-3 is not efficiently phosphorylated, the extreme concentration of GSK-3 along with AKT (or CK1 for the LRP6 chimeras) produced by dialysis against high molecular weight PEG enabled sufficient production of the phosphorylated enzyme for crystallization. Note that although the peptide is phosphorylated in the structure, we did not know what fraction of the total enzyme input to crystallization was phosphorylated.To address the reviewers’ request, we used the crystallization protocol to produce phosphorylated full-length GSK-3β, but halted the dialysis prior to crystallization. Mass spectrometry showed that this material is 24 % phosphorylated. The partially phosphorylated material shows poor activity relative to the non-phosphorylated enzyme . Although we cannot produce fully phosphorylated material, the experiment clearly shows that the in vitro phosphorylated material is inhibited. This is now described in the text and shown in Given the clear data reported here and earlier on inhibition by synthetic phosphorylated peptides in trans, we do not feel that it would be worth the considerable costs in time and money needed to repeat this experiment with the LRP6 chimeras.Additional comments for consideration:- While it certainly seems reasonable that the present work provides insight regarding recognition of substrate peptides , it may be excessive to claim that the structures presented here reveal the basis of primed substrate recognition. These peptides are inhibitors, not substrates, and indeed it seems that none of the structures reveal an ordered residue in the position expected for a phosphoacceptor. Further, intermingled discussion of the peptides as inhibitors and substrates is potentially confusing to the reader. Our advice would be to discuss implications for substrate recognition in a separate section, with clear acknowledgement of the limitations of the interpretation.We agree that the distinction of inhibitor and true substrate peptides was muddled in the original manuscript. The point was that comparison with a known active kinase like Thr197-phosphorylated PKA shows that the enzyme itself is in an active conformation, indicating that the inhibitors work as pseudo-substrates. We have carefully maintained this distinction in the revised paper. Given these structural data, as well as the comparison with known true substrate sequences (see next point), it is very likely that the binding mode visualized in these structures represents the mode of binding of true primed substrates. Moreover, the effect of F93 mutation clearly shows that the interaction with the C-loop is relevant to the action of the kinase on a true substrate.- The authors refer to the phosphorylated residue as P+4. This is understandable, given the P+4 priming phenomenon. However, is this a structurally sound interpretation? In the superposition with PKA bound to PKI, does the “P+1” in the peptides studied here superimpose with the P+1 in PKI? Are there backbone hydrogen bonds between peptide and activation loop in common between the structures described here and other kinase/peptide substrate complexes that allow the authors to define the register and conclude that the phosphorylated Ser/Thr is indeed in a P+4 position.We are not entirely sure about the reviewers’ question as to whether this is a “structurally sound interpretation”. The P+4 assignment is based on the known properties of GSK-3 substrates. The electron density of the peptide is well defined at this resolution, so there is no ambiguity regarding the direction of the polypeptide nor the visible side chain assignments. We have now added a comparison to PKA:PKI and other kinases bound to substrates, showing that the orientation and structures of these other kinases in the P+1 site is similar text and . This fu- Were the C-loop mutants studied in the context of inhibition by LRP6 or pSer9?No. The F93 mutation was made to show that this residue interacts with real substrates as part of catalysis, supporting the claim that the structures visualize an active conformation and that the inhibitors are pseudo substrates. Given the poor activity of 5 the F93 mutants, it is doubtful that we would be able to detect further inhibition by the peptides.- The authors state that this is the first kinase complexed to a primed substrate. While this may be true for Ser/Thr kinases, the authors should cite the following, “Structural recognition of an optimized substrate for the ephrin family of receptor tyrosine kinases” :4395-404), as a structure that shows a pTyr primed substrate complexed to an ephrin receptor kinase.Thank you for this suggestion; we have now cited this paper in the Introduction.- The authors state that, “Most kinase inhibitors in use today stabilize the inactive conformations...” While lapatinib, sorafenib, and imatinib are examples of this, erlotinib, gefitinib, dasatinib, stutent, and vemurafinib bind to active conformations of kinases. Thus, we would suggest modifying this point.Thank you, we have clarified this point in the last paragraph.- Is it clear from the structures that an N-tail from one enzyme is not extending into the active site of a neighbour in the crystal lattice ? The authors could comment on this. If there is any doubt in solution, they could measure the rate of GSK3 kinase activity at different phospho-GSK3 concentrations.Addressing whether the S9 interaction is truly autoinhibitory versus trans-inhibitory is an interesting question, but this was not our focus as we are interested in comparing the interaction of the N-terminal peptide (presumed to be cis) with the LRP6 peptides, which are of course part of a different molecule in vivo. To answer the reviewer’s question, we cannot rule out that the N-tail of one molecule is binding in the active site of another in the crystals, as the connection is disordered and the arrangement of molecules in the lattice might allow this to happen. We are also somewhat restricted in the range of concentrations to test GSK-3 activity due to the signal-to-noise of the assay at low concentrations and the need to keep GSK-3 soluble and to maintain steady-state conditions at high concentrations. We have noted in the Discussion that there is a “presumed” kinetic advantage of having the inhibitory peptide in cis in GSK-3.- The authors state in their Abstract and ultimate sentence that their results will aid design of GSK-3β inhibitors that are selective for specific pathways, but it is not obvious how. The authors may wish to better flesh out this claim or consider omitting it.We have clarified this proposal in the last paragraph.


Metal nanosolicoparticles are suspected to cause diseases in a number of organisms, including man. In this paper, we report the effects of nanosilver  on the early development of the zebrafish, a well-established vertebrate model. Embryos at the midgastrula stage were exposed to concentrations ranging from 100 to 0.001 mg/L to verify the effects on different endpoints: lethality, morphology, expression of cholinergic molecules, and development of the immune system. (1) Relative risk of mortality was exponential in the range between 0.001 and 10 mg/L. Exposure to 100 mg/L caused 100% death of embryos before reaching the tail-bud stage. (2) Developmental anomalies were present in the 72 h larvae obtained from embryos exposed to nanosilver: whole body length, decreased eye dimension, and slow response to solicitation by gentle touch with a needle tip, with a significant threshold at 0.1 mg/L. (3) Dose-dependent inhibition of acetylcholinesterase activity was significant among the exposures, except between 1 mg/L and 10 mg/L. (4) The distribution of CD41+ cells and of CDF/LIF-like immunoreactivity was altered according to the Ag concentration. The possible effect of nanosilver in impairing immune system differentiation through the inhibition of molecules related to the cholinergic system is discussed. One of the important problems of theoretical and practical medicine and physiology is the study of the responses of the organisms to environmental cues, with the goal of enhancing prevention of the main diseases induced by environmental stress.An emerging risk is represented by the wide diffusion of nanoparticles, such as the silver nanoparticles, which were among the first metal nanoparticles to reach the market. Manufacturers have exploited their exceptionally efficient antibacterial, antiviral, and antifungal activity , 2. As rThe first thoroughly experimented studies regard the health effects of airborne nanoparticles , while vBrachydanio rerio (Danio rerio), as a vertebrate model to test the effects of nanosilver in the aquatic environment. In this work, we will use the abbreviation ZF for zebrafish. During the last decades, this fish has been chosen as a reliable vertebrate model for understanding basic events in developmental biology.In this work, we have used the first developmental stages of the cyprinid zebrafish, This model possesses a high degree of homology to the human genome , is easyIn this model, the effects of exposures to nanosilver have been studied by several authors: silver gills toxicity was demonstrated to be effect of particulates as opposed to release of dissolved ions , 10. BarIn aquatic organisms, pollutants are taken up by entering food, gills, and skin and rapidly are diffused to blood and distributed to all the body regions, as demonstrated in sea urchin model : ingesteFor authors working on human health and mammalian models, the relationships between potentiation against impairment in immune system exerted by nanosilver are still controversial , 13. We In this light, we also identified CD41 immunoreactive (IR) cells, which have been reported to be responsible to generate RAG2+ T lymphocyte precursors  as well Danio rerio were purchased from a commercial source and reared in aquaria with 15 L ultrafiltered (mesh = 0.22 μm) freshwater. The animals were regularly fed and maintained at temperature ranging between 24 and 28°C, with natural light/dark cycle.Adult specimens of T = 28°C.The eggs, spawned and fertilized at the first daylight, were laid in trays covered with glass spheres at the bottom of aquaria. The glass spheres were used to prevent predation of eggs and embryos by adult specimens. Fertilized eggs and embryos were collected and allowed to further develop for 12 h at Ag NPs were obtained from Polytech , supplied as a 1000 ppm in deionized water suspension of metallic silver (Ag particles encapsulated in liposomes) with an NP size between 1 and 10 nm. This “nanosuspension” did not need further sonication, since it was very stable and it did not form any agglomerates . Size raAt 12 h dead and anomalous embryos were discarded, and the healthy ones were divided into multiwell dishes containing nanosilver particles (Ag) suspended in ultrafiltered freshwater at concentrations ranging from 100 to 0.001 mg/L.Control eggs were maintained in ultrafiltered fresh water for the time of the experiment. T = 25°C and the control and exposed specimens were allowed to develop for further 24 and 48 h, up to hatching. All the dishes were placed in a thermostat at After those times, the survived larvae were counted and measured. Developmental anomalies were registered and classified.After incubation, the living larvae were fixed in paraformaldehyde (PFA) 3% in phosphate buffer saline (PBS) + 70% cold methanol for 20 min and then rinsed and rehydrated in 0.1% BSA/PBS for 10 min before processing for immunohistochemical reactions.T = 5°C in the primary antibodies diluted 1 : 200 in PBS containing 0.5% BSA, 0.1% NGS. The primary antibodies were anti-leukemia inhibitory factor (CDF/LIF), raised in goat , or anti-CD41  raised in mouse. After rinsing in PBS/BSA, the samples were incubated overnight at 5°C in the secondary antibodies , 1: 300 in PBS/BSA. Nuclei were counterstained with 1 μM RNAase followed by 2 μM propidium iodide (PI). The preparations were mounted on a slide with the antifading gelvatol [Samples were incubated overnight at μL for each vial) were frozen in 100 μL of 0.1 M phosphate buffer, pH7 for 24 h. Then, they were brought to the room temperature and homogenate in an ice bath by use of a minipotter homogenisator (Braun-Melsungen). The protein content of each sample was determined by the Bradford method. Of these, a quantity corresponding to 30 mg protein was used for the measurement of ChE. The lecture of the speed of substrates cleavage was performed in 0.1 phosphate buffer, pH 8, in the presence of dithiobis nitrobenzoic acid (DTNB) and bicarbonate, according to the quantitative method suggested by Ellman et al. [λ = 412 nm. The absorbance kinetics were recorded for 3 min, for at least 15 homogenate fractions/each exposure. The amount of cholinesterase units (ChE U) was related to the protein content of each sample (30 mg) previously measured by the Bradford method. (one ChE U hydrolyzes 1 μmole of substrate per min at pH 8.0 at 37°C).The control and exposed larvae (a dry volume of 100 β-methyl)thiocholine iodide (AMTCh), 1,5-bis-pentan-3-one dibromide (BW284C51), Tetraisopropyl pyro phosphoramide (iso-OMPA), propionylthiocholine iodide (PTCh), S-butyrylthiocholine iodide (BTCh), 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), and choline chloride were purchased from Sigma.Acetylthiocholine iodide (ATCh), acetyl controls were performed by omitting the primary antibody. Aspecific reactions were blocked by use of BSA, NGS, as above described.βMeTCh and BuSCh were selected as diagnostic substrates selective for ChE, AChE, and BChE, respectively [−4 M BW284c51 (specific inhibitor of AChE), 10−4 M eserine (inhibitor of AChE and pseudocholinesterases), and 10−4 M iso-OMPA . All the inhibitors were obtained by Sigma (IT).The specificity of ChE enzyme activity was checked by AcTCh, and Acectively . A furthectively  medium tAlthough the statistic elaboration of mortality data (ANOVA) did not show significant dose dependence, nevertheless a strong trend was seen for the effects exerted by the exposure to the different Ag concentrations. Thus, we calculated the relative risk (RR), showing the probability of death occurring in the exposed group versus te control cohort of larvae. RR shows the probability of death in the exposed groups versus nonexposed groups . With tha = 0.01, in order to make up for the increased likelihood of type 1 error [For homogeneous measures , a one-way ANOVA was performed to test for differences among the effects of different nanosilver concentrations. Prior to running analyses, homogeneity of variances was tested by Levene's test; whenever necessary, data were transformed and retested. When transformation did not produce homogeneous variances, we set  1 error . Post ho 1 error .The occurrence of dead embryos for each exposure was different among the embryos exposed to the different Ag concentrations, with a trend to increase from control to the more concentrated exposures, but no homogeneity among the different groups of embryos was present. The RR elaboration of the data showed an exponential risk probability of about 1.2-folds with respect to the control for the exposure to 0.001 mg/L, average risk of 1.7-folds for the embryos exposed to 0.01 mg/L, 0.1 mg/L, and 1 mg/L, and a risk of 2.56-folds for the exposures to 10 mg/L Ag concentration . The expP ≪ 0.01) threshold of effect at 0.1 mg/L, as shown in Figures Exposed samples presented some developmental anomalies, including scarce reactivity to stimulation exerted by the gentle touch of a needle tip. If after the third touch they did not move or their swim was inefficient, we marked a no-reply in the excel column used for registration. This feature was not dose dependent: control = 17%: 0.001 = 44%; 0.01 and 0.1 mg/L = 0%; 1 mg/L = 75% and 10 mg/L = 100%. The anomalies were classified as anomalous shape of the vertebrate column, anomalies in the otoliths shape and size (not shown). The most measurable effects were represented by the larval length  and by tP < 0.01), in a dose-dependent way, except between the exposures to 1 and 10 mg/L Ag. Both acetylcholinesterase and propionylcholinesterase activities were decreased by exposures; PChE activity was always lower than that AChE . The AChP < 0.01), and no significant difference was present among 0.1, 1, and 10 mg/L exposures (Pseudocholinesterase (PChE) activity, measured by cleavage of PTChI, showed nondose-dependent decrease caused by exposure to crescent Ag concentrations, as 0.001 mg/L and controls did not differ in a significant way, while the exposure to 0.01 and 0.1 mg/L caused significantly different impairment (xposures . −4 M iso-OMPA affected the speed by a negligible percentage (activity reduced by 9.6% in controls). The inhibition percentages exerted by BW284 c51 decreased with Ag concentration, suggesting an increased amount of BChE in parallel with the decrease of AChE activity (Acetylcholinesterase activity measured by cleavage of AcTChI was impaired by BW284c51 (activity reduced by 90% in controls), while 10activity .In control samples, the CDF/LIF-like fluorescent immune reaction marked a complex net of vessels in the head, in the thymus (not shown), and the main trunk vessels. For simplicity, we showed only the trunk and tail regions, containing the supraintestinal (SILV), the dorsal longitudinal (DLLV), the paracordal (PCLV), and the intersegmental lymphatic vessel (ISLV) . In conFigures Cells showing IR to CD41 were present in the lymphatic vessels of controls and of the larvae obtained from embryos exposed to 0.001 and 0.01 mg/L Figures , includiP < 0.001) responses, as compared to controls, followed by the effects of the exposures to 1 mg/L and 10 mg/L. This suggests the presence of thresholds for the Ag effects, probably corresponding to stages of increased catalytic activity. At these concentrations, the total area of the particles surface would reach the ability to interfere in the normal chemical reactions of the organisms, such as for instance the activity of key enzymes. These thresholds were rather congruent, although slightly different among the different endpoints. The effects found in exposed larvae were mainly due to the particulate nanosilver suspension other than to the possible ionic component. Although no news are available about the Ag(+) component, the encapsulation in liposomes prevents direct contact with water. Besides, pH of freshwater is neutral, and ionic transformation is proportional to the alkalinity of the medium, as reported by [http://stud.epsilon.slu.se/2358/1/geranmayeh_oromieh_a_110315.pdf). Our results show a toxic effect and a death risk exerted by nanosilver exposure, inhibition of AChE and PseudoChE activity, and impaired recruitment of T-lymphocytes (CD41+ cells). The lethal effects showed a random occurrence, so that lethal toxicity was only demonstrated for the Ag suspension of 100 mg/L. Nevertheless, the risk of death showed a trend growing exponentially with the increasing concentration of the Ag suspension. As it concerns the morphophysiological and enzymatic responses to exposure, the controls and the samples exposed to 0.001 mg Ag/L generally presented similar effects, while the exposures to 0.01 and 0.1 mg/L caused statistically significant  or electrical events. In particular, the electrical events are related to the IP3 release and calcium dynamics (m1 and m3 AChRs) or to K+ outwards fluxes (m2 and m4 AChRs). In addition, AChE has been demonstrated to be a modulator of a number of nonneuromuscular events leading embryonic development [In vertebrates, acetylcholinesterase plays a key role in the modulation of neuromuscular impulse transmission, while pseudocholinesterases are mainly represented in blood plasma . The rolelopment , is a weelopment , is invoelopment , 36, modelopment  and cellelopment , and regelopment  and inflelopment . In theselopment  the actielopment .At the studied ZF stages, ChE activities in the zebrafish seem to be mainly represented by AChE  and, at a minor extent, by pseudocholinesterases, such as PChE, able to cleave propionylthiocholine iodide. Butyrylcholinesterase  is scarcely present at these developmental stages as shown by the scarce inhibition of AcTChI cleavage exerted by iso-OMPA in controls  but seemThe alteration found in the AChE and pseudoChE activities might be involved in the morphological and physiological responses of the zebrafish larvae. Actually, during the last 40 years, awareness is increased of the AChE's role in regulating cell-to-cell communications such as the ones occurring during embryonic development , 39, forThe distribution of AChE activity, as compared to the CDF/LIF pattern, is not completely overlapping, because in the head AChE activity is not present, except in the main sense organs Figures . In the The small size of the NPs, ranging between 1 and 10 nm, may be responsible for enhanced penetration in cells and tissues, reaching the hematopoietic tissues, directly interfering in the cell-to cell communication modulated by cholinesterase activity, and impinging on differentiation of immune cells during the first developmental events of the zebrafish. The zebrafish CD41(+) cells are known to be involved in the immune system differentiation, because, after the first wave of aorta-gonad-mesonephros emopoyesis, these cells colonize the thymus to generate rag2(+) T-lymphocyte precursors . These eα7 nicotinic receptors in macrophages have been proposed to play a role in neuroimmunomodulation of resistance and relief in mammalian inflammatory processes [The relationship between AChE activity and blood  and in trocesses . In the rocesses –19. The decrease of AChE and PChE activity at doses from 01, 1, and 10 mg/L corresponding to the doses inhibiting the presence of CD41 IR cells suggests a possible function of these enzymes also in the regulation of thymocytes recruitment during early zebrafish development, or as suggested by Koelle et al. , pseudoCThe effects of exposure to NPs show a trend to impairment of immune responses, possibly related to the degree of inhibition of AChE activity. This hypothesis paves the way to further studies on the presence of molecules related to the cholinergic system in the immune cells of different organisms and the competitive effects possibly exerted by the Ag NPs. The general mechanism of action seems to be represented by a depression of the immune system, as previously stated by Falugi and Aluigi  in sea uAs reported by different authors, the mechanism of action of the nanoparticles is largely unknown: in particular, it is not clear which are the NP characteristics triggering the organismic responses . In gene